Community-Acquired Pneumonia (Birkhäuser Advances in Infectious Diseases)

Birkhäuser Advances in Infectious Diseases BAID

Series Editors Axel Schmidt University Witten/Herdecke Faculty of Medicine Alfred-Herrhausen-Str. 50 58448 Witten Germany Stefan H.E. Kaufmann Max-Planck-Institut für Infektionsbiologie Department of Immunology Schumannstrasse 21/22 10117 Berlin Germany

Manfred H. Wolff University Witten/Herdecke Faculty of Biosciences Stockumer Str. 10 58448 Witten Germany

Community-Acquired Pneumonia Edited by N. Suttorp, T. Welte and R. Marre

Birkhäuser Verlag Basel Boston Berlin

·

·

Editors Norbert Suttorp Charité – University Medicine Berlin Dpt. of Internal Medicine Augustenburger Platz 1 D-13353 Berlin Germany

Reinhard Marre University of Ulm Medical Microbiology and Hygiene Robert-Koch-Str. 8 D-89081 Ulm Germany

Tobias Welte Medizinische Hochschule Hannover Carl-Neuberg-Str. 1 D-30625 Hannover Germany

A CIP catalogue record for this book is available from the library of Congress, Washington, DC, USA Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de

ISBN 3-7643-7562-0 Birkhäuser Verlag, Basel - Boston - Berlin The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2007 Birkhäuser Verlag, P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TFC ' Cover illustration: Colored transmission electron micrograph of Mycoplasma pneumoniae (green) demonstrating flask-shaped morphology. The background shows a chest x-ray from a patient. With the friendly permission of Kristen L. Hoek, Vanderbilt University Medical Center, Nashville, and Matthias Krüll. Printed in Germany ISBN-10: 3-7643-7562-0 e-ISBN-10: 3-7643-7563-9 ISBN-13: 978-3-7643-7562-1 e-ISBN-13: 978-3-7643-7563-8 987654321 www. birkhauser.ch

Contents List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Preface

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ix

Tobias Welte Diagnosis and treatment of community acquired pneumonia – the German perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Reinhard Marre Detection of respiratory bacterial pathogens

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Walter Hampl and Thomas Mertens Viral pathogens and epidemiology, detection, therapy and resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Mathias W.R. Pletz, Lesley McGee and Tobias Welte Resistance in Streptococcus pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57

Hans-Dieter Klenk Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Matthias Krüll and Norbert Suttorp Pathogenesis of Chlamydophila pneumoniae infections – epidemiology, immunity, cell biology, virulence factors . . . . . . . . . . . . . . . . .

83

Dina M. Bitar, Marina Santic, Yousef Abu Kwaik and Maëlle Molmeret Legionnaires’ disease and its agent Legionella pneumophila . . . . . . . . . . . 111 Sven Hammerschmidt, Gavin K. Paterson, Simone Bergmann and Timothy J. Mitchell Pathogenesis of Streptococcus pneumoniae infections: adaptive immunity, innate immunity, cell biology, virulence factors . . . . 139

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Contents

Ken B. Waites, Jerry W. Simecka, Deborah F. Talkington and T. Prescott Atkinson Pathogenesis of Mycoplasma pneumoniae infections: adaptive immunity, innate immunity, cell biology, and virulence factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Pablo D. Becker and Carlos A. Guzmán Community-acquired pneumonia: paving the way towards new vaccination concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

List of contributors Yousef Abu Kwaik, Department of Microbiology and Immunology, Room MS-410, University of Louisville, Louisville, KY 40209, USA T. Prescott Atkinson, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35249, USA Pablo D. Becker, Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany; e-mail: [email protected] Simone Bergmann, Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany Dina M. Bitar, Department of Microbiology and Department of Medical Microbiology and Immunology, Faculty of Medicine, Al-Quds University, Jerusalem, 19356, Israel; e-mail: [email protected] Carlos A. Guzmán, Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany; e-mail: [email protected] Sven Hammerschmidt, Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany; e-mail: [email protected] Walter Hampl, Institute for Virology, University Clinic of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; e-mail: [email protected] Hans-Dieter Klenk, Institut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Str. 3, 35043 Marburg, Germany; e-mail: [email protected] Matthias Krüll, Dept. Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité, Universitätsmedizin Berlin, Augustenburgerplatz 1, 13353 Berlin, Germany; e-mail: [email protected] Reinhard Marre, University of Ulm, Medical Microbiology & Hygiene, Robert-Koch-Str. 8, 89081 Ulm, Germany; e-mail: [email protected] Lesley McGee, Hubert Department of Global Health, Emory University, 1518 Clifton Road, Atlanta, GA, 30322, USA; e-mail: [email protected] Thomas Mertens, Institute for Virology, University Clinic of Ulm, AlbertEinstein-Allee 11, 89081 Ulm, Germany; e-mail: [email protected]

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List of contributors

Timothy J. Mitchell, Division of Infection and Immunity, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow G12–8QQ, UK Maëlle Molmeret, Department of Microbiology and Immunology, Room MS-410, University of Louisville, Louisville, KY 40209, USA Gavin K. Paterson, Division of Infection and Immunity, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow G12–8QQ, UK Mathias W.R. Pletz, Department of Respiratory Medicine, Hannover Medical School, Carl-Neuberg-Str.1, Hannover, 30625, Germany; e-mail: [email protected] Marina Santic, Department of Microbiology and Parasitology, Medical Faculty, University of Rijeka, Croatia Jerry W. Simecka, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA Deborah F. Talkington, Division of Foodborne, Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Ken B. Waites, Department of Pathology, WP 230, University of Alabama at Birmingham, 619 19th St. South, Birmingham, AL 35249, USA; e-mail: [email protected] Tobias Welte, Department of Pulmonary Medicine, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; e-mail: [email protected]

Preface Community-acquired pneumonia is a disease of high morbidity and mortality. Demographic changes in industrialised countries with a growing population of elderly persons will add to its significance. In the last years much progress in the field of community-acquired pneumonia has been achieved. Vaccination programs against influenza and Streptococcus pneumoniae have been established. Risk-adjusted management of patients with community-acquired pneumonia allows to identify patients in need of hospitalisation and intensive care and helps to choose an effective antibiotic therapy. “New” pathogens such as C. pneumoniae, Legionella pneumophila, Chlamydia-like organisms, the human coronavirus or the avian influenza virus have been detected. In spite of all progress, clinical diagnosis of community-acquired pneumonia is by no means trivial; detection of respiratory pathogens often fails or gives inconclusive results and duration and choice of antibiotics still is a matter of debate. Moreover, many patients progress from uncomplicated pneumonia to severe pneumonia and even to pneumonia-related septic shock despite adequate antibiotic therapy. Therefore, besides new antibiotics we definitely need a non-antibiotic approach and a better understanding of what determines individual immune responses to pneumonia is crucial. Fundamental molecular and cellular pathologic characteristics of disease must be linked with clinical aspects of infection. The present book is intended to bridge the gap between basic science, clinical research and patient management and to crosslink patient care with biology and microbiology. It gives a state of the art information on different aspects of community-acquired pneumonia and allows the reader to get data on recent developments in community-acquired pneumonia. The editors Norbert Suttorp, Tobias Welte and Reinhard Marre themselves, representing clinical medicine, clinical research, microbiology as well as cell biology, hope that this book will help to manage patients with communityacquired pneumonia and to identify promising areas of research. Berlin/Hannover/Ulm, August 2006

Norbert Suttorp Tobias Welte Reinhard Marre

Community-Acquired Pneumonia ed. by N. Suttorp, T. Welte and R. Marre © 2007 Birkhäuser Verlag Basel/Switzerland

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Diagnosis and treatment of community acquired pneumonia – the German perspective Tobias Welte Department of Pulmonary Medicine, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Abstract Current concepts of diagnosis and treatment of CAP are risk stratified and adapted to the national resistances of important pathogens. Thorough surveillance systems have to be implemented in all countries. The risk of patients can be assessed reliably with a limited number of clinical data (CRB-65 score). Extended microbiological and laboratory diagnosis is recommended for hospitaliszed patients only. Outpatient treatment can be performed with classic antibiotics like amoxicillin or doxycyclin. Macrolides are only an alternative in these patients. In the hospital, treatment has to be adapted to the severity of the disease. Further studies concerning the duration of treatment and advantageous combinations are necessary. Recommendations for treatment of CAP have to be adapted to the quickly changing epidemiology and have to be updated every 2 to 3 years.

Introduction Pneumonia is a worldwide, serious threat to health, and an enormous socio-economic burden for healthcare systems. According to recent WHO data, each year three to four million patients die from pneumonia, a large proportion of whom are children or elderly people. Pneumonia is the third most common cause of death among infectious diseases in the world [1]. Detailed epidemiological data is available from the USA, where two to three million cases of CAP occur each year, leading to around 10 million doctor-patient contacts [2]. If an estimated proportion of 20 % (half a million) of these patients were hospitalised, the incidence is 258 hospital admissions per 100,000 inhabitants. The requirement for hospitalization depends on age, with the highest rates observed for patients over age 65, among whom the necessity for hospital admissions rises by a factor of four to around 1000 per 100,000 inhabitants [3]. In total, it is estimated that the costs for pneumonia treatment reach 8 billion dollars annually in the USA.

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The largest proportion of this amount is spent on elderly and hospitalized patients. Community Acquired Pneumonia (CAP) results in high mortality (mean about 8%). In the US, CAP is the sixth most frequent reason for dying, and there is an increase of 0.5 to 1% per year [2]. The increase is caused by the growing life expectancy, by aging of the population, and by a better treatment of chronic diseases. Elderly people with concomitant diseases are more susceptible to infectious diseases [4], and have typically a spectrum of pathogens (gram negative enterobacteriaceae, staphylococci, legionella, bacteriaemic pneumococci), which is associated with higher mortality [5, 6]. While the mortality of CAP is low in outpatients (1%), it can rise to up to 12% in hospitalized patients [2].

Definition Pneumonia is an infection of the alveolar space, with accumulation of inflammatory cells and secretions in the alveoli, resulting in impaired gas exchange [8]. Each pneumonia acquired outside of a hospital is defined as community acquired pneumonia [4], while nosocomial pneumonia is caused during a stay in the hospital and up to one week after discharge. A subgroup of CAP is the ”healthcare associated pneumonia” of patients with frequent contact with the healthcare system (haemodialysis patients, patients in nursing homes) [9]. Although it is community acquired, this form is treated similar to a nosocomial infection. The approach of scientific communities to CAP is very different. A proof of the disease is the pathologic result, combined with a positive microbiologic specimen of the tissue. In the clinical routine, biopsies of the lungs cannot be obtained. The typical signs on the chest radiograph can be delayed, even with the best technical equipment. Initial diagnostics often shows no pulmonary infiltration [10]. All other signs which are typical for pneumonia, such as the typical sounds on auscultation, fever, cough and sputum expectoration, dispnoea, chest pain and serological markers of inflammation are not pathognomic, and are nearly not always present in all patients. In the English-speaking countries, the appearance of a new or progressive infiltration is absolutely necessary for a diagnosis of pneumonia. All other criteria are of minor importance for the diagnosis [11]. The European Respiratory Society (ERS) defines pneumonia not via a chest radiograph finding, since many outpatients do not receive this diagnostic and therefore chest x-ray cannot be the major criterion. The ERS defines a “lower respiratory tract infection”, according to the clinical presentation. This includes tracheo-bronchitis, influenza infection, exacerbation of COPD, and pneumonia [12]. The recommendations of the ERS are much broader and cannot been compared with the narrowly focused American recommendations for CAP.

Diagnosis and treatment of community acquired pneumonia – the German perspective

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Aetiology The spectrum of pathogens and resistances varies widely between continents and countries. Universal guidelines for diagnosing and treatment are for rough orientation only; the treatment must be adapted to the specific local situation. Even under optimal diagnostic conditions, sufficient sputum specimen can be obtained in only 50% of patients [13]. In the early phase of the infection, sputum production may still be normal. In about one-third of all cases, the specimen do not meet international quality standards, which require a high proportion of leukocytes and a low proportion of squamous cells (Bartlett-criteria [4]). Depending on the patient group (all patients, all patients with positive results in the specimen, all patients who were able to expectorate sputum, all patients who produced purulent sputum), very different distributions of pathogens has been reported. According to results from the German competence network for CAP (CAPNETZ [14]), a reliable microbiologic diagnosis can be established in only 20% of all cases [15]. Worldwide, the most important pathogen is Streptococcus pneumoniae, followed by Haemophilus influenzae and Mycoplasma pneumoniae. Legionella is rare with a frequency of 4%, but associated with an excessive mortality. This underlines the importance of the very sensitive urinary-antigen testing in cases with clinically suspected infection with legionella (Tab. 1). Infections with enterobacteriaceae are most common in patients from nursing homes, elderly patients and multi-morbid patients (cardiac and kidney diseases, neurologic disorders and chronic obstructive pulmonary disease, CODP). The mortality of these patients is much higher than in patients who are living in the normal community [16]. In the USA, Pseudomonas aeruginosa is also a typical pathogen in CAP [6], but Pseudomonas is not important in middle and northern Europe. Some studies from Italy and Spain [17] report a high prevalence of Chlamydia pneumoniae (> 10%). These results come from serologic test-

Table 1. Clinical findings in patients with legionella infection Fever

100%

Chills

73%

Cough

83%

Purulent sputum

50%

Chest pain

30%

Abdominal symptoms

30%

Polymyositis

78%

Neurological symptoms

23%

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Figure 1. Association of the consumption of penicillins and the prevalence of penicillin resistant pneumococci in Europe (modified according to [22]). AT, Austria; BE, Belgium; HR, Croatia; CZ, Czech Republic; DK, Denmark; FI, Finland; FR, France; DE, Germany; HU, Hungary; IE, Ireland; IT, Italy; LU, Luxembourg; NL, The Netherlands; PL, Poland; PT, Portugal; SI, Slovenia; ES, Spain; UK, England only.

ing. Studies using polymerase chain reaction (PCR) show in less than 3% a positive finding [15]. The titres of IgA and IgM may remain elevated, even after previous or oligosymptomatic infections. The use of serologic testing seem not to be sensible in an acute infection. Chlamydia pneumoniae might be very prevalent is special outbreaks, but presently, the importance of C. pneumoniae for CAP seems to be low. Viruses had been found in a number of studies (with or without PCR) in 10 to 1 % of all CAP cases [5, 15, 18]. The question of whether viruses are the responsible pathogen for CAP, or if the virus induced damage of the bronchial epithelia is the precursor of bacterial infection, is still open. In winter, influenza viruses are most important (70% of all viruses), underlining the importance of influenza vaccination in the elderly because of the prevalence and severity of pneumonia. Large trials revealed that vaccination results in lower rates of pneumonia [19]. Similar results could not be obtained for the vaccination against S. pneumoniae, since the vaccinate did not cover all serotypes of this pathogen. Bacteriaemic infections could be prevented, but there is no local protection [14]. American studies revealed

Diagnosis and treatment of community acquired pneumonia – the German perspective

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that vaccination of children reduced the incidence of severe pneumonia in adults, albeit different serotypes play the dominant role [20].

Resistances Problems with resistances of the most important pathogens, especially pneumococci, vary widely between different countries [21]. The main reasons are differences in the consumption of antibiotics. There is a direct correlation between the use of antibiotics and resistances in many countries (Fig. 1) [22]. The significance of pathogen resistances for the outcome of a patient is controversially discussed. Increasing resistances of pneumococci against penicillin did not affect the mortality, even when treatment with penicillin was continued. On the other hand, resistances against Cefuroxim had been found to worsen mortality of pneumonia patients [23]. Pneumococci resistant against makrolides seem to be associated with higher rates of bacteriaemia [24], but the impact on mortality in unclear. If peumococci are resistant against fluorochinolones (there has been one epidemic in Hong Kong), treatment with fluorochinolons had worsened the prognosis of some individual patients [25]. An improvement of resistances can be achieved – as documented in Scandinavia – by temporary reduction of the use of the respective classes of antibiotics [26].

Risk stratification The risk factors for an increased CAP mortality are age, the number of concomitant diseases, and the place of residence before admission to the hospital (patients from nursing homes had an eight-fold higher risk for dying than patients coming from “regular” homes) [7]. Different scores for the estimation of the prognosis of patients with CAP had been evaluated to substantiate the decision for hospital admission and the decision of where to treat the patient (regular ward, intermediate care unit, intensive care unit). Many scores designed for hospital patients (Pneumonia Severity Index, PSI [27], CURB Score [28]) have the disadvantage that they need laboratory testing, what is not available in the outpatient setting. Recent data revealed that a simple clinical score (CRB-65; C: Confusion, R: respiratory rate > 30/min, B: blood pressure < 90 mmHg, 65: age > 65 years) allows to stratify patients into a low, moderate, or high mortality risk group [29, 30]. The risk within the hospital can be best assessed with the modified ATS Score [31]. If one major criterion (septic shock requiring vasopressor therapy or mechanical ventilation) or two minor criteria (acute respiratory

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insufficiency (PaO2/FiO2 < 250), multilobular infiltrates in the chest radiograph, systolic blood pressure < 90 mmHg) are present, then intensive care treatment is necessary.

Diagnostic procedures A chest radiograph (pa (posterior/anterior) and lateral view) is highly recommended to establish the diagnosis. However, the chest radiograph does not have 100% sensitivity. High-resolution computed tomography (hrCT) might show infiltrates which had not been detected in a previous chest x-ray. If there is a clinical suspicion of pneumonia, the chest radiograph is normal, and there is no clinical improvement of the patient, a new chest radiograph should be performed 24 to 48 h later, or a chest computed tomography should be performed. Not all infiltrates in the lungs are caused by pneumonia. Congestive heart failure, tumour, pulmonary infarction, and infiltrates due to interstitial pulmonary diseases or systemic diseases (Wegener’s granulomatosis, collagenoses, and others) are important differential diagnoses for acute pneumonia. Elevated levels of C-reactive protein (CrP) or procalcitonin III (very expensive and not currently a routine procedure) are important parameters [32], even if they do not prove infection. In bacterial pneumonia, leucocytosis and an overrepresentation of young granulocytes can be expected. Leucopenia might be a sign of sepsis and might therefore be a bad prognostic factor. In patients with a CRB-65 score of 0, no further laboratory diagnostic is necessary. In hospitalized patients, the diagnostics should be focused on inflammation, and further laboratory diagnostics should be performed, depending on the severity of the disease and on concomitant problems. Microbiologic testing is recommended, depending on the severity of the pneumonia and on other risk factors. Patients with a low risk (CRB65 = 0) receive no benefit. However, this may change, if in addition to the urine legionella antigen testing other quick reacting tests become available. Preliminary results from the pneumococci antigen test do not justify a routine use of this expensive test. Microbiologic specimen from the lower airways (sputum, bronchoalveolar lavage and biopsies), pleural effusions, and blood cultures can be better and faster collected in the hospital than in the outpatient setting. Such diagnostics can be recommended for all hospitalized patients in severe cases it is mandatory. Blood cultures show positive results in 10 to 20% only and that is a proof. In severe infections, blood cultures must be obtained. If legionella infection is suspected (for clinical signs see Table 1), an antigen testing from urine specimen on Legionella pneumophilia antigen is recommended.

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Serological testing (to screen for Mycoplasma, Chlamydia and viruses) is not generally recommended since results are difficult to interpret (see above). Patients who do not improve within 72 h, or who worsen in clinical state, must be seen as non-responders [34]. In this situation, invasive diagnostic procedures (bronchoalveolar lavage, transbronchial lung biopsy), and – depending on the clinical course – further radiologic diagnostic (pleural ultrasound, CT) might become necessary.

Treatment All guidelines distinguish patients with low risk of dying (CRB-65 ) 1, CURB ) 1, PSI I und II), who might be treated as outpatients, and patients with high risk, who need hospital treatment. Further prognostic factors are concomitant diseases and the type of pathogen. Important demographic, epidemiologic, and clinical risk factors for a specific spectrum of pathogens are: – Previous antibiotic treatment increases the rate of resistant pathogens. The previous antibiotic should not be continued [22]. – Recent visits to countries with high prevalence of legionellosis (e.g. Spain, Italy) and/or contact to contaminated water has to be explored. – Patients aged 65 and above have a higher prevalence of gram-negative pathogens, especially if they have co-morbidities (heart, lung, kidney, liver), previous treatment with antibiotics, or previous hospitalisation [35]. – Patients from nursing homes or with previous hospitalization are at increased risk for infections with enterobacteriaceae and Staphylococcus aureus, and have a higher risk for aspiration pneumonia [16]. – Chronic lung diseases are associated with Haemophilus influenzae [36]. In advanced stages of COPD, in patients with cystic fibrosis or bronchiectasis, there is a frequent finding of S. aureus and Pseudomonas aeruginosa [6]. – Contact to animals: Birds: C. psittaci, sheep: C. burnetii. – Previous treatment with steroids of at least 10 mg/day prednisoloneaequivalent over at least 4 weeks, or structural lung diseases (COPD, bronchiectasis, cystic fibrosis) and a hospitalization within the last 30 days (longer than a two-day stay) are associated with P. aeruginosa and Legionella spp. [9]. The recommendations for treatment distinguish whether there are risk factors for an infection with Pseudomoas aeruginosa [6]: – Pulmonary concomitant diseases (structural defects in COPD stadium GOLD IV, bronchiectasis, cystic fibrosis).

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Table 2. Treatment recommendations for patients with low risk in the outpatient setting. Substance First choice Aminopenicillines – Amoxicillin Or Tetracycline – Doxycycline

Alternative Macrolidea – Azithromycin – Clarithromycin – Roxithromycin

Dosage (per day)

Duration of therapy

* 70 kg: 1 g t.i.d p.o. < 70 kg: 0.75 g t.i.d. p.o.

7–10 days

200 mg once initially, * 70 kg: 200 mg once daily < 70 kg: 100 mg once daily

7–10 days

500 mg once daily p.o. 2 × 500 mg p.o. 3 days, followed by 250 mg b.i.d 1 × 300 mg oral

3 days 7–10 days 7–10 days

only in countries with low macrolide resistance

a

– Hospitalization within the last 30 days (longer than two days stay). – Risk for aspiration (e.g. neurological diseases). – Treatment with a broad spectrum antibiotic over more than 7 days within the last month. Depending on the number of risk factors, the probability of Pseudomonas aeruginosa increases exponentially (to more than 50 % in three or more risk factors). The guidelines show wide difference in the recommended antibiotics. The reasons are different local resistances. The recently published recommendations of the ERS are presented here [12]. Updates of the recommendations of the ATS and ISDA are under preparation, but not finalized yet. Table 2 shows the recommended treatment of CAP patients with a low risk and treatment in the outpatient setting. A follow-up to assess the success of treatment should be performed after 72 h. Patients or persons in their environment should be advised to contact their doctor again if fever exceeds 4 days, dyspnoea gets worse, patients stop drinking, or consciousness is decreasing. Macrolides have been used to treat pathogens such as Chlamydia and Mycoplasma which did not respond to beta-lactam antibiotics. Some recently published studies [37, 38] demonstrate that patients with a low risk of dying have no worse outcome with penicillin derivatives compared with macrolides. Infections with Chlamydia seem to be rare, and infections

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Table 3. Treatment recommendations for the calculated initial therapy of hospitalized CAP patients (no severe pneumonia) without a risk of Pseudomonas infection Substances Preferred in regions with low pneumococcal resistance rate – Penicillin G – Ampicillin – Amoxicillin/Clavulansäure# – Ampicillin/Sulbactam – Cefuroxim – Ceftriaxon – Cefotaxim ± Macrolides (s. Tab. 2)* Alternatives (in regions with increased pneumococcal resistance or intolerance to preferred drugs) Fluorquinolone§ – Levofloxacin – Moxifloxacin

Dosage

Duration of therapy

1 Mill. IU t.i.d 1.5 g t.i.d i.v. 2.2 g t.i.d i.v. 3.0 g t.i.d i.v. 1.5 g t.i.d i.v. 2.0 g once daily i.v. 2.0 g t.i.d i.v.

7–10 days 7–10 days 7–10 days 7–10 days 7–10 days 7–10 days 7–10 days 7–10 days

500 mg once daily i.v. 400 mg once daily i.v.

7–10 days 7–10 days

# Can be applied as sequential treatment using the same drug * new macrolides preferred to erythromycin § within the fluoroquinolones, moxifloxacin has the highest antipneumococcal activity. Experience with ketolides is limited but they may offer an alternative when oral treatment is adequate

with Mycoplasma seem to be self-limiting in low-risk patients. In these situations, patients might be treated without intracellular active antibiotics. Doxycyclin had been accepted as first-line treatment despite insufficient clinical data, since there is no information about treatment failures due to resistance. In the outpatient setting, oral treatment is preferred. Oral cephalosporins have a low bioavailability, and there are doubts about their pharmacokinetics and pharmacodynamics in elderly people. Cephalosporins are an alternative in exceptional cases only. The recommendations for hospitalized patients are shown in Table 3. Hospitalized patients should receive parenteral treatment, due to unreliable oral pharmacokinetics in elderly and severely affected patients. If the clinical response is satisfactory, an early switch (after 72 h) to similar oral substances is possible. Treatment success is assumed if the respiratory rate is lower than 25/min, and oxygen saturation is above 90%, body temperature fell by at least 1°C, and the haemodynamic and neurological status are satisfactory, and if oral feeding is possible [39]. A number of retrospective, and one prospective study [40] show that in bacteriaemia with S. pneumoniae a combination therapy with a beta-lactam

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Table 4. Criteria for severe community acquired pneumonia (modified according to [46])* Baseline (minor) criteria assessed at admission Respiratory rate > 30 min–1 Severe respiratory failure (Pa,O2/Fi,O2 ratio > 250) Bilateral involvement in chest radiograph Involvement of more than two lobes in chest radiograph (multilobar involvement) Systolic blood pressure < 90 mmHg Diastolic blood pressure < 60 mmHg Major criteria assessed at admission or during clinical course Requirement for mechanical ventilation Increase in the size of infiltrates by * 50% in the presence of clinical nonresponse to treatment or deterioration (progressive infiltrates) Requirement of vasopressors > 4 h (septic shock) Serum-creatinine * 2 mg·dl–1 or increase of * 2 mg·dl–1 in a patient with previous renal disease or acute renal failure requiring dialysis (acute renal failure) Pa,O2, arterial oxygen tension; Fi,O2, inspiratory oxygen fraction; *, the presence of at least two minor criteria or one major criterion defines severe pneumonia, i.e., pneumonia requiring admission at the ICU.

antibiotic and a macrolide offers the best treatment success. Despite severe weakness in the design of these studies, combination therapy is recommended at least for severe infections. Mono therapy with a new fluoroquinolone (moxifloxacin or levofloxacin) seems to be as effective as combination therapy, but reliable data is missing [41]. Table 4 shows criteria for severe community acquired pneumonia (sCAP). In this form of pneumonia, broad initial treatment effective against all possible pathogens is necessary. The choice of antibiotics is guided by the probability of Pseudomonas infection (Tab. 5). In patients with sCAP, the combination therapy between a beta-lactam and an aminoglycoside or fluorochinolone is also under discussion. Two Meta analyses [42, 43] could not reveal an additional effect of aminoglycosides in infections with gram-negative pathogens. However, the data on infections with pseudomonas are not sufficient. The recommended combination of beta-lactams with pseudomonas-active fluoroquinolones seems to be of advantage, but this combination has not been tested systematically until today. One abstract, published from the ECCMID meeting 2006 [44] suggested that mono therapy with moxifloxacin, which penetrates tissue well, could be sufficient. Treatment failure is defined as lack of clinical improvement after 72 h of treatment or worsening of the clinical situation. Besides extended diagnostic procedures, which also allow the recognition of important complications of CAP (pleural empyema, lung abscess), treatment must be de-escalated target intracellular pathogens (combinations of macrolides and newer fluo-

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Table 5. Treatment recommendations for the calculated initial therapy of patients with severe CAP. Substances No risk factors for Pseudomonas – Ceftriaxon – + Makrolides (s. Tab. 2) – or + Fluorquinolone (s. Tab. 3) Risk factors for Pseudomonasa – Piperacillin/Inhibitor – Cefepime – Carbapenem – + Ciprofloxacin

Dosage

Duration of therapya

2,0 g once daily i.v.

7–10 days

4,5 g t.i.d i.v. 2,0 g t.i.d i.v. 1,0 g t.i.d i.v. 400 mg t.i.d i.v.

7–14 days 7–14 days 7–14 days 7–14 days

aminimum treatment is 7 days, maximum 14 days if there is a proof of pseudomonas or L. pneumophilia.

roquinolones). Gram-negative pathogens and Pseudomonas must be covered (Tab. 5). The problem of an optimized duration of treatment is controversially discussed. First results of short-term treatment (5 days) in patients with low risk are encouraging [45]. Hospitalized patients who were treated on a regular ward were nearly completely free of symptoms after five days [41]. The current evidence does not allow a general recommendation of such shor-t term therapy. Treatment duration of 10 days should only be exceeded in rare, exceptional cases.

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Lopez AD, Murray CC (1998) The global burden of disease. Nat Med 4: 1241– 1243 Center for Disease Control and Prevention (1997) Premature deaths, monthly mortality and monthly physician contacts-United States. MMWR Morb Mortal Wkly Rep 46: 556 Marston BJ, Plouffe JF, File TM Hackman BA, Salstrom SJ, Lipman HB, Kolczak MS, Breiman RF (1997) Incidence of community-acquired pneumonia requiring hospitalization. Arch Intern Med 157(15): 1709–1718 Bartlett JG, Dowell SF, Mandell LA, File TM Jr, Musher DM, Fine MJ (2000) Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America. Clin Infect Dis 31: 422–425 Lim WS, Macfarlane JT, Boswell TC, Harrison TG, Rose D, Leinonen M, Saikku P (2001) SCAPA: Study of Community Acquired Pneumonia Aetiology in adults admitted to hospital: implications for management guidelines. Thorax 56: 296–301

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Arancibia F, Bauer TT, Ewig S, Mensa J, Gonzalez J, Niederman MS, Torres A (2002) Community-acquired pneumonia due to gram-negative bacteria and Pseudomonas aeruginosa: incidence, risk, and prognosis. Arch Intern Med 162: 1849–1858 Fine MJ, Smith MA, Carson CA, Mutha SS, Sankey SS, Weissfeld LA, Kapoor WN (1996) Prognosis and outcome of patients with community acquired pneumonia. JAMA 275: 134–141 Osler W (1901) Lobar pneumonia. In: J Young (ed) The principles and practice of medicine. Pentland, Edinburgh & London, 126–129 American Thoracic Society; Infectious Diseases Society of America (2005) Guidelines for the management of adults with hospital-acquired, ventilatorassociated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 171(4): 388–416 Syrjala H, Broas M, Suramo I (1998) High-resolution computed tomography for the diagnosis of community-acquired pneumonia. Clin Infect Dis 27: 358–363 Bartlett JG, Breiman RF, Mandell LA, File TM (1998) Community-acquired pneumonia in adults: Guidelines for management. Clin Infect Dis 26: 811–838 Woodhead M, Blasi F, Ewig S, Huchon G, Leven M, Ortqvist A, Schaberg T, Torres A, van der Heijden G, Verheij TJ (2005) Guidelines for the management of adult lower respiratory tract infections. Eur Respir J 26(6): 1138–1180 Metlay JP, Schulz R, Li YH, Singer DE, Marrie TJ, Coley CM, Hough LJ, Obrosky DS, Kapoor WN, Fine MJ (1997) Influence of age on symptoms at presentation in patients with community-acquired pneumonia. Arch Intern Med 157(13): 1453–1459 Welte T, Suttorp N, Marre R (2004) CAPNETZ – community-acquired pneumonia competence network. Infection 4: 234–238 Welte T, Marre R, Suttorp N; for the CAPNETZ Network (2006) What is new in the treatment of community-acquired pneumonia? Med Klin (Munich) 101(4): 313–320 El-Solh AA, Sikka P, Ramadan F, Davies J (2001) Etiology of severe pneumonia in the very elderly. Am J Respir Crit Care Med 163: 645–651 Blasi F (2004) Atypical pathogenes and respiratory tract infections. Eur Respir J 24: 171–181 De Roux A, Marcos MA, Garcia E, Mensa J, Ewig S, Lode H, Torres A (2004) Viral community-acquired pneumonia in nonimmunocompromised adults. Chest 125: 1343–1351 Nichol KL, Wuorenma J, von Sternberg T (1998) Benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens. Arch Intern Med 158: 1769–1776 Whitney CG, Farley MM, Hadler J, Harrison LH, Bennett NM, Lynfield R, Reingold A, Cieslak PR, Pilishvili T, Jackson D et al (2003) Active bacterial core surveillance of the emerging infections program network. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. N Engl J Med 348(18): 1737–1746 Felmingham D, Reinert RR, Hirakata Y, Rodloff A (2002) Increasing prevalence of antimicrobial resistance among isolates of Streptococcus pneumoniae from the PROTEKT surveillance study, and compatative in vitro activity of the ketolide, telithromycin. J Antimicrob Chemother 50 Suppl S1: 25–37

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Goossens H, Ferech M, Vander Stichele R, Elseviers M (2005) Outpatient antibiotic use in Europe and association with resistance: a cross national database study. Lancet 365: 579–587 Yu VL, Chiou CC, Feldman C, Ortqvist A, Rello J, Morris AJ, Baddour LM, Luna CM, Snydman DR, Ip M et al.; International Pneumococcal Study Group (2003) An international prospective study of pneumococcal bacteremia: correlation with in vitro resistance, antibiotics administered, and clinical outcome. Clin Infect Dis 37(2): 230–237 Lonks JR, Garau J, Gomez L, Xercavins M, Ochoa de Echaguen A, Gareen IF, Reiss PT, Medeiros AA (2002) Failure of macrolide antibiotic treatment in patients with bacteremia due to erythromycin–resistant Streptococcus pneumoniae. Clin Infect Dis 35 (5): 556–564 Davidson R, Cavalcanti R, Brunton JL, Bast DJ, de Azavedo JCS, Kibsey P, Fleming C, Low DE (2002) Resistance to Levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med 346: 747–750 Seppala H, Klaukka T, Vuopio-Varkila J (1997) The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland. Finnish Study Group for Antimicrobial Resistance. N Engl J Med 337: 441–446 Fine MJ, Auble TE, Yealy DM, Hanusa BH, Weissfeld LA, Singer DE, Coley CM, Marrie TJ, Kapoor WN (1997) A prediction rule to identify low-risk patients with community acquired pneumonia. N Engl J Med 336: 243–250 Lim WS, van der Eerden MM, Laing R, Boersma WG, Karalus N, Town GI, Lewis SA, Macfarlane JT (2003) Defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study. Thorax 58: 377–382 Capelastegui A, Espana PP, Quintana JM, Areitio I, Gorordo I, Egurrola M, Bilbao A (2006) Validation of a predictive rule for the management of community-acquired pneumonia. Eur Respir J 27(1): 151–157 Bauer T, Ewig S, Marre R, Suttorp N, Welte T (2006) CURB and CRB–65 scores predict mortality in hospitalised and out-patients with communityacquired pneumonia (CAP). J Intern Med 260: 93–101 Ewig S, de Roux A, Bauer T, Garcia E, Mensa J, Niederman M, Torres A (2004) Validation of predictive rules and indices of severity for community acquired pneumonia. Thorax 59 (5): 421–427 Christ-Crain M, Jaccard-Stolz D, Bingisser R, Gencay MM, Huber PR, Tamm M, Muller B (2004) Effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, singleblinded intervention trial. Lancet 363: 600–607 Ishida T, Hashimoto T, Arita M, Tojo Y, Tachibana H, Jinnai M (2004) A 3-year prospective study of a urinary antigen-detection for Streptococcus pneumoniae in community acquired pneumonia: utility and clinical impact on the reported etiology. J Infect Chemother 10: 359–363 Menendez R, Torres A, Zalacain R, Aspa J, Martin Villasclaras JJ, Borderias L, Benitez Moya JM, Ruiz-Manzano J, Rodriguez de Castro F, Blanquer J et al.; Neumofail Group (2004) Risk factors of treatment failure in community acquired pneumonia: implications for disease outcome. Thorax 59(11): 960– 965

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Janssens JP (2005) Pneumonia in the elderly (geriatric) population. Curr Opin Pulm Med 11(3): 226–230 Sethi S, Evans N, Grant BJ, Murphy TF (2002) New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. N Engl J Med 347(7): 465–471 Mills GD, Oehley MR, Arrol B (2005) Effectiveness of beta lactam antibiotics compared with antibiotics active against atypical pathogens in non-severe community acquired pneumonia: meta-analysis. BMJ 330: 456 Guchev IA, Yu VL, Sinopalnikov A, Klochkov OI, Kozlov RS, Stratchounski LS (2005) Management of nonsevere pneumonia in military trainees with the urinary antigen test for Streptococcus pneumoniae: an innovative approach to targeted therapy. Clin Infect Dis 40: 1608–1616 Marrie TJ, Lau CY, Wheeler SL, Wong CJ, Vandervoort MK, Feagan BG (2000) A controlled trial of a critical pathway for treatment of community-acquired pneumonia. CAPITAL Study Investigators. Community-Acquired Pneumonia Intervention Trial Assessing Levofloxacin. JAMA 283: 749–755 Baddour LM, Yu VL, Klugman KP, Feldman C, Ortqvist A, Rello J, Morris AJ, Luna CM, Syndman DR, Ko WC et al (2004) Combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia. Am J Respir Crit Care Med 170: 440–444 Welte T, Petermann W, Schuermann D, Bauer TT, Reimnitz P, Moxirapid Study Group (2005) Treatment with sequential intravenous/oral moxifloxacin was associated with faster clinical improvement compared to standard therapy in hospitalized CAP patients with initial parenteral therapy. Clin Infect Dis 41(12): 1697–1705 Paul M, Benuri-Silbiger I, Soares-Weiser K, Leibovici L (2004) `-lactam monotherapy versus `-lactam-aminoglycoside combination therapy for sepsis in immunocompetent patients: systematic review and meta-analysis of randomised trials. BMJ 328: 668 Safdar N, Handelsman J, Maki DG (2004) Does combination antimicrobial therapy reduce mortality in gram-negative bacteraemia? A meta-analysis. Lancet Infect Dis 4: 519–527 Torres A, Read R, Lode H, Carlet J, Winter JH, Garau J, Welte T, Arvis P, Le Berre MA, Choudhri SH (2006) Once daily sequential intravenous/oral (IV/ PO) moxifloxacin is equivalent to IV ceftriaxone plus twice daily IV/PO levofloxacin in the treatment of severe community-acquired pneumonia requiring hospitalization: the MOTIV study. ECCMID 2006 Dunbar LM, Wunderink RG, Habib MP, Smith LG, Tennenberg AM, Khashab MM, Wiesinger BA, Xiang JX, Zadeikis N, Kahn JB (2003) High-dose, shortcourse levofloxacin for community-acquired pneumonia: a new treatment paradigm. Clin Infect Dis 37: 752–760 Niederman MS, Mandell LA, Anzueto A, Bass JB, Broughton WA, Campbell GD, Dean N, File T, Fine MJ, Gross PA et al (2001) Guidelines for the management of adults with community-acquired pneumonia. Diagnosis, assessment of severity, antimicrobial therapy, and prevention. Am J Respir Crit Care Med 163: 1730–1754

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Detection of respiratory bacterial pathogens Reinhard Marre University of Ulm, Institute of Medical Microbiology & Hygiene, Robert-Koch-Str. 8, 89081 Ulm, Germany

Abstract Microbiology of community-acquired pneumonia often is based on indirect or precarious evidence. Time and effort to detect a respiratory pathogen often is not sufficiently related to its usefulness in guiding therapy. If sputa are accepted by the laboratory for microbiological studies (Gram stain, culture), they should fulfill quality criteria such as high number of leukocytes and low number of squamous epithelial cells. Complementary tests such as antigen detection assays are useful adjuncts for diagnosing pneumococcal and Legionella pneumonia. Nucleic amplification tests help to overcome the problems in detecting Chlamydia pneumoniae, Legionella and Mycoplasma pneumoniae.

Introduction Community acquired pneumonia is caused by a wide variety of different bacterial species. Streptococcus pneumoniae is the leading pathogen, followed by Haemophilus influenzae, Staphylococcus aureus, Chlamydia pneumoniae, Mycoplasma pneumoniae and a broad spectrum of enterobacterial species. While it is generally agreed upon that S. pneumoniae is the predominant pathogen in CAP, reported incidences of other species vary considerably depending on patient population, specific epidemiological situations and microbiological aspects such as type of specimens studied, detection methods used and pathogen definition. Even in clinical trial conditions detection of a respiratory pathogen often is based on precarious evidence, namely microscopical or cultural detection of a facultative pathogen in respiratory secretions which may be heavily contaminated by bacteria of the oropharyngeal tract. Unequivocal, definitive evidence, i.e. growth in blood or other usually sterile specimens is rare and selects for severe bacteremic pneumonia and may thus not be representative. Nucleic acid amplification techniques (NAT) such as PCR to detect microbial pathogens are – in the absence of a solid reliable gold standard – not a reliable indicator

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of a causative agent. In addition, clinical patterns often do not give a clear clue about the causative micro-organism [1]. Beyond clinical trials the situation is even worse and quality of microbiology of respiratory secretions often is regarded as unsatisfactory. This resulted in the statement of Bartlett that the culture of lower respiratory specimens may result in more unnecessary microbiologic efforts than any other type of specimen [2]. Clinical relevance of microscopy of respiratory specimens too is regarded as very controversial [3] and guidelines of the ATS and the IDSA differ with respect to the evaluation of the clinical impact of a gram stain result of a sputum sample [4]. Because of these limitations and critical appraisal, microbiology of the CAP may not be useful unless performed by expert microbiologists and well trained technicians using standardized approaches for specimen workup, external and internal quality assurance with an active communication network between microbiologists and clinicians. The effects of quality on reported incidence has been illustrated by Bartlett [5], who described an apparent decrease in the yield of S. pneumoniae from Gram staining and culture from 80 to < 18% over the last 30 years. In evaluating 15 published reports from North America, it was found that S. pneumoniae incidence ranged from 20 to 60% [6], which is in agreement with the German Multicenter Network CAPNETZ in which the incidence of S. pneumoniae ranged from 28 to 48% depending of the patients population studied.

Microscopy of respiratory secretions Microscopy of respiratory sample may help in guiding initial therapy in CAP if a properly collected specimen is tested. Non-invasive samples such as sputa and upper respiratory swabs give less conclusive evidence than invasive samples (Bronchoalveolar lavages (BAL), protected specimen brushings), but are more easily available. An essential problem is that sputum may be contaminated by saliva. Several approaches have been developed to assess the quality of a respiratory sample. These have been recently described and evaluated by Sharp et al [3]. The bottom line is that the number of leukocytes related or not to the number of squamous epithelial cells are a criteria of quality (Fig. 1). Based on this criterion, the number of CAP patients with a good-quality sputum has been shown by Rosón et al to be only 40% of all patients and 60% of the patients with a sputum sample [7]. In 35% of the patients no sputum sample was available. In a study performed by Kalin et al 76% of the sputa were deemed purulent [8]. In CAPNETZ, a Germany-wide network on CAP with more than 3,000 patients sputum was available in 45 to 70% of patients. In about half of the patients with sputum, the sample was purulent. Non-purulent sputum should not be used for culture and therefore rejected. If non-purulent sputum is used for culture non or little pathogenic microorganisms (Candida

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Figure 1: Gram stain of a high-quality (purulent) (below) and a non purulent sputum (above).

Figure 2: Frequency of isolates in dependence of the quality of sputum. Filled columns: < 10 leukocytes per 10x field, empty columns 10–25 leukocytes per 10x field, shaddowed columns: > 25 leukocytes per 10x field.

18 Reinhard Marre

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albicans, Escherichia coli and other enterobacteria) can be detected in high numbers and in a high proportion of patients, thus perhaps leading to flawed therapy decisions (Fig. 2). Although these species cannot be regarded as pathogens in CAP, they may indicate altered colonization. Limiting identification and work-up of the sample may lead to improved outcome as shown convincingly by Barenfanger in the case of detection of yeasts in respiratory specimens [9]. However, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella can be detected in non-purulent sputum samples by NAT techniques. Many studies state that sputum can also safely be used for microscopic detection of bacterial pathogens [3]. Positive and negative predictive value were 90% and 62%, respectively, if patients with a definitive diagnosis of pneumococcal pneumonia (detection of S. pneumoniae in sterile body fluids or detection of the pneumococcal antigen) were evaluated, and 75 and 98% if patients with a Haemophilus influenzae pneumonia were evaluated [7]. The corresponding sensitivity values were 35% (S. pneumoniae) and 42% (H. influenzae). Including only patients with a bacteremic pneumococcal pneumonia, Musher reported a sensitivity of 57% [10]. It should, however, be kept in mind that these studies select for patients with generalized bacterial infection and that therefore microscopy may be of less value if patients with a less severe infection are included. In a meta-analysis, Reed et al. reported a range of sensitivity values from 15 to 100%, and of specificity from 11% to 100% in pneumococcal pneumonia [11], which illustrates that a Gram stain in the hands of inexperienced may yield misleading results.

Detection of pathogens by culture Cultural detection of respiratory pathogens in sterile body fluids is usually specific but limited to CAP with generalized infection. If secretions are obtained from deeper sites of infections such as bronchoalveolar lavages or fiber optic brush technique (FB) and if a cut-off level of 103 to 104 cfu/ml is defined, cultural detection has a sensitivity between 42 and 95% and a specificity between 45 and 100% (reviewed by Sharp [3]). However, BAL and FB and sterile body fluids are not available on a routine basis and therefore microbiology has also to rely on culture of sputum. On the other hand, the usefulness of sputum cultures is a matter of controversial debate. Heineman et al stated that cultural detection of pathogens in sputum is meaningless if not guided by microscopy [12]. Recent publications report either limited value or nonvalue of microbiological studies to detect a CAP pathogen [13, 14]. Other reports indicate however that sputum cultures may reveal therapeutically relevant information in individual cases and that the relevance can be increased if the sputum sample is purulent, obtained before the start of antibiotic chemotherapy and if cultured without delay.

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Streptococcus pneumoniae S. pneumoniae is the most predominant species in CAP. The incidence ranges from 20 to 60%. About 20% of these pneumonias are bacteremic. Pneumococci can be detected out of respiratory samples by microscopy and culture, in addition by blood cultures and by detection of urinary antigen. As described above, Gram stain of a sputum sample has a sensitivity of 60% and specificity of near 100% to detect pneumococci in bacteremic patients [7, 10]. The examination of a sputum Gram stain, however, is not as trivial as generally believed and requires expertise of the lab personnel and an active quality management programs. The diagnostic value of a sputum culture in bacteremic patients has been shown to be high if only bacteremic patients with purulent sputum are included. In these patients it may help to guide therapy and to narrow the spectrum of the antibiotic. The cultural isolates also allow recognizing trends in resistance development that have implications on the improvement of guidelines. Urinary antigen assays for the detection of pneumococci (Binax NOW) may overcome the limitations of sputum microscopy and culture, since it is a rapid (15 min) bedside diagnostic, not limited by the availability of purulent sputum, and does not require experienced lab personnel. The assay detects the c polysaccharide antigen that is not capsular serotype specific. Sensitivity is reported to be 70 to 75%, specificity 90 to 95% [15-17]. Patients with a positive sputum culture may be antigen positive in about only 50% [17], indicating either the low specificity of the sputum culture or the low sensitivity of the antigen assay in non-bacteremic patients. In contrast to adults, the test does not discriminate between children with and without pneumococcal pneumonia [18]. Nucleic acid amplification assays are not useful in diagnosing a pneumococcal pneumonia. When using plasma, buffy coat or urine as specimens, Murdoch et al. studied the performance of a nested PCR targeting the pneumolysin gene [19]. Plasma and buffy coat were only positive in 30% of patients with a positive blood culture, urine was positive in 2% while the antigen assay was positive in 29% of the patients. Throat swabs were positive in 55 to 56 % by PCR, irrespective of the status as a control or as patient.

Haemophilus influenzae Although H. influenzae is the second or third most common species in CAP, little attention has being paid to this species as a respiratory pathogen. Patients with H. influenzae pneumonia often are elderly with a history of chronic obstructive lung diseases, tumor pathology or other causes of impaired immunity. Non-encapsulated strains of H. influenzae

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predominate. Lethality of an invasive H. influenzae infection of this patient population is high [20]. As reported by Roson et al [7] sputum microscopy has a high sensitivity and specificity for the diagnosis of a H. influenzae pneumonia of hospitalized patients. Detection of H. influenzae by sputum culture may overestimate the incidence of this species in CAP since it is a normal inhabitant of the oropharyngeal tract. Assays to diagnose H. influenzae infection by antigen detection are to the best of my knowledge not available.

Legionella Legionella comprises a genus with about 50 different species most of which are never or rarely encountered in the human host. The overwhelming majority of Legionella infections are attributed to Legionella pneumophila serotype 1. Australia, however, may be an exemption where Legionella longbeachae is more predominant. Legionella occur in aquatic systems where it can thrive within amoebae. When inhaled by a susceptible person (more than 60 years of age, smoker, male, immunocompromised), it can cause a potentially lethal pneumonia. Legionella infections occur with an incidence of 5 to 10% in CAP in an endemic situation. Morbidity is about 20 cases of Legionella infection per one million inhabitants. At the time being there is no one-for-all easy and rapid test to detect Legionella. If a Legionella infection is suspected by the clinician, different assays should be combined. Although Legionella can be cultured from respiratory secretion when using special media containing cysteine and iron as essential growth factors, the detection rates are rather low and sensitivity may not exceed 60%. The advantage of a culture is that a Legionella isolate for further investigations (source identification) is available. Sputum Gram stain is not helpful since the Gram negative rod Legionella is stained only faintly by fuchsin, unless the stain is allowed to act for a prolonged time. Direct immunofluorescence test using polyclonal or monoclonal antibodies directed against the lipopolysaccharide can be useful in principal. Crossreaction of the polyclonal antiserum with Pseudomonas species [21], the low sensitivity, the long hands-on time and the need for experienced personnel have limited the use of this test. The introduction of the antigen detection assay (Binax or Biotest) has significantly contributed to a rapid and specific diagnosis of Legionella serotype 1 infections. Legionella infections other than Legionella pneumophila serotype 1, however, are not covered reliably. The antigen detection assay may also select for severe infections as shown by Yzerman [22]. For patients with mild Legionella infection, the test sensitivities ranged only from 40 to 53%, whereas for patients with severe Legionnaires Disease (LD) who needed immediate special medical care, the sensitivities reached 88 to 100%. Guerrero et al. reported that the Bartels EIA and the Biotest EIA

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had significantly higher sensitivity levels than the Binax NOW immunochromatographic test (65 to 70% vs. 37%) [23]. Nucleic acid amplification assays using respiratory secretions can complement the Legionella test assay system. Real-time PCR is a rapid as well as reliable method with a sensitivity of 100% when culture proven cases of Legionella are studied [24, 25]. The 16S rDNA, 5S rDNA or the macrophage infectivity potentiator (mip) gene of Legionella commonly are used as target genes. Antibody assay is available for the diagnosis of Legionnaire’s Disease (LD), but its value is limited since some LD patients do not react with a significant rise of antibodies and others have a persistant IgM antibody titer. With the advent of molecular tests the indication of serological testing in LD in acute care is reduced to near null.

Chlamydia pneumoniae Chlamydia pneumoniae was identified as pulmonary pathogen only 15 years ago. Because of its debated association with coronary heart disease C. pneumoniae has attracted considerable attention. The reported incidence ranges from 2 to 40% depending on the epidemiological situation, site and time of the investigation, detection methods used and patients included. In recent years reports with low incidences (less than 2%) seem to have increased [25–27]. The CAPNETZ trial confirmed the low incidences of C. pneumoniae infections [28]. Most assays for the detection of C. pneumoniae are not applicable for routine microbiology. Cell culture techniques to grow C. pneumoniae are restricted to specialized laboratories. Even under optimal conditions (no delay until culture, high quality specimens) cultural detection is much more futile than, for example, cultural detection of C. trachomatis. If grown in a first passage, many isolates do not outlive further passages. The C. pneumoniae-specific microimmuno-fluorescence (MIF) test has been introduced as matter of choice to prove a C. pneumoniae infection and many clinical CAP studies rely on this assay. In clinical practice, however, serodiagnosis must often rely on a single serum sample and, therefore, seroconversion and/ or rise in antibody titers are missed in many patients. In addition, about 60% of adults have antibodies against C. pneumoniae which makes it difficult to differentiate between an acute or past infection. During the last decade, molecular methods including conventional and real-time PCR assays have been developed for sensitive and specific detection of C. pneumoniae DNA in respiratory tract specimens [29]. However, there is no consensus on the most suitable specimen, the optimal DNA isolation method and target gene. Results of multicenter PCR comparison trials have been contradictory and difficult to interpret [30]. Therefore, further efforts to optimize and standardize diagnostics in C. pneumoniae are urgently needed.

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Mycoplasma pneumoniae Detection of Mycoplasma pneumoniae in CAP is dealt with in the chapter by Waites et al.

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routine microbiological diagnostics in patients hospitalized for communityacquired pneumonia. Scand J Infect Dis 34: 873–879 Theerthakarai R, El Halees W, Ismail M, Solis RA, Khan MA (2001) Nonvalue of the initial microbiological studies in the management of nonsevere community-acquired pneumonia. Chest 119: 181–184 Gutierrez F, Masia M, Rodriguez JC, Ayelo A, Soldan B, Cebrian L, Mirete C, Royo G, Hidalgo AM (2003) Evaluation of the immunochromatographic Binax NOW assay for detection of Streptococcus pneumoniae urinary antigen in a prospective study of community-acquired pneumonia in Spain. Clin Infect Dis 36: 286–292 Ishida T, Hashimoto T, Arita M, Tojo Y, Tachibana H, Jinnai M (2004) A 3-year prospective study of a urinary antigen-detection test for Streptococcus pneumoniae in community-acquired pneumonia: utility and clinical impact on the reported etiology. J Infect Chemother 10: 359–363 Murdoch DR, Laing RTR, Mills GD, Karalus NC, Town GI, Mirrett S, Reller LB (2001) Evaluation of a rapid immunochromatographic test for detection of Streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia. J Clin Microbiol 39: 3495–3498 Navarro D, Garcia-Maet L, Gimeno C, Escribano A, Garcia-de-Lomas J, and and the Spanish pneumococcal study network (2004) Performance of the binax NOW Streptococcus pneumoniae urinary antigen assay for diagnosis of pneumonia in children with underlying pulmonary diseases in the absence of acute pneumococcal infection. J Clin Microbiol 42: 4853–4855 Murdoch DR, Anderson TP, Beynon KA, Chua A, Fleming AM, Laing RTR, Town GI, Mills GD, Chambers ST, Jennings LC (2003) Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia. J Clin Microbiol 41: 63–66 Campos J, Hernando M, Roman F, Perez-Vazquez M, Aracil B, Oteo J, Lazaro E, de Abajo F, and the Group of Invasive Haemophilus Infections of the Autonomous Community of Madrid (2004) Analysis of invasive Haemophilus influenzae infections after extensive vaccination against H. influenzae Type b. J Clin Microbiol 42: 524–529 Tenover FC, Edelstein PH, Goldstein LC, Sturge JC, Plorde JJ (1986) Comparison of cross-staining reactions by Pseudomonas spp. and fluorescein-labeled polyclonal and monoclonal antibodies directed against Legionella pneumophila. J Clin Microbiol 23: 647–649 Yzerman EPF, Boer JW, Lettinga KD, Schellekens J, Dankert J, Peeters M (2002) Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires’ disease in the Netherlands. J Clin Microbiol 40: 3232–3236 Guerrero C, Toldos CM, Yague G, Ramirez C, Rodriguez T, Segovia M (2004) Comparison of diagnostic sensitivities of three assays (Bartels Enzyme Immunoassay [EIA], Biotest EIA, and Binax NOW Immunochromatographic Test) for detection of Legionella pneumophila serogroup 1 antigen in urine. J Clin Microbiol 42: 467–468 Reischl U, Linder H, Lehn N, Landt O, Barratt K, Wellinghausen N (2002)

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Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis. J Clin Microbiol 40: 3814–3817 Templeton KE, Scheltinga SA, van den Eeden WC, Graffelman AW, van den Broek PJ, Claas EC (2005) Improved diagnosis of the etiology of communityacquired pneumonia with real-time polymerase chain reaction. Clin Infect Dis 41: 345–351 Tsolia MN, Psarras S, Bossios A, Audi H, Paldanius M, Gourgiotis D, Kallergi K, Kafetzis DA, Constantopoulos A, Papadopoulos NG (2004) Etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections. Clin Infect Dis 39: 681–686 Weigl JA, Puppe W, Grondahl B, Schmitt HJ (2000) Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction. Eur J Clin Microbiol Infect Dis 19: 336–343 Wellinghausen N, Straube E, Freidank H, von Baum H, Marre R, Essig A (2006) Low prevalence of Chlamydia pneumoniae in community-acquired pneumonia. Int J Med Microbiol; in press Dowell SF, Peeling R, Boman J, Carlone GM, Fields BS, Guarner J, Hammerschlag MR, Jackson LA, Chou-Chou K, Maass M et al (2001) Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin Infect Dis 33: 492–502 Apfalter P, Blasi F, Boman J, Gaydos CA, Kundi M, Maass M, Makristathis A, Meijer A, Nadrchal R, Persson K et al (2001) Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens. J Clin Microbiol 39: 519–524

Community-Acquired Pneumonia ed. by N. Suttorp, T. Welte and R. Marre © 2007 Birkhäuser Verlag Basel/Switzerland

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Viral pathogens and epidemiology, detection, therapy and resistance Walter Hampl and Thomas Mertens Institute for Virology, University Clinic of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

Abstract Worldwide community-acquired pneumonia (CAP) is one of the most frequent infectious diseases and a leading cause of death. Several studies have shown that a pathogen could be identified only in 50 to 60% of all patients, although in children < 6 month infectious agents can be detected in about 90%. Viral infections are most frequent in children < 2 years (80%), whereas bacterial infections increase with age. RSV, influenzaviruses, rhinoviruses, parainfluenzaviruses and adenoviruses are the most common viruses associated with CAP in children. Among adenoviruses a predominance of adenovirus 7 has been reported in several countries with emergence of highly pathogenic variants with significant lethality in young children. Many childhood respiratory infections are caused by more than one pathogen and up to 30% mixed viral / bacterial infections can be observed. CAP in immunocompetent adults is rare, whereas persons with underlaying diseases have an increased incidence of CAP. In the elderly, RSV, influenzaviruses, parainfluenzaviruses and less frequent adenoviruses are predominant viruses causing pneumonia. Less frequently associated with CAP are the newly discovered human metapneumovirus and the coronaviruses NL63 and HKU1. Hantaviruses, involved in the hantavirus pulmonary syndrome, belong to the emerging pathogens to date in North, Middle and South America. For optimum diagnosis the whole spectrum of potential respiratory viral agents should be included and multiple diagnostic techniques have to be used. In view of the high relevance of influenzavirus for CAP influenza vaccination is highly advisable for prevention of CAP, especially in high-risk groups.

Introduction Viral infections are involved in 10–25% of CAP. Frequently mixed infections, viral/viral or viral/bacterial, are detected. CAP in infants and young children is most commonly due to viral infections, with the predominance of respiratory syncytial virus (RSV). In developing countries the incidence of viral pneumonia is higher and is a relevant reason for death of young children [1, 2].

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Viral pathogens and epidemiology The major viral pathogens are summarized in Table 1. Usually they cause mild self-limited illness, mainly restricted to the upper respiratory tract. The leading viral pathogens for severe disease are RSV and influenzaviruses. In the past two decades some new, mostly zoonotic viral pathogens have emerged like SARS-coronavirus, avian influenzaviruses and hantaviruses associated with hantavirus pulmonary syndrome (HPS), which has crossed species barriers. Numerous other viruses occasionally can cause severe respiratory disease in the lower respiratory tract, but they are better known for other clinical manifestations (Tab. 2). Respiratory virus infections are more common in winter and early spring, but some cause respiratory illness without a clear seasonal pattern. The frequency of detection of respiratory viruses varies in different studies due to methodological problems. Many studies did not include all known respiratory viruses. Additionally, the prevalence of viruses may vary over time and between different geographical areas. In immunocompromised patients exogenous respiratory viruses may persist with prolonged shedding. Individuals with a subclinical infection may be an undetectable source of transmission. Only a few studies have investigated respiratory viruses in asymptomatic humans with very variable results. An age-dependent occurrence of asymptomatic respiratory infections has been reported with the highest frequency of 68% in young children newborn to 4 years.

Factors influencing occurrence of community-acquired pneumonia (CAP) Virus-associated CAP occurs mainly in infants and young children, in elderly and frail adults, in persons with underlying diseases and in immunocompromised patients. Even “harmless” agents like rhino- or coronaviruses frequently induce acute disorders or exacerbations in people with chronic cardiopulmonary diseases or asthma. The main host and environment related risk factors have been investigated, but the influence of various other factors, which can predispose CAP is controversial. In general, the viral-associated CAP decreases with age. In several recent studies in developed countries a pathogen was detected in 79% to 85% in immunocompetent children with CAP; 25% to 62% of the patients had evidence of a single viral infection, 8% to 11% for viral/viral infection and 23% to 43% a viral/bacterial infection. Inflammation and disease were more severe in viral/bacterial infection [3–6]. In nonimmunocompromised adults the demonstrated viral etiology is less common. Roux et al. [7] identified a pathogen only in 38% of the cases with a proportion of viral infections of approximately 20% (single viral infection 9%) with influenzavirus

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29

Table 1. Major respiratory viruses Virus family / Subfamily Adenoviridae Coronaviridae

Genus

Virus type

Mastadenovirus

Human adenovirus (1 – 7, 14 and 21) Human coronavirus (HCoV-OC43, -229E, -NH, -NL63, -HKU1) SARS-coronavirus Influenzavirus A, B, C Avian influenzavirus A (e.g. H5N1)

Respirovirus Rubulavirus Pneumovirus Metapneumovirus Rhinovirus Enterovirus

Parainfluenzavirus 1, 3 Parainfluenzavirus 2, 4a/b Respiratory syncytial virus (A, B) Human metapneumovirus (A, B) Human rhinovirus (HRV 1-102) Human enterovirus (Cox A10, 16, 21, Cox B16, ECHO 4, 9, 11, 25, 32, HEV 68, 69, HRV 87)

Orthomyxoviridae Paramyxoviridae Paramyxovirinae Pneumovirinae Picornaviridae

Table 2. Other respiratory viruses Virus family / Subfamily Bunyaviridae

Genus

Virus type

Hantavirus

Paramyxoviridae Herpesviridae Alphaherpesvirinae

Morbillivirus

e.g. Sin-Nombre virus, Andes virus, Choclo virus Measles virus

Betaherpesvirinae Gammaherpesvirinae

Herpes simplex virus (HSV) Varicella-zoster virus (VZV) Cytomegalovirus (CMV) Human herpes virus 6 (HHV6) Epstein-Barr virus (EBV)

(rare)

(very rare)

as the most frequently identified viral agent. In this study chronic heart failure (CHF) was a risk factor for virus infections.

Viruses causing CAP Adenovirus (Adv) The virus is composed of nonenveloped, icosahedral particles containing a double-stranded linear DNA genome. They are 70–90 nm in diameter and consist of 252 capsomers (hexons and pentons) with filamentous glycoproteins (fibers) on the penton bases, which display characteristic different lengths. Human adenoviruses have been classified into six species (subgen-

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era) A-F on the basis of antigenicity and other biological properties (hemagglutination, tumorogenicity in animals). This is in quite good agreement with classification based on genomic differences, like DNA homology of less than 20% between viruses of the different subgenera. Serotype-specific epitopes are predominantly found on the hexon capsomere and the terminal part of the fiber, and are defined by the quantitative neutralization test. At present, 51 serotypes have been identified by neutralizing antibodies and nine of these are documented as respiratory pathogens. Adenoviruses are very resistant, also against proteolytic enzymes in the intestinal tract. The species C adenoviruses (1, 2, 5 and 6) are endemic and are responsible for approximately 60% of all human adenovirus infections (Adv 6 only for 4%) and for more than 80% of the adenovirus infections (most commonly Adv 1 and 2) early in life, whereas they cause 15% of symptomatic lower respiratory tract infections. After primary infection, C viruses may be shed in feces for months or even years. Following the initial infection the C viruses establish a lifelong, asymptomatic, persistent infection, with currently unknown state of viral persistence. Probable places of persistence are tonsils and adenoids. New data suggest that human mucosal T-lymphocytes may harbor C adenoviruses in a latent form [8]. Premature infants are at high risk to develop disseminated neonatal adenovirus infection with pneumonia and high lethality [9]. In infants and children adenovirus infections primarily occur between 6 month and 5 years and are responsible for 4–10% of childhood pneumonias. Outbreaks in predominantly healthy children are most frequently associated with type 7, followed by types 3 and 21 [10–12]. For Adv 7-infected children at high risk the mortality rate is up to 40% and 12% in healthy children [13]. Adv differ in their ability to induce inflammatory response in lung tissue, but particularly Adv 7 is involved in severe lung inflammation and neutrophil infiltration [14]. The incidence of Adv infection may vary and depends on the detection assays and the study population. In general it is higher in children (21%) than in adults (9%) [15, 16]. In 50% of the described cases a viral coinfection was recognized [8, 17]. It is likely that Adv infections were caused also by reactivation of endogenous virus. Further, these infections are not linked to seasonal pattern. Adenovirus epidemics do occur in human populations living in crowded conditions with poor hygiene. A large outbreak, predominantly caused by types 4 and 7, was observed between 1950 and 1960 in 10% of the recruits in the US military. Ninety percent of the Adv-infected persons developed pneumonia. Vaccination against adv 4 and 7 since 1971 has effectively reduced illness from these serotypes [18]. Recent epidemiological studies have shown that latent subgroup C viruses are involved in some chronic diseases in immmunocompetent patients which are at high risk for pneumonia induced by other viral pathogens [19, 20].

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Table 3. Examples of epidemiological data of adenovirus pneumonia Patients Age / no manifestation 7588 < 5 years / ARD 1640

< 5 years / LRTI

75

5–14 years / CAP

Risk factor mostly none mostly yes immunodeficient None

Viral infections % ~ 40

Pneumonia Adv sero- Study % type 0,4 ? [17]

43–50

4–10

7, 3, 2

[13]

65

12

?

[21]

Therapy and resistance Disseminated adenovirus infections are difficult to treat and the agents used, ribavirin and cidofovir, yielded variable results. The benefit of intravenous ribavirin (a synthetic guanosine analog) treatment in life-threatening disease especially in immunocompromised patients, seemed to be better if antiviral therapy was started early. Other reports could not show a clear beneficial effect. In vivo as well as in vitro data suggest that susceptibility to ribavirin is highly dependent on the virus species [22, 23]. Viruses of subgroup C were shown to be sensitive to ribavirin, whereas serotypes of the subgroups A, B, D, E and F were resistant. Despite a high level and long treatment in some patients no changes in sensitivity of the virus isolates against ribavirin to date have been reported. Cidofovir is a nucleotide analogue of cytosine with an effective in vitro activity against different DNA viruses, adenoviruses included. Treatment is indicated for severe, disseminated Adv infections, but results are varying and treatment is limited by severe nephrotoxicity. Emergence of resistant Adv has been observed only in experimental systems.

Human coronavirus (HCoV) Coronaviruses are spherical, pleomorphic enveloped viruses with a diameter of 80–200 nm. They possess the largest genomes of all RNA viruses. The single-stranded positive RNA is associated with the nucleoprotein N forming the helical nucleoprotein complex. It is surrounded by the envelope, which contains three characteristic surface structures, the S protein, the membrane protein M and the envelope protein E. Oligomeres of the S protein are formed to spikes on the virion surface and resemble a solar corona. The S protein determines cell tropism, is responsible for pathogenicity and is the strongest inducer of neutralizing antibodies. Some coro-

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Table 4. Examples of epidemiologial data of coronavirus pneumonia Patients Age / no manifestation 501 < 2 to > 65 years ARTI 316 < 65 years ARTI 418 > 65 years CAP

Risk factor mostly none

Viral infections % 37

Pneumonia % 0,4

CHF, COPD

~ 30

0,6

cardiopulmonary

?

2,4

HCoV

Study

-OC43

[25]

-OC43, -229E -HKU1

[24] [29]

naviruses contain a fourth envelope protein with a hemagglutinating and esterase activity. Four serogroups have been distinguished containing three human pathogens: group 1 with the prototype HCoV-229E, group 2 with the known HCoV-OC43. In 2003 the severe acute respiratory syndrome (SARS) led to the detection of a novel coronavirus. The consecutive extensive research in this field led to the discovery of further coronaviruses (see the following). After rhinoviruses, coronaviruses are most frequently associated with the “common cold” (15–30%) in young adults, and they seem not to pose a risk for healthy elderly. Non SARS coronaviruses rarely cause pneumonia. However, they may cause diseases of the lower respiratory tract (LRT) in infants, immunocompromised patients, patients with underlying diseases and in frail older adults. During endemic outbreaks of HCoV-OC43 and -229E only elderly patients at risk rarely develop pneumonia [24, 25]. HCoV-NL63 (first described in 2004 in the Netherlands), but although widespread within the human population it is seldom responsible for pneumonia in children [26–28]. Recently, a novel coronavirus HCoV-HKU1 from an 71-year-old patient with COPD and pneumonia was described in Hong Kong [29]. HKU1-associated pneumonias so far described, occur from winter to spring, predominantly in the elderly (80% were > 65 years) with comorbidity [30]. In November 2002, a new emerging disease, SARS was described in China as a contagious, potentially lethal atypical pneumonia. The SARSCoV originated from animal viruses, which could be the result of recombination between mammalian and avian coronaviruses. By June 2003, worldwide 8447 cases of this illness from > 30 countries were registered with more than 800 deaths (lethality 9.5%). SARS-CoV seems to have a distinct cell entry pathway. The initial infection is possible with an extreme low infectious dose resulting in the generation of proteases in the lung, which are responsible for a 100- to 1000-fold more efficient rate of infection [31]. In younger children SARS often induces a relatively mild and nonspecific respiratory illness [32, 33]. In a Chinese study the overall mortality rate was 19.7%, but increased to 78.6% in the patient group with serious underlying diseases [34].

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Therapy and resistance There is no information about antiviral treatment of HcoV infections and the rare cases of HCoV associated pneumonia. At present also no standard antiviral therapy can be recommended for SARS. Ribavirin and corticosteroids used in severely ill patients seemed to be effective and a better outcome was reported after combination therapy with lopinavir/ritonavir, ribavirin plus steroid [35]. Nothing is known about emergence of resistant virus variants.

Human influenzavirus / avian influenzavirus Human and avian influenzaviruses are described in separate chapters within this book. Only the epidemiological association with CAP and the virological diagnosis are discussed in this chapter. Children (1 to 5 years) and adults with comorbidity, predominantly chronic heart disease and broncho-pulmonary dysplasia, pregnant women in the second or third trimester and persons > 65 years are at high risk for influenza virus-associated pneumonia. Using sensitive methods it has been shown that the prevalence of influenza infections in older children with CAP may be higher, and mixed infections, mostly viral/bacterial infections were documented in up to 35% [3, 21]. Influenza A virus-associated pneumonia in pregnancy is accompanied by higher morbidity and lethality and infants may be born preterm with low weight. Although pneumonia in elderly patients may present only with few respiratory signs, recovery is prolonged, especially in the frail elderly, where the incidence of pneumonia is highest [7, 36]. Mixed infections are more common in the older age groups, but single virus infections are seen increasingly in the patients over 65 years [37]. Influenza B virus-associated pneumonia is rare and has been reported in single cases in children [38, 39].

Table 5. Examples of epidemiologial data of influenzavirus pneumonia Patients no 514 126 241 147 75 338

Age / manifestation CAP 0–1 year 1–< 5 years 5–16 years 5–14 years CAP > 65 years CAP

Risk factor

Viral infections %

Pneumonia %

10% premature 23% comorbidity

Study [40]

none

42 21 6,8 65

0,8 5,8 1,4 6,7

[21]

CHF

18

11,5

[7]

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Walter Hampl and Thomas Mertens

Parainfluenza virus (HPIV) HPIV are pleomorphic enveloped viruses of between 150 and 300 nm. They contain a helical nucleocapsid with single-stranded negative RNA. It encodes at least six structural proteins and two nonstructural proteins. Important are the two envelope glycoproteins HN (hemagglutinin-neuraminidase) and F (fusion), responsible for neutralizing antibodies. Within the family of the paramyxoviruses only HPIV express neuraminidase activity. Four major HPIV serotypes exist which are divided into subtypes and genotypes with steadily occurring antigenic variations. Types 1, 2 and 3 are distributed worldwide, while type 4 is predominantly spread in America. The four serotypes are distinct in their epidemiological and clinical behavior. HPIV 3 is endemic throughout the year, but there are yearly epidemic outbreaks from winter to spring. HPIV 1 and 2 are associated with a biennial epidemic pattern with the peak from fall to winter. Parainfluenzaviruses 1 to 3, particularly type 3, seem to have the highest virulence and the capacity to persist. The majority of type 1 infections occur in children between the second and third year of life, whereas 60% of type 2 are in children younger than 5 years with a peak incidence between the first and second year. They are most commonly associated with croup or laryngitis, but may also cause pneumonia. Type 1 can be found in hospitalized, previously healthy adults and may be involved in bacterial pneumonias. HPIV-3 infections occur in 40% of young infants in the first year of life and in most children in the first two years. Pneumonia with type 3 is seen primarily in the first 6 months of life, similar to RSV, but with lower frequency [41]. Like RSV HPIV can reinfect both children and adults with predominantly mild respiratory tract symptoms accompanied by low and short virus shedding [42, 43]. In childhood CAP parainfluenzavirus 1–3 infections beside RSV and influenzaviruses are the most common viral pathogens, which are involved in up to 10% of the diseases [3, 4, 40, 44]. Parainfluenza virus infections have been reported in the elderly with pneumonia in up to 12% of cases [43]. Viral CAP in adults with comorbidity like COPD or CHF is characterized by a more severe clinical outcome [7, 45]. Viral/bacterial coinfections (approximately 20%) are observed frequently.

Therapy and resistance Ribavirin has in vitro activity against HPIV and both aerosolized and intravenous ribavirin have been used. There are anecdotal reports about reduction of clinical signs and viral load in immunocompromised patients, when

Viral pathogens and epidemiology, detection, therapy and resistance

35

treatment was started early after onset symptoms. Ribavirin resistant virus variants have not been described.

Respiratory syncytial virus (RSV) RSV is a negative-strand RNA virus. It is pleomorphic and has a size of 150–300 nm in diameter. The genome encodes eight structural and two nonstructural proteins. The helical capsid is surrounded by an envelope with three surface glycoproteins, the fusion (F) protein which mediates membrane fusion with the host cell resulting in viral penetration, the G protein which is responsible for attachment to the host cell, and an SH protein with unknown function. In contrast to other paramyxoviruses a hemagglutinin and neuraminidase function do not exist. The G protein has the highest degree of antigenic diversity in RSV. It accounts for the strain-specific epitopes and allows the classification into two antigenic groups RSV-A and RSV-B with multiple genotypes [46]. Immunologically important are the F and the G glycoproteins inducing protective neutralizing antibodies. However, immune-protection is not complete and early reinfections occur. The G glycoprotein is produced as a membrane bound and a secretioned form. The secretioned form is able to modulate the innate immune response to RSV and priming with this protein increases the severity of illness after RSV reinfection. In children with severe disease caused by RSV the type 2 Th-cell response seems to be dominant. The G protein induces an unbalanced Thcell response, which in the lung could result in airway hyperresponsiveness, mucus hypersecretionion, and inflammation, and may contribute to peripheral blood and pulmonary eosinophilia [47]. RSV infections occur worldwide peaking in the winter months in temperate climates and in the rainy season in tropical climates. A and B viruses and their multiple variants generally circulate simultaneously within epidemical outbreaks. Yearly outbreaks are possible, because the pattern of the circulating RSV strains changes, depending on the local strain-specific immunity in the human population [48]. RSV is the most important cause of acute respiratory tract viral infection in infants. Primary infections are symptomatic with a spectrum of clinical manifestations from mild upper tract illness to life-threatening pneumonia. RSV accounts for 50% of all cases of pneumonia during the first 2 years of life. The peak incidence of RSV lower respiratory tract infection (LRTI) is between 1 and 6 months of age. Maternal RSV-specific antibodies rapidly decrease after birth to approximately 6% at 3 months. This may be the reason for the high frequency of RSV infections before 3 months of age [49]. Premature infants (28 to 32 weeks) are at risk for 12 to 6 months after birth. All children get infected once until the

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Walter Hampl and Thomas Mertens

age of 2 years, but 50% of them already had experienced re-infections [4, 21, 40, 50–52]. There are numerous independent risk factors for severe RSV infection including genetic factors that are under discussion. In immunocompromised children, RSV-related mortality (with very high RSV load) was 15%, and for children with primary immunodeficiencies 40% [53]. In immunocompetent adults younger than 60 years RSV reinfections are generally mild and may contribute to 2–4% of the lower respiratory tract infections. In elderly people reinfections can induce life-threatening pneumonitis. Similar to children there may exist specific risk factors for severe RSV disease. In different studies in adult and elderly patients 3–10% developed RSV diseases depending on risk factors. The infections accounted for 11% of hospitalizations for pneumonia [54-56]. The development of a prophylactic vaccine against RSV was pursued with a high priority. The formalin-inactivated RSV vaccine developed in the 1960s unfortunately led to a more severe lung disease in vaccinated children after a subsequent RSV challenge [57].

Therapy and resistance Ribavirin is licensed to treat severe RSV-associated diseases in children. Immune globulin for intravenous administration and a humanized monoclonal antibody preparation (palivizumab (Synagis)) are designed to prevent or reduce the severity of RSV infection. It has been shown that in RSV-infected infants with lower respiratory tract disease ribavirin aerosol therapy improves clinical outcome, but may be effective in adults as well. Despite high dose and prolonged treatment in some patients no ribavirinresistant RSV variants have been isolated [58].

Human metapneumovirus (HMPV) This recently identified paramyxovirus is most closely related to the pneumovirus RSV, but HMPV differs from RSV in two aspects: it is lacking the nonstructural proteins (NS1 and NS2) and it has a different gene constellation. HPMV is classified into two main lineages, A and B, based on sequence analyses of the F gen. Further sequence analysis of different HMPV genes including the G gene are required for refined characterization of the virus isolates. The two major groups with numerous genotypes cocirculate throughout the year. Sometimes even genetically distinct strains of HMPV are circulating during the same year. Most infections have been detected during late winter and early spring following the peak activity of both RSV and influenzavirus. The virus was first detected in young children in 2001 in the Netherlands [59], but serological studies showed that the virus has been circulating in

Viral pathogens and epidemiology, detection, therapy and resistance

37

Table 6. Epidemiologial data of metapneumovirus pneumonia Patients no 90 208 145

Age / manifestation 0–1 years ARD < 3 years ARD adults CAP+EA

Risk factor

Pneumonia % 10

Study

none

Viral infections % 47

10%

78

1

[62]

COPD / CHF / asthma

19

2,8

[64]

[61]

humans for at least 50 years. HMPV has a worldwide distribution and association with respiratory illness in all age groups. It causes upper respiratory tract infections, but is also associated with lower respiratory tract infections. It has been suggested that most severe HMPV infections occur in children < 2 years of age and seem to peak in the third and fifth months of life, somewhat later than RSV. It is found less frequently in hospitalized children than RSV and the clinical course may be milder. Based on the presence of HMPV antibodies approximately 55% of children at the age of two and 100% at the age of 5–10 years had a HMPV history. HMPV like RSV may cause clinical important reinfections in late childhood and adult life, but the highest infection rate was found in young adults. HMPV also can be responsible for pneumonia in premature born babies [60] and other persons at risk. Although in the study of Maggi et al. [61] the number of infants with age less than 2 years is small, the majority of the HMPV-infected children developed pneumonia. The incidence in this study was higher (33%) than in other reports, but the difference is related to the difference in the population of children studied [62, 63]. The rate of bronchopneumonias was higher in children with isolated HMPV infection than in children with mixed infections. Surprising in this study [61] was the detection of HMPV RNA in plasma of 41% of HMPV-infected children, like was shown also for RSV infections. HMPV pneumonia clinically cannot be distinguished from RSV and influenza, but the disease seems to be somewhat less severe and there is a greater percentage of cases with underlying diseases (25%) compared to the influenza- or RSV-associated pneumonia (< 10%). Small studies demonstrated that HMPV is a relatively important viral pathogen, which can also lead to pneumonia (14%), especially in elderly and adults with underlying disease like COPD, CHF, or asthma [64]. Additional studies will be needed, especially year-long active surveillance over consecutive years with analysis of more data from future respiratory seasons to fully define the clinical and epidemic impact of HMPV infections.

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Therapy and resistance No antiviral agents or antibody preparations are currently available for the treatment of HMPV infection. It was shown that ribavirin and a polyclonal intravenous immunoglobulin had equivalent in vitro activity against both HMPV and RSV. Treatment may be considered for severe HMPV infection in immunocompromised patients [65]. No reports about resistant HMPV exist.

Picornaviruses (human rhinovirus (HRV) / human enterovirus (HEV)) Picornaviruses are nonenveloped particles and with a diameter of 30 nm they are very small. They consist of a single positive-strand RNA genome surrounded by an icosahedral capsid composed of 60 protomers. Each protomer consists of three nonglycosylated surface proteins (VP1 to VP3) and internal proteins (VP4). In the center of each protomeric unit is a canyon where antigenic sites and structures can be found binding to receptors of the target cells. There are over 100 immunologically distinct rhinovirus serotypes and two species A and B can be distinguished. Rhinoviruses are acidlabile and differ in membrane receptor recognition. Further, more than 60 enterovirus serotypes exist. Rhinoviruses are ubiquitous and infections by relatively low infectious doses occur throughout the year. They are the most important pathogens of the “common cold,”, in 80–90% of humans during the peak season in late autumn. Rhinoviruses are able to infect the lower respiratory tract. It has been shown in experimental infections that a lower respiratory infection with rhinoviruses (detected in bronchial biopsy samples) during “common cold” is not unusual. Picornaviruses are the most frequently detected virus in respiratory tract infections in the first year of life. In hospitalized infants with acute expiratory wheezing illness respiratory picornaviruses (rhino- and enteroviruses) are found in 42% of the cases. In older children picornaviruses are predominant with 65% at the age of 1–2 years and with 82% in children older than 3 years [66, 67]. Earlier studies have not included rhinoviruses, but current data suggest that they are rarely involved in pneumonia in infants, young children and in older adults. Rhinoviruses can cause pneumonia in children of the age group 0–6 months, but the highest isolation rate was found in children between 6 and 12 months with the same frequency as RSV [4, 24, 68, 69]. Coinfections of rhinoviruses and bacteria were found in pediatric patients with CAP in about 10%. The inflammatory response to rhinovirus infection is strong and several cytokines are related to pneumonia like IL-6, which may be an important factor in rhinovirus pathogenesis [68, 70]. In

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39

Table 7. Examples of epidemiologial data of picornavirus pneumonia Patients no

Age / manifestation

502 178 184 96 254 316

ARTI 0–6 months 6–12 months > 12 months 0,1–17 years CAP > 65 years ARTI

Risk factor

Viral infections % 43

Pneumonia % 3

?

46 40 38 62

2,8 4.9 1 23

[4]

CHF, COPD

30

0,5

[24]

56% passive smoking

Study [69]

a recent review about enterovirus-associated respiratory tract diseases Rotbart et al. [71] reported, that 13% of infected patients presented with pneumonia. In contrast, Kellner et al. [69] in a prospective study recovered enteroviruses only sporadically from children with upper respiratory tract disease. However, case reports exist about fatal pneumonias in congenital and neonatal echovirus-infected infants. In these rare and sporadic cases a maternal disease has been reported in 59 to 68% [71–75]. Epidemic outbreaks of hand, foot, and mouth disease with enterovirus 70/71 in children have been observed, where after CNS involvement pulmonary edema appeared [76]. Using modern diagnostic methods it is increasingly recognized, that rhinovirus and enterovirus infections are the most common reasons for unnecessary antibiotic therapy.

Therapy and resistance The most promising antiviral of the so called WIN compounds is pleconaril with a broad potent anti-EV and anti-RV activity. It binds to hydrophobic sites in the base of the capsid canyons and inhibits uncoating of the capsid in all enteroviruses. Rhinoviruses of the species B, have a significant reduced susceptibility to pleconaril. For therapeutic application it will be important to differentiate between natural occurring resistance to pleconaril in B rhinoviruses and the emergence of RV resistance under pleconaril treatment [77]. Pleconaril has been shown to be effective in experimental studies and to significantly reduce clinical symptoms in treated adult volunteers. It is available for life threatening enterovirus infections in immunocompromised patients. Ten percent of the enterovirus isolates have been shown to be resistant to pleconaril and some patients did not respond (resistant virus has been identified). Ruprintrivir is an inhibitor of the virally encoded 3C

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Walter Hampl and Thomas Mertens

protease, which is still under investigation for treatment of rhinovirus infection in immunocompetent patients [78].

Hantavirus (HV) Hantaviruses are enveloped, predominantly negative-strand RNA viruses (ambisense). The genome of these viruses consists of three different singlestranded RNA segments. The segments L, M and S encode the viral polymerase, a glycoprotein processed into G1 and G2 glycoproteins located in the envelope, and a nucleocapsidprotein. Virus particles are of spherical shape with a diameter of 80–120 nm, but also elongated forms are seen (170 nm). Hantaviruses are important zoonotic pathogens, primarily of rodents. The infections are per-sistent and most of them seem to be asymptomatic in the natural rodent hosts. Antibodies against hantaviruses are also present in nonnatural hosts, other wild and domestic animals. Beside the transmission to humans through human/rodent contacts, infections are acquired by inhalation of virus-contaminated aerosols of rodent excreta (saliva, urine or feces) [79]. An occasional transmission may occur from person to person as was documented for Andes virus in Argentina. Some hantaviruses belong to the so called emerging pathogens. They cause an influenza-like acute pulmonary disease in North, Middle and South America, the hantavirus pulmonary syndrome (HPS). The first HPS was recognized in 1993 in the USA and was caused by a number of hantavirus variants, e.g. Sin Nombre virus (SNV). Symptoms of HPS may vary, depending on the virus genotype. In 1995, cases of HPS were reported in South America and a new strain – the Andes hantavirus – was identified. In late 1999 and early 2000 first outbreaks through an again novel hantavirus, the choclo virus, were documented in Central America. The overall mortality was about 44%, but is declining as a consequence of better recognizing less severe forms of the infections and a better medical management. Confirmed cases of HPS so far include children, but in the majority of cases adults (19 to 58 years). SNV accounts for a small number of pediatric cases in the US, whereas Andes virus was found in pediatric patients (Chile, South Argentina) in a higher proportion (16%) [80–82]. The incubation period for Andes virus in Chile was 5 to 25 days. Febrile prodromi last for approximately 4 days followed by a rapid progression to moderate to severe respiratory distress.

Therapy and resistance For hantavirus diseases no established specific therapy is currently available. Ribavirin shows activity against HV, but this has not been well documented in studies. Emergence of resistant HV is unknown.

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Nonconventional respiratory viruses Measles virus In developing countries the frequent complications of measles virus infection are responsible for the mortality rate. Measles virus infection causes a transient and strong immunosuppression, which is the reason for the increased susceptibility to other viral or bacterial infections. In 3-4% of the infected patients measles virus can cause pneumonia, which can be present either as primary measles virus pneumonia or as atypical measles virus pneumonia, but most patients develop secondary bacterial pneumonia. Measles pneumonia rarely occurs in young adults as shown in 1976 to 1979, when a measles outbreak in US Air Force recruits was responsible for 106 cases with pneumonia (3.3%), of which two-thirds had a secondary bacterial infection [83]. During pregnancy measles virus infections induce a higher number of pneumonias in the mother [84, 85]. There are a few reports about preterm and newborn infants in which measles pneumonia was observed [86].

Herpes viruses After primary infection herpes viruses are able to persist lifelong in their human host, typically as latent infection, but they also reactivate under immunosuppression and then some of them, like CMV, HSV, VZV, EBV and HHV6, may induce pneumonia. HSV-1 pneumonia may occur occasionally in high-risk persons. Rarely disseminated HSV infections during pregnancy have been reported, as in the case of a previously healthy womn with a fatal progressive HSV-2 pneumonia in the third trimester of pregnancy [87]. Ramsey et al. reported 20 patients with pneumonia, in which they could differentiate between cases with focal HSV pneumonia as a result of HSV spreading to the lung parenchyma and cases with interstitial pneumonia as a result of a hematogenous dissemination of HSV [88]. In immunocompetent adults primary VZV infection is uncommon, but the incidence is increasing (5-10%) and VZV pneumonia is the main complication (incidence 5.5%–16.5%) with high mortality. Some patients may develop secondary bacterial pneumonia. Most patients (76.7%) do have at least one known risk factor like pregnancy, smoking or chronic obstructive pulmonary disease [84, 89–91]. VZV beside influenzavirus is the most common pathogen, causing pneumonia during pregnancy, predominantly in the second and third trimester. The risk of primary VZV infection for VZV pneumonia during pregnancy (0.1–18.3%) is higher if patients are smokers or manifest multiple (> 100) skin lesions. The mortality rate before a possible antiviral intervention was significantly higher in pregnancy (41% versus

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1.5–12.1%) [89, 92, 93]. VZV pneumonias as consequence of a primary VZV infection in childhood are rare [94]. CMV-infected preterm infants can develop a chronic lung disease, which may be associated with CMV pneumonia with high mortality. Ganciclovir treatment has been shown to rapidly improve symptoms, a fact that supports CMV causality of pneumonia [95–97]. Extremely rare are reports about CMV pneumonia in previously healthy adults [98]. In patients with lymphoma CMV pneumonia is less common and the incidence after chemotherapy and corticosteroid application is approximately 1% with a mortality rate of 30% [99]. Two genetically distinct variants HHV-6A and -6B do exist, but in primary infection the variant -6B is dominant. During primary infection a severe respiratory disease is an extremely rare complication. In a case report from Knox et al. [100] a fatal pneumonitis due to HHV-6 infection is documented in an infant with severe T lymphocytopenia. Whereas for immunocompromised patients (BMT) HHV-6 pneumonia is documented, in immunocompetent patients an etiologic role of the reactivated HHV-6 infection in pneumonia is not clearly defined [101, 102]. Another report of an extremely rare case with HHV-6-associated pneumonia is documented by Merk et al. [103], where an apparently immunocompetent young women developed a fatal pneumonia. A mild asymptomatic pneumonitis is described in 5–10% of the cases with infectious mononucleosis, but a severe pneumonitis as result of primary EBV infection is rare in immunocompetent patients [84, 104, 105]. In patients with lymphocytic interstitial pneumonia EBV DNA has been found in lung tissues of infants, children and of adults as well [106–108]. In autopsy cases with diffuse interstitial pneumonia EBV DNA could be detected in leukocytes and pneumocytes and frequently in airway epithelial cells [109]. The EBV-associated lymphocytic interstitial pneumonia without HIV infection has been reported predominantly in adults. This is also true for a rare chronic interstitial lung disease due to EBV, mainly seen in adults, but also shown in two infants in early months of life [106, 110].

Diagnosis of viral pulmonary infection Especially in adult patients respiratory virus infections are extremely underdiagnosed since adequate and possible virological diagnosis is not routinely performed. For diagnosis of acute respiratory infections the detection of infectious viruses or viral components (proteins, nucleic acid) is the method of choice, whereas antibody detection is not relevant and should only be used in selected patients at later timepoints after infection using paired sera.

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Specimen Nasopharyngeal aspirate (NPA) is the gold standard for the detection of all major respiratory viruses, predominantly adopted in infants and children. NPA is taken by suction of cell-containing mucosal secretion from the nasopharyngeal area. Approximately 0.5 ml fluid should be collected into 2 ml of viral transport medium. Nasal lavage can be obtained with less discomfort for the patient by gently instilling 2 ml PBS at room temperature into each nostril and by simultaneously suctioning into a sterile trap; the sensitivity for virus detection is comparable to that of NPA, but is controversially reported for RSV [111, 112]. Nasopharyngeal swab, taken usually from adults, is obtained by deep bilateral nasal and posterior pharynx swabs with sterile cotton swabs, which should then be placed in 3 ml viral transport medium (VTM) [113]. Calcium alginate swabs or swabs with wooden sticks may contain substances which inactivate some viruses and inhibit PCR testing and should not be used! Tracheal aspirate can be collected from intubated patients after instillation of 4–10 ml sterile normal saline into the endotracheal tube and by suctioning into a sterile trap; this is the most easily obtained specimen from lower respiratory tract in these patients [114]. Induced sputum should be collected in the morning after rinsing mouth and throat with sterile hypertonic saline. Thereafter sputum has to be collected in a sterile container. Induced sputum represents a more complete sampling of the respiratory tract and is comparable with nasopharyngeal aspirate specimens. While induced sputum might be used for virus detection from the lower respiratory tract, the obligate admixture of saliva with the accompanied flora is an obvious disadvantage. Nevertheless, with minimal saliva contamination it is a valuable alternative material to BAL [115, 116]. Bronchioalveolare lavage (BAL) has a high diagnostic yield for infectious pathogens predominantly in immunocompromised patients with lung infiltrates. Transbronchial biopsy (TBB) / lung biopsy. This invasive technique can in addition to BAL significantly improve the diagnostic yield [117]. EDTA blood in immunosuppressed patients may facilitate diagnosis of some viruses (e.g. adenovirus, CMV) and disseminated virus infections and determination of the virus load may be predictive for disease and outcome; furthermore, virus load is important for monitoring of therapy.

Methods for detection of viruses Direct virus detection (rapid antigen detection / nucleic acid assay) Enzyme/immunoassays (EIA) for rapid viral antigen detection in airway secretions exist for almost all major respiratory viruses, but have been often

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evaluated only for children and may vary in sensitivity and specificity (range between 60 and 95%). Results from EIAs for detection of single respiratory viruses are usually available between 10 min to 3 h. Alternatively a screening for respiratory viruses in an airway secretion can be achieved by indirect immunofluorescent antigen assay (IFA) (sensitivity 85–95%; specificity 95–99%) using a pool of monoclonal antibodies, whose specificity is directed against RSV, influenzaviruses A, B, parainfluenzaviruses 1, 2, 3 and adenovirus. The examination of several cell spots of a cytospin preparation on slides with pooled and single antisera allows viral antigen detection and typing within 2 h. The results are strongly dependent on the quality of the clinical material. For elderly hospitalized and immunocompromised patients with virus associated LRTI the rapid virusantigen detection is insensitive. When rapid conventional methods are not available, like for coronaviruses, metapneumoviruses, rhinoviruses or enteroviruses, the PCR has to be established.

PCR The PCR may be used alternatively to the mentioned antigen detection assays. It offers a great sensitivity and can be used for a wider range of viral pathogens. In principle PCR can detect and differentiate several viruses simultaneously in a single reaction mixture as multiple RT-PCR (multiplex PCR), but it provides a lower sensitivity than PCR for single pathogens and is more difficult to establish. For early detection and monitoring of viral infections the quantitative PCR represents a sensitive technique that delivers results within 3 h. For diagnosis of disseminated virus infections with pulmonary manifestations frequently caused by adenoviruses, RSV in infants or nonconventional respiratory viruses, like CMV additional analysis of plasma or EDTA blood is helpful.

Detection of infectivity (short term culture / isolation) Clinical specimen namely secretions of the respiratory tract can be used for an infectivity assay in centrifuge enhanced shell vial culture (SVC). After inoculation of airway specimens on different cell systems, which are susceptible for many of the respiratory viruses, cell cultures are evaluated for early virus infection after 24–72 h with a pool of virus-specific monoclonal antibodies and afterwards for typing with the respective individual monoclonal antibodies by IFA. Once isolated, the virus must be typed again preferentially with IFA using virus-specific monoclonal antibodies or otherwise increasingly by PCR.

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Conventional cell culture with virus isolation has no impact on clinical decision and management of patients during hospitalization. Virus isolation is useful for a characterization of virus strains involved in epidemic outbreaks.

Antibody detection Serology is not useful during the acute phase of infection. Due to the delayed onset of the antibody response, approximately at the end of the first week of disease serological methods can detect antibodies. To use only serological methods for diagnosis of viral infections in CAP is not at all sufficient. Paired serum samples may be used to detect seroconversion or a fourfold increase in antibody titer comparing the first and the second serum. Serology in addition is of limited value in newborns with maternal antibodies, in immunocompromised patients, in the elderly and patients receiving blood products. Antibody detection can be done with the complement fixation test (CFT, increasingly obsolete), ELISA-IgG, -IgA, -IgM, immunofluorescent assays, immunoblot and neutralization test.

Diagnostic comments on specific viruses Adenovirus Viruses can be detected in 25 to 72% of patients with disseminated infection in peripheral blood for more than 3 weeks. Detection of virus from multiple sites correlates with more severe disease and higher mortality. It is essential to monitor viral load during antiviral therapy. Phenotyping of isolates can be done by neutralization test, by hemagglutination test or by serotype-specific monoclonal antibodies and finally by PCR. Group-specific antigens are used in CFT for antibody detection in the second week of disease. CFT is insufficient to detect antibodies in infants. IgG antibodies are detectable by ELISA at the end of the second week, whereas IgM antibodies are not detectable in all cases with primary infection [10].

Coronavirus HCoVs antigen can be detected by IFT using rabbit antisera (no monoclonals available). Coronaviruses RNA can be detected with RT-PCR also in stool. SARS-CoV can be detected in plasma, but in urine and stool longer than 4 weeks. Virus load is unusually low in the early phase of SARS. A

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chip-based test detecting 10 respiratory viral pathogens including coronaviruses has been developed. Non-SARS human coronaviruses are difficult to isolate. With SARS-HCoV no IgG and IgM antibodies can be found within the first 7 days of disease, seroconversion may be delayed up to 8 weeks and IgG does not persist [34, 118, 119].

Influenzavirus Rapid antigen detection tests are available (15 min) which differentiate between A and B viruses and some now include avian influenza virus H5N1. Sensitivity and specificity range from 60–95% and 52–99% respectively. RTPCR is much more sensitive and allows virus typing. Virus isolation is still important for characterization of circulating influenza strains. Antibody detection with ELISA-IgG, -IgA and IFA-IgG, -IgA is possible, but shows low levels of both IgG and IgA, a delayed peak antibody titer and shorter persistence of antibodies in elderly patients.

Parainfluenzavirus and respiratory syncytial virus In adults and elderly people virus shedding is lower and shorter. RT-PCR is more sensitive in patients with parainfluenzavirus and RSV infections, particularly reinfections, and it might be useful especially for rapid diagnosis in elderly. For rapid RSV antigen detection in pediatric patients different antigen detection assays (20 min) with sensitivities between 61 to 92% and specificities between 93 to 98% are available [120]. Alternatively the rapid immunofluorescent antigen assay can be used. Since RSV is thermolabile, it is important to use a qualified transport medium, and to have a cooled and short transport until processing (30 min). The shell vial culture assay shows the highest sensitivity (94.3%) and specificity (96.9%) for detecting RSV from NPA in children younger than 1 year. Only 41% of RSV-infected children could be identified by serology and antibodies titers that develop are usually low, although both serum and secretory antibodies are produced [121].

Human metapneumovirus Commercial monoclonal antibodies are available for IFA. The sensitivity and specificity of this test is 73.3 and 97% respectively and results agree with RT-PCR in 89.6% [122].

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Isolation of HMPV is difficult and regardless of CPE the use of RT-PCR to enhance the HMPV identification in cell culture is indicated. No commercial tests are available for antibody detection.

Picornaviruses (rhinovirus / enterovirus) Due to more than 100 distinct serotypes a rapid detection of rhinovirus capsid antigen cannot be developed. The application of molecular assays has markedly increased the detection rate of picornaviruses in acute respiratory infections. Rhinovirus culture is the “gold standard,” but it takes 3 to 7 days. The sensitivity of PCR is superior to the infectivity detection in the cell culture. No reliable typespecific serological test for rhinovirus infections exists. Entervoirus serology by neutralization tests is possible but difficult and not completely reliable.

Hantavirus Currently no antigen detection assay is available. RT-PCR for hantavirus RNA detection is useful to detect infection and to identify the viral genotype. RT-PCR for viral RNA detection can be done from whole blood or serum during the acute phase in the first 10 days of illness. Virus isolation is inefficient and there is no routine assay. Serum specimens were tested for IgM and IgG antibodies by ELISA using Sin Nombre virus antigen, according to the guidelines of the US Centers for Disease Control (CDC). Positive results indicate infections with new world hantaviruses. IgM antibodies can be detected in all acute cases, maximal IgG level occurs during the first week of illness and are detectable after disease for a relatively long period.

HSV, VZV and CMV Since HSV, VZV and CMV pneumonia can be effectively treated it is important to rapidly investigate specimens from the lower respiratory tract, because pneumonia can only be established on the basis of BAL or lung tissue examined by PCR (HSV, VZV) and/or shell vial culture (HSV). Serologic tests are not relevant in case of HSV reactivation. Primary VZV infection can be confirmed by IgM detection and by IgG seroconversion. In premature infants with risk for CMV pneumonia CMV DNA can be found directly by PCR in urine and in the pharynx. Virus detection in air-

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way specimens (virus load in BAL) and lung tissue will be positive before seronconversion.

Summary In various studies and case reports it has been shown that respiratory viral pathogens are frequently involved in community-acquired pneumonia. Age groups at risk are the very young and elderly, as well as persons with comorbidity, where immune response is restricted. The frequency of detection of respiratory viruses varies in different studies due to methodological problems, patient selection and the fact that only detection of specific viruses was performed. With modern diagnostic tools it has been increasingly realized that rhinovirus and enterovirus infections are the most common reasons for unnecessary antibiotic therapy, often also adenovirus infections. Future studies have to consider the whole range of viral agents that can be involved in community-aquired pneumonia. In recent decades several new respiratory viruses have been described and it is reasonable to assume that new viruses or virus types from different virus families will emerge in the future, often after having crossed a host species barrier with high pathogenic potential for severe pulmonary diseases. Most of the relevant infections can be diagnosed by virus detection. Several studies in children have documented a significant proportion (25 to 30%) of viral/bacterial infections beside isolated virus infections. Children with isolated virus infection tended to be younger than coinfected children. The implications of coinfecting agents in epidemiology, pathogenicity and clinical outcome have to be elucidated.

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100 Knox KK, Pietryga D, Harrington DJ, Franciosi R, Carrigan DR (1995) Progressive immunodeficiency and fatal pneumonitis associated with human herpesvirus 6 infection in an infant. Clin Infect Dis 20: 406–413 101 Cone RW, Huang ML, Hackman RC (1994) Human herpesvirus 6 and pneumonia. Leuk Lymphoma 15: 235–241 102 Plummer G, Benyesh-Melnick M (1964) A plaque reduction neutralization test for human cytomegalovirus. Proc Soc Exp Biol Med 117: 145–150 103 Merk J, Schmid FX, Fleck M, Schwarz S, Lehane C, Boehm S, Salzberger B, Birnbaum DE (2005) Fatal pulmonary failure attributable to viral pneumonia with human herpes virus 6 (HHV6) in a young immunocompetent woman. J Intensive Care Med 20: 302–306 104 Gautschi O, Berger C, Gubler J, Laube I (2003) Acute respiratory failure and cerebral hemorrhage due to primary Epstein-Barr virus infection. Respiration 70: 419–422 105 Ankermann T, Claviez A, Wagner HJ, Krams M, Riedel F (2003) Chronic interstitial lung disease with lung fibrosis in a girl: uncommon sequelae of EpsteinBarr virus infection. Pediatr Pulmonol 35: 234–238 106 Mueller GA, Pickoff AS (2003) Pediatric lymphocytic interstitial pneumonitis in an HIV-negative child with pulmonary Epstein-Barr virus infection. Pediatr Pulmonol 36: 447–449 107 Kaan PM, Hegele RG, Hayashi S, Hogg JC (1997) Expression of bcl-2 and Epstein-Barr virus LMP1 in lymphocytic interstitial pneumonia. Thorax 52: 12–16 108 Marzouk K, Corate L, Saleh S, Sharma OP (2005) Epstein-Barr-virus-induced interstitial lung disease. Curr Opin Pulm Med 11: 456–460 109 Oda Y, Okada Y, Katsuda S, Nakanishi I (1994) Immunohistochemical study on the infection of herpes simplex virus, human cytomegalovirus, and Epstein-Barr virus in secondary diffuse interstitial pneumonia. Hum Pathol 25: 1057–1062 110 Pfleger A, Eber E, Popper H, Zach MS (2000) Chronic interstitial lung disease due to Epstein-Barr virus infection in two infants. Eur Respir J 15: 803–806 111 Balfour-Lynn IM, Girdhar DR, Aitken C (1995) Diagnosing respiratory syncytial virus by nasal lavage. Arch Dis Child 72: 58–59 112 Heikkinen T, Marttila J, Salmi AA, Ruuskanen O (2002) Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses. J Clin Microbiol 40: 4337–4339 113 Falsey AR, Formica MA, Walsh EE (2002) Diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-PCR to viral culture and serology in adults with respiratory illness. J Clin Microbiol 40: 817–820 114 Akhtar N, Ni J, Stromberg D, Rosenthal GL, Bowles NE, Towbin JA (1999) Tracheal aspirate as a substrate for polymerase chain reaction detection of viral genome in childhood pneumonia and myocarditis. Circulation 99: 2011–2018 115 Simpson JL, Moric I, Wark PA, Johnston SL, Gibson PG (2003) Use of induced sputum for the diagnosis of influenza and infections in asthma: a comparison of diagnostic techniques. J Clin Virol 26: 339–346 116 Xiang X, Qiu D, Chan KP, Chan SH, Hegele RG, Tan WC (2002) Comparison of three methods for respiratory virus detection between induced sputum

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and nasopharyngeal aspirate specimens in acute asthma. J Virol Methods 101: 127–133 Jain P, Sandur S, Meli Y, Arroliga AC, Stoller JK, Mehta AC (2004) Role of flexible bronchoscopy in immunocompromised patients with lung infiltrates. Chest 125: 712–722 Woo PC, Lau SK, Wong BH, Tsoi HW, Fung AM, Kao RY, Chan KH, Peiris JS, Yuen KY (2005) Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 43: 3054–3058 Niedrig M, Leitmeyer K, Lim W, Peiris M, Mackenzie JS, Zambon M (2005) First ex-ternal quality assurance of antibody diagnostic for SARS-new coronavirus. J Clin Virol 34: 22–25 Gregson D, Lloyd T, Buchan S, Church D (2005) Comparison of the RSV respi-strip with direct fluorescent-antigen detection for diagnosis of respiratory syncytial virus infection in pediatric patients. J Clin Microbiol 43: 5782–5783 Brandenburg AH, Groen J, van Steensel-Moll HA, Claas EC, Rothbarth PH, Neijens HJ, Osterhaus AD (1997) Respiratory syncytial virus specific serum antibodies in infants under six months of age: limited serological response upon infection. J Med Virol 52: 97–104 Ebihara T, Endo R, Ma X, Ishiguro N, Kikuta H (2005) Detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofluorescent-antibody test. J Clin Microbiol 43: 1138–1141

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Resistance in Streptococcus pneumoniae Mathias W. R. Pletz1, Lesley McGee2 and Tobias Welte1 1Department

of Respiratory Medicine, Hannover Medical School, Carl- Neuberg-Str.1, Hannover, 30625, Germany; 2Hubert Department of Global Health, Emory University, 1518 Clifton Road, Atlanta, GA, 30322, USA

Abstract Streptococcus pneumoniae is a leading cause of community-acquired lower respiratory tract infections, sinusitis, meningitis, and bloodstream infections. Pneumococci are Gram positive, encapsulated bacteria and exhibit more than 90 different capsular serotypes. Resistance to penicillin in clinical isolates was reported anecdotally as early as 1965, but was not considered a major concern until the mid-1990s. In the 1990s, there was a tremendous global increase in resistance to penicillins and this led to the increased use of macrolides and tetracyclines to treat infections. After several years, the resistance rates to these antibiotics began to increase as well. Currently, fluoroquinolones are used most frequently to treat community-acquired respiratory infections in adults and resistance rates globally are still low. Pneumococci are naturally competent bacteria and frequently acquire resistance by intraspecies or interspecies gene transfer. Resistance to `-lactams is due to the acquisition of different mutations within the pencillin-binding proteins that have been demonstrated to originate from the less pathogenic viridans streptococci. Other mechanisms of antibiotic resistance include enzymes and efflux pumps on mobile genetic elements (e.g. erm and mef), or resistance arising through spontaneous mutations. Clinical studies show that resistance, particularly to penicillins, is not always related to clinical failure. The global increase in resistance rates in pneumococci is in part due to the spread of a limited number of highly sucessful multiresistant pneumococcal clones. Isolates belonging to a specific clone, defined by sequence types according to multilocus sequencing, often exhibit the same serotype. However, capsular switching due to genetic rearrangements within the same clone has been observed. The recently introduced seven-valent conjugated pneumococcal vaccine has been shown to decrease disease and carrier rates of the included serotypes. Since some of the multiresistant clones exhibit vaccine serotypes, resistance rates to penicillin, macrolides and fluoroquinolones have been decreasing since the introduction of this vaccine.

INTRODUCTION S. pneumoniae is the most frequent pathogen causing community-acquired pneumonia and for many years penicillin or ampicillin were the drugs of choice for all pneumococcal infections.

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In contrast to other bacterial species, such as staphylococci or Gramnegative bacteria, pneumococci had remained susceptible to almost all antibiotics for decades. Penicillin-resistant strains were selected in the laboratory already in the 1940s, but it took more than 30 years before the first clinical isolates with penicillin-resistance were detected. The subsequent global emergence of penicillin-resistance in the 1980s and 1990s led to the increased use of macrolides, other non-beta lactam antibiotics and the fluoroquinolones. Due to these new selective pressures, resistance to macrolides and tetracyclines emerged rapidly. For both classes it could be demonstrated that the increase in resistance rates paralleled the use of these individual classes of antibiotics [1]. It had also been shown that this trend could be reversed when the use of the concerned antibiotic was restricted [2]. This data explains, in part, the considerable differences in rates of antibiotic resistance published between different countries and reflects the antibiotic prescription behavior. In general, antibiotic resistance in countries where antibiotics can be purchased without a prescription (over the counter) as in Spain and Mexico, are higher than in countries where antibiotics can only be prescribed by physicians. Recently, resistance to fluoroquinolones has emerged, but there are global differences reported with individual cases described in Austria and rates as high as 13.3% in Hong Kong [3, 4]. Due to the tremendous cost for the development of antibiotics and the challenges associated with the safety of newly developed drugs, many pharmaceutical companies have discontinued their development of new antibiotics. During the last few years only a limited number of new antibiotics were licensed, such as telithromycin, linezolid and quinupristin-dalfopristin. Clinical pneumococcal isolates with resistance to quinupristin-dalfopristin, telithromycin and linezolid have already been reported [5–7]. In this context it is also of concern that pneumococci with tolerance to vancomycin, an antibiotic used as a reserve against multi-resistant Gram-positive bacteria, have already been documented [8]. Resistance rates parallel antibiotic usage and exhibit therefore tremendous regional differences and trends [3, 9]. Besides numerous local and regional surveillance studies, several large longitudinal surveillance projects monitor globally the trends of antibiotic resistance in pneumococci (i.e. the Alexander project, Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin – PROTEKT). Table 1 shows resistance rates in eight European countries according to results of the PneumoWorld study from 2001–2003 [3].

Genetic background Bacterial resistance may be “intrinsic” or an inherent – naturally occurring property of an organism, i.e., these organisms may lack the appropriate drug-susceptible target or possess natural barriers that prevent the agent

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Table 1. MIC90 and antibiotic resistance of 2,279 isolates of S. pneumoniae in eight European countries according to Reinert et al. [(3] (Abbreviations: I, intermediate; R, resistant) Country (no. of strains)

Antibiotic

Austria (n = 160)

Penicillin G

0.03

Cefuroxime

0.06

0.6

Clarithromycin

0.25

10.0

Belgium (n = 148)

Germany (n = 530)

Portugal (n = 174)

Spain (n = 310)

Switzerland (n = 52)

4.4

1

0

Tetracycline

4

10.6

Penicillin G

0.125

11.5

Cefuroxime

0.25

8.8

32

23.7

Levofloxacin

1

0.7

Tetracycline

32

23.7

Penicillin G

2

47.6

Cefuroxime

8

39.1

Clarithromycin

32

46.1

Levofloxacin

1

0.9

Tetracycline

32

40.4

Penicillin G

0.03

6.0

Cefuroxime

0.06

1.5

1

10.6

Clarithromycin

Italy (n = 462)

% Resistance (I+R)

Levofloxacin

Clarithromycin

France (n = 443)

MIC90

Levofloxacin

1

0.4

Tetracycline

4

11.3

Penicillin

0.25

13.0

Cefuroxime

0.5

7.4

Clarithromycin

32

35.5

Levofloxacin

1

1.3

Tetracycline

32

23.8

Penicillin G

2

19.0

Cefuroxime

8

14.4

Clarithromycin

1

10.3

Levofloxacin

1

1.2

Tetracycline

8

12.1

Penicillin G

2

61.9

Cefuroxime

8

43.9

Clarithromycin

32

43.6

Levofloxacin

1

1

Tetracycline

32

47.1

Penicillin G

0.5

17.3

Cefuroxime

2

11.5

0.25

17.3

Levofloxacin

1

0

Tetracycline

4

13.5

Clarithromycin

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from reaching its target. Acquired resistance results either from spontaneous mutations in existing DNA or the acquisition of foreign genetic material by genetic elements such as plasmids and bacteriophages (horizontal gene transfer). To date, no plasmids have been detected in any resistant S. pneumoniae strain and genetic changes conferring resistance are therefore all chromosomal. Pneumococci are one of the few bacterial species that are naturally competent and are able to acquire resistance genes through horizontal gene transfer, usually via the process of transformation. Pneumococci are able to bind, internalize and integrate free DNA by recombination without antecedent stimulation as it is, for example, necessary for E. coli. The origin of the genetic material for these transformations may be from the same (intraspecies recombination) or a different – but closely related – species (interspecies recombination). The results of interspecies recombination result in mosaic genes, which are genes consisting of both pneumococcal sequences and sequences from other species. The higher the degree of homology between the donor and recipient DNA the higher is the likelihood of successful homologous recombination. Therefore the amino acid sequence of the donor and recipient DNA is often identical. In contrast, due to the degeneration of the genetic code the 3rd nucleotide of a codon coding for the same amino acid can be different. The resulting “codon usage bias” is species specific. A characteristic feature of integrated DNA derived from another species is a repeated mismatch of the 3rd nucleotide leading to a decreased similarity of the recombined sequence when compared to a reference sequence (Fig. 1). Viridans streptococci have been frequently observed as a donor species for the transfer of genetic resistance determinants to S. pneumoniae. Viridans streptococci belong to the commensal flora and are exposed to the antibiotics administered to the host and resistant strains should therefore be easily selected. Since viridans streptococci are to a high degree homologous with S. pneumoniae, recombination between the two species exhibits a high success rate. In addition, both species colonize the human nasopharynx and so contact between the two species is guaranteed, which is necessary for the exchange of genetic material. Since commensal bacteria rarely cause disease, they are not routinely tested for resistance and surveillance studies addressing resistance in commensal bacteria are rare. In pneumococci acquisition of foreign genetic material and the consequent spread of resistant strains seems to be the primary cause for the increase in resistance to different antibiotics.

Spread of resistance in pneumococci – the clone concept The global spread of penicillin-resistant pneumococci has been documented by numerous surveillance studies. Genetic fingerprinting using techniques like pulsed-field gel electrophoresis of pneumococcal DNA digested by

Resistance in Streptococcus pneumoniae

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Figure 1. Alignment of PBP 2b of a penicillin-resistant S. pneumoniae strain (#402) with the susceptible pneumococcal laboratory reference strain R6. The red part of the sequence is derived from S. oralis. (Courtesy of Dr B. Beall, CDC)

the restriction enzyme SmaI, has revealed that the majority of the resistant clinical isolates belong to a small number of highly successful clones, some of which have spread globally [10]. More recently, another tool to investigate the genetic relatedness of resistant isolates became available, namely multi-locus sequence typing (MLST) [11]. MLST is based on the sequencing of seven highly conserved “housekeeping genes.” The sequences at each of the seven loci are compared with all of the known alleles at that locus which is available at the pneumococcal MLST web site (http: //spneumoniae.mlst. net/). The alleles at each of the seven loci define the allelic profile of each isolate and their sequence type (ST). If isolates have identical sequence types, or differ at less than three loci, they are considered to be related. Many studies have used PFGE for the rapid clustering of genetically related isolates and MLST to assign nonsubjective and electronically portable clone identifiers to clusters of related isolates. In order to create a nomenclature to monitor the international spread of resistant pneumococci, in 1997 the Pneumococcal Molecular Epidemiology Network (PMEN) was established under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antibiotic-resistant pneumococcal clones (http: //www.sph.emory.edu/PMEN/) [12]. Currently, the PMEN describes 26 international clones, some of which have a global distribution and have been reported in many countries with varying antibiograms. The global spread of the PMEN-clone 1, Spain 23F-1, is illustrated in Figure 2. Pneumococci have the ability to switch their capsular serotypes which can be due to mutations or the exchange of capsular genes. For surveillance it is therefore not appropriate to equal serotype and sequence type (clone) since capsular variants of clones exist, e.g., 19F capsular variants of PMEN-clone 1 is designated Spain 23F-1-19F.

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Figure 2. Global distribution of the PMEN strain Spain23F-1.

Serotype switching serves also as a mechanism to avoid the selective pressure due to vaccination with vaccines containing only a limited number of serotypes. PMEN clones are spreading more successfully than other pneumococci. The reason for that fitness advantage is not yet clear, but it has been observed that resistance to some classes of antibiotics has emerged rapidly once it has been introduced into these clones [13].

Beta-lactams Beta-lactams bind and inactivate the so-called penicillin-binding proteins (PBPs). These proteins are enzymes that catalyze the terminal stages of murein synthesis which is the main component of the bacterial cell wall [14]. The action of `-lactams leads to cell death, i.e. the action is bactericidal. Most of the bacterial species exhibiting `-lactam resistance secrete or contain `lactamase enzymes that cleave the `-lactams. In contrast to other bacterial species, `-lactam resistant pneumococci do not express any `-lactamases. The pneumococcal mechanism of resistance to `-lactams consists of multiple mutations within several penicillin-binding proteins (PBPs)[15]. These mutations mediate changes in the structure of the penicillin-binding domain of the PBP leading to a decreased affinity to `-lactams. Six types of PBPs are found in pneumococci: PBP 1a (92–100 kDa), PBP 1b (89–95 kDa), PBP 2x (85 kDa), PBP2a (80–81 kDa), PBP 2b (77–78 kDa), and PBP 3 (43–52 kDa) (K 64, 117). High-level resistance to third-generation cephalosporins has occurred primarily by the development of altered forms of PBP1a and 2x, whereas high-level penicillin resistance additionally requires alterations of PBP2b [16]. It has been found that the genes of low-affinity forms of

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PBPs 1a, 2b and 2x from clinical isolates contain sequence blocks with up to 20% divergence in their nucleotide sequence. Such sequence blocks have not evolved by spontaneous mutations and the majority of these alterations do not confer amino acid substitutions (silent mutation). The mosaic blocks are shared between resistant S. pneumoniae and the closely related viridans streptococci (S. oralis, S. mitis). Based on these observations, it has been assumed that `-lactam resistance developed originally in viridans streptococci and was transferred to pneumococci by interspecies recombination [17]. This hypothesis is supported by the fact that genes adjacent to the pbps (e.g., ddl) are frequently part of the recombination and appear in resistant strains. This was recently observed in very high level penicillin resistant (MIC * 8 mg/l), invasive pneumococcal isolates from the CDC’s ABC Surveillance study. The ddl locus in two of these strains was from S. oralis, suggesting that the origin of very-high-level resistance in some strains may result from the transformation and incorporation of resistance determinants from viridans group streptococci [18]. There is evidence that altered PBPs from resistant pneumococci have a preference for branched peptides in regard to the cell wall synthesis [19]. This is in contrast to PBPs from susceptible strains which use primarily linear stem peptides. A newly identified protein MurM which, together with MurN, is involved in the synthesis of short peptide branches in the pneumococcal cell wall. Cells in which MurM was inactivated produced cell walls without branches and also completely lost penicillin resistance [20]. Currently, the role of MurM in `-lactam resistant strains is the subject of further studies.

Macrolides Macrolides consist of saccharides that are attached to 14-membered (erythromycin, clarithromycin), 15-membered (azithromycin) or 16-membered rings. The use of the latter is usually restricted to veterinary purposes. Macrolides act bacteriostatically by binding to the 23S ribosomal RNA of the 50S subunit of the bacterial ribosome and consequently blocking the elongation step of protein synthesis. There are two main mechanisms of macrolide resistance: active efflux and target site modification. Active efflux is performed by an energy dependent cell membrane transporter protein that is encoded by the mef gene, which is located on a conjugative transposon. The mef gene provides lower levels of resistance and confers the so-called M-phenotype, i.e. resistance primarily to macrolides. Two variants of the mef gene, mef(A) (primarily found in S. pyogenes) and mef(E) have been identified in pneumococci. However, due to confusion in the nomenclature many authors do not differentiate between the two and call it mef(A) regardless of the variant. Target site modification is performed by a methylase enzyme which adds a CH3-group to an adenine residue on the

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23S rRNA. The methylation blocks the binding site of not only macrolides, but also lincosamides and streptogramins and the resulting phenotype is called MLSB. The methylase enzyme is encoded by the erm gene which is also located on a transposon. Among bacterial species there exists several variants of the erm gene, in pneumococci primarily the erm(B) gene is found. Some macrolide resistant pneumococci have been documented to exhibit both the erm and mef genes. These strains appear to be highly clonal and most belong to the PMEN clone Taiwan19F-14 [21]. A third, currently emerging mechanism of resistance to macrolides consists of mutations conferring amino acid substitutions in the ribosomal proteins and nucleotide mutations in the 23S rRNA itself. Frequently, the ribosomal proteins L4 and L22 are involved and mutations in the rRNA and ribosomal proteins can confer new resistance phenotypes with combined resistance to macrolides and lincosamides (ML) or macrolides, ketolides and streptogramins (MKSB). Usually, the decrease in susceptibility to ketolides (for example telithromycin) is less than the decrease in susceptibility for other MLKS(B) agents [22].

Tetracyclines Tetracyclines are antibiotics derived from Streptomyces species and inhibit bacterial protein synthesis by blocking attachment of the aminoacyl-tRNA to the ribosome. Resistance to tetracyclines in pneumococci is conferred by ribosomal protection proteins Tet(M) and Tet(O). These proteins are homologous to the elongation factors EF-Tu and EF-G. It is assumed that Tet(M) and Tet(O) induce the detachment of tetracyclines from the bacterial ribosome, the detailed mechanism of action is not known. Tet(M) and Tet(O) are encoded by the tet(M) and tet(O) genes that are located on a transposon. In species other than pneumococci, resistance to tetracyclines is frequently mediated by plasmids.

Fluoroquinolones Resistance to fluoroquinolones in pneumococci is caused by efflux and/or by mutations in the quinolone resistance-determining regions (QRDR) of the genes coding for type II topoisomerase enzymes: DNA gyrase and topoisomerase IV. Mutations conferring resistance occur in a stepwise fashion, with mutations observed in either parC or gyrA (depending on the selecting fluoroquinolone) or both, leading to decreased fluoroquinolone susceptibility [23]. Strains usually become fully fluoroquinolone resistant with the addition of a mutation in the other target gene (either gyrA or parC) and mutations in parE and gyrB may contribute to resistance in some isolates.

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QRDR mutations can arise spontaneously or be transferred by the integration of foreign genetic material. In comparison to `-lactam resistance, horizontal gene transfer seems to play a minor role in fluoroquinolone resistance. Studies addressing that question found evidence for horizontal gene transfer in 0–11% of fluoroquinolone resistant isolates and interestingly, this ratio seems to be higher in respiratory isolates than in invasive isolates [24–26]. This discrepancy might be explained by the hypothesis that interspecies recombination takes place in the nasopharynx, which is colonized by both pneumococci and viridans streptococci. One could speculate that the antecedent nasopharyngeal colonization period for invasive strains is shorter than that of respiratory isolates. Therefore, there may be less time for interspecies recombination in invasive isolates. The impact of efflux on fluoroquinolone resistance seems to be more limited and selective. To date, no highly resistant isolate has been found with efflux as the only mechanism of resistance. Fluoroquinolones with a small molecule size, e.g. ciprofloxacin, seem to be affected to a higher extent than larger molecules such as moxifloxacin. A putative efflux pump, PmrA was described by Gill et al., that exhibits homology to the multidrug efflux pumps NorA and Bmr [27]. PmrA consists of 12 transmembrane segments as efflux proteins of the proton-dependent pumps. In addition, a recent article describes the presence of a non-PmrA pump in S. pneumoniae. Many studies have addressed the epidemiology of PmrA by comparing ciprofloxacin MICs in the absence and the presence of the pump inhibitor reserpine. In contrast to Mef(A), PmrA seems not to be encoded by a resistance gene but rather over expression. However, little is know about the mechanism of the expression regulation of PmrA.

Resistance to new antibiotics Since 1962, only a few classes of novel antibiotics have been introduced, and all since 1999, including the streptogramins (quinupristin/dalfopristin), the oxazolidinones (linezolid), and the lipopeptides (daptomycin). All other recently introduced drugs, such as tigecycline and ertapenem are derived from antibiotic classes that are already in use. The ketolides (e.g., telithromycin) were developed from the macrolides to overcome antibiotic resistance in pneumococci and are characterized by the lack of the L-cladinose sugar at position 3 of the erythronolide A moiety, which is replaced by a keto group. Farrell and Felmingham recently reported only 10 isolates to be telithromycin resistant among a worldwide collection of 13,874 S. pneumoniae isolates isolated between 1999 and 2003. The strains isolated in France, Italy, Spain, Hungary, and Japan had telithromycin MICs of 4 to 8 +g/ml and showed an erm(B) genotype [28]. Reinert et al. recently described a telithro-

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mycin resistant isolate in Germany (MIC 8 +g/ml) that exhibited in addition to the erm(B) genotype, mutations in the L4 (S20N) protein [6]. The S20N L4 alteration has been shown to contribute to the increase in macrolide MICs in pneumococci. It may be assumed that a combination of erm(B) and L4 mutations confer ketolide resistance. Also a single base deletion within the 23S-rRNA has been detected to confer resistance to macrolides and telithromycin [29]. Another clinical isolate resistant to telithromycin and fluoroquinolones with a still unidentified mechanism was recently found in Argentina [30]. Streptogramin (quinupristin-dalfopristin, Q/D) resistance among Grampositive cocci has been very uncommon [31]. Two clinical isolates among 8,837 (0.02%) S. pneumoniae isolates were discovered in 2001 to 2002 with Q/D MICs of 4 +g/ml. Each had a 5-amino-acid tandem duplication (RTAHI) in the L22 ribosomal protein gene (rplV) preventing synergistic ribosomal binding of the streptogramin combination [7]. Recently, two clinical S. pneumoniae isolates, identified as nonsusceptible to linezolid and as resistant to macrolides and chloramphenicol, were found to contain 6-bp deletions in the gene encoding riboprotein L4 [5].

Clinical relevance of resistance MIC based breakpoints have been suggested by different organizations to distinguish between susceptible, intermediate and resistant strains. The breakpoints published by the CLSI (formerly the NCCLS) are widely used in routine clinical laboratories and in surveillance studies. However, in some cases these breakpoints are unable to predict clinical failure in patients who were discordantly treated. Pharmacodynamic parameters are superior to MIC in predicting bacteriological eradication that is related to clinical outcome. To calculate these parameters, ideally antimicrobial susceptibility (measured by MIC) is compared to the achievable unbound fraction of antibiotic at the site of infection. Since the latter is difficult to measure, usually serum/plasma concentrations obtained from clinical studies with healthy volunteers are used instead. This may lead to some limitations since recent pharmacokinetic studies have clearly demonstrated the there can be tremendous differences in the concentration of an antibiotic between healthy subjects and severely ill patients, in regard to the patient’s age and the body site.

Clinical relevance of `-lactam resistance In pneumococcal pneumonia caused by penicillin-resistant strains, no association between treatment failure and discordant therapy could be observed in several studies, as long as the administration was intravenous

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and drugs were penicillin, ampicillin, amoxicillin, cefotaxime or ceftriaxone. The only studies that have found such an association were not stratified for the severity of disease or did not document the susceptibility of the pneumococci to the drug that failed. The basis of that seeming contradiction is high serum concentrations of these `-lactams and relatively low breakpoints defining resistance [32]. The main PK/PD parameter of `-lactams which are time-dependent killing antibiotics is the proportion of time of the dose interval during which the plasma concentration exceeds the MIC (T > MIC) [33]. Pharmacodynamic calculations predict that high doses of intravenous penicillin remain useful for the treatment of pneumococcal pneumonia up to the MICs of 4 mg/l (non-susceptibility according to CLSI is defined by MIC, > 0.12 mg/l). Pharmacodynamic also predicts that `-lactams with less anti-pneumococcal activity may lead to failure. Examples of such failures have been documented for cefazolin, cefuroxime and ticarcillin. Only a few data are available on the clinical failure of oral `-lactam therapy. However, due to the decreased bioavailability of ampicillin and amoxicillin such failure could be expected and the failure of low-dose amoxicillin prophylaxis to prevent penicillin-resistant pneumonia in patients with sickle cell disease has been documented. In a recent cohort study presented at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (45th, ICAAC, Washington, D.C., 2005) Iannini et al. investigated treatment failure in outpatients with pneumococcal pneumonia (abstract # 896). In patients treated orally with cephalosporines they found cephalosporine resistance associated treatment failure, however, this was not found in outpatients treated orally with penicillins. This might be also caused by the unfavourable bioavailability of cephalosporins. In another abstract presented at the same conference File et al. described the eradiaction of penicillin resistant pneumococci (amoxicillin MIC, ) 4mg/l) in 43 out of 44 patients with pneumonie by amoxicillin/clavulanic acid (abstract # 707). Since pneumococci do not express `-lactamases this effect has to be contributed primarily to amoxicillin.

Macrolides The clinical relevance of macrolide resistance has been a matter of debate. After the first break through bacteraemias during macrolide treatment, it was thought that only resistant strains harboring the erm(B) mechanism could cause treatment failure [34]. It has since become clear that also strains with the efflux mechanism can exhibit high MIC’s resulting in treatment failure. Of particular concern is the recent observation that mutations in the ribosomal proteins or the 23S rRNA itself can develop during macrolide treatment and confer MIC’s high enough to cause treatment failure [35].

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Clinical relevance of fluoroquinolone-resistance and first step mutants Whereas most studies have shown no increase in mortality in patients with invasive disease infected with penicillin-resistant pneumococci, FQ resistance has been associated with clinical failure [36, 37]. In 2002, Davidson et al. published a paper on a topic that was receiving broad attention [38]. They described four patients treated with levofloxacin with pneumococcal pneumonia in whom treatment failure due to fluoroquinolone resistance was observed. Two of the patients died. Prior to this publication several reports had documented treatment failure due to fluoroquinolones resistance, however, most treatment failures occurred with ciprofloxacin, a fluoroquinolone with weak antipneumococcal activity. Meanwhile, several other reports of fluoroquinolone resistance associated treatment failure have been published and were reviewed in a recent paper by Fuller and Low [39]. During the stepwise acquisition of mutations leading to full resistance, so called “first-step mutants” are evolving. These first-step mutants are frequently phenotypically susceptible and therefore cannot be detected by routine resistance testing [40]. Their detection therefore requires the sequencing of the topoisomerase genes, a method outside the scope of most routine susceptibility testing in clinical laboratories, but an important method for surveillance laboratories. Once a mutation in one of the target enzymes is present there is a significant increased likelihood for the acquisition of mutations in the second target enzyme leading to complete resistance [41]. Croisier et al. exposed susceptible first-step mutants with mutations within parC to levofloxacin and moxifloxacin in a rabbit model, matching human pharmacokinetics and found that double mutants (i.e. containing mutations in parC and gyrA) resistant to these agents were selected under both treatment regimens [42]. This is particularly likely to occur when large pneumococcal populations, as observed in COPD for example, are exposed to fluoroquinolones [42].Therefore, FQ treatment of infections caused by first-step mutants can lead to the selection of resistant isolates resulting in treatment failure and a general increase in FQ resistance. Some authors consider first-step mutants to be a key parameter for the spread of FQ resistance and estimate that their prevalence may be several times higher than the prevalence of phenotypically resistant pneumococci [40, 43].

Limiting the spread of resistance – the impact of vaccines The main mechanism driving the increase of antibiotic resistance in pneumococci is clonal spread. Even if “clone” does not equal “serotype”, the most frequent isolates of the same clone exhibit also the same serotype. The majority of the serotypes exhibited by the main antibiotic resistant pneumococcal clones are covered by the 23-valent non-conjugated pneu-

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mococcal polysaccharide vaccine. The vaccine was demonstrated to reduce the severity of pneumococcal vaccine types but, because the vaccine does only induce a B-cell response, it cannot prevent the colonization of the nasopharynx by strains exhibiting the vaccine serotypes. In contrast, the seven-valent conjugated vaccine does induce also a T-cell response. Studies have shown that the overall prevalence of strains of vaccine serotypes (both in the vaccine target group and all age groups) has been reduced since the introduction of the conjugate vaccine in 2000 in the United States [44]. Obviously the immunological response introduced by the vaccine enables carrier to clear strains of vaccine serotypes. Since most of the vaccine serotypes are exhibited by antibiotic resistant clones, antibiotic resistance should decrease. This has already been demonstrated for penicillin and macrolide resistance [44–46]. Surveillance data indicate that the rise of fluoroquinolone resistance seems also to plateau in the United States after the introduction of the vaccine [47]. However, little is known about the long-term impact of the vaccine, and the extent to which vaccine serotypes will be replaced by nonvaccine types is still not clear. Pneumococci are naturally transformable, and capsular switching is a well-documented mechanism for the potential evasion of the host immune response. Reports of the emergence of penicillin-nonsusceptible clones of NVT sharing genetic relatedness with internationally established clones targeted by the vaccine [48, 49] have indicated the need to track phenotypic and genotypic changes within invasive pneumococci.

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minants (erm(B) and mef(A)) in South Africa. Antimicrob Agents Chemother 45(5): 1595–1598 Edelstein PH (2004) Pneumococcal resistance to macrolides, lincosamides, ketolides, and streptogramin B agents: molecular mechanisms and resistance phenotypes. Clin Infect Dis 38 (Suppl 4): S322–327 Pan XS, Ambler J, Mehtar S, Fisher LM (1996) Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob Agents Chemother 40(10): 2321–2326 Balsalobre L, Ferrandiz MJ, Linares J, Tubau F, de la Campa AG (2003) Viridans group streptococci are donors in horizontal transfer of topoisomerase IV genes to Streptococcus pneumoniae. Antimicrob Agents Chemother 47(7): 2072–2081 Bast DJ, de Azavedo JC, Tam TY, Kilburn L, Duncan C, Mandell LA et al (2001) Interspecies recombination contributes minimally to fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob Agents Chemother 45(9): 2631–2634 Pletz MW, McGee L, Beall B, Whitney CG, Klugman KP (2005) Interspecies recombination in type II topoisomerase genes is not a major cause for fluoroquinolone resistance in invasive Streptococcus pneumoniae isolates in the United States. Antimicrob Agents Chemother 49: 779–780 Gill M, Brenwald NP, Wise R (1999) Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob Agents Chemother 43: 187–189 Farrell DJ, Felmingham D (2005) The PROTEKT global study (year 4) demonstrates a continued lack of resistance development to telithromycin in Streptococcus pneumoniae. J Antimicrob Chemother 56(4): 795–797 Canu A, Malbruny B, Coquemont M, Davies TA, Appelbaum PC, Leclercq R (2002) Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob Agents Chemother 46(1): 125–131 Faccone D, Andres P, Galas M, Tokumoto M, Rosato A, Corso A (2005) Emergence of a Streptococcus pneumoniae clinical isolate highly resistant to telithromycin and fluoroquinolones. J Clin Microbiol 43(11): 5800–5803 Hancock RE (1997) Peptide antibiotics. Lancet 349(9049): 418–422 Feldman C (2004) Clinical relevance of antimicrobial resistance in the management of pneumococcal community-acquired pneumonia. J Lab Clin Med 143(5): 269–283 Craig WA (1998) Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 26(1): 1–10; quiz 11–12 Nuermberger E, Bishai WR (2004) The clinical significance of macrolide-resistant Streptococcus pneumoniae: it’s all relative. Clin Infect Dis 38(1): 99–103 Perez-Trallero E, Marimon JM, Iglesias L, Larruskain J (2003) Fluoroquinolone and macrolide treatment failure in pneumococcal pneumonia and selection of multidrug-resistant isolates. Emerg Infect Dis 9(9): 1159–1162 Ewig S, Ruiz M, Torres A, Marco F, Martinez JA, Sanchez M et al (1999) Pneumonia acquired in the community through drug-resistant Streptococcus pneumoniae. Am J Respir Crit Care Med 159(6): 1835–1842 Goldstein EJ, Garabedian-Ruffalo SM (2002) Widespread use of fluoroqui-

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nolones versus emerging resistance in pneumococci. Clin Infect Dis 35(12): 1505–1511 Davidson R, Cavalcanti R, Brunton JL, Bast DJ, de Azavedo JC, Kibsey P et al (2002) Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med 346(10): 747–750 Fuller JD, Low DE (2005) A review of Streptococcus pneumoniae infection treatment failures associated with fluoroquinolone resistance. Clin Infect Dis 41(1): 118–121 Lim S, Bast D, McGeer A, de Azavedo J, Low DE (2003) Antimicrobial susceptibility breakpoints and first-step parC mutations in Streptococcus pneumoniae: redefining fluoroquinolone resistance. Emerg Infect Dis 9(7): 833–837 Gillespie SH, Voelker LL, Ambler JE, Traini C, Dickens A (2003) Fluoroquinolone resistance in Streptococcus pneumoniae: evidence that gyrA mutations arise at a lower rate and that mutation in gyrA or parC predisposes to further mutation. Microb Drug Resist 9(1): 17–24 Croisier D, Etienne M, Bergoin E, Charles PE, Lequeu C, Piroth L et al (2004) Mutant selection window in levofloxacin and moxifloxacin treatments of experimental pneumococcal pneumonia in a rabbit model of human therapy. Antimicrob Agents Chemother 48(5): 1699–1707 Pletz MW, Shergill AP, McGee L, Beall B, Whitney C, Klugman KP (2006) Prevalence of first-step mutants among levofloxacin-susceptible invasive Streptococcus pneumoniae in the United States. Antimicrob Agents Chemother 50(4): 1561–1563 Whitney CG, Farley MM, Hadler J, Harrison LH, Bennett NM, Lynfield R et al (2003) Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. N Engl J Med 348(18): 1737–1746 McEllistrem MC, Adams JM, Patel K, Mendelsohn AB, Kaplan SL, Bradley JS et al (2005) Acute otitis media due to penicillin-nonsusceptible Streptococcus pneumoniae before and after the introduction of the pneumococcal conjugate vaccine. Clin Infect Dis 40(12): 1738–1744 Stephens DS, Zughaier SM, Whitney CG, Baughman WS, Barker L, Gay K et al (2005) Incidence of macrolide resistance in Streptococcus pneumoniae after introduction of the pneumococcal conjugate vaccine: population-based assessment. Lancet 365(9462): 855–863 Pletz MW, McGee L, Jorgensen J, Beall B, Facklam RR, Whitney CG et al (2004) Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States: evidence for clonal spread and the impact of conjugate pneumococcal vaccine. Antimicrob Agents Chemother 48(9): 3491–3497 Porat N, Arguedas A, Spratt BG, Treffler R, Brilla E, Loaiza C (2004) Emergence of penicillin-nonsusceptible Streptococcus pneumoniae clones expressing serotypes not present in the antipneumococcal conjugate vaccine. J Infect Dis190: 2154–2161 Pai R, Moore MR, Pilishvili T, Gertz RE, Whitney CG, Beall B and the Active Bacterial Core Surveillance Team (2005) Postvaccine genetic structure of Streptococcus pneumoniae serotype 19A from children in the United States. J Infect Dis 192: 1988–1995

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Influenza Hans-Dieter Klenk Institut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Str. 3, 35043 Marburg, Germany

Abstract Influenza-A-viruses have a wide host range and occur with a wide spectrum of variants defined by 16 HA and 9 NA subtypes. All of these subtypes occur in birds, whereas only some of them have so far been observed in man, pig, horse, and a number of other mammals. In contrast, influenza-B and C-viruses occur only in man, and there are no subtypes of these viruses. Influenza-A-viruses occasionally can be transmitted from aquatic birds, their natural reservoir, to terrestrial birds and mammals. On rare occasions, they adapt to the new species and establish thus new virus lineages. Adaptation requires multiple mutations and it may involve gene reassortment after co-infection with another virus. By these mechanisms, viruses with new surface glycoproteins and therefore a distinct change in antigenicity are generated. If a new virus with such an antigenic shift occurs in man, it causes a pandemic. Antigenic drift, unlike antigenic shift, is characterized by slight changes in antigenicity resulting from successive mutations in HA and NA. Antigenic drift is responsible for the annual human epidemics. It occurs not only with influenza-A-viruses, but also with influenza-B viruses. Influenza is a highly contagious disease that is transmitted by aerosols. Virus replication occurs in airway epithelia and reaches its peak 2–3 days after infection. Symptoms typically include high fever, chills, headache, sore throat, dry cough, myalgias, anorexia, and malaise. Complications include primary viral pneumonia, secondary bacterial pneumonia, or combined bacterial and viral pneumonia. Serious complications of influenza most often occur in people 65 years of age and older, in the very young, and in those of any age with underlying chronic cardiac, pulmonary, or metabolic disease. Vaccination is the most potent instrument for influenza control. Prime candidates for vaccination are persons at risk for complications and individuals who might transmit influenza to such persons. Inactivated vaccines obtained from infected chicken embryos are most commonly used. Neuraminidase inhibitors are the influenza antivirals of choice. Application is limited to a relatively small time window shortly before or after infection.

Introduction Influenza viruses are segmented negative stranded RNA viruses. They have a high genetic variability and occur with a large number of different

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variants in man and animals. Human influenza viruses are unique in their ability to cause recurrent seasonal epidemics of different severity as well as global pandemics during which acute febrile respiratory disease occurs explosively in large parts of the population. Influenza outbreaks can be traced back to the Middle Ages and antiquity. Particularly disastrous was the Spanish influenza which cost more than 40 million lives in 1918 and 1919. In the 1930s, influenza viruses have been identified in man and pigs. Twenty years later it was discovered that influenza viruses occur also in birds. It was then possible to systematically investigate these pathogens. Despite detailed knowledge on structure and replication of influenza viruses, many aspects of epidemiology and pathogenesis are still poorly understood.

Classification and structure of influenza viruses Influenzaviruses belong to the family orthomyxoviridae. There are three genera or types: influenza-A-viruses, influenza-B-viruses, and influenza-Cviruses. Influenza-C-viruses do not play a major role as human pathogens. The genome of influenza-A and B-viruses consists of eight single-stranded RNA molecules of negative polarity each of which encodes one or two viral proteins. The entire genome of influenza-A and B-viruses has a length of 13,600 and 14,600 nucleotides, respectively. The individual RNA segments have a circular structure resulting from base pairing at the 3’ and 5’ ends. The segmentation of the genome is of particular biological significance, since it is a precondition for the high genetic variability of these viruses. The RNA genome forms together with the polymerase proteins PB1, PB2, and PA the helical nucleocapsid core of virus particles. It is surrounded by the lipid-containing viral envelope that is lined on its inner side by the matrix protein M1 and has glycoprotein spikes on its outer surface. Influenza-A and B-viruses have hemagglutinin (HA) spikes with receptor binding and fusion functions and neuraminidase (NA) spikes with receptor destroying activity. The fusion activity of HA depends on proteolytic cleavage by cellular proteases and a pH dependent conformational change. This activation process results in exposure of the fusion domain of HA and its penetration into the target membrane and thus in membrane fusion. There are a number of other proteins. With influenza-A-viruses, these are the M2 protein that forms an ion channel in the viral envelope, the NS2 protein present in small amounts in virions, and the non-structural protein NS1 (Fig. 1). The surface glycoproteins of influenza viruses are the subtype and strain specific antigenic determinants. They are the major viral components responsible for eliciting protective immunity. Matrix and nucleocapsid proteins show type-specific antigenicity and allow the serological differentiation of types A, B, and C [1].

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Figure 1. Structure of influenza-virus. Upper panel: Electronmicrograph of a virus particle (diameter 100 nm). The spikes at the surface (length 10 nm) can be seen. Lower panel: Virus model. The envelope contains hemagglutinin (HA) and neuraminidase (NA) spikes as well the M2 protein. The interior of the particle is formed by ribonucleoproteins, consisting of the genomic RNA-segments, the nucleocapsid protein NP, and the polymerase proteins PB1, PB2, and PA. The inner side of the envelope is lined by the matrix protein M1. Virions contain also small amounts of the NS2 protein.

Replication cycle HA initiates infection by mediating binding to neuraminic acid-containing receptors and membrane fusion following endocytosis. The M2 ion channel plays an important role in uncoating by lowering the pH within the virus particle and, thus, allowing dissociation of the internal components. The nucleocapsid complexes are then transported into the nucleus, where transcription and replication take place. The genomic RNA (vRNA) serves as template for two different RNA species: complementary RNA (cRNA) which is a complete copy of vRNA, and mRNA with a cap structure at the 5’ end and with the 3’ terminal nucleotides of cRNA being replaced by a poly-A-tail. Non-coding sequences at the 3’ end of vRNA serve as primers for mRNA synthesis. The cRNA is the template for new vRNA molecules. The cap structures of the viral mRNAs are derived from cellular mRNA molecules. Viral mRNA utilizes the cellular translation machinery for the

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synthesis of viral proteins. The virus enhances the coding capacity of its genome by several mechanisms. These include splicing, expression of two cistrons in tandem position, and start of translation at two different initiation codons. Translation of nucleocapsid protein, polymerase proteins, matrix protein, and two non-structural proteins NS1 and NS2 occurs on free polysomes. Ribonucleoproteins are assembled in the nucleus and subsequently exported into the cytoplasm. The envelope proteins are translated at the rough endoplasmic reticulum and then transported by the exocytotic apparatus to the plasma membrane where virus particles are assembled in a budding process. The neuraminidase mediates virus release by removing receptors from the infected cell [2].

Epidemiology The epidemiology of influenza is largely determined by its high genetic variability. This is particularly obvious with influenza-A-viruses. They have a wide host range and occur with a wide spectrum of variants defined by 16 HA and 9 NA subtypes. All of these subtypes occur in birds, whereas only some of them have so far been observed in man, pig, horse, and a number of other mammals. In fact, aquatic birds appear to be the natural reservoir of influenza-A-viruses. In contrast, influenza-B and C-viruses occur only with man, and there are no subtypes of these viruses. The host barrier is not an insurmountable obstacle for influenza-A viruses. Thus, they occasionally can be transmitted from aquatic birds, their natural reservoir, to terrestrial birds and mammals with transient outbreaks of disease. On rare occasions, they adapt to the new species and thus establish new virus lineages. Adaptation requires multiple mutations and it may involve gene reassortment after co-infection with another virus. By these mechanisms, viruses with new surface glycoproteins and therefore a distinct change in antigenicity are generated. If a new virus with such an antigenic shift occurs in man, it causes a pandemic. There were three pandemics in the last century. After the Spanish influenza (H1N1) in 1918, an antigenic shift in HA and NA gave rise to the Asian influenza (H2N2) in 1957, and another shift in HA to the Hong Kong influenza (H3N2) in 1968. In 1977 the H1N1 subtype re-emerged and co-circulates now in man with H3N2 and influenza-B virus. More recently avian viruses of subtypes H5, H7, and H9 have been transmitted, but not yet adapted to man. This includes H5N1 viruses that emerged in 1997 in South-East Asia and caused large outbreaks in domestic and wild birds, and are now spreading into many other parts of the world (Fig. 2). Antigenic drift, unlike antigenic shift, is characterized by slight changes in antigenicity resulting from successive mutations in HA and NA. Antigenic drift is responsible for the annual human epidemics. It occurs not only with influenza-A-viruses, but also with influenza-B viruses [1].

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Figure 2. Influenza-A periods in man. Circulation periods of H1N1, H2N2, and H3N2 viruses are shown. In recent years, transmission of avian strains of subtypes H5, H7, and H9 to man have also been observed. These viruses have not adapted yet to man. Of particular concern, however, are the H5N1 viruses (“bird flu viruses”) that are now endemic in birds in Asia from where they rapidly spread to other parts of the world.

Pathogenesis Influenza viruses exhibit large differences in organ tropism and serverity of disease. In man and other mammals, influenza usually causes respiratory disease. In contrast, most avian viruses cause asymptomatic enteric infection, whereas a few H5 and H7 viruses lead to fowl plague or bird flu, a highly lethal systemic disease. Experimental studies at the molecular level have shown that many biological properties of influenza viruses contribute to their pathogenicity, such as replication efficiency, organ tropism, spread of infection, and sensitivity to host defense mechanisms. Thus, the immune status of the human population and the extent of the antigenic differences between viruses are largely the reason that new pandemic viruses induce more severe disease than interpandemic ones. In each phase of the viral life cycle, there are specific interactions between viral proteins and host factors, such as cell receptors, nuclear proteins, and proteases. The role of these interactions in pathogenesis is particularly evident with proteolytic activation of HA. The highly pathogenic avian viruses are activated by the protease furin that is ubiq-

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Figure 3. Cells infected with influenza virus in human airway epithelium. Ciliae are stained black. Cells infected with influenza virus A/Memphis/14/96 (H1N1) are stained brown. Virus infection targets specifically non-ciliated cells [5].

uitous and therefore promotes rapid virus spread within the organism. In contrast, the viruses causing local infection including the human influenza viruses are activated by proteases confined to specific tissues. Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae as well as several other bacteria secrete also HA activating proteases. After co-infection with these microorganisms, influenzavirus infections show therefore a particularly severe course of disease [3]. Other mechanisms that contribute to pathogenicity are the interferon antagonism of the NS1 protein and an activity optimum of the viral polymerase probably also depending on host factors. Examination of tissues obtained from patients revealed that viral replication can occur throughout the respiratory tract. These studies also indicated that ciliated epithelial cells are a primary site of infection, whereas recent studies carried out on cultured airway epithelia revealed that human influenza viruses target specifically non-ciliated cells (Fig. 3). Inflammation of the larynx, trachea, and bronchi, and desquamation of ciliated columnar epithelium into the lumen of the bronchus was observed in individuals with uncomplicated acute influenza infections.

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Regeneration of the respiratory epithelial cells takes ca. 3–4 weeks, during which time pulmonary function abnormalities may persist. In these typical cases of influenza in which infection is confined to the respiratory tract, prostration, fever, and myalgia often seem to be disproportionate to objective clinical signs or observed pathological changes. Lungs from fatal cases of primary viral pneumonia most notably show hyaline membrane coverage of alveolar walls together with extensive intra-alveolar edema and hemorrhage. Tracheitis and bronchitis are also observed. Patients with secondary bacterial pneumonia have changes characteristic of bacterial pneumonia in addition to the tracheobronchial findings of influenza.

Immune response Innate immunity is an important defense mechanism early in infection. There are profound changes in the expression of a large number of cellular genes involved in interferon production and signaling, apoptosis, and oxidative stress. Influenza-infected patients trigger the activation of antiviral genes and production of cytokines, such as interleukin (IL)6, IFN-alpha, and IL-8. IL-8 is correlated with lower respiratory tract involvement. Influenza viruses have evolved mechanisms to counter the innate antiviral responses of the host. The viral NS1 protein antagonizes interferon production and the activation of PKR in influenza-infected cells [1]. The adaptive immune response to infection with influenza viruses leads to induction of both virus-specific B- and T-cell immune responses that clear infection and generate long-lasting specific immunologic memory. Antibodies are made against the viral external glycoproteins HA and NA as well as the internal type-specific proteins NP and M1. Neutralizing antibody directed against the HA is the primary immune mediator of protection from infection and clinical illness due to influenza viruses. Antibodies and T-cells play complementary roles in clearing the infection and promoting recovery. CD4 + (Th1 and Th2) and CD8 + T-cell responses to influenza are type-specific and are largely cross-reactive among influenza A viruses of different subtypes. In naturally infected humans the CD4 + cell response recognizes epitopes on the internal proteins NP and M1 as well as the surface proteins NA and HA. The CD8 + cytotoxic T lymphocyte response is directed to multiple surface and internal influenza proteins, in which many epitopes are potentially recognized, as determined by their binding to class I MHC molecules. Recent animal model studies have shown that both CD4 + and CD8 + T-cells can contribute to immunity to influenza viruses. The increased severity of influenza infections in geriatric patients has been correlated with a combination of functional impairments in their T-cell responses to influenza [4].

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Clinical manifestations Influenza is a highly contagious disease that is transmitted by aerosols. Virus replication occurs in airway epithelia and reaches its peak 2–3 days after infection. Patients shed virus for about 7 days, with primary infections for up to 2 weeks. Early symptoms in adults typically include fever, chills, headache, sore throat, dry cough, myalgias, anorexia, and malaise. A fever of 38–40°C that peaks within 24 h of onset is common, but peaks as high as 41°C can also occur. Pyrexia typically lasts 3 days, but may last from 1 to 5 days or longer. Other symptoms that occur less frequently include substernal soreness, photophobia and other ocular symptoms, nausea, abdominal pain, and diarrhea. Although most symptoms typically resolve within a week, cough and malaise may persist for 1 or more weeks after fever has subsided. In children, symptoms are similar to those in adults, but gastrointestinal symptoms such as vomiting, abdominal pain, and diarrhea are seen more frequently. Maximum temperatures also tend to be higher in children than in adults, and febrile convulsions can occur. In addition, myositis, croup, and otitis media occur more frequently in children. Influenza infection of neonates can be life-threatening and may be manifest only as an unexplained febrile illness. Complications of the upper respiratory tract after influenza infection include bacterial sinusitis and otitis media. Lower respiratory tract complications include exacerbation of chronic obstructive pulmonary disease and chronic congestive heart failure, croup, bronchitis, bronchiolitis, wheezing attacks in asthmatics, and pneumonia (primary viral pneumonia, secondary bacterial pneumonia, or combined bacterial and viral pneumonia). Primary influenza pneumonia develops abruptly and progresses rapidly. It has been a frequent cause of death in the 1918 pandemic and is also responsible for the high case fatality rate of the human infections with the ongoing H5N1 outbreak. However, with ordinary influenza epidemics, this type of pneumonia is uncommon and occurs mainly among those at increased risk for complications of influenza. Rapid respiration rate, tachycardia, cyanosis, high fever, and hypotension are frequent symptoms. Diffuse pulmonary infiltrates and acute respiratory failure with a high mortality rate are also features of this disease. Combined viral and bacterial pneumonia is more common than primary viral pneumonia and may be clinically indistinguishable from it. Secondary bacterial infections typically occur 5–10 days after initial onset of influenza symptoms and are responsible for most pneumonias during influenza epidemics. Productive cough, pleuritic chest pain, and chills are common symptoms of this type of pneumonia. Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae are the organisms most commonly involved. These illnesses respond to appropriate antimicrobial agents and have a lower case fatality rate than primary viral pneumonia. Other reported but less frequent complications of influenza include myositis, myocarditis and pericarditis, acute renal failure, encephalopathy,

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encephalitis, transverse myelitis, Guillain-Barré syndrome and a range of other neurological complications, Reye’s syndrome, and toxic shock syndrome. Higher rates of spontaneous abortion, stillbirths, and premature births were reported among pregnant women during the major pandemics of 1918/19 and 1957/58. Serious complications of influenza most often occur in people 65 years of age and older, in the very young, and in those of any age with underlying chronic cardiac, pulmonary, or metabolic disease. Complications in elderly people, particularly among those with pulmonary, cardiovascular, or other chronic diseases, account for most of the mortality in influenza epidemics [4].

Laboratory diagnosis Virus is isolated from nose or throat secretions by inoculation of cell cultures (MDCK cells) or embryonated eggs. Isolation is usually only successful within the first days of infection. Rapid diagnosis is possible by antigen detection in cells obtained from throat swabs. Viral RNA can be detected by PCR in clinical specimens. This approach is now replacing virus culture as “gold standard” for virus detection, because it is more sensitive, more rapid, and allows more precise characterization. Serological diagnostics involves complement fixation, hemagglutination inhibition, neutralization, and enzyme immunoassays. Antibody titres increase rapidly in the course of infection and decrease within a couple of weeks to low levels. It is often impossible to detect a fourfold increase in titre in blood samples taken with an interval of 2 weeks, since high antibodies reach high titres already in the acute disease phase. Therefore a single high antibody titre matched by the typical clinical symptoms is usually considered to be sufficient for the diagnosis of the infection.

Prophylaxis and therapy Vaccination is the most potent instrument for influenza control. Prime candidates for vaccination are persons at risk for complications and individuals who might transmit influenza to such persons. Inactivated vaccines obtained from infected chicken embryos are most commonly used. They contain HA and NA antigens as protective components and are each year formulated according to the circulating influenza-A and B strains. Because of antigenic drift, vaccination protects only against known viruses. Vaccinations should therefore be done every year. Current vaccines protect 70–90 percent of healthy adults. With older people, i.e. the most important target group, effectiveness is lower. Live vaccines are only available in some countries, such as the USA. Development of pandemic vaccines is a particular challenge, since

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they will have to be manufactured within a very short time period in very large quantities. Antiviral compounds were previously available only in the form of amantadine and rimantadine. Both compounds block the M2 ion channel of influenza-A-viruses and are therefore not effective against influenza-Bvirus infections. Prophylactic application provides 70–90 percent protection. Concerns about side effects involving the central nervous system have limited the use of amantadine. Development of amantadine resistant virus strains, which have emerged recently with increasing frequency, is another problem. The neuraminidase inhibitors zanamivir and oseltamivir that interfere with virus spread compare favorably with amantadine by having less side effects and a wider therapeutic spectrum that includes also influenza-B viruses. The neuraminidase inhibitors are therefore now the influenza antivirals of choice. However, as is the case with amantadine, application is limited to a relatively small time window shortly before or after infection, and there is increasing evidence for the development of virus resistance to these drugs, too. Nevertheless, neuraminidase inhibitors are expected to play an important role in the control of a future pandemic.

References 1

2 3 4 5

Cox NJ, Neumann G, Donis RO, Kawaoka Y (2005) Orthomyxoviruses: influenza. In: BWJ Mahy, V ter Meulen (eds): Topley and Wilson’s microbiology and microbial infections, Virology, Vol. 1. Hodder Arnold, London, 634–698 Noah DL, Krug RM (2005) Influenza virus virulence and its molecular determinants. Adv Virus Res 65: 121–145 Klenk H-D, Garten W (1994) Host cell proteases controlling virus pathogenicity. Trends Microbiol 2: 39–43 Nicholson KG (1998) Human influenza. In: KG Nicholson, RG Webster, AJ Hay (eds): Textbook of influenza. Blackwell Science, Oxford, 219–264 Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk H-D (2004) Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci USA 101: 4620–4624

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Pathogenesis of Chlamydophila pneumoniae infections – epidemiology, immunity, cell biology, virulence factors Matthias Krüll and Norbert Suttorp Dept. Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité Universitätsmedizin Berlin, Augustenburgerplatz 1, 13353 Berlin

Abstract Chlamydophila (Chlamydia) pneumoniae, a Gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen causing sinusitis, pharyngitis, bronchitis and pneumonia. Repetitive or chronic persistent infections have been associated with an increased risk for asthma, chronic obstructive pulmonary disease (COPD) or vascular lesions. Although the genome of C. pneumoniae has been sequenced completely this information has not led yet to an understanding of the mechanisms of infection and target cell activation nor to the identification of potential chlamydial virulence factors. In this review we will give an overview on the pathogenesis of C. pneumoniae-induced acute and chronic infections.

Epidemiology, diagnosis and clinical manifestation Chlamydophila pneumoniae, a Gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen causing sinusitis, pharyngitis, bronchitis and pneumonia [1–3]. The majority of C. pneumoniae infections are subclinical, but severe pulmonary infection and profound lymphocytic alveolitis are observed [4]. In addition, chronic-persistent or recurrent infections may be an important risk factor for adult-onset asthma [5], chronic obstructive lung disease (COLD, [6]) and development of vascular lesions [7–10]. Regarding vascular diseases, the field, however, is troubled by the “hen vs. egg” problem and causative proof is difficult because besides different anti-chlamydial isotypes of antibodies there are no good markers to differentiate among new vs. old (IgM vs. IgG) as well as acute vs. chronic persistent (IgM vs. IgA) C. pneumoniae infection.

Epidemiology C. pneumoniae was first isolated 1965 from the conjunctiva of a Taiwanese child (strain TW183, [11]). The first respiratory strain (AR 39) could be

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isolated 1983. Both strains had a DNA-homology of > 99.5% and were established in 1986 as a new (third) human-pathogenic chlamydia species “Chlamydia pneumoniae,” synonym TWAR [1]. Until now about 50 strains have been isolated worldwide, DNA-homology between these strains is 94100%. Chlamydiae have been placed in their own order, Chlamydiales, with one family, Chlamydiaceae. Molecular evaluation of rRNA sequences confirms that chlamydiae are bacteria, but with only very distant relationship to other bacterial divisions [12, 13]. Although it has been proposed that Chlamydia trachomatis and Chlamydia pneumoniae represent different genera [14], their gene content and genome organization are extremely similar [15] as are their structure and biology [16]. The newly proposed nomenclature – as used throughout this chapter – “Chlamydophila pneumoniae”, however, has not been generally accepted [17]. Chlamydophila pneumoniae is found worldwide. Seroepidemiologic surveys have demonstrated that more than 70% of all adults have been exposed to this organism during their lifetimes [18]. Age-specific prevalence rates start to rise early in childhood, although there is only little disease associated with these early timepoints of infection. The most rapid rise (in the Western world) in age-specific prevalence occurs during the ages of 5- to 20-years. In spite of a high percentage of C. pneumoniae-seropositive adults reinfections are common. According to these seroepidemiologic studies, C. pneumoniae infection seems to be both endemic and epidemic with frequent reinfection during a lifetime. Currently available data suggest that C. pneumoniae is primarily transmitted from human to human without any animal reservoir. Transmission seems to be inefficient, although household outbreaks with high transmission rates are reported [19].

Diagnosis For differential diagnosis of an acute, repetitive or chronic/persistent C. pneumoniae infection the kinetics of the antibody expression has to be taken into consideration. In patients with primary infection IgM antibodies appear about 2–3 weeks post infection and remain elevated/detectable up to 2–6 months. IgG antibodies may not reach high titers until 6-8 weeks after onset of clinical symptoms. C. pneumoniae does not induce a persistent protective immunity and reinfections are common. In case of reinfection, the level of IgG antibodies increase rapidly within 1–2 weeks while IgM antibodies may not appear again [19]. Until now there is no reference test for validating a chronic/persistent infection by means of serological testing. Persistently elevated IgG or the presence of IgA antibodies has been suggested as potential serological marker. Several studies have proposed that high IgA titers might be a better marker for a chronic infection than IgG

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titers because serum IgA has a half- life of about 5–7 days, whereas IgG has a half-life of weeks to months [9, 20]. Further evaluation, however, and confirmation by other tests have to be awaited. A lack of validated and standardized diagnostic techniques in diagnosis of acute, recurrent or chronic persistent infection with C. pneumoniae has made interpretation of published data difficult. Recommendations for standardized approaches were recently published by the Centers for Disease Control and Prevention (CDC, Atlanta) and the Laboratory Centre for Disease Control (Ottawa, Canada) in cooperation with members of the C. pneumoniae-study group [21]. Diagnostic approaches include serological testing, isolation/culture, nucleic acid-based amplification techniques such as PCR, and tissue diagnostics like immunofluorescence, immunohistochemistry (IHC) or in situ hybridization.

Serological testing The microimmunofluorescence (MIF) test, developed by Wang et al. in the 1970s [22] is currently the serological testing method of choice for diagnosis of acute C. pneumoniae infection. This test allows the quantitative determination of antibody reactivity to formalinized elementary bodies from C. pneumoniae, C. trachomatis and C. psittaci fixed as dots on a single glass slide. Dilution of sera are placed over the antigen dots and incubated. Use of the MIF test allows definition of criteria for serologic evidence of acute infection (defined by a four-fold rise in IgG between acute and convalescent samples or an acute IgM titer * 1: 16), or past exposure (indicated by an IgG titer * 1: 16). Kits based on the MIF format are commercially available, however, it should be noted that the quality of commercially available MIF kits varies and interpretation of the results is subjective [23]. Due to the lack of species specificity or sensitivity, other serological tests like complement fixation (CF), whole fluorescence, ELISA and EIA cannot currently be endorsed [24, 25].

Culture Chlamydophila pneumoniae is an obligate intracellular pathogen and must be cultivated on specific eukaryotic host cells. All currently established culture techniques involve inoculation of specimens onto a human cell line via centrifugation, incubation for up to 72 h and subsequent staining with fluorescent-labeled species-specific antibodies to visualize intracellular inclusion bodies [8, 26, 27] (see Fig. 1). Sensitivity is low due to the complexicity of the assay. Specificity is dependent on the ability of the lab staff to distinguish between true chlamydia inclusions and artifacts.

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Figure 1. Chlamydia pneumoniae strain TW183 inclusion bodies in freshly isolated human airway epithelial cells. Inclusion bodies were visualized, using a fluorescein isothiocyanate (FITC)–conjugated, genus-specific monoclonal antibody (DakoCytomation, Hamburg, Germany).

PCR Although many in-house PCR techniques for detection of C. pneumoniae have been published [28-31], sensitivity and specificity remain almost unknown. Due to a broad variety of methods for specimen-collection and specimen-processing, primer design, nucleic acid extraction, amplification product detection and identification of false-positive and false-negative results, no commercially standardized tests have been approved by the FDA.

Tissue diagnostics/immunohistochemistry (IHC) Tissue diagnostic methods offers the advantage of preserving tissue morphology and permitting localization of the infectious agent to specific areas and cells. IHC has been the most frequently used in published studies [32, 33]. Detection rates are higher than those of PCR. This is attributed to a faster degradation of DNA, difficulties of extracting DNA from tissues and the presence of PCR inhibitors. The most widely used IHC-technique is the avidin-biotinylated immune-complex method. However, interpretation

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of IHC-results is a critical challenge due to difficulties in distinguishing between true- and false-positive results of the staining. Specific recommendations for standardized culture/isolation or detection tests (e.g., specimen-collection, -transport and -processing conditions) have been suggested elsewhere (s.a., [21]). Taken together, diagnosis of C. pneumoniae infection is difficult because cell culture techniques are not available for routine clinical use and nonculture techniques using antigen detection methods (IHC) or DNA probes have not been developed for commercial use. The MIF test is currently the serological testing method of choice for diagnosis of acute C. pneumoniae infection (“gold standard”), although the test is technically complex, requiring experience in fluorescence microscopy, interpretation is therefore subjective.

Clinical manifestation Chlamydophila pneumoniae causes acute respiratory diseases and is responsible for approximately 5% of bronchitis and sinusitis cases and 510% of community acquired pneumonia (CAP) cases in adults worldwide [34–37] (see Fig. 2). However, a broad geographic diversity and periodicity with higher incidence rates of C. pneumoniae-mediated CAP-cases for 2 or 3 years followed by 4 or 5 years with low incidence has been suggested [18]. A recently submitted study from the German “community acquired pneumonia-network, CAPNETZ”-study group, including more than 4000 CAP-patients in Germany demonstrated, that between 2001 and 2004 using recommended standardized diagnosis protocols (MIF test, PCR from BALfluid) C. pneumoniae could be identified as causative agent in < 1 % of all CAP-cases (personal communication N. Suttorp, speaker of CAPNETZ). C. pneumoniae infection has also been implicated in the pathogenesis of asthma in both adults and children. This hypothesis is based on clinical studies and on the evidence of specific IgE production, direct epithelial damage, induction of T-cell immunopathologic diseases, and vascular smooth cell infection. In addition, asthma patients, especially those with moderate asthma, had significantly higher serum IgA antibody levels to chlamydial heat shock protein 60 (cHsp60) than healthy controls [5, 38–41]. Moreover, recurrent or chronic persistent C. pneumoniae infection seems to be common in patients with chronic bronchitis whether exacerbated or not, and is characterized by a strong humoral immune response to this intracellular pathogen, which is present in the majority of patients with severe chronic bronchitis. Increased antichlamydial IgG and IgA indicated acute exacerbations with C. pneumoniae in COPD patients [5, 42-44]. Some authors suggest a possible role of this organism in the etiology of lung cancer; future studies, using measures of chronic C. pneumoniae status, however are necessary, to substantiate these data [45, 46].

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Figure 2. Chest x-ray of a 28-year-old man reveals basal infiltration in both lungs as well as a generally pronounced interstitial pattern. Chlamydophila pneumonia infection could be confirmed by MIF test and PCR from BAL-fluid.

Chronic-persistent or recurrent Chlamydophila pneumoniae infections may be a trigger and promoter of inflammation which may cause vascular lesions and atherosclerosis [8, 9, 31, 47, 48]. The theory is supported by a serological association between C. pneumoniae infection and coronary heart disease as well as other vascular diseases (arterial occlusive disease, carotid artery stenosis and stroke [9, 20, 49, 50]), the demonstration of C. pneumoniae in atherosclerotic plaques by electronmicroscopy, immunocytochemistry, PCR, and isolation of viable chlamydia (indicating a productive chlamydial infection [8, 10, 29, 31, 51, 52]), and different animal models, demonstrating that intranasal infection of mice and rabbit with C. pneumoniae leads to pneumonia, perimyocarditis, septic circulatory dysregulation and – more delayed – systemic spread of chlamydia into spleen, lymphnodes, peritoneum and atherosclerotic plaques of arterial blood vessels [7, 53–56]. Current antibiotic treatment options for acute chlamydial infection, however, were

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proven to be ineffective with respect to clinical outcome in different groups of atherosclerotic patients (WIZARD, AZACS, ACES or PROVE-IT study [55, 57–62]). Interpretation of these clinical trials is challenging as definite markers of chronic vascular C. pneumoniae infection are still missing and serologic testing seems to be inaccurate in patient populations with high chlamydial IgG seroprevalence. Moreover and probably more important, C. pneumoniae in the persistent state, as observed in blood monocytes, shows high resistance against macrolides, tetracyclines and rifampicin [63–65]. This observation may explain the ineffectiveness of all interventional studies in atherosclerotic patients. Several recent studies have suggested a chronic/persistent C. pneumoniae infection as a possible risk factor for central nervous system diseases like Alzheimer’s disease or multiple sclerosis, data, however, are poor and clear evidence is still missing [66, 67].

Innate and adaptive immunity Little is known about a humoral or cell-mediated immunity (“CMI”) induced during acute or chronic/persistent infection with Chlamydophila pneumoniae. Most of the data have been elaborated using other Chlamydiae species (C. trachomatis, C. psittaci). As intracellular bacteria, Chlamydiae pose an extra challenge for the defense mechanisms of the host. While importance of anti-chlamydial antibodies still is controversially discussed [68], cell-mediated immune responses are decisive, at least in mice. CMI against C. pneumoniae, however, is weak since recurrent infections as well as persistency of viable pathogens in different target cells are common phenomena.

Innate immunity C. pneumoniae is able to infect and to replicate in a multitude of target cells [69–71]. Subsequently, different intracellular signal transduction pathways are activated to induce a proinflammatory phenotype (for review see [72]). However, there is still limited knowledge of the mechanisms of Chlamydiae entry into host cells. Wuppermann et al. showed that heparan sulfate-like glycosaminoglycans (GAG) might act as possible chlamydial receptors on the surface of epithelial cells (HEp-2 cells), the importance of the estrogen receptor complex is controversially discussed [73, 74]. In a recent paper, Puolakkainen et al. demonstrated, that C. pneumoniae uses the mannose 6phosphate/insulin-like growth factor 2 receptor for infection of endothelial cells, additional studies, however are necessary to confirm these results [75]. Several recent studies demonstrated the involvement of extracellular Tolllike receptor-2 (TLR2) and -4 (TLR4) in initiation of innate immune cell

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activation by C. pneumoniae or chlamydial components [76–80]. Moreover, we were able to demonstrate that the recently identified nucleotide-binding oligomerization domain (Nod) proteins might act as intracellular receptors during C. pneumoniae-infection of target cells [81]. C. pneumoniae is internalized by macrophages as well as by nonprofessional phagocytes (innate cells), where it survives and replicates [64]. In these cells IFN-a synergizes with bacterial products to activate various bactericidal or bacteriostatic mechanisms [82]. IFN-a is a strong activator of indoleamine 2,3-dioxygenase (IDO), limiting the availability of L-tryptophan to intracellular microorganisms [83]. Induction of IDO has been demonstrated to inhibit chlamydial growth in vitro [84, 85]. IFN-a can also activate inducible nitric oxide synthase (iNOS) which catalyzes production of NO from L-arginine. Inhibition of chlamydial growth through induction of iNOS has also been reported [86]. Moreover, stimulation of neutrophils or monocytes with IFN-a induced transcription of the gp91 component of NADPH oxidase (ox) mRNA with subsequent enhanced respiratory burst of phagocytic cells [82]. In addition, Rottenberg et al. could demonstrate that IFN-a-receptor-double knock-out mice (IFN-a-R–/–) showed a dramatically increased susceptibility to C. pneumoniae, mediated via a reduced iNOS-mRNA accumulation, independent of diminished levels of specific antibodies. An increased susceptibility of iNOS–/– mice substantiated the protective role of this enzyme-activity during infection with C. pneumoniae [87]. These data suggested a relevant protective role of IFN-a-dependent innate mechanisms of protection. For a more extended view of the signal transduction pathways involved in Chlamydia-mediated innate immune cell activation see Figure 3, in addition we would like to refer to our recent review [72].

Adaptive immunity In C. trachomatis infection models, both CD4+ and CD8+ cells have been shown to confer protection, although the former are considered of major importance [88, 89]. In C. psittaci infection, CD8+ rather than CD4+ cells have been reported to confer protection in mice [90]. Since the overall DNA homology between C. pneumoniae and C. trachomatis or C. psittaci is less than 5 or 10%, respectively [15] the parameters of infection identified with the later two cannot be directly extrapolated to C. pneumoniae. Most data have been acquired using mouse models for C. pneumoniaemediated pneumonia [91, 92]. The situation, however, is complicated due to mouse strain-specific differences studying mechanisms of adaptive immunity. In addition, importance for a protective role of T-cell subspecies during primary infection and reinfection still is controversially discussed. Using C57BL/6J mice genomically lacking T-cell coreceptors or cytokine receptors, Rottenberg et al. demonstrated that CD4 + T-cells played a dual

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Figure 3. Scheme of the supposed signaling cascades induced by C. pneumoniae infection in human cells (TyrKi, tyrosin kinase; ERK, extracellular regulated kinase; PKC, protein kinase C; reproduction from [75] courtesy of the authors and of Schattauer Verlag, Germany).

role, one deleterious, promoting bacterial growth and disease early after infection, but also participating in the control of bacterial growth at later timepoints as well as in protection against reinfection. The early damaging effect of CD4 + cells in the absence of CD8 + cells was associated with enhanced IL-4 and interleukin10 (IL-10) mRNA levels and delayed IFN-a mRNA accumulation in the lungs of mice [87]. CD8 + T cells inhibited this CD4 + activity. The CD8 + T-cell mediated protective immunity during early stages of primary infection was perforin independent and associated with an altered cytokine balance as indicated by increased IL-4 and IL-10 and delayed accumulation of IFN-a mRNA in CD8–/– mice. The early higher susceptibility of CD8-deficient mice correlated with an immune deviation from a normal Th1 response to a Th2 cytokine pattern [87]. These results were confirmed by Wizel et al. demonstrating the importance of peptidespecific CD8 + T-cell lines in local and systemic compartments after primary (intranasal) infection with C. pneumoniae. These CTL lines suppressed chlamydial growth in vitro by direct lysis of infected target cells and by secretion of IFN-a. In addition, they were able to identify 18 H-2(b) binding peptides representing sequences from 12 C. pneumoniae antigens [93]. The importance of CD8 + CTL during reinfection could be confirmed by Penttilä et al. They demonstrated in a BALB/c nude mice model with absence of

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all T-cells, that the overall clearance kinetic after primary C. pneumoniae infection was not dependent on either CD4 + or CD8 + cells alone [94], but, after reinfection, acquired immunity was strongly CD8 dependent with an enhanced IFN-a-production and an increased local lymphoid reaction in the lungs [94, 95]. Analyzing lymphocytes from male patients with respiratory tract infection Halme et al. demonstrated a C. pneumoniae-specific CMI response during acute, primary infection early after onset of disease symptoms and simultaneously with a humoral response (increase of C. pneumoniaespecific IgM-antibodies). C. pneumoniae-induced lymphocyte activation involved CD8 + T-cells in the early phase of infection and CD4 + cells in the later stage [96]. The mechanism underlying the protection mediated by the CD8 + cells in C. pneumoniae infection is unclear. CTL specific for C. trachomatis have been demonstrated in C. trachomatis-infected mice [97, 98]. CD8 + cells may function by secreting cytokines such as IFN-a. Rottenberg et al. showed that C57BL/6 mice produce IFN-a in response to C. pneumoniae primary infection and suggested a IFN-a-mediated protection mechanisms [82]. These results were supported by Vuola et al. demonstrating an altered bacterial load in anti-IFN-a-treated C57BL/6 mice with markedly exacerbated signs of pulmonary inflammation [99]. Results are different in BALB/c mice. Pentillä et al. and Vuola et al. demonstrated an IFN-a-independent cellular response in BALB/c mice [95, 99]. Interestingly although BALB/c mice appear not to be dependent on IFN-a during primary infection, they do not develop a typical Th2 type response either [99]. During reinfection, neutralization of IFN-a exacerbated the infection in both strains [82, 95]. CD8 + cells may therefore at least partially function through IFN-a production, however actively IFN-a-producing cells have also been demonstrated in CD8-depleted mice, in which the acquired immunity seen during reinfection is abolished [94]. Thus, IFN-a is an important, but not the only effector mechanism in acquired immunity. Overall, a different mouse model of C. pneumoniae-infection demonstrated that immunity is critically dependent on CD8 + CTL. In a recent study the group of Wizel et al. was able for the first time to successfully immunize C57BL/6 mice with a CD8 + T-cell heptaepitope based DNA vaccine to induce a protective immunity against C. pneumoniae [100]. These results were confirmed by Pentillä et al., demonstrating that DNA immunization is a promising possibility for developing much wanted vaccines against important chlamydial pathogens [101]. Further studies are now required to elaborate the optimal design of (multicomponent) anti-C. pneumoniae vaccines for humans. Infection with C. pneumoniae induces a strong serum response. Little is known about the immunogenic antigens of C. pneumoniae. The 40 kDa major outer membrane protein (MOMP) is the most important immunodominant structure during C. trachomatis-infection; during infection with C.

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pneumoniae it is a relatively immunorecessive antigen [102, 103]. This lack of antigenicity has not been resolved yet. Among the different antigens of C. pneumoniae the 60 kDa heat-shock protein 60 (cHsp 60, GroEL-1) has been suggested to be a key player during C. pneumoniae-induced humoral immunity, further studies, however, are required to identify the importance of this antigen during acute and chronic infection (see also the chapter on “virulence factors”) [102, 104]. Antibodies to different structures on the C. pneumoniae elementary body can neutralize the organism in cell culture, however, the epitope specificity of these neutralizing monoclonal antibodies to specific C. pneumoniae proteins remains undefined at a molecular level [105]. Moreover, in vivo, protective effects of antibodies seem to be weak since reinfections or chronic chlamydial infections are common despite high antibody titers [18, 19]. Under some circumstances, antibody response during C. pneumoniaeinfection might be immunopathological; for details please see chapter “virulence factors”.

Cell biology Monocytes, macrophages, smooth muscle cells, endothelial cells, human airway epithelial cells (HAEC), BEAS-2B (bronchoepithelial cell line), HEp-2 and Hela-229 cells have all been shown to be susceptible for C. pneumoniae infection [69-71]. Following inhalation, bronchial epithelial cells, however, are the first line of defense in getting in contact with C. pneumoniae and respiratory epithelium has been identified as the primary target of infection [70, 71]. Little is known about Chlamydiae-induced epithelial cell alterations and C. pneumoniae-mediated interactions among all cell types involved in orchestrating airway inflammation (e.g. lymphocytes, macrophages, granulocytes). Chlamydiae have a unique development cycle with two functionally and morphologically distinct forms, the condensed, “spore-like” infectious but metabolically inactive elementary body (EB, 0.3 +m) and the labile, noninfectious metabolically active reticulate body (RB, 0.9 +m, see Fig. 4) Infection or invasion is an active process requiring the existence of viable chlamydia, heat- or UV-inactivated bacteria are not able to invade target cells. However, there is still limited knowledge of the mechanisms of Chlamydiae entry into host cells. The chlamydial growth cycle is initiated when an infectious elementary body (EB) attaches to a susceptible target cell, promoting entry into a host cell-derived phagocytic vesicle. EB are internalized, dissociated from the endocytotic pathway by actively modifying the vacuole to become fusogenic with exocytic vesicles. Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydia to establish themselves in a site that is not destined to fuse with lysosomes [63, 106]. Coombes and Mahony suggested a receptor-

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Figure 4. Transmission electron micrographs of Chlamydophila pneumoniae-infected HL cells 72 h postinfection. Large inclusions filled with C. pneumoniae particles are found in the HL cells. Mature EBs are small with an electrodense core (thick arrow), while RBs are large with a coarse inner structure (thin arrow, reproduction from [157] courtesy of the authors and the American Society for Microbiology).

mediated induction of specific cell signaling by Chlamydiae as an essential step in C. pneumoniae invasion of epithelial cells [107]. After internalization EB then develop into reticular bodies (RB), a process which could be detected metabolically within 15 min and microscopically 12-15 h after addition of Chlamydiae to HEp-2 and Hela-229 cells [70, 71]. RBs are first observed dividing by binary fission after about 12 h. As the RBs multiply, the inclusion membrane expands to accommodate the increasing numbers of bacteria. After about 18-24 hpostinfection, the first RBs begin differentiating back into EB which accumulate in the lumen of the inclusion as the remainder of the RBs continue to multiply. Forty-eight to 72 h after completing the development cycle, depending primarily on the infecting species, infectious EB are released either by lysis of the host cell or by fusion of the inclusion membrane with the plasma membrane to release the content of the inclusion into the environment (for review see [108, 109]). Under some conditions, however, such as in the presence of IFN-a, or penicillin or depletion of essential nutrients (iron, tryptophan) Chlamydiae

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can achieve a state of intracellular chronic, persistent infection in which they remain viable, but metabolically quiescent and do not replicate [63, 110, 111]. Because of the reduced or negative ribosomal cell activities, these bacteria have no adequate targets for the known chlamydia-targeting antibiotics [64, 112]. Persistent Chlamydiae fail to complete development from RBs into EBs are enlarged and morphologically aberrant, and form small inclusions. Moreover, they exhibit characteristic gene and protein expression profiles showing reduced levels of outer membrane proteins like the major outer membrane protein OmpA or OmcB and substantially increased levels of Heat shock protein 60 (Hsp 60) [113]. Chlamydiae may be reactivated from persistence by removal of the inducing stimulus. A second strategy of Chlamydiae to prolong intracellular survival is to inhibit pro-apoptotic pathways of the infected host cells. It is thought that C. pneumoniae can protect infected cells by inhibiting the release of cytochrome c from mitochondria and upregulate the expression of the antiapoptotic mediators IAP and MCL-1. Thus, protection against apoptosis may be another strategy that the Chlamydiae use to maintain a persistent, chronic infection. For a more extensive view of the mechanisms involved in Chlamydia interference with apoptosis signaling in host cells please refer to the review of Byrne et al. [114].

Virulence factors Although the genome of C. pneumoniae was sequenced completely in 1999 (1.23 × 106 nt encoding for approx. 1052 proteins, Gen-Bank No.: AE001363, [15]), until now little is known about structures on the chlamydial surface (proteins, glycolipids) initiating and mediating bacterial contact to target cells and inducing subsequent target cell activation. A multitude of the identified sequences from the chlamydial genome encode for proteins of bacterial metabolism. Other sequences demonstrated homologies to known proteins and virulence factors of other bacterial pathogens. Most of these factors of C. pneumoniae and C. trachomatis have now been identified by proteom-analysis of both species [115, 116]. Proteins of the “outer membrane complex” (OmpA/B, Omp3, OmcB, POMP), chlamydial lipopolysaccharide (cLPS), chlamydial heat-shock-proteins (e.g. chsp60/GroEL-1), a type III secretion apparatus (TTS), the “chlamydial protease- or proteasome-like activity factor” (CPAF) or peptidoglycans and peptidoglycan-like structures are likely candidates as possible virulence factors.

Chlamydial envelope/outer membrane complex The chlamydial outer membrane complex is composed primarily of three proteins specifically the outer membrane protein A, OmpA, formerly

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referred to as major outer membrane protein (MOMP) and two cysteinerich proteins, the outer membrane complex B protein (OmcB) and the outer membrane complex A protein (OmcA) [16]. The gene that encodes MOMP, ompA [117] exhibits extensive DNA sequence variations that is confined mainly to four variable segments or domains (VS or VD 1 to VD 4) that contain subspecies- and serovar-specific antigenic determinants [118]. Until recently, OmpA was the only protein unequivocally shown to be expressed on the surface of all chlamydia. The protein is the most important immunodominant structure in all chlamydial strains except C. pneumoniae [102, 103]. Until now, this lack of antigenicity has not been resolved. It might be possible that either the exposed variable regions of C. pneumoniae OmpA are not recognized by human or animal immune systems, or that surface exposure of the OmpA is somehow masked, perhaps by one of the polymorphic outer membrane proteins (POMP) as suggested by Christiansen et al. [103]. OmpA of the chlamydial elementary body is the main component for chlamydial protection against the environment outside the host, defense against the host immune response, and attachment to host cells [108]. OmcB, encoded by omcB, does not appear to be surface exposed but is thought to form a supramolecular lattice in the periplasm. Another important difference is that OmcB is extremely highly conserved (for review see [119]). Stephens et al. could demonstrate that the 60 kDa cystein-rich outer chlamydial membrane complex protein OmcB is also able to bind heparin [120]. This protein therefore might be the link mediating chlamydial attachment and initial steps of invasion into target cells. Although recent studies indicated a functional peptidoglycan (PG) pathway in chlamydia [121, 122], a clear cut biochemical evidence for the synthesis of peptidoglycans in chlamydia is missing [123, 124]. Chlamydia, however, are sensitive to antibiotics that inhibit peptidoglycan synthesis [125]. This phenomenon has been referred to the “chlamydial anomaly,” Recent studies, using genomics or proteomics suggested the expression of peptidoglycan-like structures, not on the surface of elementary bodies, but – after invasion of the target cells – during cell division on the subsequently developed reticular bodies [121, 126]. Elucidating the existence of PG in Chlamydia is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

Chlamydial heat shock protein 60 (chsp, GroEL-1) Heat shock proteins (Hsps) belong to a family of evolutionarily highly conserved proteins, which are produced by eucaryotic and procaryotic cells during a variety of conditions such as heat shock, nutrient deprivation, infections, and inflammatory reactions, functioning to stabilize cellular proteins. Several studies using neutralizing monoclonal antibodies or purified recombinant chlamydial heat-shock protein 60 (cHsp60, GroEL-1),

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however, suggested that this protein can act as an extracellular agonist and might be a key player in activation of different intracellular signal transduction pathways with a subsequent expression of a profound and prolonged proinflammatory phenotype in treated cells [76, 79, 127]. Moreover, it has recently been demonstrated, that GroEL-1 is highly expressed in IFN-a-induced persistent infections of tissue culture cells [63, 128]. Sasu et al. demonstrated that GroEL-1 is a potent inducer of human vascular smooth muscle cell proliferation and that this effect is mediated by rapid TLR4-mediated activation of ERK1/2 [80]. We were able to show that purified recombinant GroEL-1 induced a rapid phosphorylation of ERK1/2 and p38-MAPK with subsequent enhanced release of IL-8 from human umbilical vein endothelial cells (HUVEC) [72]. Moreover, GroEL1 protein has been shown to stimulate a hyperinflammatory response in animal models [129–131]. The responses were mediated via a TLR2- and TLR4-dependent fashion similar to the whole microorganism and differed markedly from responses induced by endotoxin or CpG oligonucleotides [77, 78]. Several groups have demonstrated that elevated IgA antibody-titers of chlamydial heat shock proteins are predictors of chronic chlamydial infections like bronchial asthma, COPD, arteriosclerosis or pelvic inflammatory disease, PID [41, 104, 127, 131]. The molecular mechanisms, however, by which C. pneumoniae might contribute to development of chronic diseases remain unclear. Due to the close structural homology between human and bacterial Hsps and their highly immunogenic nature, Hsps – especially Hsp60 – have been proposed as key antigens in the development of autoimmune diseases [132]. Bachmaier et al. showed that a peptide from the murine heart muscle-specific _-myosin heavy chain that has sequence homology to the Hsp60 of C. pneumoniae, C. psittaci, and C. trachomatis, was shown to induce autoimmune inflammatory heart disease in mice, suggesting that Chlamydia-mediated heart disease is induced by antigenic mimicry of a heart muscle-specific protein [133]. In addition, Kol et al. demonstrated that cHsp60, produced in large amounts during chronic chlamydial infections, colocalizes within plaque macrophages with human Hsp60 [127]. Human Hsp60, when expressed by heat-shocked endothelial cells, can provoke an autoimmune reaction mediating endothelial cytotoxicity [134]. Chlamydial Hsp60 might therefore augment atherosclerosis and/or stimulate humoral and cellular immunity in atheroma [127, 134].

Chlamydial LPS Although chlamydia are Gram-negative bacteria, the common LPS group antigen of all chlamydial species (cLPS) differs significantly from LPS of other Gram-negative pathogens. In 1998, a first monoclonal antibody was

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isolated which recognizes LPS and neutralizes the infectivity of C. pneumoniae strain TW183 [135]. This antibody, however, does not neutralize other strains of C. pneumoniae suggesting the presence of more than a genus-specific epitope on cLPS. Until now, at least two important differences between cLPS and LPS from of , for example, enteric bacteria have been demonstrated: 1) the core trisaccharide 3-deoxy-D-manno-octulosonic acid (KDO) structure of chlamydial LPS contains a 1-8 linkage, a genus specific epitope as well as a 1–4 linkage similar to that of other bacteria, encoded by a single multifunctional KDO transferase [136–138], and 2) the chlamydial LPS has low endotoxic activity, although inducing some cytokines [139, 140]. Immunogold studies suggested that surface exposure of LPS is greater on RBs than on EBs [141, 142]. Moreover, using different antibodies which recognized either RB or EB Birkelund et al. suggested that epitope exposure, or the chemical structure of LPS might differ during the development cycle [143]. cLPS can be released from intracellular, intrainclusion Chlamydiae to the inclusion membrane, to the host cell cytoplasm and surface, and to surrounding infected cells [144–146]. Although this release might have an impact on the pathogenesis of chlamydial infections and the host’s immune disposition of infected cells, several studies have demonstrated that cLPS plays only a minor role for target cell activation [79, 138].

Chlamydial protease- or proteasome-like activity factor (CPAF) In 2001, Zhong et al. demonstrated for the first time that a chlamydial species, C. pneumoniae, secreted a protease into the cytoplasm of infected target cells. This protease, “chlamydial protease- or proteasome-like activity factor” (CPAF) split host cell transcription factors necessary for MHC-I (RFX5) and -II (“upstream stimulatory factor 1”, USF-1) antigen presentation. Shaw et al. and Dong et al. showed that CPAF is secreted by different chlamydial species [147, 148]. Heuer et al. suggested that CPAF could be located in the inclusion lumen or associated with bacteria during the first 48 h of an acute infection. Seventy-two hours and later, CPAF was present predominantly in the cytoplasm of the infected cells. Translocation of CPAF into cytoplasm correlated in time with degradation of the transcription factor RFX5 [149]. CPAF does not preexist in chlamydial organisms and synthesis requires organism replication in cells. Moreover, mice inoculated with viable chlamydial organisms produced a strong antibody response to CPAF. In addition, sera from women diagnosed with C. trachomatis cervicitis displayed higher levels of antibodies to CPAF than to either chlamydial major outer membrane protein or heat shock protein 60. This sera neutralized the proteolytic activity of CPAF in vitro, suggesting that CPAF is both produced and immunogenic during human chlamydial infection [150, 151].

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Type III secretion system (TTS) The first genetic evidence that Chlamydia might have a type III secretion (TTS) system was presented by Hsia et al. in 1997, when they described four genes homologous to structural and regulatory components of a contact-dependent or TTS apparatus share high homology with TTS systems of other bacterial pathogens [152]. Subsequently, these results could be confirmed by genome and proteom analysis as well as by microscopic observations [15, 115, 153, 154]. Since TTS systems have been shown to play a major role in the pathogen-host interaction in several other pathogens like, for example, Shigella or Salmonella, the TTS may also act as a key virulence mechanism of Chlamydia [155]. One can speculate about a role for TTS, both in the initial stages of infection where Chlamydia first comes into contact with the host cell as well as in the intracellular phase of chlamydial development using the structure of the TTS system to translocate different effectors into the host cell, depending on the phase of the developmental cycle. TTS genes expressed in the mid- to late stage of the developmental cycle appear to be down-regulated by IFN-a treatment [156]. This suppression may play a role in maintaining C. pneumoniae in a persistent or altered state within the host cell. It will therefore be important to determine what structures are present in C. pneumoniae and what role each of them plays in the development and possible persistence of Chlamydia.

Future research directions Overall, the data presented suggest that Chlamydophila pneumoniae are able to infect a multitude of target cells and subsequently to activate and trigger a cascade of early and prolonged signal transduction events. Additional studies are required to determine the relationship between distinct steps of initial attachment, the chlamydial development cycle, importance of different chlamydial virulence factors and initiation of host cell signaling pathways that could lead to target cell damage and inflammation which in turn may result in acute diseases like bronchitis or (community-acquired) pneumonia or may promote chronic diseases like COPD, bronchial asthma or atherosclerosis. New techniques of biochemical and genetical analysis (genomics, proteomics) are now available to improve our understanding about pathomechanisms of infection and inflammation and offer unprecedented opportunities to address many fundamental questions regarding chlamydial interactions with the host cells. These results subsequently will direct new research avenues in terms of diagnostics, therapeutics, as well as vaccination strategies. Especially chronic persistent infections with metabolically aberrant chlamydiae that are refractory to current antimicrobial treatment schemes are a challenge for the development of new therapeutic

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strategies. In addition, vaccination against Chlamydophila pneumoniae (e.g., via DNA immunization as recently demonstrated [101]) could be a beneficial approach for either preventing or controlling infection by this human respiratory pathogen.

Acknowledgements The authors apologize for not citing more original manuscripts due to space limitations and hope that the cited reviews will provide more detail. This work was in part supported by the Deutsche Forschungsgemeinschaft to M.K. and N.S. (Kr 2197/1-2), as well as by the Bundesministerium für Bildung und Forschung (BMBF) to N.S. (CAPNETZ).

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Legionnaires’ disease and its agent Legionella pneumophila Dina M. Bitar1, Marina Santic2, Yousef Abu Kwaik3 and Maëlle Molmeret3 1Department

of Microbiology and Department of Medical Microbiology and Immunology, Faculty of Medicine, Al-Quds University, Jerusalem, 19356, Israel; 2Department of Microbiology and Parasitology, Medical Faculty, University of Rijeka, Croatia; 3Department of Microbiology and Immunology, Room MS-410, University of Louisville, Louisville, KY 40209, USA

Abstract Legionella pneumophila, the agent responsible for Legionnaire’s disease, is a facultative intracellular pathogen that can replicate within protozoa and macrophages. Protozoa are considered to play a central role in the pathogenesis and ecology of L. pneumophila. In humans, L. pneumophila reaches the lungs, where it is ingested by alveolar macrophages. Unlike phagosomes containing inert particles or avirulent bacteria, the L. pneumophilacontaining vacuoles avoid fusion with lysosomes, recruiting rough endoplasmic reticulum and mitochondria. The formation of this specialized vacuole is directed by the type IV secretion system encoded by the dot/icm genes in mammalian and protozoan cells. Killing of mammalian cells by L. pneumophila has been proposed to occur through induction of apoptosis during the early stages of the infection. A rapid induction of necrosis by L. pneumophila also occurs upon entry into the post-exponential phase of growth within both macrophages and protozoa, when the bacteria become cytotoxic. Before the lysis of the mammalian or protozoan plasma membrane, the bacteria egress into the cytoplasm. In vivo, clearance of Legionella from the lungs depends on the host production of IFN-a in A/J mice, while in BALB/c mice IFN-a is not produced. Intracellular replication of L. pneumophila is inhibited in IFN-a-activated mouse and human primary macrophages. Both antigen-specific humoral and cell-mediated immune responses are induced during Legionella infection. Although Legionella-specific antibodies are produced during human or murine infection, acquired cell-mediated immune response is believed to play a stronger role in Legionella clearance. Both macrophages and DCs are able to present microbial antigens on major histocompatibility class I and class II molecules, which stimulate antigen-specific T-cell response. Identification of antigens and determination of vesicular trafficking mechanisms involved in processing and presentation remain to be understood in greater detail.

Introduction The first recognized outbreak of pneumonia due to Legionella pneumophila occurred in Philadelphia, during the summer of 1976 among 180 persons attending the 56th annual American Legion Convention. Twenty-nine patients died and the disease became known as Legionnaires’ disease [1].

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Guinea pigs were infected with postmortem lung tissue from the patients with fatal Legionnaires’ disease, and embryonated yolk sacs were inoculated with spleen homogenates from the infected guinea pigs. In January of 1977, a Gram-negative bacterium was isolated and designated L. pneumophila [2]. The source of the infection during the Legionnaires’ convention was later found to be the air conditioning system in the hotel. It has been documented that the hallmark of Legionnaires’ disease is the intracellular replication of L. pneumophila in the alveolar spaces. At least 48 species of legionellae have been identified, some of which are associated with disease while others are environmental isolates and whether they can cause disease is not known [3]. L. pneumophila is responsible for more than 80% of cases of Legionnaires’ disease, and among the 13 serogroups of L. pneumophila, serogroup 1 is responsible for more than 95% of Legionnaires’ disease cases. It is estimated that L. pneumophila is responsible for at least 25,000 cases of pneumonia/year in the US, which is most probably an underestimate due to the difficulty in bacterial isolation from clinical samples. Since L. pneumophila is the most frequent cause of Legionnaires’ disease, most pathogenic and environmental studies have focused on L. pneumophila. Free-living amoebae are important predators controlling microbial communities. They are ubiquitous and have been isolated from various natural sources such as soil, freshwater, salt water, dust, and air. Although their presence in soil is limited, they have been implicated in the stimulation of phosphorus and nitrogen turnover and thus play an important role in soil ecosystems [4]. Free-living amoebae are also frequently isolated from anthropogenic ecosystems, such as tap water, air conditioning units and cooling towers, feeding on the microbial biofilms present in those systems [5, 6]. However, several bacteria have developed mechanisms to survive phagocytosis by free-living amoebae and are able to exploit them as hosts [7, 8]; see review in [9]. Transient association with amoebae have been reported for a number of different bacteria including Legionella pneumophila, mycobacterium sp., Francisella tularensis, or Escherichia coli O157, among others [8, 10– 12]. As most of these bacteria are human pathogens, amoebae have been suggested to represent their environmental reservoirs, acting as “Trojan horses” of the microbial world [9, 13]. To date only the interaction of L. pneumophila, a facultative intracellular pathogen of humans causing Legionnaire’s disease, with free-living amoebae has been studied in greater detail. L. pneumophila has a very similar intracellular fate within both mammalian and protozoan cells. Intracellular multiplication of Legionella with protozoa such as Acanthamoeba polyphaga and macrophages requires the Dot/Icm secretion system for biogenesis of the phagosome and intracellular replication [14, 15]. The dot/icm genes are located in two different regions. The region I includes seven genes (dotA–D, icmV,W,X) and the larger region II contains the remaining 17 members (icmT,S,R,Q,P,O,N,M,L,K,E, G,C,D,J,B,F) [16–19]. The dot/icm genes of the Type IV secretion system are

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thought to encode proteins involved in the translocation of effector molecules into the host cell that will prevent the bacteria from being killed [16, 20–23]. The dot/icm loci are highly similar to the transfer region of plasmid R64 and other IncI1 plasmids, which suggests that dot/icm virulence genes share a common ancestor with plasmid conjugation system [15, 24–28]. It is not known whether the dot/icm genes derive from a single plasmid, which has been separated into the two gene clusters, or there were the result of multiple gene transfer events. However, only Coxiella burnettii has homologs of the full icm/dot genes, which are contained in a single locus [29-33]. Besides the dot/icm Type IVB secretion system, L. pneumophila possess a second Type IV secretion system, the lvh/lvr genes that surprisingly are not involved in its virulence [26]. The Type IV secretion system has been shown to be the main virulence system of L. pneumophila.

Legionellae facultative intracellular pathogen of free-living amoebae In 1980, Rowbotham described the ability of L. pneumophila to multiply intracellularly within protozoa [7, 8]. Since then, L. pneumophila has been described to multiply in many species of protozoa, and this host-parasite interaction is central to the pathogenesis and ecology of L. pneumophila. At least 14 species of amoebae and 2 species of ciliated protozoa have been shown to support intracellular replication of L. pneumophila [34, 35]. Among the most predominant amoebae in water sources are Hartmannellae and Acanthamoebae, which have also been isolated from water sources associated with Legionnaires’ disease outbreaks [34]. Interaction between L. pneumophila and protozoa is considered to be central to the pathogenesis and ecology of L. pneumophila [8, 9, 36]. In humans, L. pneumophila reaches the lungs after inhalation of contaminated aerosol droplets [34, 37]. The main sources of contaminated water droplets are hot water and air conditioning systems, but the bacteria have been isolated from fountains, spas, pools, dental and hospital units and other man-made water systems [37]. No person to person transmission has ever been described. Once in the lungs, L. pneumophila are ingested by alveolar macrophages, which are thought to be the major site of bacterial replication. This results in an acute and severe pneumonia. Approximatively one-half of the 48 species of Legionella have been associated with human disease. L. pneumophila is responsible for 95 % of cases of Legionnaires’ disease. However, all the Legionella species under appropriate conditions may be capable of intracellular growth and infliction of human disease. Infections due to less common species of legionellae are not frequently diagnosed and reported, and are less studied than L. pneumophila (review in [38, 39]). In addition to recognized Legionella species, a number of Legionellarelated bacteria designated Legionella-like amoebal pathogens (LLAPs) [8, 40] have been described [41]. Interestingly, many LLAPs have been asso-

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ciated with Legionnaires’ disease [42, 43]. In contrast to other Legionella species, however, most of LLAPs cannot be cultured in vitro on artificial media, but are isolated by co-culture with protozoa [34]. Considering that approximately 50% of the 0.5 million annual cases of pneumonia in the US are of unknown etiology, the LLAPs may be responsible for at least some of these cases. The recent developments in using the polymerase chain reaction for bacterial identification in environmental samples will facilitate better identification of legionellae and LLAPs. Further cellular and molecular biology studies are needed to better understand the intracellular life of these endosymbionts.

The role of amoebae in persistence of Legionella in the environment It is most likely that the association of legionellae with protozoa is a major factor in continuous presence of the bacteria in the environment. Many strategies have been used to eradicate legionellae from sources of infection in water and plumbing systems that have been associated with disease outbreaks. These strategies include chemical biocides such as chlorine, overheating of the water, and UV irradiation [44–46]. Such interventions have been successful for short periods of time after which the bacteria reappear in these sources [46, 47]. Thus, eradication of L. pneumophila from the environmental sources of infection requires continuous treatment of the water with agents such as monochloramine or copper-silver ions in addition to maintenance of the water temperature above ~55 °C [45, 48–50]. Compared to in vitro-grown L. pneumophila, amoebae-grown bacteria have been shown to be highly resistant to chemical disinfectants and to treatment with biocides [51]. Amoebae-grown L. pneumophila have been shown to manifest a dramatic increase in their resistance to harsh environmental conditions such as fluctuation in temperature, osmolarity, pH, and exposure to oxidizing agents [52]. Protozoa have been shown to release vesicles containing L. pneumophila that are highly resistant to biocides [53]. The ability of L. pneumophila to survive within amoebic cysts, further contributes to resistance of L. pneumophila to physical and biochemical agents used in bacterial eradication [51, 54]. It is likely that eradication of the bacteria from the environment should start by preventing protozoan infection, an integral part of the infectious cycle of L. pneumophila. Extracellular L. pneumophila is more susceptible to environmental conditions and is not protected from biocides and disinfectants.

The role of amoebae in pathogenesis of Legionella There are many lines of evidence to suggest that protozoa play major roles in transmission of L. pneumophila as infectious particles for Legionnaires’

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disease ([7]; review in [9]). First, many protozoan hosts have been identified that allow intracellular bacterial replication, the only documented means of bacterial amplification in the environment [34, 36, 55]. Second, in outbreaks of Legionnaires’ disease, amoebae and bacteria have been isolated from the same source of infection and the isolated amoebae support intracellular replication of the bacteria [56]. Third, following intracellular replication within protozoa, L. pneumophila exhibit a dramatic increase in resistance to harsh conditions including high temperature, acidity, and high osmolarity, which may facilitate bacterial survival in the environment [57-59]. Fourth, intracellular L. pneumophila within protozoa are more resistant to chemical disinfection and biocides compared to in vitro-grown bacteria [51, 54, 60]. Fifth, protozoa have been shown to release vesicles of respirable size that contain numerous L. pneumophila. The vesicles are resistant to freeze-thawing and sonication, and the bacteria within the vesicles are highly resistant to biocides [53]. Sixth, following their release from the protozoan host, the bacteria exhibit a dramatic increase in their infectivity for mammalian cells in vitro [61]. In addition, it has been demonstrated that intracellular bacteria within H. vermiformis are dramatically more infectious and are highly lethal in mice [62]. Seventh, the number of bacteria isolated from the source of infection of Legionnaires’ disease is usually very low or undetectable, and thus, enhanced infectivity of intracellular bacteria within protozoa may compensate for the low infectious dose [63]. Eight, viable but non-culturable L. pneumophila can be resuscitated by co-culture with protozoa [64]. This observation may suggest that failure to isolate the bacteria from environmental sources of infection may be due to this “dormant” phase of the bacteria that cannot be recovered on artificial media. Ninth, there has been no documented case of bacterial transmission between individuals. The only source of transmission is environmental droplets generated from man-made devices such as shower heads, water fountains, whirlpools, and cooling towers of air conditioning systems [34]. These findings indicate a rather sophisticated host-parasite interaction and a tremendous adaptation of legionellae to parasitize protozoa. This host-parasite interaction is central to the pathogenesis and ecology of these bacteria.

Intracellular infection of L. pneumophila Entry of L. pneumophila into protozoa The attachment and entry mechanisms of L. pneumophila into its protozoan hosts and variations in their mechanisms have been reported for both amoeba Hartmanella vermiformis and Acanthamoeba spp. Attachment of L. pneumophila to H. vermiformis is mediated by adherence to a protozoan receptor characterized as a putative galactose/N-acetyl-galactosamine (Gal/

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GalNAc) lectin [65–67]. Host protein synthesis by A. polyphaga is not required for invasion by L. pneumophila whereas it is required for invasion of H. vermiformis [66]. Integrins are heterodimeric protein tyrosine kinase receptors that undergo tyrosine phosphorylation upon engagement to ligands, which subsequently results in recruitment and rearrangements of the cytoskeleton. Interestingly, attachment of L. pneumophila to the Gal/GalNAc of H. vermiformis triggers signal transduction events in H. vermiformis that are manifested in dramatic tyrosine dephosphorylation of the lectin receptor and other proteins [65, 68]. Similar observations have been obtained upon infection of H. vermiformis by another species. of legionellae, L. micdadei [69]. Among the L. pneumophila-induced tyrosine dephosphorylated proteins in H. vermiformis are the cytoskeletal proteins paxillin, vinculin, and focal adhesion kinase [65, 68]. Tyrosine phosphatases have been shown to disrupt the cytoskeleton in mammalian cells. Thus, the induced tyrosine phosphatase activity in H. vermiformis is probably manifested in disruption of the protozoan cytoskeleton to facilitate entry through a cytoskeleton-independent receptor-mediated endocytosis. Interestingly, in addition to these manipulations of the signal transduction of H. vermiformis by L. pneumophila, bacterial invasion is also associated with specific induction of gene expression in protozoa, and inhibition of this gene expression blocks entry of the bacteria [70]. Following this initial host-parasite interaction, uptake of L. pneumophila by A. castellanii occurs by coiling phagocytosis [11, 12]. However, the uptake of L. pneumophila by H. vermiformis occurs mainly through cup-shaped invaginations (or zipper phagocytosis) on the surface of the amoeba, in addition to occasional coiling phagocytosis [11, 12]. Human macrophages are able to phagocytose heat- or formalin-killed L. pneumophila by coiling phagocytosis [71], which indicates that coiling phagocytosis does not play a role in the intracellular fate of L. pneumophila. However, the infectivity of A. castellanii and macrophages by L. pneumophila has been shown to be similar [72]. In addition, the adherence receptors of macrophages used by L. pneumophila does not seem to affect its intracellular survival profoundly as the bacterium multiplies within phagocytes after entry under different opsonizing or non-opsonizing conditions [73–76]. Uptake of L. pneumophila by another protozoan host, A. polyphaga, is not completely blocked by Gal or GalNAc and is associated with partial tyrosine dephosphorylation of a 170 kDa protein, which may be related to the Gal/ GalNAc lectin of H. vermiformis [66]. Thus, entry of the bacteria into A. polyphaga is partially mediated by the Gal/GalNAc lectin and additional receptors may be involved for bacterial attachment and entry. The heterogeneity in the uptake mechanisms of L. pneumophila into H. vermiformis and A. polyphaga has been confirmed using invasion defective mutants of L. pneumophila. Several mutants that were severely defective in attachment to A. polyphaga exhibited minor reductions in attachment to H. vermiformis [66].

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The mode of entry into mammalian and protozoan host cells have been shown to occur by both dot/icm dependent and independent mechanisms [72, 77, 78]. Studies have shown that phagocytosis of wild-type L. pneumophila is more efficient than uptake of dot/icm mutants within macrophages and A. castallanii, indicating that this mechanism is independent of adherence receptors [72, 77]. However, when the hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.4 buffer, a treatment which enhances the resistance to acid, hydrogen peroxise and antibiotics stress, entry into A. castellanii and macrophages do not require functional dot/icm genes [78]. In addition, a “repeats in structural toxin” (RTX) gene, rtxA has also been shown to play a role in adherence and entry and replication within human macrophages, A. castellanii and in vivo [79, 80]. These data indicate the remarkable adaptation of L. pneumophila for attachment and invasion into different host cells.

Bacterial invasion of macrophages cells and genetic susceptibility Invasion and intracellular replication of L. pneumophila within pulmonary cells in the alveoli is the hallmark of Legionnaires’ disease [81]. Uptake of L. pneumophila by monocytes and macrophages has been shown to occur through conventional and coiling phagocytosis [71, 82-85]. It has been recently shown that the enhanced phagocytosis of L. pneumophila by mammalian cells is dot/icm-dependent [72]. Interestingly, the dot/icm genes delay uptake and induce macropinocytosis in A/J mice macrophages [77]. With the exception of A/J mice, most of the inbred mouse strains are not permissive to L. pneumophila infection, neither are macrophages isolated from these mice [86, 87]. Macropinosomes containing L. pneumophila in A/J mice macrophages are induced transiently and shrink rapidly (5-15 min) [77], and this mode of uptake is linked to the lgn1 locus on chromosome 13 of mice [77, 88, 89]. In macrophages of non permissive strains of mice, the macropinocytic uptake of L. pneumophila is reduced [77]. The lgn1 allele causes the bacteria to behave as if they are lacking the dot/icm system [77]. Thus, the lgn1 allele is required for dot/icmdependent macropinocytosis and delayed uptake by mice macrophages [77]. Whether this mode of uptake plays a role in subsequent trafficking of L. pneumophila is not known. The intracellular growth of others species than pneumophila are not under lgn1’s control [90]. The mouse lgn1 region includes six copies of the neuronal apoptosis inhibitory protein (naip) gene. These Naip proteins have been shown to be direct inhibitors of caspase 3 and 7 [91]. Recently, naip5, also known as birc1e (baculoviral inhibitory apoptosis protein repeat-containing 1), has been shown to be the allele within lgn1 responsible for susceptibility to Legionella [92, 93]. The mechanism by which naip5 regulates susceptibility to L. pneumophila is not yet known.

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Manipulation of vesicle traffic by the Dot/Icm system and biogenesis of a replicative ER-derived vacuole L. pneumophila utilize the Dot/Icm secretion system to transfer macromolecules into the host cell to evade endocytic fusion [25, 27]. The dot/icm loci may be involved in the insertion of a pore in the host cellular membrane through which the effector proteins are exported into the host cell [94, 95]. The effector molecules involved in intracellular trafficking and evasion of the lysosomal fusion within mammalian cells are cis-acting on the phagosome, but do not alter endocytic fusion in the rest of the cell [96]. With few exceptions, the function of individual Dot/Icm proteins is unknown. Several studies demonstrated that upon internalization of L. pneumophila by the host cell, the Legionella-containing vacuole recruits organelles such as vesicles, mitochondria and ER [11, 12, 97, 98] (Fig. 1). Within 5 min following entry of the bacteria into host cells, the L. pneumophila phagosome is surrounded by host cell vesicles, and RER [98-100]. It has been shown that the Legionella-containing phagosome within mammalian macrophages and protozoa does not fuse to lysosomes [12, 71, 101, 102]. Interestingly, examination of the intracellular infection of macrophages, alveolar epithelial cells, and protozoa by another legionellae spp., L. micdadei, showed that within all of these host cells, the bacteria were localized to RER-free phagosomes [103]. Whether other legionellae species replicate within RER-free phagosomes is still to be determined. Recent studies suggest that fusion [104], or exchange of lipid bilayer with ER vesicles on the L. pneumophila-containing phagosome [99] allows the phagosomal membrane to become as thin as the ER membrane with similar characteristics [99, 104]. Within 5 min of uptake, host vesicles come into contact with wild-type Legionella-containing phagosomes and flatten along the surface of the phagosome, and this process is completed within 15 min [99], and is dot/icm-dependent [99]. This is consistent with earlier studies that have shown that after 4 h of infection, there are only few vesicles associated with the phagosomal membrane, but there are ribosomes studding the phagosomal membrane [98]. It is likely that the recruitment of the ER may be involved in the biogenesis of the phagosome that is dependent on the type IV secretion system, since the dot/icm mutants are unable to recruit RER and their phagosomes fuse to the lysosomes [105]. In addition, it has been shown that the L. pneumophila-containing phagosome is a transitional ER (tER)-derived organelle [106]. Its biogenesis involves intercepting early secretory vesicles exiting from tER [106]. Similar to its intracellular fate within macrophages, L. pneumophila is enclosed, after entry into amoebae, in a phagosome surrounded by host cell organelles such as mitochondria, vesicles, and a multilayer membrane derived from the rough endoplasmic reticulum (RER) of amoeba [11, 98–100]. A few hours after internalization and formation of the ER-derived replicative organelle, bacterial replication is initiated. Both, evasion of endocytic fusion and recruitment of early secretory vesicles as they exit

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Figure 1. L. pneumophila are contained within intact phagosome at 8 h post-infection. Representative electron micrographs of L. pneumophila-infected macrophages 8 h post-infection are shown in (a) and (b). The membrane of the LCP is intact as indicated by the thin arrows. Lpn for L. pneumophila, N for nucleus. Adapted from [140].

the ER is controlled by the Dot/Icm type IV secretion system [11, 98, 99, 106–108]. Most or all of the Dot/Icm structural proteins have been shown to be essential for biogenesis of the LCP and for intracellular multiplication within both protozoa and mammalian cells [25, 27]. The role of the RER in the intracellular infection is unknown, but the RER is not required as a source of proteins for the bacteria [109]. Recently, the soil amoeba, Dictyostelium discoideum has been studied as a new host model for Legionella, in particular to understand how Legionella establish its replicative niche within phagocytic host cells. The advantages of using this model for the infection of Legionella is that D. discoideum can be genetically manipulated, that cellular markers are commercially available, and more importantly, the growth of L. pneumophila is similar to that in macrophages and fresh-water amoebae including the requirement for a functional dot/icm Type IV secretion system [110, 111]. D. discoideum is found in soil as a unicellular free-living amoeba that feeds on bacteria. Under starvation conditions, the organism undergoes a complex developmental cycle during which it aggregates to form a multicellular motile phototactic slug. This slug can develop into a fruiting body forming viable spores supported by a column of stalk. In a rtoA mutant of D. discoideum where vesicle trafficking event is lowered, the intracellular growth of L. pneumophila is depressed [112]. In addition, Cytoskeleton-associated proteins and calcium-binding

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proteins of the ER, calnexin and calreticulin, specifically influence the uptake and the intracellular growth of L. pneumophila within D. discoideum [113]. Therefore, ER recruitment plays an important role in the intracellular multiplication of L. pneumophila within this host. One of the hypotheses regarding the role of the recruitment of ER to the LCP is that autophagy mechanism involved in cellular homeostasis was highjacked by Legionella in order to establish its replicative niche. As macroautophagy genes have been identified in D. discoideum, they have been used to show that macroautophagy is dispensable for the intracellular multiplication of L. pneumophila in D. discoideum [114]. Therefore, it is rather clear that ER-derived vesicles and proteins are part of a system that leads to the establishment of the replicative vacuole of L. pneumophila but autophagy does not appear play a role in this mechanism within Legionella protozoan hosts.

Role of the dot/icm genes in evasion of the endocytic pathway The main virulence system of L. pneumophila is the dot/icm Type IV secretion system. Because the Dot/Icm secretion system is ancestrally related to Type IV secretion systems that mediate conjugal DNA transfer between bacteria [16], L. pneumophila may utilize this transporter to transfer macromolecules into the host cell to evade endocytic fusion [104]. The Dot/Icmmediated transfer is thought to occur through the insertion of a pore in the host cellular membrane through which the effector proteins are transported [94, 95]. With few exceptions, the function of individual Dot/Icm proteins is unknown. However, the dot/icm genes are present in all tested Legionella species [115]. Most of the dot/icm genes required for intracellular growth within human cells, are also required for intracellular growth in the protozoan host Acanthamoeba castellanii [15]. Although some loci have been shown to be only essential for the intracellular growth of L. pneumophila in macrophages [116], numerous loci have been identified as essential for survival and intracellular replication of L. pneumophila in A. polyphaga or H. vermiformis and in macrophages [14, 117, 118]. D. discoideum has been shown to support intracellular multiplication of L. pneumophila [110, 111, 119]. As stated earlier, the intracellular fate of L. pneumophila is very similar in infected D. discoideum to that in macrophages, including the recruitment of RER, evasion of lysosomal fusion [119], and dependence of intracellular growth on dot/icm gene functions [119]. The similarity between the infection by L. pneumophila of different protozoa, supports the idea that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa. The Type IV secretion system of L. pneumophila is though to be an effective apparatus to translocate effector molecules into the host cell and modulate the host cell physiology in order for the bacteria to establish a replicative

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niche by recruiting ER-derived vesicles and evading the endocytic pathway. Genetic screens that enable the identification of the set of 26 dot/icm genes have failed to identify the genes encoding the Type IV secretion system substrates secreted into host cells [17, 19, 27]. Different strategies have been used for this purpose. First, strategies, which are not based on intracellular growth defect of the bacteria, but based on homology to eukaryotic protein have allowed the identification of Type IV secretion system effectors such as RalF, LepA and LepB [20, 21]. Most of these substrates do not express an intracellular replication deficient phenotype (or a minor one) within both mammalian and protozoan host cells, which explain why they had not been isolated previously in genetic screens for intracellular growth mutants. RalF protein, containing a Sec7-homology domain is produced by L. pneumophila and is injected into the host cells by the Dot/Icm transporter, functioning as an exchange factor that activates members of the ARF protein family [21, 120]. LepA and LepB, which have a weak homology to SNAREs have been shown to be delivered to host cells by a Type IV secretion system-dependent mechanism. The double mutant of the paralogues lepA lepB exhibited a defect in release of L. pneumophila-containing “fecal” or “respirable” vesicles from A. castellanii and D. discoideum [20]. Second, a strategy based on that fact that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator and mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability, has allowed identifying an effector called LidA. This substrate LidA, identified by a complex genetic screen has been shown to be associated with the cytoplasmic face of the L. pneumophila-containing phagosome [23]. Third, an interbacterial protein transfer assay is another strategy used to identify the substrate SidC (substrate of icm/dot complex) identified among other Sid substrates also secreted into macrophages [22]. Finally, some substrates have been identified using chaperone proteins belonging to the dot/icm genes secretion system, as bait. IcmS has been hypothesized to serve as a chaperone for secreted substrates since it is predicted to be located in the bacterial cytoplasm, has no homology to bacterial conjugal transfer proteins and has similar biochemical properties of secretion chaperones from the type II secretion system [121, 122]. IcmS has been used as a bait to identify potential secreted substrates such as SidE family proteins (SdeA, SdeB, SdeC, SidE) [123] family proteins have been shown to be secreted by L. pneumophila at early stages of infection of macrophages across the phagosomal membrane and have been shown to be required for full virulence in Acanthamoeba castellanii although individual deletions of a number of substrates had a modest or no effect in intracellular replication [22] probably resulting from the export of functionally redundant proteins by the Type IV secretion system. In addition, as the complex IcmS-IcmW form a stable protein complex and play an important role in subtrate translocation, IcmW has also been used as a bait in a yeast two-hybrid system to identify substrate proteins translocated into host cells by the type IV secretion sys-

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tem [124]. IcmW-interacting proteins (Wips) have been recovered including SidG and SidH identified previously with the interbacterial protein transfer screen [22] as well as WipA, which is translocated into mammalian host cells. A paralogue called wipB, which is also translocated into macrophages by the Type IV secretion system, was found in the Legionella genome data base and the double mutant wipA wipB had no growth defect in A. castallanii [124]. The functions of the identified substrates are under investigation. There are probably hundred of effectors that need to be identified in order to understand how Legionella establish a successful replicative niche within its host cells. Some of these substrates may also be host-dependent and be functional only in one of the various host cells of Legionella.

Growth phase-regulated virulence L. pneumophila obtained from post-exponential cultures, expresses traits that are correlated with virulence, in contrast to exponentially-growing bacteria, [7, 8, 57, 58]. During the replication phase, L. pneumophila are sodium resistant and aflagellated [7, 8, 57, 58]. When L. pneumophila egress from host cells, the bacteria are flagellated and sodium-sensitive [7, 8, 57, 58]. It has been hypothesized that amino acid limitation in vitro induces the virulent phenotype [57, 58]. When L. pneumophila enters into post exponential growth phase or is subjected to amino acid limitation, the bacteria accumulate the stringent response signal, guanosine 3’,5’-bispyrophosphate (ppGpp) through the ppGpp synthetase, RelA [125]. The accumulation of ppGpp increases the amount of the stationary-phase sigma factor RpoS, which triggers the expression of the stationary-phase genes [125]. A rpoS mutant of L. pneumophila replicates within HL60 and THP-1 monocytic cell lines, but is attenuated in A. castellanii [126]. In bone marrow-derived macrophages from A/J mice, L. pneumophila rpoS mutants replicate poorly because they traffic rapidly to a late endosome-like compartment [125]. These phenotypic differences in different host cells may be due to different parental strains used to construct the rpoS mutants. It has been shown that some genotypic and phenotypic differences exist between the AA100, JR32 and LP01 stains, which are the most commonly used in virulence studies [127] and AA100 is clearly the most virulent. Therefore, the differences between L. pneumophila strains and/or between the host cells may explain the different intracellular growth observed for the rpoS mutants. Sodium sensitivity and maximal expression of flagellin also requires RpoS [125]. Therefore, some RpoS-regulated traits could be critical for efficient transmission or infection [126]. L. pneumophila in post-exponential phase becomes cytotoxic by an RpoS-independent pathway [125]. It is proposed that when nutrient levels and other conditions are favorable, L. pneumophila replicates within host cells, and when amino acids become rare, intracellular bacteria express several traits that permit escape from the host cell,

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survival in the environment and the transmission to a new host. However, the intracellular signals that trigger phenotypic transition at the post-exponential phase are still to be determined. Although a previous study has shown that RpoS, which accumulates when RelA is activated, is required for intracellular growth in A. castellanii [126], Zusman et al. have shown that relA gene product is dispensable for intracellular growth in HL-60-derived human macrophages and in A. castellanii [128]. Moreover, it has also been shown that RelA and RpoS have minor effects on expression of some of the dot/icm genes [128]. Thus, the role of RpoS in the intracellular infection seems to be host cell-specific, but that may be due to differences between the parental strain used to construct the rpoS mutants. Interestingly, it has been shown that the conserved RNA binding protein CsrA is a global repressor of phenotypic transition of L. pneumophila at the post-exponential phase [129]. Overexpression of CsrA in L. pneumophila blocks many phenotypic traits that are expressed at the post-exponential phase such as flagellation and reduction of cell size [129]. This is also associated with reduction in transcription of fliA and flaA [129]. In addition, the global response two-component regulator of L. pneumophila, LetA/S, is involved in regulation of phenotypic transition, since letA mutants of L. pneumophila exhibit reduced infectivity and are more resistant to oxidative and acid stress in addition to a severe defect in intracellular replication in Acanthamoeba [130, 131]. However, the letA mutant is not defective for intracellular replication in human-derived macrophages, similar to the rpoS mutant and mutants in the type II secretion system [131]. Thus, RpoS and LetA/S regulate phenotypic transition of L. pneumophila at the post-exponential phase and both are essential for expression of L. pneumophila genes required for replication in the protozoan host but not in human-derived macrophages. Since both regulatory systems are triggered by ppGpp synthesized by RelA, and RelA is not required for the intracellular infection of macrophages or amoeba, it is not clear how other regulatory pathways contribute to the regulation of phenotypic transition.

Cytolysis of the host and bacterial egress A fundamental step in the pathogenic life cycle of intracellular bacteria is the ability to lyse the host cell and to egress. Apoptosis and necrosis are the two commonly observed types of cell death. Necrosis is characterized by physical damage causing cell death. Apoptosis is a regulated suicide program of the cell manifesting morphological and biochemical features distinct from those of necrosis [132]. Killing of mammalian cells by L. pneumophila has been proposed to occur in two phases [133, 134]. In the first phase, L. pneumophila induces apoptosis in macrophages, monocytes and alveolar epithelial cells during the early stages of the infection [133,

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Figure 2. L. pneumophila is cytoplasmic at 24 h post-infection. Representative electron micrographs of L. pneumophila-infected macrophages at 24 h post-infection are shown in (c) and (d). L. pneumophila (Lpn) are cytoplasmic where bacteria are surrounded by numerous vesicles (V), lysosomal contents (white arrows), mitochondria (M), and amorphous elements (A). No distinguishable phagosomal membrane surrounds the bacteria. N for nucleus. Adapted from [107].

135–137], which is mediated through the activation of caspase-3 [134]. Induction of apoptosis is largely independent of the bacterial growth phase [134]. The second phase is mediated through rapid induction of necrosis by L. pneumophila upon entry into the post-exponential phase of growth when the bacteria become cytotoxic [138, 139]. Our working model of bacterial egress can be presented in three steps. First, upon exiting the exponential phase of intracellular growth, an „egress pore“ is inserted into the phagosomal membrane leading to its disruption. Second, the bacteria egress into cytoplasm. Third, disruption of organelles and the plasma membrane occurs, culminating in lysis of the host cells and bacterial egress. A detailed ultrastructural analysis of late stages of intracellular replication has been performed to examine egress of L. pneumophila from both macrophages and amoebae by electron microscopy [140]. The membrane of the L. pneumophila-containing phagosome (LCP) within both macrophages and Acanthamoeba polyphaga is intact up to 8 h postinfection [140]. However, at 12 h, the majority of the LCPs are disrupted within both hosts, while the plasma membrane remains intact [140]. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic [140] (Fig. 2). Thus, by 18 h–24 h postinfection, the majority of the remaining host cells harbor cytoplasmic bacteria and this transient cytoplasmic presence of L. pneumophila precedes lysis of the plasma membrane. Interestingly, within both macrophages and amoebae, bacterial replication proceeds in the cytoplasm.

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Therefore, the phagosomal membrane is disrupted first, rather than by simultaneous lysis of both the phagosomal and the plasma membranes. These disruptions of LCP may be the result of multifactorial events linked to apoptosis, the pore forming activity (PFA) of the Type IV secretion system and mechanical pressure due to the increase of the phagosomal size [95].

Immunity to intracellular L. pneumophila The Toll-like receptors As the interaction between host phagocytes and Legionella results in the induction of the host cell response that is thought to activate the innate immune system through the stimulation of TLRs on the surface. It leads to the production of pro-inflammatory cytokines that recruit lymphocytes to the infection sites and activate macrophages. Macrophages express both TLR2 and 4, which can recognize different bacterial products (peptidoglycan, LPS, etc.). However, surprisingly TLR2 plays a major role in the response to Legionella, whereas TLR4, which recognizes predominantly LPS, is less involved in the process [141, 142]. In addition, TLR5 may also be important in immunity to Legionella as TLR5 recognizes a conserved region in most bacterial flagellin including Legionella flagellin.

The cytokine production Neutrophils, macrophages and dendritic cells have been shown to produce cytokines after Legionella infection, which is comparable to the cytokine production involved in the Th1 response [143, 144]. IL-12 is produced by many cells, including macrophages, DC and neutrophils after exposure to bacteria and has been shown to have an important role in eliminating Legionella in vivo [145]. IL-18 is also crucial in the induction of IFN-a production from T-cells, B cells and NK cells in vitro and during in vivo infection with Legionella [146]. When they are depleted independently or together, the levels of IFN-a drop significantly, which lead to a decreased ability of clearing Legionella from the lungs of the mice [146]. Intracellular replication of L. pneumophila is inhibited in gamma interferon (IFN-a)activated bone marrow-derived mouse macrophages and IFN-a-activated human monocyte-derived macrophages in a dose-dependent manner. This inhibition of intracellular replication is associated with the maturation of the LCP into a phagolysosome [147]. Together with IFN-a, TNF-_ plays also a role in the clearance of Legionella from the lungs via activation of macrophages. Added independently or together, both IFN-a and TNF-_ can also significantly restrict the intracellular growth of Legionella [148, 149].

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The clearance of Legionella from the lungs depends on the host production of IFN-a in A/J mice [150]. Additionally, Legionella infection cannot be cleared efficiently in BALB/c mice, which do not produce IFN-a compared to infection in mice producing IFN-a [151]. Similarly, when human monocytes and alveolar macrophages are treated with IFN-a, it results in dosedependent restricted growth of Legionella [152]. Restriction of Legionella growth is, in part, due to low availability of intracellular iron, a process mediated by the transferring receptor which is downregulated in IFN-a activated monocytes [153]. A recent study also showed that activated macrophages infected by L. pneumophila can downregulate T-cell responses via production of prostaglandins, which may play a role in limiting unnecessary immune-mediated damage of host tissues [154]. Activated macrophages can also produce nitric oxide after bacterial infection, which has a direct lethal effect on many pathogens. In the case of Legionella infection, the role of NO may not be direct. Inhibitors of NO synthesis had an effect intracellular replication of L. pneumophila in BALB/c alveolar macrophages, but not in A/J mice macrophages. It had, however, a effect on the Legionella infection of A/J mice model [149, 151, 155-157].

Acquired immune response Both antigen-specific humoral and cell-mediated immune responses are induced during Legionella infection. Although Legionella specific antibodies are produced during human infection, or in the guinea pigs model, their role in controlling Legionella infection has not been clearly demonstrated [158]. Antibody opsonization does not inhibit intracellular multiplication of L. pneumophila [159]. However, when CD4 and CD8 T-cells are depleted in mice, susceptibility to Legionella infection is enhanced, suggesting that acquired cell-mediated immune response play a role in Legionella clearance [160]. Both macrophages and DCs are able to present microbial antigens on major histocompatibility (MHC) class I and MHC class II molecules, which stimulate antigen specific T-cell response. DCs appear to play an important role in producing antigen-specific immune responses and in priming T-cells. DCs are able to restrict the intracellular growth of Legionella without preventing traffic of Legionella-containing vacuole or bacterial proteins synthesis. Therefore, resctriction of growth is possible without the Legionella vacuole fusing to lysosomes, suggesting that the range of proteins presented by DCs to naïve T-cells is very similar to those presented by macrophages [161, 162]. Taken together, these data showed that Legionella antigens synthesized in a non-degradative ER-derived vacuole are most likely processed and loaded onto MHC class II molecules for presentation to CD4 + T-cells [161]. In addition, it has recently been shown that in mice primary macrophages, trafficking of Legionella-containing phagosome to lysosomes is required for

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optimal antigen processing and MHC-II presentation to CD4 T-cells [163]. Identification of antigens and determination of vesicular trafficking mechanisms involved in processing and presentation remain to be understood in greater detail.

Conclusions L. pneumophila has become a paradigm of an intracellular pathogen that manipulates several processes involved in endocytosis and vesicle traffic between host cell organelles, and many of these manipulations are controlled by the Dot/Icm secretion system and the effectors exported through this system. This organism avoids phagosomes-lysosomes fusion within the first few minutes of the intracellular infection and the phagosomes are converted into endoplasmic reticulum-derived organelles that support intracellular replication [99]. This process involves intercepting early secretory vesicles exiting from the tER [106]. After 4 h, L. pneumophila starts to replicate within this replicative organelle. Between 8 h and 18 h, the phagosomal membrane is gradually disrupted and the bacteria become cytoplasmic and are dispersed among cytoplasmic organelles such as mitochondria and lysosomes prior to lysis of the host cell [140]. Legionella infection triggers both antigen-specific humoral and cell-mediated immune responses, but the cellmediated responses appear to play a greater role in Legionella clearance from the lungs. Understanding the unique intracellular fate at the molecular and the cellular level will unravel fascinating aspects of the intricate balance in the evolution of this host-parasite interaction and the ability to control Legionella propagation in human and in environment.

Acknowledgements Our work is supported by Public Health Service grant RO1AI43965 awarded to Y.A.K.

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mophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. J Bacteriol 184(1): 67–75 Fettes PS, Forsbach-Birk V, Lynch D, Marre R (2001) Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation. Int J Med Microbiol 291(5): 353–360 Lynch D, Fieser N, Gloggler K, Forsbach-Birk V, Marre R (2003) The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii. FEMS Microbiol Lett 219(2): 241–248 Gal-Mor O, Segal G (2003) The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii. Microb Pathog 34(4): 187–194 Cohen JJ (1993) Overview: Mechanisms of apoptosis. Immunol Today 14: 126–130 Gao L-Y, Abu Kwaik Y (1999) Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity. Infect Immun 67: 862–870 Gao L-Y, Abu Kwaik Y (1999) Activation of caspase-3 in Legionella pneumophila-induced apoptosis in macrophages. Infect Immun 67(9): 4886–4894 Hagele S, Hacker J, Brand BC (1998) Legionella pneumophila kills human phagocytes but not protozoan host cells by inducing apoptotic cell death. FEMS Microbiol Lett 169(1): 51–58 Molmeret M, Zink SD, Han L, Abu-Zant A, Asari R, Bitar DM, Abu Kwaik Y (2004) Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome. Cell Microbiol 6(1): 33–48 Zink SD, Pedersen L, Cianciotto NP, Abu-Kwaik Y (2002) The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages. Infect Immun 70(3): 1657–1663 Alli OAT, Gao L-Y, Pedersen LL, Zink S, Radulic M, Doric M, Abu Kwaik Y (2000) Temporal pore formation-mediated egress from macrophages and alveolar epithelial cells by Legionella pneumophila. Infect Immun 68: 6431–6440 Molmeret M, Alli OA, Zink S, Flieger A, Cianciotto NP, Kwaik YA (2002) icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect Immun 70(1): 69–78 Molmeret M, Bitar DM, Han L, Kwaik YA (2004) Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga. Infect Immun 72(7): 4040–4051 Arnoult D, Tatischeff I, Estaquier J, Girard M, Sureau F, Tissier JP, Grodet A, Dellinger M, Traincard F, Kahn A et al (2001) On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death. Mol Biol Cell 12(10): 3016–3030 Akamine M, Higa F, Arakaki N, Kawakami K, Takeda K, Akira S, Saito A

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(2005) Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila. Infect Immun 73(1): 352–361 Tateda K, Matsumoto T, Ishii Y, Furuya N, Ohno A, Miyazaki S, Yamaguchi K (1998) Serum cytokines in patients with Legionella pneumonia: relative predominance of Th1-type cytokines. Clin Diagn Lab Immunol 5(3): 401–403 Tateda K, Moore TA, Deng JC, Newstead MW, Zeng X, Matsukawa A, Swanson MS, Yamaguchi K, Standiford TJ (2001) Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia. J Immunol 166(5): 3355–3361 Gao L-Y, Stone BJ, Brieland JK, Abu Kwaik Y (1998) Different fates of Legionella pneumophila pmi and mil mutants within human-derived macrophages and alveolar epithelial cells. Microb Pathog 25: 291–306 Brieland JK, Jackson C, Hurst S, Loebenberg D, Muchamuel T, Debets R, Kastelein R, Churakova T, Abrams J, Hare R, O’Garra A (2000) Immunomodulatory role of endogenous interleukin-18 in gamma interferonmediated resolution of replicative Legionella pneumophila lung infection. Infect Immun 68(12): 6567–6573 Santic M, Molmeret M, Abu Kwaik Y (2005) Maturation of the Legionella pneumophila-containing phagosome into a phagolysosome within gamma interferon-activated macrophages. Infect Immun 73(5): 3166–3171 Skerrett SJ, Bagby GJ, Schmidt RA, Nelson S (1997) Antibody-mediated depletion of tumor necrosis factor-alpha impairs pulmonary host defenses to Legionella pneumophila. J Infect Dis 176(4): 1019–1028 Brieland JK, Remick DG, Freeman PT, Hurley MC, Fantone JC, Engleberg NC (1995) In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide. Infect Immun 63: 3253–3258 Brieland J, Freeman P, kunkel R, Chrisp C, Hurley M, Fantone J, Engleberg NC (1994) Replicative Legionella pneumophila lung infection in intratracheally inoculated A/J mice: A murine model of human Legionnaires’ disease. Am J Pathol 145: 1537–1546 Brieland J, McClain M, Heath L, Chrisp C, Huffnagle G, LeGendre M, Hurley M, Fantone J, Engleberg C (1996) Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect Immun 64(7): 2449–2456 Nash TW, Libby DM, Horwitz MA (1988) IFN-gamma-activated human alveolar macrophages inhibit the intracellular multiplication of Legionella pneumophila. J Immunol 140: 3978–3981 Byrd TF, Horwitz MA (1989) Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J Clin Invest 83: 1457–1465 Neild AL, Shin S, Roy CR (2005) Activated macrophages infected with Legionella inhibit T cells by means of MyD88-dependent production of prostaglandins. J Immunol 175(12): 8181–8190 Gebran SJ, Yamamoto Y, Newton C, Klein TW, Friedman H (1994) Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J

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Pathogenesis of Streptococcus pneumoniae infections: adaptive immunity, innate immunity, cell biology, virulence factors Sven Hammerschmidt1, Gavin K. Paterson2, Simone Bergmann1 and Timothy J. Mitchell2 1Research

Center for Infectious Diseases, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany; 2Division of Infection and Immunity, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow G12–8QQ, UK

Abstract During the past two decades the intense study of the infection process of Streptococcus pneumoniae has elucidated multifaceted interactions of the human pathogenic bacterium with the host. A broad spectrum of pneumococcal virulence factors, which are adapted successfully to different host niches, is involved either predominantly in nasopharyngeal colonization or subsequently in dissemination and transmigration of host tissue barriers. The severe course of infections becomes manifest in invasive diseases like pneumonia, meningitis and septicaemia. To escape the risk of increasing antibiotic resistance and to combat the threat of pneumococcal infections pneumococcal vaccines have been developed. The carrier protein of the current available heptavalent vaccine is not derived from pneumococci therefore it is thought to substitute this carrier by a highly conserved and immunogenic pneumococcal-specific protein. S. pneumoniae is a versatile microorganism and has evolved numerous successful strategies to colonize its host and to evade host defence mechanisms. In this report we discuss the bacterial repertoire of virulence factors and provide insights into the surface protein variability. In addition, we show the impact of these virulence factors on interactions with host components, including cellular receptors and how the function of these proteins contributes to colonization and virulence of S. pneumoniae. The non-invasive and invasive infections are accompanied by immune responses of both the innate and adaptive immune system. These two systems operate in concert to combat infections, but pneumococci have developed highly sophisticated mechanisms to subvert the host immune system. We introduce pattern recognition receptors that recognize specific structures of pneumococci and stimulate thereby host defence mechanisms.

Introduction Streptococcus pneumoniae (the pneumococcus) is a serious human pathogen causing local infections including otitis media and sinusitis and lifethreatening invasive diseases, such as lobar pneumonia, sepsis and men-

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ingitis [1]. The burden of disease is highest in the youngest and elderly population and in patients with immunodeficiencies. The pneumococcus is the prime cause of community-acquired pneumonia in adults and accounts for 50–75% cases. The incidence of pneumococcal pneumonia remains high, between 68–260 cases per 100,000 and year [2]. Despite the use of antibiotics and availability of vaccines the mortality rate remains high. Each year, 1 million children younger than 5 years die from pneumonia and invasive diseases [3]. Even more, community-acquired pneumococcal meningitis has a very high case-fatality rate. The survivors often develop long-term clinical sequelae including hearing loss, neurological deficits, and neurophysiological impairment [4]. The infections caused by pneumococci are preceded by an asymptomatic carrier status and accompanied by the transmigration of tissue barriers by the pathogen. The clinical outcome of disease has been shown to be dependent on both the pathogen and host susceptibility for the individual pathogen. Pneumococci are endowed with a multitude of factors that contribute to the pathogenic potential of this versatile pathogen. Biological activities that have been attributed to these virulence factors include the subversion of host immunity and adoption of host-protein functions to facilitate adherence and invasion. Pneumococci disseminate and gain access to the ear, lungs, blood or meninges. The adaptation of pneumococci to different host milieus including the nasopharynx has been correlated with a differential expression of several pneumococcal factors [5–9]. A comprehensive understanding of critical steps during pneumococcal pathogenesis including colonization, progression to pneumonia, dissemination in the bloodstream, and transition of the blood-brain-barrier is crucial to combat the threat of pneumococcal infections and hence, reduce the mortality exacted by this pathogen. This review will evaluate our current understanding of the mechanisms employed by pneumococci to encounter the human host.

Nasopharyngeal colonization S. pneumoniae is a complex microorganism divided into over 90 serotypes depending on the antigenic structures of their capsular polysaccharides [10]. Pneumococci of different serotypes are able to simultaneously colonize the nasopharyngeal cavity of healthy individuals [11]. The rates of carriage and acquisition depend on age, genetic background, socioeconomic conditions and geographical area [12]. Europe and the United States show similar serotype distributions with minor differences in several serotypes. Disease is most commonly due to strains representing 20 of the > 90 different pneumococcal serotypes and these 20 serotypes have been covered by the 23-valent polysaccharide (PS) vaccine [13, 14]. By contrast, the serotype distribution in Asia is slightly different, which had resulted in only < 70% effectiveness of the 23-valent conjugate PS vaccine [15]. The current vaccine, a seven-

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Figure 1. Adherence of Streptococcus pneumoniae to human epithelial cells after 4 h infection as seen by high-resolution field emission scanning electron microscopy. (Image courtesy Manfred Rohde, German Research Centre for Biotechnology, Braunschweig, Germany.)

valent conjugate vaccine (Prevnar, Wyeth, USA), covers the most prevalent serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. It is noteworthy that invasive disease originates from nasopharyngeal colonization with the homologous serotype [16]. Interestingly, certain serotypes including 14 and 18C clones have a high potential to cause invasive disease, whereas the most commonly carried serotypes 6B, 19F, and 23F are least invasive. By contrast, infrequent colonizers and non-vaccine serotypes including serotypes 8, 38, 33F appear to be more invasive [12, 17].

Transition from colonization to pneumonia and invasive infections The mechanisms of how the pneumococcus makes the transition from a commensal with asymptomatic carriage to a virulent pathogen causing disease have not yet been fully explained. It is, however, known that the local immune response has an important regulatory role during colonization and subsequent infections [18]. The successful conversion of the commensal to an invasive microorganism is accompanied by the transmigration of tissue barriers and the subsequent adaptation of the pathogen to different host niches. A prerequisite for transmigration is the ability of the pneumococcus to adhere to mucosal cells of the respiratory tract. It has been shown that pneumcoccal colonization of the nasopharynx correlates with observed differences in colony phenotypes. S. pneumoniae undergoes spontaneous, reversible opacity phase variation with a frequency of 10–3 to 10–6 resulting in opaque and transparent colonies [19]. Differences in colonial opacity have previously been shown to correlate with different levels of capsular polysaccharide (CPS) [19]. The transparent phenotype produces lower amounts of CPS and has an enhanced ability to reside on mucosal surfaces when tested in several animal models [20, 21]. By contrast, the opaque variant is more virulent in systemic infections [22]. The higher amounts of

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CPS produced by these variants make them more resistant to complementmediated opsonophagocytotic killing [22]. Although higher amounts of CPS have been demonstrated to block pneumococcal adherence, a basal level of encapsulation is essential for colonization [23, 24]. Strikingly, electron micrographs illustrated that the intimate contact of pneumococci with host cells is associated with a reduction of encapsulation [25]. Pneumococci spread presumably to the lungs by aspiration and attachment of pneumococci has been indicated to bronchial epithelial cells [26] or components of the basement membrane including laminin, collagen and fibronectin [27, 28]. Binding is promoted by an impaired ciliary beating frequency due to the damage caused by smoking or the release of pneumolysin, a cytolysin of pneumococci. Pneumolysin inhibits the normal beating of cilia on epithelial and endothelial cells thereby facilitating penetration of pneumococci into the bloodstream [29, 30]. The effect of the hydrogen peroxide released by pneumococci, which is as toxic as pneumolysin to ependymal ciliary cells, is masked in the presence of pneumolysin [31]. Within the alveoli pneumococci adhere to type II pneumocytes and can spread rapidly into the blood by crossing the vascular endothelium [32]. Three stages of lesions were distinguished during pneumococcal pneumonia: (1) engorgement, (2) red hepatization, and (3) gray hepatization. Engorgment is associated with the accumulation of a serous exudate in the alveoli and is followed by a leakage of erythrocytes into the alveoli. At the stage of gray hepatization bacterial multiplication peaks, fibrin is formed through the procoagulant activity induced by pneumococci and polymorphonuclear leukocytes (PMNs) recruited to the infection site start to control pneumococcal multiplication [32]. Clearance of pneumococci by opsonophagocytosis is further dependent on complement and facilitated by anti-capsular antibodies [15]. One of the remarkable features of pneumococcal pneumonia is that the lungs of patients who withstand the inflammatory response of the host and survive almost invariably return to normal, irrespective of the severity of the systemic or pulmonary condition when the disease was at its peak [33].

Cell wall structures and virulence factors of S. pneumoniae Pneumococci are encased by a capsular polysaccharide that has been recognized as a sine qua non of virulence. Survival in the bloodstream depends on the expression of capsule. The degree of activation of the classical or alternative pathways of complement, the deposition and degradation of complement components, and the degree of protection against complement-mediated opsonophagocytosis is determined by the biochemical structure of the CPS rather than by the thickness of the CPS [15]. The layer underneath the capsule, the pneumococcal outer cell wall, is comprised of peptidoglycan, teichoic (TA) and lipoteichoic acids (LTA),

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Figure 2. Infections caused by pneumococci. Depicted are encapsulated pneumococci (arrowheads) in the cerebrospinal fluid (top left panel) and the histopathology of purulent meningitis caused by pneumococci (top right panel). The photomicrograph of a section of the subarachnoid space, in hematoxylin-eosin stain, shows the infiltration of leukocytes (arrowheads) and purulent meningitis (arrows). Image courtesy Roland Nau, University of Göttingen, Germany. Chest X-ray of a lobar pneumonia (bottom, left panel) and section of lung representing a stage of hepatization.

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and phosphorylcholine (PCho). The PCho is covalently linked to the TA and LTA, which differ only in their attachment to the pneumococcal cell wall. Although PCho is part of the cell wall of other respiratory pathogens including Neisseria spp., Haemophilus influenzae, and Pseudomonas, only pneumococcal PCho anchors a special class of proteins, the choline-binding proteins (CBP) non-covalently on the surface. CBPs have a modular organization and consist in general of a leader peptide, a biologically active N-terminal domain and the conserved choline-binding domain (CBD) that targets the aminoalcohol PCho. The CBD, generally located at the carboxy terminus and preceded by a proline-rich sequence, consists of highly homologous 20-amino acid long repetitive sequences. Pneumococci can produce 13 to 16 different CBPs and the number of produced CBPs depends on the pneumococcal strain. To date, the extensively characterized CBPs include the pneumococcal surface protein A (PspA), the pneumococcal surface protein C (also referred to as CbpA or SpsA), and four cell wall hydrolases. In general, the bacterial cell wall hydrolases (CWHs) are endogenous enzymes that specifically cleave covalent bonds of the cell wall. The pneumococcal CWHs are: the major autolysin LytA (Nacetyl-muramoyl-L-alanine amidase), a `-N-acetylglucosamidase (LytB), a `-N-acetylmuramidase (LytC; lysozyme), and a phosphorylcholine esterase (Pce or CbpE). LytA has been characterized in detail and recently, the structural analysis of the CBD of LytA demonstrated that this module adopts a peculiar solenoid structure [34]. LytB is highly expressed in the early exponential growth phase and has been shown to be important for cell separation of pneumococci. In contrast to other CBPs, LytB and LytC possess the CBD as an N-terminal domain [34]. Apart from these, CbpD, CbpG, CbpJ, CbpI and, PcpA represent further members of this family [35, 36]. Three clusters of surface proteins can be distinguished by genome analysis: the lipoproteins including peptide permeases and ATP binding cassette (ABC) transporter (42 in R6 and 47 in TIGR4), the above mentioned family of CBP (10 in R6 and 15 in TIGR4) and proteins with an LPxTG motif (13 in R6 and 19 in TIGR4). The latter represent typical Gram-positive surface proteins that are covalently anchored in the cell wall after cleavage of the LPxTG sequence by a transpeptidase, designated sortase. Strikingly, many of these LPxTG proteins contain enzymatic activity including neuraminidases, hyaluronidase, IgA1-protease and zinc metalloproteases (ZmpB, ZmpC, and ZmpD). In addition to these predicted surface proteins, nonclassical surface proteins lacking a classical leader peptide and membrane anchoring motifs, have been identified on the pneumococcal surface. These proteins have received considerable attention for their contribution to virulence of pneumococci and other pathogenic bacteria. To date, the mechanism of secretion and anchoring of these proteins that include the enolase, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the PavA (pneumococcal adherence and virulence factor A) remain unknown for pneumococci and many other Gram-positive pathogens.

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Another important molecule that is associated with pneumococcal virulence, but not surface-exposed due to the lack of secretion signal, is pneumolysin. This hemolysin is intracellularly produced by pneumococci and released by the action of cell-bound autolysin. Pneumolysin is a sulfhydryl(thiol)-activated pore-forming cytolysin that binds to cholesterol in the plasma membrane of host cells [37]. Soluble pneumolysin monomers form ring-shaped oligomeric pores [38, 39]. The observation of the poreforming process by cryo-electron microscopy has indicated that the membrane bound form with an intact bilayer is the prepore and that the conformational transition from the prepore to the pore form is accompanied with a separation of monomers and a substantial refolding of protein domains [40]. The multiple biological activities of pneumolysin have been shown to interfere with eukaryotic host-cell function and the immune system.

Cellular biology of pneumococcal infections Bacteria attach to eukaryotic host cells via surface-exposed adhesins which specifically interact with cellular host receptors or adhesive glycoproteins of the extracellular matrix (ECM) which connect the microorganism with cellular receptors [41]. It has been shown that pneumococci translocate the respiratory barrier and gain access to the blood circulation through the intracellular route [42]. However, the strategies used by pneumococci for translocation are not yet fully elucidated and a paracellular route of entry cannot be excluded as an alternative route.

Adherence to host cells In the early stages of the infectious process pulmonary epithelial cells and vascular endothelial cells are targeted by pneumococci. Attachment to resting lung cells and vascular endothelial cells occurs via the recognition of two classes of glycoconjugates. The disaccharides N-acetyl-D-galactosamin `13/4 galactose (GalNAc (`1-3/4) Gal) and sialylated N-acetyl-D-glucosamine `1-3 galactose (GlcNAc (`1-3) Gal) are recognized by pneumococcal virulence determinants that have not been identified so far [43, 44]. Binding of pneumococci to resting cells was completely abolished by a combination of these two carbohydrates as represented by asialo-GM and globoside [45]. It is has been suggested that the multiple neuraminidases (NanA, NanB and probably NanC) of pneumococci cleave terminal sialic acid (N-acetylneuraminic acid) from glycolipids, glycoproteins, and oligosaccharides on host-cell surfaces and body fluids thereby enhancing intimate adherence. In fact, NanA has been shown to cleave the terminal sialic acids of lipooligosaccharides from Haemophilus influenzae and Neisseria meningitidis [46]. Because terminal sialic acids protect these respiratory pathogens against

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complement mediated phagocytosis, desialylation of competitors may provide an advantage during colonization of host niches. In addition, NanA has been shown to be implicated in desialylation of human proteins exhibiting sialic acid including the secretory component, lactoferrin and IgA2 [47]. These human proteins are pneumococcal host targets and the removal of sialic acid may facilitate bacterial persistence in the respiratory tract. NanA sequence diversity is restricted to regions that are not required for enzymatic activity and has been suggested to provide an important advantage in evading the adaptive immune response [48]. In a chinchilla infection model, loss of NanA has been shown to impair pneumococcal persistence in the nasopharynx and middle ear [49]. In contrast, the nanA knockout was not attenuated in an intraperitoneal infection model [50]. A similar role is suggested for the hyaluronidase (Hyl), which hydrolases primarily hyaluronan. Although hyl knockout strains are attenuated in an intraperitoneal mouse infection model [51], the precise function of the Hyl during pathogenesis has yet to be clarified. S. pneumoniae produce up to four zinc metallo proteases including IgA1protease, ZmpB, ZmpC, and ZmpD, which are anchored to the cell wall by an N-terminal LPxTG motif. The IgA1-protease is produced virtually by all pneumococci and Weiser and co-workers [52] demonstrated that cleavage of surface-bound serotype-specific IgA1 by the IgA1-protease markedly enhanced adherence of pneumococci to host cells. It has been assumed that bound Fab fragments neutralize the negatively charged capsule and negate the anti-adhesive effects of the capsule. A role in colonization has also been suggested for the lipoprotein SlrA, and the choline-binding proteins CbpD and CbpG, which is thought to be a serine protease [35]. Recent reports indicated that CbpD is a competencestimulating-peptide-inducible protein and a function as a murein hydrolase has been proposed. CbpD has been demonstrated to assist LytA in competence-induced cell lysis [53]. A further study provided experimental evidence that the biological activity of CbpD is involved in the ability of competent bacteria to trigger release of virulence factors from non-competent S. pneumoniae [54]. The surface-exposed lipoprotein SlrA is a functional, cyclophilin-type peptidyl-prolyl isomerase. The deficiency in SlrA caused a less efficient nasopharyngeal colonization of mice, and this has been attributed to the decreased ability of the knockout mutants to adhere to non-professional cells [55]. The pneumococcal surface adhesin A (PsaA), which is the substratebinding lipoprotein of an ATP binding cassette (ABC)-type manganese transport system [56] has also been demonstrated to affect adherence of pneumococci. Mutations in psaA caused pleiotropic effects including reduced adherence of pneumococci to host cells, attenuation in an intranasal and intraperitoneal mouse infection model and increased sensitivity to oxidative stress [9, 57] . After detecting the pleiotropic effects it was assumed that PsaA does not function itself as an adhesin, however, a recent

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report has demonstrated that antibodies against PsaA reduce the adherence of pneumococci to nasopharyngeal epithelial cells [58]. This is consistent with the finding that mucosal immunization of mice with PsaA is highly protective against pneumococcal carriage [59] and that psaA is upregulated during atttachment of pneumococci to epithelial cells [7, 9].

The major pneumococcal adhesin The pneumococcal surface protein C (PspC; also known as SpsA or CbpA) is a multifunctional CBP of pneumococci; PspC promotes uptake of pneumococci into nasopharyngeal epithelial cells and interferes with components of the innate immune system. PspC promotes pneumococcal adherence via a human specific interaction with the secretory component (SC), which is found on the polymeric immunoglobulin receptor and secretory forms of IgA and IgM on mucosal surfaces [60–62]. The SC consists of five Ig-like ectodomains (D1 to D5) and independent reports have indicated that the human specificity of the PspC-SC interaction is determined by amino acid differences in the ectodomains D3 and D4 of the SC [63, 64]. Despite such human specificity, loss of function in PspC has been shown to reduce colonization of infant rats [65] and pIgR knockout mice [62]. The underlying mechanisms of these in vivo effects are poorly defined. However, PspC has also been shown to bind to immobilized sialic acid and lacto-N-neotetraose [65] and to interact with complement components including C3 and factor H [66, 67]. The amino-terminal part of most of the PspC molecules contains repeated domains, designated R1 and R2. Structural analysis of R1 (aa175 to 285) and R2 (aa327 to 442) of PspC derived from TIGR4 (PspC allele PspC3.4 [68]) demonstrated that the R domains adopt an unusual and simple structure comprised of three _-helices. These R domains of PspC contain the minimal and conserved SC-binding site Y/RRNYPT [61]. The structure indicated that bundling of the helices through _-helix/_-helix interactions results in a flat, raft-like structure in which the critical residues YPT of the minimal SC-binding motif are located in a loop between helix 1 and helix 2 and form a “tyrosine fork” structure [69].

Interaction with ECM components and recruitment of proteolytic activity There is experimental evidence that binding of microorganisms to ECM components may be of importance for pathogenesis. Pneumococci have been shown to bind to various ECM components, however, the impact of these interactions on colonization are not yet clarified. The PavA protein (pneumococcal adherence and virulence factor A) has been identified as a

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pneumococcal adhesin for fibronectin and, probably more important, as a crucial virulence determinant in pneumococcal infections. In a systemic and experimental mouse meningitis model of infection the pneumococcal pavA knockout of strain D39 was substantially attenuated [70, 71]. In addition, PavA is most likely involved indirectly and in a fibronectin-independent manner in pneumococcal adherence to host cells. However, the precise function of PavA has yet to be clarified, because deficiency in PavA does not affect expression and function of known virulence factors [71]. S. pneumoniae acquires host proteolytic activity by binding plasmin(ogen), and interestingly, the glycolytic enzymes enolase and GAPDH were identified as plasmin(ogen)-binding proteins displayed on the cell wall [72, 73]. The enolase has been shown to potentiate degradation of extracellular matrix (ECM), dissolution of fibrin and pneumococcal transmigration [74]. The key pneumococcal binding site in enolase responsible for plasminmediated ECM degradation has been attributed to the nonameric peptide “FYDKERKVYD” [75]. Crystal structure analysis indicated an octameric composition of the pneumococcal enolase and depicted the nonameric plasminogen-binding site on the surface of the protein [76]. The importance of enolase in virulence has been demonstrated in intranasal mice infections of mice. Enolase mutants with functionally inactive plasminogen-binding sites were significantly attenuated compared to the isogenic D39 parental strain [75].

Invasion of host cells by pneumococci The proportion of internalized pneumococci in resting pulmonary cells was only 0.1 % of the adherent bacteria [77]. In contrast, activation of vascular endothelial cells with thrombin or tumor necrosis factor-_ (TNF-_) caused a substantial increase in pneumococcal uptake. The cell activation is associated with an increase in expression of novel cell adhesion molecules such as receptor for the platelet-activating factor (PAF). The PAF receptor is rapidly internalized after interaction with its ligand PAF and pneumococci have been shown to engage the upregulated PAF receptor for internalization [77]. It has been shown that the cell wall structural component phosphorylcholine (PCho), which is also present in PAF, function as an adhesin for the PAF receptor. Binding and uptake of pneumococci to activated endothelial cells was inhibited by PAF antagonists, purified pneumococcal cell wall components, or anti-PCho antibodies. The absence of PCho in pneumococci reduced adherence to levels indicated for resting cells, indicating that PCho directly interacts as an adhesive molecule with the PAF receptor [77]. Binding of pneumococci via PCho to activated cells was inhibited by glycoconjugates N-acetyl-glucosmamine or lacto-N-neotetraose. These sugars showed no effect on adherence of pneumococci to resting cells. This implicates that pneumococci most likely bind to the PAF receptor at two

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Figure 3. Surface proteins of pneumococci contributing to colonization and invasion of host cells. A. The extracellular matrix (ECM) of the mucosal cavity represents the first mechanical barrier for pneumococcal colonization. Colonization of pneumcocci is facilitated by cleavage of bound IgA1 by the IgA1-protase and by removing of terminal sialic acids by NanA. The glycolytic enzymes enolase and GAPDH bind human plasminogen (PLG), which is converted to the protease plasmin (PA) and promotes degradation of various ECM compounds. ZmpC activates the matrix metallo protease 9 (MMP-9) which has collagenase activity and degrades the ECM. The non-classical surface associated protein PavA has been shown to bind to immobilized fibronectin (Fn) and to modulate pneumococcal adherence. B. The intimate contact of pneumococci with cellular receptors is mediated by adhesins. The major adhesin of pneumococci identified is PspC, which interacts with the ectodomain of the human polymeric immunoglobulin receptor (pIgR). PCho mediates pneumococcal adherence to stimulated cells via an interaction with upregulated platelet activating factor receptor (PAFr). It is thought that the lipoprotein PsaA may also function as an adhesin, but the cellular receptor is not defined yet. Reprinted from Microbiology SGM [209] with permission of the publisher.

sites: one shared with the natural ligand PAF and the other at a site of glycosylation of the receptor. Alternatively, the glycosyl determinant can be located on a putative co-receptor that interacts with the PAF receptor. The PCho-PAF receptor interaction represents further a specific mechanism for pneumococcal targeting of the blood-brain barrier (BBB) and transmigration of pneumococci across the BBB [24].

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The efficiency of colonization and invasion of brain microvascular endothelial cells correlates with the higher amounts of PCho on transparent pneumococci. In addition, the amount of PCho is modulated by the phosphorylcholine esterase activity of Pce (also referred to as CbpE) which removes PCho from the cell wall and causes changes in colony phenotype [78, 79]. The crystal structure reveals that Pce belongs to the metallo-`lactamase family and that only PCho residues that are located at the end of the teichoic acid chains are accessible to the catalytic center of Pce [80, 81]. It is thought that Pce may have a dual function and favor both colonization and invasive infection by modulating the amount of PCho on the pneumococcal cell wall. The physiological significance of the PCho-PAF receptor interaction was demonstrated in a rabbit model of pneumonia. The PAF-receptor antagonist blocked activation-dependent adherence and transition of pneumococci from the alveolus into the blood. This resulted in reduced nasal colonisation and attenuated development of pneumococcal bacteremia [77].

Cell signaling induced by S. pneumoniae The PAF receptor is a G-protein coupled receptor and binding of PAF activates phopholipase C [82]. In contrast, internalization of pneumococci via the PAF receptor was independent of the G-protein pathway and failed to induce signal transduction [77]. The endocytosis of pneumococci requires both the PAF receptor as a portal of entry and `-arrestin. It has been demonstrated that pneumococci induced the translocation of `-arrestin at the plasma membrane, where it colocalized with the PAF receptor. This event caused a G-protein independent activation of the MAP kinase ERK-1/ ERK-2 and pneumococi moved into clathrin-coated vesicles. At least half of the pneumococci proceeded through Rab5 to Rab7 marked endosomes toward lysosomes. Other vacuoles acquire Rab11, which is consistent with the known recycling of the bacteria to the apical surface [83]. In pneumococcal pneumonia massive leukocyte recruitment to the lung is observed [84]. Invasion of leukocytes is accompanied by secretion of proinflammatory and chemotactic cytokines by lung epithelium and cells of the innate immune response including alveolar macrophages. It has been shown that the pneumococcal cell wall mediated signaling induces the expression of transcription factor NF-kB and induces the production of TNF-_, IL-8, IL-6 and IL-8 [85-87]. Schmeck and co-workers demonstrated that in pneumococcal pneumonia the induction of NF-gB and p38 MAPK signaling pathways contribute to the secretion of IL-8 and GM-CSF. Activation of NF-gB was IgB-kinase dependent, but activation was independent of p38 MAPK. It has been demonstrated that p38 MAPK did not affect inducible nuclear translocation of NF-gB/RelA to the IL-8 promotor but regulates IL-8 transcription on the level of phosphorylated RelA at the promoter.

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Both, p38 MAPK and NF-gB have been shown to be required to modulate the host response at the transcriptional level in response to pneumococci infection [88].

Pulmonary virulence determinants Large-scale in vivo screens including signature-tagged mutagenesis (STM), differential fluorescence induction (DIF) and whole-genome microarray analysis have provided an extended list of putative pulmonary virulence factors [5–9]. These approaches confirmed already known virulence factors including CbpD, ComD, Hyl, IgA1-protease, LytA, NanA, Pneumolysin, PcpA, PsaA, PspA, PspC, sortase, and ZmpB. In addition, a role in pathogenesis has been suggested for ABC transporters such as the glutamine transporters, proteases such as HtrA and PrtA, proteins involved in metabolic pathways and regulatory elements such as the transcription factor RlrA. The importance of individual virulence determinants for pneumococcal pneumonia has further been assessed in in vivo studies. Pneumococci produce several proteases that are surface-exposed and implicated in pneumococcal virulence. Intranasal infection experiments confirmed the significant contribution of IgA1-protease and ZmpB to pneumococcal virulence [89]. ZmpC was characterized in TIGR4 as a bacterial zinc metallo protease cleaving human matrix metalloproteinase 9 (MMP-9) and inactivation of the zmpC gene in serotype 19F impaired virulence in a pneumonia mouse model [90]. The HtrA (high-temperature requirement A) functions in a temperature-dependent manner as a molecular chaperone or heat shock-induced serine protease, and is regulated by the CiaRH two-component system. HtrA has been shown to be implicated in resistance against oxidative stress, colonizing the nasopharynx of rats and in pneumococcal pneumonia. Moreover, htrA knockouts induced lower levels of inflammatory cytokines IL-6 and TNF-_ in the lungs during pneumonia compared to the isogenic wild type D39 [91–94]. PrtA is a further surfaceexposed serine protease and prtA knockouts have been shown to be attenuated in an intraperitoneal mouse infection model [95]. The highly variable pneumococcal surface protein A (PspA) is expressed virtually by all important clinical serotypes and has significant immune protective potential. Loss of function has been shown to attenuate virulence and increase complement receptor-mediated clearance of pneumococci [96]. PspA is a lactoferrin-binding protein [97] and Shaper et al. [98] demonstrated that expression of PspA protects against the bactericidal effect of apolactoferrin. In the presence of antibodies recognizing PspA this effect is abrogated, suggesting that binding of apolactoferrin to PspA blocks the bactericidal activity of apolactoferrin. Regarding the cell wall hydrolases of S. pneumoniae, loss-of-function of LytB or LytC significantly reduced nasopharyngeal colonisation of rats [35].

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Table 1. Pulmonary virulence factors

Toxins enzymes/ proteases

choline-binding proteins

Biological activities

Refs.

Pneumolysin

inhibition of ciliar mobility, cytotoxic and cytolytic activities

[29, 30, 122]

HtrA

resistance against oxidative stress, induction of inflammatory cytokines

[91–93]

Hyaluronidase

hydrolysis of hyaluronan

[51]

IgA1-protease

enhancement of bacterial adherence to host cells

[52]

LytA, LytB, LytC

cell wall hydrolases

[34, 35]

Neuraminidase (NanA)

cleavage of terminal sialic acids of [46–49] human IgA secretory component, lactoferrin and IgA2

PrtA

serine protease

[95]

ZmpB

impact on pneumococal virulence

[89]

ZmpC

cleavage of metalloproteinase 9 (MMP-9)

[90]

CbpD

competence-induced cell lysis by murein hydrolytic activity, impact in allolysis

[53, 54]

Pce

phosphorylcholine esterase

[77, 78]

PcpA

unknown

[36]

PspA

binds lactoferrin and protects against bactericidal effects of apolactoferrin, inhibition of complement-induced clearance

[97, 98, 127, 128]

PspC

pneumococcal adhesin for pIgR

[60]

regulatory components

ComD RlrA

transformation competence factor transcriptional regulator

[5, 211] [5]

lipoproteins

PsaA

Mn-transporter and putative adhesin

[56]

Regulation of gene expression and virulence The pneumococcal virulence determinants can be regulated by one-component regulatory systems including luxS, rlrA, regM/R, and mgrA, or by one of the 14 two-component regulatory systems (TCS) that have been identified in S. pneumoniae [99, 100]. TCS respond to environmental changes and mediate, therefore, the adaptation of pneumococci to their different microenvironments. Throup et al. [101] and others have demonstrated the impact of TCS on pneumococcal virulence in a mouse infection model. The CiaRH was the first TCS identified (and is required for efficient nasopharyngeal colonization and important for protecting cells from stress of competence development [102, 103]). As mentioned above, CiaRH regulates HtrA,

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which is thought to be the key component in the contribution of CiaRH to virulence [93]. The major adhesin PspC is upregulated during colonization [7] and pspC expression has been shown to be influenced by the TCS RR06/HK06 [104]. However, in vitro and in vivo infections suggested that this TCS is also important for the regulation of other yet unknown virulence factors interfering with colonisation and invasion. Recently it has been shown that the phosphorylated VicR (YycF) response regulator of the TCS VicRK (YycF) binds to a region upstream of pspA and regulates expression of pspA [105]. As opacity variation accounts for changes of a number of cell surface-exposed components including capsule, PspC, PspA and PCho it is implicated in pneumococcal colonization and invasive diseases. Capsule regulation has been indicated under in vivo and in vitro conditions. Recombinant exchanges and spontaneous sequence duplications within type 3- and 8 specific capsule genes cause high-frequency serotype and phase variations, respectively [106-108]. Proteins such as CpsB and CpsD which influence production of capsular polysaccacharide regulate the amount of capsule at the post-translational level. Both have been demonstrateed to modulate the phosphorylation of CpsD or other substrate molecules [109–112].

The pneumococcus and the innate immunity of the host Innate immunity covers a diverse array of host defenses including mucocilliary clearance, complement, neutrophils and macrophages. It acts as a non-specific defense able to recognize and respond rapidly against a broad range of microbes. Unlike adaptive immunity, which involves the clonal expansion of T- and B-cells specific to the pathogen, innate recognition is achieved through a limited set of germline encoded receptors and does not possess immunological memory. The two systems do not however operate in isolation as the innate immune system plays crucial roles in the initiation, development and effector stages of adaptive immunity.

Complement and the pneumococcus The complement system comprises over 30 serum and membrane proteins which, when activated, form a cascade of reactions contributing to the elimination of invading microorganisms. The binding and activation of complement components to the surface of a microbe leads to opsonophagocytosis and the induction of inflammation. For some organisms, but not the pneumococcus, complement can destroy the microbe directly through lysis by the membrane attack complex. Complement contributes to and links both innate and adaptive immunity. Three pathways of activation exist (reviewed in [113], in brief these are: 1) The classical pathway

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activated by antibody: antigen complexes for example, antibody binding to the microbial surface. Non-antibody-dependent activation can also occur, such as the binding of acute phase proteins to the microorganism, 2) the lectin pathway, which is triggered by mannose-binding-lectin recognition of carbohydrate on the microbial surface, and 3) the alternative pathway is continuously activated at low levels, but only amplifies on foreign surfaces. The crucial contribution of complement to innate and adaptive responses to pneumococcal infection has been long established both in animal models and humans with genetic complement deficiencies (for recent overview see [114]). However, only recently, through the use of a panel of gene knock-out mice lacking various complement components, has the relative importance of individual activation pathways been assessed in innate immunity to pneumococcal infection [115]. The classical pathway of complement activation was found to be the dominant complement pathway for innate immunity to the pneumococcus in mice [115]; the specific loss of which resulted in significantly increased disease severity. Natural IgM antibodies, possibly to C polysaccharide (teichoic acid), contributed to this activation of the classical pathway as shown with the use of +s–/– knock-out mice which lack such antibodies [115]. However, the activation of the classical pathway during this innate immune response was only partially dependent on natural antibodies and other activation pathways also contributed. These were proposed to include acute phase proteins such as C-reactive protein or direct binding of complement component C1q to the pneumococcal surface [115]. The alternative pathway also contributed to protective innate responses, but to a lesser degree than seen for the classical pathway, while the role for the lectin pathway appeared negligible. In line with this latter finding, genetic mannose binding lectin deficiency is associated with only a small (but significant) increased susceptibility to pneumococcal disease in humans [116]. Regardless of the activation pathway, the deposition and activation of complement component C3 on the bacterial surface is a key step in the complement cascade leading to elimination of the microbe. In accordance with this crucial role of complement in innate immunity, the pneumococcus has evolved several mechanisms to resist its affects. The capsule is a key factor in this resistance, acting not only to limit access to cell bound complement, but also reducing the amount of complement deposited [117]. The pneumococcal surface protein, PhpA (also called PhtB and BVH-11 [118], has been found to possess C3 degrading activity [119, 120] and so PhpA may contribute to preventing complement-mediated clearance. In addition to its cytolytic activity the toxin pneumolysin, a major pneumococcal virulence factor, has multiple other biological activities (for review see [121]). Recently pneumolysin has been shown to confer protection from complement mediated clearance [122]. Deletion of pneumolysin caused an increase in C3 deposition on pneumococcal cells in vitro. Showing that

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this activity of pneumolysin contributed to virulence, absence of complement in gene knock-out mice reduced the importance of pneumolysin to pneumococcal virulence. Furthermore, this effect of pneumolysin in vitro and in vivo was specific to the classical and not the alternative activation pathway [122]. The mechanisms by which pneumolysin achieves this affect are as yet unconfirmed. However, this specific evasion of the classical pathway is in agreement with the ability of pneumolysin to activate this pathway in the complement; evasion in this instance may ironically be the result of complement activation rather than inhibition. Released pneumolysin may result in complement activation away from the bacterial cell thus protecting it and also consuming the available complement components. In addition, increased complement activation may contribute to host tissue damage thereby promoting bacterial pathogenesis. Furthermore, the surface proteins, PspA and PspC also contribute to complement resistance. The PspC molecules are divided into different groups. Interestingly, both the classical PspC proteins containing a CBD and the PspC-like Hic (PspC 11.4) bind the complement factor H [123]. Hic is produced predominantly by serotype 3 pneumococcal strains that are negative for SC binding. Hic contains an LPxTG motif that anchors the protein in a sortase-dependent manner to the peptidoglycan of the cell wall. Factor H is a fluid phase regulator of the alternative complement pathway and consists of 20 short consensus repeats (SCRs). Hic interacts with the SCRs 8-11 and 12-14 of factor H [124], whereas a role for the SCRs 6 to 10 and 13-15 of factor H was suggested for the interaction with PspC [67, 125]. Recruitment of factor H by Hic has been shown to efficiently prevent activation of C3b and complement mediated opsonophagocytosis of pneumococci [124]. The improved survival of pneumococci expressing PspC or Hic in a systemic mouse infection model provides further evidence for the versatility and importance of PspC in different host niches [126]. PspA contributes to inhibition of complement receptor-mediated clearance of pneumococci. Tu et al. [127] have demonstrated a delay of wild type pneumococci clearance compared to the isogenic pspA mutant. It is thought that PspA probably inhibits the factor B-mediated complement activation and functions as an inhibitor of C3b deposition [127]. The inhibitory effect by PspA has been shown for PspA from family 1 and family 2 [128]. In addition, PspA expression decreases C3 binding on pneumcococi, indicating that PspA may also inhibit complement deposition via the classical pathway [96].

Pattern recognition receptors Key components of the innate immune system are so-called pathogen recognition receptors (PRRs). These can be located on the hosT-cell surface, intracellularly, or be secreted and act to initiate the recognition and response to microbes and in some cases host products [129]. The microbial

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components recognized by PRRs are referred to as pathogen-associated molecular patterns (PAMPs) so-called because they are typically invariant structures found among many microbes, but absent in eukaryotes. Examples include lipopolysacchride (LPS) and peptidoglycan from bacteria, doublestranded RNA from certain viruses and mannan from fungi. C-reactive protein (CRP) is a well-known example of a PRR involved in the response to this pneumococcus. This soluble protein binds PCho in the pneumococcal cell wall, inducing complement activation leading to bacterial clearance [130, 131]. Additional PRRs, important in pneumococcal infection have recently been described including members of the toll-like receptor (TLR) family, LBP, and the cytosolic PRR Nod proteins.

Consequences of pneumococcal recognition by Toll-like receptors The TLR family of PRRs has received much attention due to their importance in the response to a wide range of microbes (for review see [132, 133]). Their major function is as PRRs to recognize microbes and initiate an inflammatory response leading to eradication of infection. At least ten TLRs have been described in humans and mice and several have been implicated in pneumococcal infection in animal models. Furthermore, descriptions of genetic defects in TLR signaling associated with increased susceptibility to pneumococcal disease show these receptors have relevance to human infection [134, 135]. TLR2 recognizes both pneumococcal lipoteichoic acid (LTA) and cell wall peptidoglycan [136-139]. Interestingly, despite numerous studies supporting the recognition of bacterial peptidoglycan via TLR2, this interaction has recently been challenged [140]. Knock-out TLR2–/– mice display increased disease severity and decreased survival times compared with wild type mice in a pneumococcal meningitis model [141]. This greater susceptibility correlated with heightened bacterial levels in the brain, but appeared independent of systemic disease as both strains showed similar bacterial levels in the blood. In agreement with these data, Koedel et al. [142] also found TLR2–/– mice had enhanced disease and increased bacterial levels in the brain in their meningitis model, which in turn have contributed to an enhanced inflammation in the brain later in infection. The role of TLR2 has also been investigated in experimental pneumococcal pneumonia [143]. Comparison between wild type and TLR2–/– mice following intranasal infection revealed only a modest contribution for this receptor in the host response and no changes in bacterial clearance and morbidity with compared with their wild type counterparts. On the basis of these data, TLR2 does not appear to play a key role in host resistance to pneumococcal pneumonia. Interestingly, the stimulation of isolated alveolar macrophages in vitro to produce TNF-_ in response to heat-killed pneumococci was entirely dependent on TLR2. However, immunohisto-

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chemical staining of infected lungs from TLR2–/– mice showed these cells were producing TNF-_ comparable with wild type mice. Presumably in the setting of the intact animal other host innate immune factors such as other PRRs and complement mask the loss of TLR2, rendering its influence minimal in pneumococcal pneumonia. Following intraperitoneal infection TLR2 knock-out mice have slightly reduced survival times compared with wild type [144]. Thus, TLR2 has a protective role in this model of systemic infection, although, as with pneumonia, the defect was arguably minor. Furthermore, in a model of nasopharyngeal colonization TLR2 knock-out mice had impaired clearance of pneumococci [145] showing that TLR2 is of relevance not only to disease states, but also carriage. The recognition of pneumococcal peptidoglycan probably involves interaction between TLR2 and 6 as shown for Staphylococcus aureus peptidoglycan [146]. Indeed, confirming a role for this receptor in pneumococcal recognition, expression of a double negative TLR6 mutant inhibited TNF-_ production in response to stimulation by the pneumococcus in a macrophage cell line [146]. A role exists for TLR1 in the recognition of pneumococcal LTA, whereby monoclonal antibodies against this receptor inhibited LTA induced TNF-_ production from human peripheral blood mononuclear cells [136]. The importance of these interactions between the pneumococcus and TLR1 and 6 has not yet been assessed in an infection model.

Recognition of pneumolysin by TLR4 Through the recognition of LPS, TLR4 is a key component of the innate response to Gram-negative infections. A role for this receptor has also been extended to the pneumococcus with the finding that the in vitro pro-inflammatory effect of pneumolysin on macrophages was TLR4-dependent [147]. Subsequently, pneumolysin has been shown to directly interact with TLR4 [148]. This inflammatory activity was not dependent on the pore-forming or complement-activating activities of pneumolysin because the PdT pneumolysin mutant, that lacks these properties, was also active in these studies. The significance of this interaction during colonization was studied by comparing wild type and TLR4 deficient mice in a nasopharyngeal carriage model [149]. In the absence of functional TLR4, mice were more heavily colonized and much more likely to develop invasive disease. Thus, through its recognition of pneumolysin, TLR4 acts in the nasopharynx to limit pneumococcal proliferation. While the inflammatory response to pneumolysin may contribute to this protection, it has also been shown that pneumolysin-TLR4 signaling can induce hosT-cell apoptosis in vitro and in vivo [148]. This also appears to be a protective host response as the inhibition of apoptosis rendered mice more susceptible to death following pneumococcal infection [148]. Interestingly, a similar model of colonization using the same mouse strains found no difference in the clearance of pneumococci between wild

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Fig. 4: Key pathogen recognition receptors involved in the recognition and initiation of the immune response towards the pneumococcus. Abbreviations: LBP, LPS-binding protein; LTA, lipoteichoic acid; TLR, toll-like receptor. Reproduced with permission from reference [210].

type and TLR4 deficient mice [145]. The use of different bacterial strains in these studies may explain this apparent conflict, but this remains to be tested. In pneumococcal pneumonia TLR4 also plays a protective role [150]. In this experimental model the absence of functional TLR4 rendered mice more susceptible to morbidity with increased bacterial counts in the lungs. The effects however, were modest with the effect on death rate only apparent at low doses and with no significant impact on pulmonary inflammation. Furthermore, the significance of TLR4 in pneumococcal infections appears restricted to the airway surfaces as earlier work found the absence of TLR4 made no difference to survival rates and blood bacterial counts after intravenous infection of mice [151].

TLR signaling Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the signaling cascade activated by engagement of TLRs or IL-1 family recep-

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tors [152]. In agreement with a role for TLRs in innate protection against pneumococcal infection, MyD88–/– mice show enhanced susceptibility to S. pneumoniae in different infection models [144, 153, 154]. Providing relevance to human infection, deficiency in IL-1 receptor-associated kinase 4 (IRAK4), also a mediator in the TLR/IL-1 receptor signaling pathway, results in increased susceptibility to pneumococcal disease [135]. As does a distinct but as yet undefined mutation in this signaling pathway [134].

Pneumococcal cell wall recognition by LBP In addition to TLR2, the pneumococcal cell wall (PCW) is recognized by the soluble acute phase protein, LBP (LPS binding protein) [138]. This protein has previously been found to bind LPS and enhance its inflammatory activity. It has now also been found to bind purified PCW and whole pneumococci and the addition of LBP accentuated the inflammatory activity of PCW in vitro [138]. In a mouse meningitis model LBP gene-knock out mice showed significantly reduced meningeal inflammation following challenge with purified cell wall or live pneumococci. Recognition of PCW by LBP appears independent of cell wall phosphorylcholine and teichoic acid, with the glycan backbone seemingly a crucial structure for this interaction [138]. Significance to human infections was shown by enhanced LBP levels in the CSF of pneumococcal patients compared with controls. Furthermore, PCW coprecipated with LBP in the CSF from a patient showing this interaction occurs in human infection [138]. LBP also contributes to the response to pneumococcal LTA [137]. However, LBP gene knock-out mice do not have altered susceptibility or responses to pneumococcal pneumonia [155]. Given that LBP acts to facilitate recognition of TLR2 ligands, this phenotype is compatible with the modest effect seen with TLR2 knock-out mice in similar infections [143]. Interestingly, LBP levels in lavage fluid increased seven-fold during experimental pneumococcal infection [155]. This was a much greater increase than seen with parallel Klebsiella pneumoniae pneumonia [155]. However, unlike pneumococcal pneumonia, endogenous LBP contributed significantly to protection against K. pneumoniae pneumonia [155]. Clearly, this highlights that functional importance cannot easily be predicted merely from expression levels alone.

CD14 Membrane bound and soluble CD14 act as a co-receptor to enhance the response to LPS [117, 156, 157]. CD14 also contributes to the recognition of the pneumococcus in vitro [136, 139, 158]. A role in infection has also been confirmed by the recent finding that CD14 gene knock-out mice show exacerbated pneumococcal meningitis [159].

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Nod proteins and the pneumococcus The cytosolic proteins, Nod1 and Nod2 are additional PRRs acting within host cells to recognize and respond to microbial products [160]. In addition to toll-like receptors, a role for Nod proteins in the recognition and response to pneumococci has also been shown [161]. Transfection with Nod2, but not Nod1, conferred responsiveness to cells following pneumococcal exposure as judged by NF-gB activation [161]. In line with the role of Nod proteins in the response to intracellular material, the recognition of pneumococcus by Nod2 was dependent on internalization of the bacteria. Nod2 has previously been shown to be responsive to a muramyldipeptide conserved in multiple peptidoglycans and this was the suggested mechanism for its recognition of the pneumococcus [161]. The availability of Nod1 and 2 knock-out mice will allow a fuller appreciation of these genes in the host response to the pneumococcus [162–164].

Recognition of capsule Recognition of the pneumococcal polysaccharide capsule by PRRs has received much attention of late. Kang et al. [165] showed that the C-type lectin SIGN-R1 expressed by macrophages, particularly in the marginal zone of the mouse spleen, bound capsular polysaccharide from several different serotypes as well as whole pneumococcal cells. Lanoue et al. [166] demonstrated a functional significance of this interaction with the generation of SIGN-R1 gene knock-out mice. Following intraperitoneal infection with either serotype 2 or serotype 14 pneumococci, these mice displayed increased susceptibility to infection compared with their wild type counterparts. Absence of SIGN-R1 caused a decrease in survival rate, shorter time to death, increased sickness scores and increased bacterial levels in the blood. A defect in the ability of macrophages in the peritoneum and spleen to bind and phagocytose the pneumococcus was likely to be a major contributor to the increased susceptibility of the knock-out mice [166]. The role of SIGN-R1 in pneumococcal pneumonia has also been investigated with the use of SIGN-R1 deficient mice [167]. When infected intranasally with a serotype 3 strain, the SIGN-R1 knock-out mice showed increased bacterial levels in the lungs compared with wild type. This was accompanied by a higher incidence of bacteremia and increased bacterial counts in the blood and spleen. Interestingly, alveolar macrophages do not express SIGN-R1 and expression was not induced following pneumococcal infection [167]. This suggests the protective role of SIGN-R1 in pneumonia does not occur within the lungs themselves. One potential mechanism for the increased bacterial growth in the absence of SIGN-R1 was found to be reduced levels of anti-phosphorylcholine IgM. In addition, systemic disease in this model was probably exacerbated by defective phagocytosis by mac-

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rophages in the peritoneum and spleen as described by [166]. Therefore, in both pulmonary and systemic infections, SIGN-R1 is instrumental in host resistance to pneumococcus. Another macrophage receptor, MARCO has also recently been identified as important in pneumococcal infection [168]. Genetic deletion of this scavenger receptor rendered mice more susceptible to pneumococcal pneumonia with impaired bacterial clearance from the lungs and increased morbidity [168]. Isolated alveolar macrophages from the knock-out mice were impaired in their ability to bind and phagocyctose the pneumococcus in vitro and this was likely a key factor in the increased susceptibility to infection. Interestingly, reduced phagocytosis was not due solely to reduced bacterial binding and so a role for MARCO appears to exist not only in binding, but in subsequent bacterial uptake. The pneumococcal ligands recognized by MARCO have not yet been identified. Previously pneumococcal capsular polysaccharide has been shown to activate macrophages, [169] an activity partially dependent on CD14, whether or not it also involves SIGNR1 and MARCO remains to be determined.

Surfactant proteins Pulmonary surfactant is a mixture of lipids and proteins that act to prevent alveoli from collapsing during expiration. In addition, the surfactant proteins (SP) SP-A and SP-D play a role in innate immunity against a variety of pathogens acting by binding microbes and promoting their phagocyctosis or by modulating immune cell function [170, 171]. In the case of pneumococcal infection, SP-D knock-out mice show enhanced susceptibility to intranasal infection [172]. While SP-A has recently been shown to promote phagocyctosis of S. pneumoniae by rat and mouse alveolar macrophages in vitro [173].

Innate immunity and interaction between the pneumococcus and other microbes For ease of study, most work on the interaction of the pneumococcus with the innate immune system has employed pure cultures. However, the mucosa of the upper respiratory tract is colonized by a diverse array of microbial species. Indeed, analysis of DNA from human airway surface fluid suggested the presence of > 500 bacterial species [174]. Concurrent stimulation of the innate immune system by multiple species appears to have distinct effects from single species interactions [175, 176] and this has recently been shown to have relevance to the pneumococcus [177]. Co-stimulation of human respiratory epithelia cells in vitro by S. pneumoniae and Haemophilus influenzae, also an inhabitant of the upper respiratory tract, resulted in synergistic production of IL-8 [177]. This extended to a mouse colonization model

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with a synergistic effect on MIP-2 production and inflammatory influx into the upper airways. This synergy was independent of TLR2 and TLR4 but involved NF-gB translocation to the nucleus and phosphorylation of p38 MAPK. With regards to the microbial products involved, pneumolysin could substitute for the pneumococcus, but the PdB pneumolysin toxoid, lacking cytolytic activity, was inactive. It was therefore speculated that the pore forming activity of pneumolysin lead to enhanced delivery of microbial products such as the soluble inflammatory protein, SCF from H. influenzae, into the host cell where recognition by Nod1 and 2 would result in increased stimulation [177]. This pro-inflammatory activity of pneumolysin is therefore distinct from its effects on macrophages that were mediated through TLR4 and were independent of pore-forming activity. The significance of pneumococcal H. influenzae interactions have recently be examined in a co-colonization mouse model [178]. In contrast to what might be expected based on in vitro studies with these bacteria [46, 179], co-coloniation in vivo resulted in rapid clearance of the pneumococcus. This effect was dependent on the innate immune system in the form of neutrophils and complement, with the depletion of either abolishing the competitive effect. Activation of peritoneal neutrophils with heat-killed H. influenzae caused an increase in their ability to kill the pneumococcus, but had no effect on their ability to kill H. influenzae [178]. The basis of this activity is not yet clear. Thus, interactions with the innate immune system can have a significant effect during competition between the pneumococcus and other microbes in the nasopharynx. Another important microbial interaction is that of the pneumococcus and influenza A. Subsequent to influenza A outbreaks secondary pneumococcal infection is an important cause of morbidity and mortality. This heightened susceptibility to pneumococcal disease can be reproduced in animal models, allowing investigation of the mechanisms involved. While viral neuraminidase contributes to this phenomenon by exposing pneumococcal receptors [20, 180], alterations in the immune response also seem to contribute. Prior influenza A infection in mice primes for an exaggerated inflammatory response to subsequent pneumococcal infection [181]. Increased levels of IL-10 in this response likely contribute to increased susceptibility as neutralization of this cytokine improved disease outcome [181]. In vitro exposure to both influenza A and pneumococcus results in a synergistic inflammatory response from human middle ear epithelial cells [176]. Microarray gene expression analysis of these cells following influenza A infection provides insight into the possible mechanisms behind this synergy [182]. For example, it was found that tlr2 expression was up-regulated by influenza A infection. This may make the cell more responsive to stimulation by pneumococcal peptidoglycan and LTA [182]. It is therefore clear that the interaction of the pneumococcus with the innate immune system is greatly influenced by the presence of other organisms such as H. influenzae and influenza A.

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CD4 +cells in immunity to the pneumococcus The function of CD4 + T-cells in adaptive immunity is well established. Interestingly, they appear also to contribute to early resistance to pneumococcal infection independently of their role in adaptive antigen-specific responses [183]. Intranasal infection of MHCII knock-out mice, which display a significant decrease in CD4 + T-cell levels, revealed a key role for these cells in the early response to pneumococcal pneumonia. These mice displayed increased susceptibility to infection as evidenced by increased bacterial counts in the lung and blood compared with their wild type counterparts. Indeed, the increased susceptibility was so great that it resulted in 100% mortality in the knock-out mice by three days post-infection, whereas all wild type mice survived the challenge. In accordance with previous data showing T-cell migration to infected lung tissue in pneumococcal pneumonia [184, 185], purified CD4 + T-cells migrated to the pneumococcus in vitro. This migration was associated with T-cell activation and interestingly occurred only in response to in vivo and not to in vitro grown bacteria. Pneumolysin plays a significant part in this migration as pneumolysin-deficient pneumococci stimulated significantly less cell migration. How pneumolysin stimulates these T-cells is unclear but the recent description of TLR4 expression by T-cells may be of relevance [186]. Recently Malley et al. [147] have demonstrated a crucial role for CD4 + T-cells in antibody independent acquired immunity to pneumococcal colonisation. How CD4 + T-cell migration and activation in response to the pneumococcus as described in pneumonia relates to this acquired immunity to colonisation is as yet unclear.

The pneumococcus and the adaptive immunity of the host During carriage and invasive disease the pneumococcus encounters the protective effect of host immunoglobulins. The pneumococcal surface is covered by a capsular polysaccharide which is involved in pneumococcal colonization and, more importantly, is a key virulence factor in invasive diseases [23, 187]. A correlation between decrease in pneumococcal carriage with rising levels of both mucosal and serum antibodies to pneumococcal surface polysaccharides has been described [145]. However, capsular polysaccharides do not yield an anamnestic response, due to the inability of polysaccharides to recruit cognate CD4 + T-cell help through T-cell receptor recognition of peptide-major histocompatibility class II complexes (MHCII) on the surface of antigen-presenting cells [188]. McLay and coworkers demonstrated in mouse infection studies that the lack of memory response by capsular polysaccharides can be overcome by the use of conjugated vaccines that elicit a different IgG subclass response to polysaccharides. Co-expression of surface polysaccharides with proteins has been assumed to mediate cognate CD4 + T-cell help for polysaccharide-specific B-cells [188]. The development

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of a seven-valent conjugate vaccine has resulted in high immunogenicity, but immune protection was restricted only to the seven serotypes, which were represented by the polysaccharides [189]. Efforts have been made to overcome the problem of variable polysaccharide determinants and to develop potent protein vaccines that have the capability to cover most pneumococcal serotypes. A number of pneumococcal cell-surface or secreted components have been shown to induce opsonophagocytic antibodies or to offer at least partial protection in murine infection models. The protein antigens currently under investigation include the phosphorylcholine epitope found on lipoteichoic acid (LTA), PspC, pneumolysin, PpmA, PsaA, PspA and some surface proteins identified by whole-genome-approaches [97, 123, 190-198]. Some of the identified protein vaccine candidates have been investigated in an experimental model of human carriage within the nasopharynx to circumvent the limitations given by murine models of pneumococcal infections [196]. In contrast to the low level of antibody response to pneumococcal polysaccharides, serum IgG and secretory IgA-response was detected to an N-terminal region of PspA and also to PspC of the inoculum strain during experimental carriage of type 23F and 6B pneumococci in adults. The specific antibody titers even exceeded basal levels of humans with pre-existing antibody response [196, 199]. Former studies described serum IgG titers to PsaA and pneumolysin emerging with age and exposure to pneumococci. In contrast, no antibody response to PsaA and pneumolysin was detected in the carriage studies with adults [196, 200, 201]. A stated problem of the human carriage model concerns the high variability of pneumococcal surface proteins including PspC that has been described as strain-to-strain diversity and may account for the low IgG titers against different serotypes. The functional organization of PspC proteins among different strains is similar, but the molecular weight of PspC has been shown to vary between 59 kDa to 105 kDa. PspC is divided into 11 groups due to differences in the N-terminal domain [68, 202]. Further studies are required to evaluate the impact of the described interaction of PspC with the secretory IgA and with free secretory component for the immune status of the host. A further example of the high strain-depending variability in protein structure and in immunogenicity is PspA, which is divided into two different protein families and subdivided into six clades [203]. To further explore the mechanism contributing to natural carriage and clearance of carriage, a mouse model of pneumococcal colonization was developed. In this model pneumococcal carriage induced a mucosal and serum antibody response to pneumococci and to PspA. But no correlation was detected between the density of colonization and amounts of detected mucosal or serum antibodies [145]. The relationship between systemic and local antibody production and carriage in children was studied in in vitro studies after antigen stimulation by Zhang and co-workers [204]. Serum, saliva and cell culture superna-

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tants of adenoidal mononuclear cells of children undergoing adenectomy were assayed for antibodies to the pneumococcal proteins: PspC, pneumolysin, PsaA, PspA. In this study carriage rates fell with age and serum levels of anti-PspC, Ply and PspA were rising. The results revealed that antibody production to PspC and pneumolysin have the potential to protect children aged 2 years and older against pneumococcal colonization. Other highly conserved vaccine candidates like LTA, PsaA, and PpmA were tested, but no antibody response was detected [196]. Further studies are required to correlate the described data of immunogenicity of vaccine candidates during human carriage with protective potential against colonization. The development of antibodies against pneumococcal surface associated proteins including enolase, IgA1.protease, SlrA and PpmA has recently been investigated in relation to pneumococcal carriage and otitis media in children [205]. These studies have indicated that enolase, IgA1-protease, SlrA and PpmA are immunogenic proteins, but no significant correlation between antibody titers and pneumococcal carriage or infection has been found. In contrast to the high structural variability of PspC and PspA, enolase and IgA1- are highly conserved proteins that share strong homology to other bacterial species colonizing the same nasopharyngeal niche like N. meningitidis and H. influenzae. The presence of cross-reactive epitopes enhances the basal titer of antibodies and might explain the little impact of current pneumococcal colonization on serum concentrations of anti-IgA1protease and anti-enolase antibodies [205]. Future challenges of vaccine development might be the identification of a surface exposed pneumococcal protein with serotype-unspecific conserved immunogenic domains that show no cross reaction with other bacterial or eukaryotic components.

Global analysis of host responses The advent of microarray technology allows greater insight into the hostcell response to the pneumococcus. The response of the human monocytic cell line THP-1 has been assessed by microarray analysis following exposure to S. pneumoniae and an isogenic mutant lacking Pneumolysin [206]. After 3-hour exposure to the pneumococcus, expression differences were revealed in 182 host genes from the 4133 examined, illustrating the potential for large-scale expression changes induced by the pneumococcus [206]. Of these 182 genes, 142 were responsive to pneumolysin showing the dominant nature of this virulence factor in host responses. While this study will not be comprehensive in fully documenting the host response it illustrates the complexity of the interaction between host-cells and the pneumococcus. An important future challenge will be to understand the significance of these expression changes in the disease process.

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Host gene expression changes in an infection model has been investigated in a rat model of otitis media [207]. Twelve hours following pneumococcal challenge, 280 genes in the middle ear (effusion and mucosa) showed a greater than two-fold change in expression compared with mock infected controls. This represented ~24% of the genes examined, again showing the ability of the pneumococcus to induce large-scale changes in host gene expression. Such data allows the pneumococcal response pathways to be mapped and for the identification of previously unrecognised responses. For example, it was found that the transcription factor fra-1, implicated in bone proliferation was upregulated during experimental otitis media. This provides a candidate mechanism to explain clinical features of otitis media involving the bone seen in both human patients and animals [207]. The global response to nasopharyngeal colonization has also been investigated in a mouse model [208]. Up-regulation of siderocalin, an iron sequestering host defense protein was noted in the nasal mucosa. How the pneumococcus causes this up-regulation of siderocalin is unclear with the effect still seen in mice deficient for either TLR2 or 4. Interestingly, this response could not be replicated in vitro, again showing the complexity of the immune response and the value of whole animal systems [208]. S. pneumoniae is resistant to siderocalin and its upregulation may be advantageous to the pneumococcus by inhibiting potential competitors in the nasopharynx [208].

Conclusion Pneumococcus is a versatile microorganism causing local infections and severe invasive diseases as well. The dramatic clinical outcome of pneumococcal pneumonia, bacteriaemia and meningitis, respectively, is the result of massive inflammatory host responses. The infections caused by this pathogen can be controlled by the innate and antibody-mediated host defense mechanisms. However, the pneumococcus has developed several sophisticated mechanisms to overcome the defense mechanism and is able to subvert host protein functions for its survival and dissemination. Another threat is the increasing rate of antibiotic resistant isolates that requires the development and use of cost effective pneumococcal vaccines or new therapeutics. A further understanding of the pneumococci-host interaction may aid to develop a better vaccine against pneumococcal infections.

Acknowledgements The work in the group of Sven Hammerschmidt is supported by grants of the German Research Foundation (DFG-SFB 479 to S.H.) and Federal Ministry of Education and Research (grant 01KI0430 to S.H., Competence Network CAPNETZ). The work in the group of Tim J. Mitchell is supported

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by the Wellcome Trust, MRC, BBRSC, the Egyptian government and the European Union. Our apologies to authors of primary articles we have failed to discuss in detail or to cite due to limitations on space. The authors are grateful to Roland Nau (University of Göttingen, Germany) for providing histopathological micrographs and Manfred Rohde (German Research Centre for Biotechnology, Braunschweig, Germany) for providing electron micrographs.

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Pathogenesis of Mycoplasma pneumoniae infections: adaptive immunity, innate immunity, cell biology, and virulence factors Ken B. Waites1, Jerry W. Simecka2, Deborah F. Talkington3 and T. Prescott Atkinson4 1Department

of Pathology, University of Alabama at Birmingham, Department of Pathology, WP 230, 619 19th St. South, Birmingham, AL 35249, USA; 2Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA; 3Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA; 4Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35249, USA

Abstract Mycoplasmas represent the smallest self-replicating organisms. They are unique among bacteria in that they lack a cell wall and require sterols for growth. The limited metabolic and biosynthetic activities of mycoplasmas have complicated development of accurate means for laboratory detection and hampered understanding of their roles as human pathogens. Mycoplasma pneumoniae was first identified and characterized in the 1960s and shown to be a common cause of upper and lower respiratory disease in children and adults. Serious infections requiring hospitalization, while rare, occur in persons of all age groups, and may affect multiple organ systems. Severity of disease appears to be related to the degree to which the host immune response reacts to the infection. Extrapulmonary complications involving all of the major organ systems can occur in association with M. pneumoniae infection as a result of direct invasion and/or autoimmune response. Evidence is accumulating for this organism’s contributory role in chronic lung conditions such as asthma. Serology has been the most common means for laboratory detection of M. pneumoniae infection due to the slow growth that makes culture impractical. Newer diagnostic methods utilizing nucleic acid amplification offer the advantages for rapid detection and are likely to become increasingly important in the future, but these techniques have not achieved widespread utilization thus far due to the lack of commercially sold products and non-standardized methodology. Management of M. pneumoniae infections can usually be achieved with macrolides, ketolides, tetracyclines, or fluoroquinolones. As more is learned about pathogenesis and immune response elicited by M. pneumoniae, improved methods for diagnosis and prevention of disease due to this organism are anticipated.

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Cell biology of Mycoplasma pneumoniae Mycoplasmas represent the smallest self-replicating organisms capable of cell-free existence, both in cellular dimensions and genome size. Individual spindle-shaped cells of M. pneumoniae are 1–2 +m long and 0.1–0.2 +m wide. Accordingly, the M. pneumoniae cell volume is less than 5% of that of a typical bacillus. Typical colonies of M. pneumoniae rarely exceed 100 +m in size and require examination under a stereomicroscope to visualize their morphologic features. The M. pneumoniae genome was sequenced in 1996 and shown to consist of 816,394 basepairs with 687 genes [1], about onesixth the size of Escherichia coli. The small genome of M. pneumoniae and its limited biosynthetic capabilities are responsible for many of the biological characteristics and requirements for complex medium supplementation in order for the organism to be cultivated in vitro. Mycoplasmas cannot synthesize peptidoglycan cell walls. Lack of a rigid cell wall makes them pleomorphic and unable to be classified in the manner of conventional eubacteria. Mycoplasmas are not found freely living in nature since they depend on a host cell to supply the necessary nutrients. Another characteristic of the genus Mycoplasma is the requirement for sterols in artificial growth media, supplied by the addition of serum. Sterols are necessary components of the triple-layered mycoplasmal cell membrane providing structural support to the osmotically fragile organisms. Although mycoplasmas can flourish within an osmotically stable environment in their eukaryotic host, they are extremely susceptible to desiccation. This explains the need for close contact for transmission of infection from person to person by airborne droplets. Another structural component that is important for extracellular survival is a protein network that provides a cytoskeleton to support the cell membrane. M. pneumoniae also produces capsular material that may have a role in cytadherence. M. pneumoniae possesses very limited metabolic and biosynthetic activities for proteins, carbohydrates, and lipids. It scavenges for nucleic acid precursors and apparently does not synthesize purines or pyrimidines de novo. Fermentation of glucose to lactic acid by means of substrate phosphorylation mediated by phosphoglyceric acid kinase and pyruvate kinase activities are means of ATP generation. M. pneumoniae possesses all reactions of glycolysis, but the tricarboxylic acid cycle and a complete electron transport chain containing cytochromes are absent. M. pneumoniae reduces tetrazolium and this property has been used historically to distinguish it from commensal oropharyngeal mycoplasmas. Reproduction occurs by binary fission, during which the attachment organelle migrates to the opposite pole of the cell during replication and before nucleoid separation. Neither genomic analysis nor electron microscopy has demonstrated the presence of structures such as flagella or pili, suggesting that gliding motility occurs by an unknown mechanism involving the attachment organelle [2].

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In addition to the crossing of the mucus barrier that protects the respiratory epithelium, gliding motility may contribute to the ability of M. pneumoniae to travel down respiratory cilia to attach to respiratory epithelial cells.

Epidemiology and transmission of infection M. pneumoniae infections can involve both the upper and lower respiratory tract and occur both endemically and epidemically worldwide in persons of all ages. Climate, seasonality, and geography are not thought to be of major significance, although most outbreaks in the USA tend to occur in the late summer and early fall. Foy [3] reported that M. pneumoniae was responsible for 15-20% of all cases of community-acquired pneumonia (CAP) between 1962 and 1975 in Seattle, Washington, USA. Additional retrospective serological studies performed in Denmark showed a pattern of M. pneumoniae infections over a 50-year period from 1946 through 1995 with endemic disease transmission punctuated with cyclic epidemics every 3 to 5 years [4]. The long incubation period and relatively low transmission rate have been implicated in the prolonged duration of epidemics of M. pneumoniae infections. A study performed in the USA during the 1990s detected M. pneumoniae in 23% of CAP in children 3-4 years of age [5]. Another study from France [6] documented its occurrence in children less than 4 years of age without significant differences in infection rates for other children or adults. These findings may reflect the greater number of young children who attend day care centers on a regular basis than in previous years, and the ease with which young children share respiratory secretions with older household members or contacts. Marston [7] reported that M. pneumoniae was definitely responsible for 5.4% and possibly responsible for 32.5% of 2,776 cases of CAP in hospitalized adults in Ohio, USA. Extrapolation of data nationally provides an estimated 18,700 to 108,000 cases of CAP in hospitalized adults due to M. pneumoniae annually. Since the majority of CAPs are treated as outpatients, the total number of pneumonias due to M. pneumoniae is almost certainly many times greater. An additional striking finding was their observation that mycoplasmal pneumonia in hospitalized adults increased with age and it was second only to Streptococcus pneumoniae in elderly persons. The P1 adhesin is a 170 kD transmembrane protein that is concentrated on the adhesive tip of M. pneumoniae and serves an essential function in cytadherence. Different P1 subtypes may operate in cycling times of M. pneumoniae epidemics. Gene divergences within the P1 adhesin and development of subtype-specific antibodies following initial infection might also contribute to the frequency of reinfections due to another subtype. Studies using a variety of genotypic methods to characterize over 200 M. pneumoniae isolates collected over several years from multiple countries showed

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that most of the isolates could be classified into two subtypes based on the sequences of the P1 adhesin gene, the ORF6 gene, the P65 gene, and by a typical DNA restriction fragment pattern [8, 9]. One or the other of the two subgroups tended to predominate in specific regions. M. pneumoniae is not considered part of the normal flora and its detection by culture can usually be considered abnormal and of etiologic significance if detected in a person with a clinical condition known to be caused by the organism. However, it can persist for variable periods in the respiratory tract following infections that resolved clinically with appropriate antimicrobial therapy, providing a reservoir for spread of the organism to others.

Cytadherence and other virulence factors The initial step in the pathogenesis of mycoplasmal respiratory disease involves M. pneumoniae adherence to ciliated respiratory epithelium. This intimate interaction damages respiratory epithelial cells through the production of toxic substances, such as hydrogen peroxide and superoxide radicals, leading to oxidative stress. Subsequent development of the host inflammatory response may actually have the greatest impact on disease. In fact, the host’s bronchoepithelial cells may contribute to the development of the inflammatory lesions through the release of cytokines in response to infection or other stimuli. Release of interleukin (IL)-8, from human bronchoepithelial cells may occur in response to stimulation with mycoplasma membranes [10]. Similarly, human lung alveolar type II pneumocytes (A549 cells) infected with M. pneumoniae show an increase IL-8, tumor necrosis factor-alpha (TNF-_), and IL-1` mRNA [11], supporting the idea that the adherence to human airway epithelial cells leads to production of cytokines and recruitment of lymphocytes and other inflammatory cells, and that these cytokines subsequently modulate the activity of the inflammatory infiltrates. The P1 protein adhesin is also immunogenic, and it is the target for antibodies that develop in the course of natural infection. The host-cell ligand for mycoplasmal adhesins has not been characterized conclusively, though sialoglycoconjugates and sulfated glycolipids have been implicated [12]. At least six other proteins (HMW1, HMW2, HMW3, P90, P40 and P30) are known to participate in adhesion. All are localized to the terminal tip of the organism, and most are likely involved in the architecture of the attachment organelle and localization of P1. P30 appears to be involved with gliding motility. HMW1, HMW2, and HMW3 are critical in the formation and stabilization of the attachment organelle, including localization of other adhesin-related proteins. Once this polar structure is established, an independently assembled complex of proteins B, C and P1 is drawn to the structure to complete formation of the functional terminal attachment

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Figure 1. Transmission electron micrographs of Mycoplasma pneumoniae grown in culture demonstrating flask-shaped morphology and the prominent adhesin tip (Courtesy Kristin Hoek and Leigh Milligan, UAB High Resolution Imaging Facility).

organelle shown as an electron dense region in the flask-shaped organism by electron micrography (Fig, 1). Two proteins, elongation factor TU and pyruvate dehydrogenase E1` are involved in binding M. pneumoniae to fibronectin [13]. Mammalian cells parasitized by M. pneumoniae can exhibit a number of cytopathic effects as a result of the local damage following cytadherence. Cells may lose their cilia entirely, appear vacuolated, show a reduction in oxygen consumption, glucose utilization, amino acid uptake, and macromolecular synthesis, ultimately resulting in exfoliation of all or parts of the infected cells. These subcellular events result in some of the clinical manifestations of respiratory tract infection such as the persistent, hacking cough. Dallo [14] recently described the ability of M. pneumoniae to survive, synthesize DNA, and undergo cell replication in artificial cell culture systems over a 6-month period. Intracellular sequestration could facilitate the establishment latent or chronic states, circumvent mycoplasmacidal immune mechanisms, facilitate the ability to cross mucosal barriers and gain access to internal tissues, and impair efficacy of some drug therapies, accounting for difficulty in eradicating the mycoplasmas in clinical conditions. However, the extent to which M. pneumoniae invades and replicates intracellularly in vivo is not known. High-frequency phase and antigenic variation of surface adhesin proteins made possible by DNA rearrangements in truncated and sequence-related copies of the P1 adhesin genes that are dispersed throughout the genome may also be a means for M. pneumoniae to evade the host immune response [15].

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Innate immunity to M. pneumoniae disease Elements of the host innate immune system are activated by M. pneumoniae and the attendant inflammation accounts for many of the initial signs and symptoms accompanying the early stages of the evolving infection. Once M. pneumoniae reaches the lower respiratory tract, the organism may be opsonized by complement or antibodies. Macrophages become activated, begin phagocytosis, and undergo chemotactic migration to the site of infection. There is in vitro evidence that the organism is susceptible to complement-mediated cytolysis, probably through both the alternative and classical pathways [16, 17]. Therefore, it is possible that complement plays a significant role in inhibiting growth of the organism, particularly after inflammation induces an exudate. While there are reports of invasive infections with M. pneumoniae in hypogammaglobulinemic individuals, there are none relating similar occurrences in patients with complement deficiencies alone. A recent study identified a highly significant association between mannose binding lectin deficiency and invasive infections with M. pneumoniae in patients with concomitant antibody deficiency [18]. A series of surfactant proteins (SP-A–SP-D) are also capable of binding and regulating the growth of microorganisms through lectin-like activity. SP-A exhibits calcium-dependent binding and growth inhibition of M. pneumoniae [19]. Interaction of M. pneumoniae with mast cells via a sialic acid-dependent binding mechanism was reported to induce cytokine production, especially IL-4, IL-5, and TNF-_ [20, 21], and is dependent upon the presence of the P1 adhesin. M. pneumoniae lipoproteins may interact with toll-like receptors 2 (TLR2) and/or 6 (TLR6) of respiratory epithelial or other cells, as found with other mycoplasmas [22, 23]. As a result of TLR interaction, cells are stimulated to produce cytokines or begin apoptosis, thus contributing to the pathogenesis of disease. M. pneumoniae may induce cellular activation during co-culture with a variety of other immunologic cells, including lymphocytes, macrophages, and respiratory epithelial cells. The nonspecific nature of the cellular targets of activation by M. pneumoniae could explain the broad range of inflammatory and autoimmune phenomena following acute infection.

Adaptive immunity and interaction of M. pneumoniae with host immune cells Adaptive immunity, characterized by both B and T lymphocyte responses, has a major impact on the progression of M. pneumoniae respiratory disease. While data are limited on the role of lymphoid responses in human M. pneumoniae infections, studies of other mycoplasmal respiratory diseases in animals indicate that some immune responses will be beneficial in controlling or preventing infection, while others contribute to disease severity.

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Local (mucosal) immune responses may be most effective in protecting the host from mycoplasma infection since resistance to M. pneumoniae seems to be most closely associated with IgA responses, but immunity is often short-lived, as humans are susceptible to repeated infections, despite the development of complement fixing and growth inhibiting antibody responses [15]. Immune responses that develop after infection often fail to eliminate the mycoplasma, indicating that adaptive immunity apparently has a limited effect on clearance of an established infection, thus leading to asymptomatic carriage for variable periods of time. M. pneumoniae infections in adults may be asymptomatic, perhaps reflecting some degree of protective immunity against reinfections over time. Persons with impaired ability to produce antibody, such as in congenital hypogammaglobulinemia, can suffer from chronic respiratory disease due to M. pneumoniae, suggesting antibody and other immune responses have a limited, but significant, role in recovery from disease [24]. In addition, hypogammaglobulinemic persons are more susceptible to extrapulmonary complications [25]. These observations suggest that antibody responses are important in controlling infection and preventing dissemination of mycoplasmas from the respiratory tract. Cytokine production and lymphocyte activation may either minimize disease through the enhancement of host defense mechanisms, or exacerbate disease through the development of immunologic hypersensitivity, worsening damage to the respiratory epithelium. The more vigorous the cell-mediated immune response and cytokine stimulation, the more severe the clinical illness and pulmonary injury. Lymphoid infiltration characteristic of M. pneumoniae disease suggests that lymphocyte activation contributes to the development of inflammatory responses. M. pneumoniae infection of laboratory rodents reveal CD4 + T helper (Th) cells within the inflammatory infiltrates in lung [26], and mycoplasma-specific Th cell responses are found in peripheral blood from humans [27]. CD8 + T cells also increase in lungs of mice after intranasal infection with M. pneumoniae [28] and T-celldepleted hamsters develop less severe M. pneumoniae respiratory disease [29]. Mycoplasma pulmonis infection of severe combined immunodeficiency (SCID) mice or T-cell-deficient mice results in milder lung lesions [30], and both T-cell populations modulate disease severity without directly affecting the number of mycoplasmas in the lungs. CD4 + Th-cells promote the inflammatory responses in rodent lungs, whereas CD8 + T-cells dampen these responses [31]. Most likely, a similar dichotomy of T-cell activity occurs in human M. pneumoniae disease. Depletion of CD4 + T-cells in mice results in a decreased cytokine responses and lymphocyte accumulation in the lungs after experimental M. pneumoniae infection [28]. Thus, populations of CD4 + T- and CD8 + T-cells most likely have contrasting roles in M. pneumoniae pulmonary disease, with the actions of a population of proinflammatory CD4 + Th-cells being suppressed by mycoplasma-specific CD8 + T-cells. Furthermore, CD4 + T-lymphocyte, B lymphocyte, and plasma cell

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accumulation in the lung is responsible for the radiographic manifestation of pulmonary infiltrates and is associated with lymphocyte proliferation, production of immunoglobulins, and release of TNF-_, interferon gamma (IFN-a), and various interleukins [15]. Host responses that develop after M. pneumoniae infection likely contribute to extrapulmonary complications. Examples are the association between M. pneumoniae infection and increased severity of asthma and production of anti-erythrocyte autoantibodies “cold agglutinins,” once used as a diagnostic indicator of M. pneumoniae infection. Overall, adaptive immune responses generated after M. pneumoniae infections have contrasting impacts on the pathogenesis of infection. Beneficial effects of host resistance lead to containment of the infection and ultimately recovery in many persons. Relative persistence of M. pneumoniae in the host leads to the provocation of ineffective immune-mediated inflammatory responses, regulated by opposing T-cell activities. Complicating the impact of these responses is their contribution to other adverse reactions, such as asthma and autoimmune-like effects.

Clinical and laboratory aspects of M. pneumoniae infections Following a 2–3-week incubation period, symptoms may develop over 1–2 days and consist of worsening nonexudative pharyngitis, nasal and sinus congestion, low-grade fever, and cough. In 1–2 weeks, more seriously affected individuals develop tracheobronchitis and primary atypical pneumonia (PAP), a syndrome consisting of fever, cough, musical inspiratory rhonchi reflecting mucus secretion in large airways, and occasionally bilateral expiratory wheezing. Acute lower respiratory symptoms may progress to respiratory distress with hypoxemia and necessitate hospital admission, but such severe cases represent only 5–10% of affected individuals. Patients with pre-existing pulmonary disease such as asthma or chronic bronchitis may be more severely affected. Antibiotic therapy with appropriate drugs results in clinical improvement, but the organism tends to persist in the airways. The cough, often productive, typically continues for several weeks and abnormalities in pulmonary hyperresponsiveness can persist for months. Diagnosis of PAP is often presumptive because of difficulties in microbiological confirmation. Chest radiographs typically reveal only perihilar and bibasilar streaky infiltrates, findings less impressive than symptoms would suggest. Small pleural effusions occur in 5-20% of patients. Clinical presentation may be similar to what is also seen with other pathogens, particularly Chlamydophila pneumoniae, various respiratory viruses, and even bacteria such as S. pneumoniae. M. pneumoniae may also be present in the respiratory tract concomitantly with other pathogens. About one-third of persons with mycoplasmal infections have leukocytosis and an elevated

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erythrocyte sedimentation rate. Sputum gram stain may show mononuclear cells or neutrophils and normal flora. There are no hepatic or renal abnormalities typical of M. pneumoniae infection, although the hemolytic anemia that develops in some patients may be reflected in the hemogram.

Autoimmunity and extrapulmonary manifestations Autoimmune reactions are believed to be responsible for many of the extrapulmonary complications that are associated with as much as 25% of M. pneumoniae infections. They may occur as a result of molecular mimicry in the ascending paralysis of Guillain-Barré Syndrome that results from autoantibody-mediated destruction of peripheral nerve fibers. These antibodies may also recognize glycolipids extracted from M. pneumoniae. Another mechanism is illustrated in hemolytic anemia due to cross-reacting antibodies against erythrocyte antigens as seen in cold-agglutinin disease. The PCR assay has greatly enhanced understanding of how M. pneumoniae can disseminate throughout the body. The presence of M. pneumoniae in blood, synovial fluid, cerebrospinal fluid, pericardial fluid, and skin lesions has been documented by PCR and/or culture. Thus, direct invasion must always be considered [15]. The frequency of direct invasion of these sites is unknown because the organism is rarely sought and most reported extrapulmonary syndromes have been attributed to M. pneumoniae infection based on serology. Extrapulmonary manifestations have been reviewed in depth elsewhere with original citations [15] and are summarized in Table 1.

Pathologic aspects of lung disease due to M. pneumoniae Acute perivascular and peribronchial cell infiltration results in destruction of respiratory epithelium. There are also neutrophilic accumulations within the airways early in disease. At later stages, massive mononuclear cell infiltration, of which T-cells are a major component, occurs. Ulceration with destruction of ciliated epithelium of bronchi and bronchioles, edema, bronchiolar and alveolar infiltrates of macrophages, lymphocytes, and neutrophils, increased numbers of plasma cells, and deposition of fibrin. Type II pneumocyte hyperplasia with diffuse alveolar damage and bronchiolitis obliterans have been described [15]. Pleura may contain patches of fibrinous exudates. Pleural effusions sometimes occur in association with more severe cases complicated by long-term sequelae such as pleural scarring, bronchiectasis, and pulmonary fibrosis. Lung abscesses may also occur. Immunosuppressed persons may lack pulmonary infiltrates, further attesting to the importance of the host immune response in lesion development.

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Table 1. Extrapulmonary manifestations of M. pneumoniae infection resulting from autoimmune reaction and/or direct invasion Organ system Neurologic

Musculoskeletal

Skin

Cardiovascular

Hematologic

Renal

Gastrointestinal and other

Manifestations Ascending paralysis, encephalitis, aseptic meningitis, transverse myelitis, cerebellar syndrome, polyradiculitis, cranial nerve palsies, optic neuritis, choreoathetosis, leukoencephalopathy, acute psychosis Myalgias, arthralgias, polyarthropathies, septic arthritis, reactive (inflammatory noninfectious) arthritis, osteomyelitis, rhabdomyolysis

Erythematous maculopapular and vesicular rashes, StevensJohnson Syndrome, conjunctivitis, ulcerative stomatitis Pericarditis, myocarditis, pericardial effusion, cardiac tamponade

Comment Nervous system complications are among the most common and most severe manifestations. They often occur within 1–2 weeks of respiratory infection and in some cases without any apparent preceding respiratory symptoms. Invasive joint infections occur more commonly in persons with antibody deficiency, but they have also been described in immunocompetent persons. Recent data suggest a possible association with adult and juvenile rheumatoid arthritis. Dermatological disorders are the most common type of extrapulmonary manifestations.

Cardiac involvement occurs in up to 8.5% of serologically diagnosed infections and is more common in adults than children. Hemolytic anemia, cold agglu- Cross-reactive antibodies against tinin disease, aplastic anemia, erythrocyte antigens or plasma von thrombotic thrombocytopenic Willebrand factor-cleaving protease purpura, disseminated intrahave been implicated as causative facvascular coagulation tors. Acute glomerulonephritis, Antibody-mediated pathogenesis is renal failure, tubulointerstitial believed to predominate, but myconephritis, IgA nephropathy plasma antigen has been detected in damaged kidney tissue by immunohistochemistry. Nausea, vomiting, diarrhea, These nonspecific complaints can be cholestatic hepatitis, pancreati- associated with respiratory disease. tis otitis media, myringitis Mechanisms have not been carefully investigated.

Chronic lung disease associated with M. pneumoniae infection M. pneumoniae has been isolated in increased frequency from stable asthmatics. It has been linked with exacerbations of existing asthma, as well as the subsequent development of asthma in previously healthy persons. Kraft [32] detected M. pneumoniae by PCR in respiratory secretions of 42% stable adult asthmatics versus 9% of healthy controls. In another study, throat cultures for M. pneumoniae were positive in 24.7% of children and adults

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with asthma exacerbation, compared with 5.7% of healthy controls [33]. In another study, 21% of adults having a flare of their asthma were found to have high levels of M. pneumoniae-specific IgM, suggesting exacerbation occurred in the context of acute infection [34]. Macrolide treatment of asthma patients in whom M. pneumoniae has been detected resulted in improvement in pulmonary function tests in comparison with asthma patients who did not have evidence of the organism in their airways [35], owing perhaps to both the antibacterial as well as anti-inflammatory effects of these drugs. Mycoplasmas have been detected by PCR in airways even when culture and serology are negative, suggesting that low numbers of organisms may evade detection by the immune system [35]. Lack of a measurable serologic response may also facilitate the organism’s persistence in the lower respiratory tract. M. pneumoniae is known to induce a number of the inflammatory mediators implicated in the pathogenesis of asthma that may play a role in exacerbations. IgE triggering of mast cell degranulation is a key event in allergic asthma, and mycoplasma-specific IgE responses may elicit a similar response [20]. Mycoplasma IL-4 stimulation may enhance the asthma-promoting IgE responses against potential allergens. M. pneumoniae can be associated with significantly greater numbers of mast cells in humans with chronic asthma [36], and experimental evidence from a rodent mast cell line suggests that the organism can induce activation of mast cells [20, 21]. Koh [37] showed that levels of IL-4 and the ratio of IL-4/IFN-a were significantly higher in children with M. pneumoniae than those with pneumococcal pneumonia or uninfected controls, suggesting a TH2-like cytokine response representing a favorable condition for IgE production. Gump [38] reported that mycoplasmal infections could be associated with some cases of chronic obstructive pulmonary disease (COPD) exacerbation. Subsequent studies, summarized in a recent review [15] have reported serologic evidence of acute M. pneumoniae infection in 6-14% of COPD exacerbations. Evidence of concomitant respiratory pathogens in some cases complicates understanding of the significance of M. pneumoniae in this context. Bacterial infection is the main cause of progressive pulmonary failure in patients with cystic fibrosis. Serological examinations have been the sole means of assessing the presence of M. pneumoniae in persons with cystic fibrosis and the methods employed present some difficulty in proper interpretation to define a recent infection. Limited findings to date suggest mycoplasmas may occur but are fairly uncommon causes of respiratory complications in persons with cystic fibrosis [15]. More work must be done to clarify the importance of M. pneumoniae in the epidemiology and pathogenesis of exacerbations of chronic lung diseases using a more comprehensive diagnostic strategy that would include direct tests for the presence of the organisms by PCR, culture, and serology.

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Measurement of the immune response as a means for diagnosis Serology has been the most common laboratory means for diagnosis of M. pneumoniae infections. Although culture and PCR are also used, persistence of the organism for variable lengths of time following acute infection makes it difficult to assess the significance of a positive culture or PCR assay without additional confirmatory seroconversion. The length of time necessary for culture (sometimes 6 weeks or more) makes it impractical for patient management and it is not widely available except in specialized reference laboratories. Detailed information on laboratory diagnosis of M. pneumoniae infection is available elsewhere [15] and only pertinent summary information is discussed here. M. pneumoniae has both lipid and protein antigens which elicit antibody responses that can be detected after about 1 week of illness, peaking at 36 weeks, followed by a gradual decline, allowing several different types of serological assays, based on different antigens and technologies. Serology is a useful epidemiologic tool in circumstances where the likelihood of mycoplasmal disease is high, but it is less suited for assessment of individual patients. Its main disadvantage is the need for both acute and convalescent paired sera collected 2 to 3 weeks apart that are tested simultaneously for IgM and IgG to confirm seroconversion. This is especially important in adults over 40 years of age who may not mount an IgM response, presumably because of reinfection. Moreover, IgM antibodies can sometimes persist for several weeks to months, making it risky to base diagnosis of acute infection on a single assay for IgM alone. Antibody production may also be delayed in some infections, or even absent if the patient is immunosuppressed. False-negative tests for IgM can also occur if serum is collected too soon after onset of illness. Since M. pneumoniae is a mucosal pathogen, IgA is typically produced early in the course of infection. Measurement of serum IgA may therefore be a better approach for diagnosis of acute infection because of its rapid rise and decline, but very few commercial assays include reagents for its detection. Complement fixation (CF) was the first method developed for serological testing for M. pneumoniae. CF measures mainly the early IgM response and does not differentiate among antibody classes, which is desirable to differentiate acute from remote infection. CF suffers from low sensitivity and specificity because the glycolipid antigen mixture used may be found in other microorganisms, as well as human tissues, and even plants. Cross-reactions with Mycoplasma genitalium are well recognized. In most clinical laboratories CF has been replaced by alternative techniques with greater sensitivity and specificity, many of which have been developed and sold as commercial kits. Immunofluorescent antibody (IFA) assays, direct and indirect hemagglutination using IgM capture, and other particle agglutination antibody assays (PAs) have been developed to detect antibody to M. pneumoniae. Enzyme immunoassays (EIAs)

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have become the most widely used commercial methods for detection of M. pneumoniae. EIAs are more sensitive for detecting acute infection than culture, and can be comparable in sensitivity to PCR, providing a sufficient time has elapsed since infection for antibody to develop and the patient has a functional immune system. These assays may be qualitative or quantitative, may or may not require specialized equipment for performing the assay and reading the results, and can be performed with very small volumes of serum. The need for acute and convalescent sera has remained the obvious limitation for prompt point-of-care diagnosis. However, qualitative rapid point-of care serologic assays that detect both IgM and IgG or IgM alone in an easy-to-read format without the need for any instrumentation have been developed and shown to compare favorably with other commercial assays [15]. The variability of results from comparative studies underscores the need for improved serological reagents for detecting acute M. pneumoniae infection [39]. The best commercial EIA for individual patient diagnosis depends on the age of the patient, timing of serum collection, whether paired sera are available, equipment available, and experience of the laboratory personnel. However, maintaining a large variety of different assays within one laboratory is not practical or cost-effective.

Use of the PCR assay for detection of M. pneumoniae Gene targets used in PCR assays for M. pneumoniae include 16S rRNA, P1 adhesin, ATPase operon gene, and tuf gene. Real-time PCR assays have also been described [15]. The sensitivity of PCR is very high, corresponding to a single organism when purified DNA is used. Other advantages are the potential ability to complete the procedure in one day, the requirement of only one specimen containing organisms that do not have to be viable, as well as the ability to detect nucleic acid in preserved tissues. Comparison of PCR with culture and/or serology has yielded varied results that are not always in agreement. Positive PCR results in culture-negative persons without evidence of respiratory disease suggest inadequate assay specificity, persistence of the organism after infection, or asymptomatic carriage, perhaps in an intracellular compartment that does not yield culturable organisms. Quantitative studies may be useful in drawing conclusions. Positive PCR results in serologically-negative persons may be due to an inadequate immune response, early successful antibiotic treatment, or to the collection of specimens before specific antibody synthesis could occur. Negative PCR results in culture or serologically proven infections raise the possibility of inhibitors or other technical problems with the assay. If antibiotics have been administered, PCR results may be negative even though serology is positive. Ideally, positive PCR assays should be confirmed by a second unrelated target gene. Thus

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far, there are no commercial PCR kits for detection of M. pneumoniae, but several are in development.

Treatment of M. pneumoniae infections Administration of antimicrobials to patients with M. pneumoniae infections will generally produce satisfactory results with a marked reduction in duration of respiratory symptoms. Management has been guided primarily by well-known and consistent susceptibilities to a variety of drugs. Macrolides are the treatments of choice, but tetracyclines and fluoroquinolones are also effective, as is the new ketolide telithromycin. Most clinical trials evaluating treatments for CAP identified small numbers of cases proven to be due to M. pneumoniae by serologic diagnosis, though some recent studies incorporated culture and/or PCR. Use of serology alone precludes determination as to whether a treatment regimen actually eradicates the organism, thus very little data are available regarding microbiological efficacy of any regimen. A summary of clinical trials evaluating various treatments for CAP in which outcome data specific for M. pneumoniae were included can be found in a recent review [15]. A recent study from Japan found that macrolide resistance in M. pneumoniae due to transition mutations in domain V on the 23S rRNA gene occurred in 6% of 195 clinical isolates from patients with acute infections [40]. The clinical significance of this resistance is uncertain, but it suggests the need to monitor clinical isolates for such resistance. High-dose steroids have been reported to be useful in treatment of encephalitis in children with complicated M. pneumoniae infection [41]. Plasmapheresis and intravenous immunoglobulin therapy might be considered if steroid therapy is ineffective in these settings.

Summary M. pneumoniae is a common cause of CAP in both children and adults. Serious infections requiring hospitalization, while rare, occur in persons of all age groups, and may affect multiple organ systems. Severity of disease appears to be related to the degree to which the host immune response reacts to the infection. Extrapulmonary complications involving all of the major organ systems can occur in association with M. pneumoniae infection as a result of direct invasion and/or autoimmune response. Evidence is accumulating for this organism’s contributory role in chronic lung conditions such as asthma. Effective management of M. pneumoniae infections can usually be achieved with macrolides, ketolides, tetracyclines, or fluoroquinolones. As more is learned about pathogenesis and immune response elicited by M. pneumoniae, improvement in methods for diagnosis and prevention of disease due to this organism are anticipated.

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Acknowledgments Portions of the work summarized in this chapter were supported by U.S. Public Health Service grants HL73907-01A1 to T.P.A. and grants HL069431, AI42075, and AI055907 to J.W.S.

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dependent adhesion of Mycoplasma pneumoniae to purified glycoproteins. J Biol Chem 264: 9289–9293 Dallo SF, Kannan TR, Blaylock MW, Baseman JB (2002) Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. Mol Microbiol 46: 1041–1051 Dallo SF, Baseman JB (2000) Intracellular DNA replication and long-term survival of pathogenic mycoplasmas. Microb Pathog 29: 301–309 Waites KB, Talkington DF (2004) Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 17: 697–728 Bredt W, Kist M, Jacobs E (1981) Phagocytosis and complement action. Isr J Med Sci 17: 637–640 Loos M, Brunner H (1979) Complement components (C1, C2, C3, C4) in bronchial secretions after intranasal infection of guinea pigs with Mycoplasma pneumoniae: dissociation of unspecific and specific defense mechanisms. Infect Immun 25: 583–585 Hamvas RM, Johnson M, Vlieger AM, Ling C, Sherriff A, Wade A, Klein NJ, Turner MW, Webster AD (2005) Role for mannose binding lectin in the prevention of mycoplasma infection. Infect Immun 73: 5238–5240 Phiboonpocanum S, Chiba H, Mitsuzawa H, Martin W, Murphy RC, Harbeck RJ, Voelker RB (2005) Surfactant protein A binds Mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of desaturated phosphatidylglycerols. J Biol Chem 280: 9–17 Hoek KL, Cassell GH, Duffy LB, Atkinson TP (2002) Mycoplasma pneumoniae-induced activation and cytokine production in rodent mast cells. J Allergy Clin Immunol 109: 470–476 Hoek KL, Duffy LB, Cassell GH, Atkinson TP (2005) A role for the Mycoplasma pneumoniae adhesin P1 in Interleukin (IL)-4 synthesis and release from rodent mast cells. Microb Pathog 39: 149–158 Into T, Nodasaka Y, Hasebe A, Okuzawa T, Nakamura J, Ohata N, Shibata K (2002) Mycoplasmal lipoproteins induce toll-like receptor 2- and caspasesmediated cell death in lymphocytes and monocytes. Microbiol Immunol 46: 265–276 Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K, Morr M, Muhlradt PF, Akira S (2000) Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J Immunol 164: 554–557 Taylor-Robinson D, Webster AD, Furr PM, Asherson GL (1980) Prolonged persistence of Mycoplasma pneumoniae in a patient with hypogammaglobulinaemia. J Infect 2: 171–175 Johnston CL, Webster AD, Taylor-Robinson D, Rapaport G, Hughes GR (1983) Primary late-onset hypogammaglobulinaemia associated with inflammatory polyarthritis and septic arthritis due to Mycoplasma pneumoniae. Ann Rheum Dis 42: 108–110 Hayakawa M, Taguchi H, Kamiya S, Fujioka Y, Watanabe H, Kawai S, Kobayashi H (2002) Animal model of Mycoplasma pneumoniae infection using germfree mice. Clin Diagn Lab Immunol 9: 669–676

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Community-acquired pneumonia: paving the way towards new vaccination concepts Pablo D. Becker and Carlos A. Guzmán Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany

Abstract Despite the availability of antimicrobial agents and vaccines, community-acquired pneumonia remains a serious problem. Severe forms tend to occur in very young children and among the elderly, since their immune competence is eroded by immaturity and immune senescence, respectively. The main etiologic agents differ according to patient age and geographic area. Streptococcus pneumoniae, Haemophilus influenzae, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV-3) are the most important pathogens in children, whereas influenza viruses are the leading cause of fatal pneumonia in the elderly. Effective vaccines are available against some of these organisms. However, there are still many agents against which vaccines are not available or the existent ones are suboptimal. To tackle this problem, empiric approaches are now being systematically replaced by rational vaccine design. This is facilitated by the growing knowledge in the fields of immunology, microbial pathogenesis and host response to infection, as well as by the availability of sophisticated strategies for antigen selection, potent immune modulators and efficient antigen delivery systems. Thus, a new generation of vaccines with improved safety and efficacy profiles compared to old and new agents is emerging. In this chapter, an overview is provided about currently available and new vaccination concepts.

Introduction The mucosa of the human respiratory tract represents a primary target for a large number of microbial pathogens. Typically, colonization is an asymptomatic process, resulting from the interplay between bacterial factors and host clearance mechanisms. Clinical illness may result from either the local release of bacterial toxins or the systemic dissemination of the pathogen after breaching the mucosal barrier. In the course of respiratory infections adaptive immune responses could be significantly impaired. This might lead to more severe forms of disease or to super-infections, which in turn complicate the clinical management of the patient. The most severe forms of respiratory infection tend to occur in very young children and among the

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elderly, in whom immune competence is eroded by immaturity or immunesenescence, respectively. In addition, patients who are immunocompromised, as a result of disease or therapeutic interventions, have the greatest risk of developing a fatal infection. Despite the availability of new antimicrobials and effective vaccines, community-acquired pneumonia remains a common and serious illness. In fact, it is a leading contributor to the nearly 4 million deaths occurring each year due to respiratory infections, especially in children from developing countries [1, 2]. The main causative agents of pneumonia differ according to the patient age and the geographic area. In addition, there are relatively few comprehensive studies on the specific aetiology of pneumonia [2] due to (i) overlaps in the clinical manifestations of the different syndromes, (ii) difficulties in establishing the precise aetiology, and (iii) frequent occurrence of co-infections. However, Streptococcus pneumoniae, Haemophilus influenzae, respiratory syncytial virus (RSV), and parainfluenza virus type 3 (PIV-3) have been identified as the main agents responsible for acute respiratory infections in children, whereas influenza virus related pneumonia is the leading cause of disease-related deaths in the elderly. In addition, the availability of new and more sensitive diagnostic tests have contributed to the identification of hitherto unknown lower respiratory pathogens, such as the human metapneumonovirus (hMPV) and novel coronaviruses causing the severe acute respiratory syndrome (SARS). Significant efforts have been invested in the last two decades to develop new diagnostic tools, to elucidate the molecular mechanisms of microbial pathogenesis and to understand host clearance mechanisms. This resulted in an improved knowledge on host responses to infection and immuno-pathogenesis, which in turn have facilitated the establishment of new prophylactic and therapeutic interventions. However, despite our accomplishments in vaccine development, there are many pathogens for which vaccines or adequate therapies are not available or the existent ones are suboptimal. The main approach applied for vaccine development has radically changed in recent years. Whole cell vaccines are systematically being replaced by subunit vaccines, in which purified antigens or their coding genes are exploited in combination with new adjuvants and/or delivery systems. As a result, many of the vaccines under development will exhibit consistently improved stability, safety and efficacy profiles. They will also be amenable for mucosal administration, thereby mimicking natural infections.

Currently available vaccines Influenza vaccines Influenza A viruses are the most commonly responsible for severe respiratory illness in humans, followed by influenza B. The population’s susceptibly

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to infection is renewed annually, because of the rapid antigenic variation of this virus. The antigenic variation is due to the accumulation of point mutations in the two major surface glycoproteins of the virus, haemagglutinin (HA) and neuraminidase (NA). This can lead to an antigenic drift of the virus, which often leaves current influenza vaccines outdated and ineffective. Antigenic shift can also occur due to the segmented nature of the viral genome that favours the emergence of re-assorted strains, in which an entire glycoprotein can be acquired from a different animal influenza virus. Both types of variation represent a critical bottleneck for the establishment of a robust vaccination strategy against influenza. In fact, when an influenza virus with the capacity to spread from person-to-person and a complete new glycoprotein subtype suddenly emerges, a worldwide pandemic outbreak can result [3].

Inactivated vaccines against influenza The earliest vaccines against influenza were whole cell vaccines obtained in the 1940s by inactivating viruses grown in the allantoic cavity of embryonated chicken eggs with formalin. While contemporary inactivated influenza vaccines are still produced in embryonated eggs, improvements in manufacturing have resulted in a highly purified and less-reactogenic detergentsplit product. Three viral strains are selected on the basis of the previous year’s surveillance data on the most prevalent subtypes, therefore, vaccine composition may vary from year-to-year. Vaccination has a high benefit:cost ratio, since influenza-related illness (e.g., hospitalizations and deaths) are effectively prevented [1]. The world’s total vaccine production is approximately 300 million doses, with a maximum capacity of 900 million doses. However, the World Health Organization (WHO) estimates that there are about 1.2 billion people at high risk for severe influenza outcomes (e.g., elderly over 65 years of age, infants, health care workers, children and adults with underlying cardiopulmonary disease). Furthermore, the global infrastructure would not be able to handle the timely manufacturing and distribution of a vaccine for a pandemic outbreak [4]. One alternative would be to lower the quantity of antigen per dose and add adjuvants to the vaccine formulation, but this needs to be tested in clinical trials [5]. Another solution would be to improve current vaccine production technologies (i.e., egg-derived vaccines). However, there is the limited number of egg producers and viral strains can emerge, which could not be easily adapted to embryonated eggs. To overcome these problems, several pharmaceutical companies have embarked themselves on projects for the development of vaccines produced by growing the virus on cell lines. The influenza virus can be adapted to grow on a variety of mammalian cell lines, including Vero, PER.C-6, and Madin-Darby canine kidney (MDCK) cells [6–8].

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This strategy would also improve the possibility of up-scaling vaccine production in face of a pandemic spread. Alternatively, it would be possible to develop a vaccine against any influenza virus, such as the avian H5N1 strain, by using reverse genetics techniques [9] (see below in advances in vaccinology).

Live attenuated vaccines against influenza Cold adaptation was found to be a reliable and efficient procedure for the derivation of live attenuated viral vaccine strains for humans. Cold-adapted (ca) virus strains can grow in primary chick kidney cells or embryonated eggs at 25–33°C, however, they exhibit a reduced replication at 37°C. The process of genetic re-assortment with the transfer of the six internal genes from a stable attenuated ca master donor strain of influenza A or B to the new prevailing wild-type epidemic strain has been used to generate attenuated cold-reassorted vaccines with the proper level of attenuation, genetic stability and immunogenicity, which show low or absent transmissibility [10]. MedImmune and Wyeth have developed along these lines a trivalent live ca vaccine (Flumist) for intranasal spray delivery, which was licensed in 2003. In contrast, to parenterally-administered vaccines, this formulation triggers immune responses resembling those observed after natural infections [11]. Despite the moderate hemagglutination-inhibiting antibody titres observed in vacinees, Flumist showed 92% efficacy over a 2-year period in children, including protection against antigenic variants that circulated during the second year [12–14]. This ca vaccine also stimulated the production of nasal IgA, as well as T-cell and interferon responses [15]. The cell-mediated immunity against virus matrix and nucleoprotein antigens may favour viral clearance and early recovery from illness [3]. The Advisory committee on Immunization Practices has recommended its use only in persons from 5 to 49 years of age, since side-effects were observed in young children (wheezing, nasal congestion) and there are no data available for elderly [16, 17]. Despite its remarkable genetic stability, this vaccine has to be kept at – 18°C. Thus, a new heat stable derivative has recently been developed, which showed good efficacy in clinical trials [1]. A live vaccine based on a master virus strain developed at the Institute of Applied Microbiology (Austria) by growing wild influenza virus in Vero cells at 25°C was also demonstrated to be safe, well-tolerated and immunogenic after intranasal immunization in young adults [18].

Subunit and DNA vaccines against influenza A number of subunit- or DNA-based vaccines are also in various stages of development. An influenza vaccine formulated in virosomes has been

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commercialized by Berna Biotech (Inflexal® V); it contains the surface spikes of the three currently circulating influenza virus strains inserted in vesicle membranes of the three corresponding virus types (for more details see section “Pseudoviruses as antigen delivery systems”) [19]. This company has also developed a virosome-based nasal formulation. However, it was withdrawn from the market due to the presence of sideeffects (i.e., Bell’s palsy), which was assumed to be linked to the presence of the Escherichia coli heat labile toxin (LT) as adjuvant. Two companies, Yeda and BionVax, are also developing a peptide-based influenza vaccine for nasal administration, which showed protective efficacy in humanized mice [1]. A subunit vaccine containing recombinant HA protein produced using a baculovirus system was successfully tested in a phase II trial in 64 to 89-year-old volunteers. An epidermal DNA-based influenza vaccine, which contained the HA gene from A/Panama/2007/99 delivered by particle-mediated epidermal delivery was also tested in humans by PowderJect [20]. Serum haemagglutination-inhibition antibody responses were observed in volunteers receiving a single dose of 1, 2 or 4 +g of DNA, with the strongest and most consistent responses in subjects vaccinated with the highest dosage. Some immunization approaches aim at the development of a universal vaccine with a broad spectrum of protective activity against different influenza strains [21]. Among them, the use of the highly conserved transmembrane M2 protein of the virion can be mentioned. A recombinant particulate vaccine has been engineered by genetically fusing copies of the M2 to the hepatitis B core antigen (HBc). The M2-HBc fusion protein spontaneously assembled into virus-like particles (VLP), which provided complete protection against a lethal challenge with influenza virus A in mice [22, 23]. Promising results were also obtained after vaccination with a M2 peptide conjugated with a Neisseria meningitidis outer membrane protein complex (OMPC) in monkeys [24].

Vaccines against the parainfluenza virus The human PIV (hPIV) consist of four serotypes, with hPIV-3 being the second leading cause of bronchitis and pneumonia in infants. No vaccine has been licensed to date against PIV, however, several approaches are currently under investigation. The initial attempts to provide protection by using vaccines based on formalin-inactivated viruses failed. Subsequent work demonstrated that the glycoproteins haemagglutininneuraminidase (HN) and F, which are responsible for virus attachment and fusion, are able to stimulate the elicitation of neutralizing antibodies in animals. However, their poor immunogenicity in naïve subjects led to the currently favoured approach, which is based on the use of live attenuated PIV.

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Live attenuated PIV vaccines have been developed from both human and bovine strains, which are amenable for delivery by the intranasal route. Candidate vaccines should be able to replicate and induce a protective immune response in young infants, even in the presence of maternally acquired antibodies. Two main attenuated strains have been studied in detail. One is the hPIV-3 strain cp45, which was selected after 45 passages of the virus in African green monkey cells at low temperature. The other is a bovine PIV (bPIV)-3 strain, which is antigenically related to the hPIV-3, and replicates poorly in humans. Both cp45 and bPIV-3 have been evaluated in phase I/II trials in sero-positive and sero-negative children and in young infants. They were found to be over-attenuated in sero-positive children, but immunogenic in sero-negative children and infants [25]. However, the magnitude of the anti-HN response was lower in children who received the bPIV-3 vaccine [25]. This prompted the engineering of chimeric bovine/human PIV-3 candidates (e.g., hPIV-Nb strain in which the human nucleocapsid is replaced by the bovine counterpart, or a bPIV3 strain that expresses the F and HN proteins of hPIV-3). Attenuated, chimeric viruses that contain PIV-3 cp45 internal genes with the F and HN genes from either PIV-1 or PIV-2 have also been tested in hamsters [26]. Berna Biotech is also developing a virosomal formulation of the PIV-3 [1].

Vaccines against the respiratory syncytial virus Using the successful approach of the influenza vaccine, a formalin-inactivated candidate against the respiratory syncytial virus (RSV) was tested in children in the 1960s. The consequence was the hospitalization of 80% of vaccinees and two deaths [1]. Moreover, vaccinated children also suffered more severe disease on subsequent exposure to the virus, as compared to unvaccinated controls [27]. This demonstrated that the elicitation of a strong immune response is not sufficient to confer protection against disease, and can even lead to immuno-pathological reactions. Thus, it is essential to stimulate the “right” type of immune response. In the particular case of RSV, host responses play an important role in the pathogenesis of the disease, thereby making the development of a preventive vaccine extremely difficult. In addition, naturally acquired immunity to RSV is neither complete nor long-lasting, and recurrent infections often occur [28]. However, older children and adults are usually protected, suggesting that protection against severe disease develops after several consecutive infections. Passive immunization with RSV-neutralizing immune globulins was also shown to prevent RSV infection in newborns with underlying cardiopulmonary disease [29]. This demonstrates that antibodies play a major role in protection against this disease, whereas T-cell immunity targeted to internal viral proteins appears to contribute to clearance.

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Subunit vaccines against RSV Although live attenuated vaccines seem to be preferable for immunization of naïve infants, subunit vaccines may be of choice for elderly, high-risk children and pregnant women. Candidate subunit vaccines based on purified F proteins (PFP-1, -2 and -3) were demonstrated to be safe and immunogenic, even during pregnancy [30]. Maternal immunization using a PFP-based vaccine could be an interesting strategy to protect infants younger than 6 months of age [25]. However, no significant protection was reported in a phase III trial performed on children 1–12 years of age with cystic fibrosis after vaccination with a subunit vaccine based on PFP-3 [31]. A formulation based on surface glycoproteins F and G together with the virion matrix protein M from RSV-A was tested in healthy adult volunteers in the presence of either alum or polyphosphazene as an adjuvant. Short-live neutralizing antibody responses to RSV-A and RSV-B were detected in 76–93% of the vaccinees, suggesting that annual boosting will be needed [32–34]. The central domain of the G protein of RSV-A is relatively conserved among viruses from the groups A and B. Thus, a recombinant vaccine candidate, BBG2Na, was developed by fusing the G2Na domain to the albumin binding region of streptococcal protein G. This candidate was shown to be moderate immunogenic in adult human volunteers, but its clinical development was interrupted due to the appearance of purpura in vaccinees [1].

Live attenuated vaccines against RSV The main two difficulties associated with the generation of live attenuated vaccines against RSV are over- or under-attenuation of the virus and limited genetic stability. Temperature-sensitive (ts), ca and cold-passaged (cp) mutant viral strains have been generated. Despite the attenuation shown in adults and sero-positive children, cpts mutants still caused moderate congestion in the upper respiratory tract of sero-negative infants (1–2 months old) [35]. Recombinant RSV vaccines with deletions in non essential genes (e.g., SH, NS1 or NS2), which also carry cp and ts mutations in essential genes are currently being evaluated [1]. Through recombinant DNA technology chimeric viruses were engineered, which contain the genes of hPIV-3 surface glycoproteins F and NH together with those of RSV glycoproteins F and G in a bPIV-3 genetic background. One of these candidates was found to be attenuated and able to induce the elicitation of immune responses against both hPIV-3 and RSV in rhesus monkeys [36]. Similarly, a bPIV-3 genome was engineered to express hPIV-3 F and HN proteins and either native or soluble RSV F protein [37]. The resulting strain, which induced RSV neutralizing antibodies and protective immunity against RSV challenge in African Green monkeys, needs to be tested for safety and efficacy against RSV and PIV-3 in infants.

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Vaccines against the severe acute respiratory syndrome (SARS)associated coronavirus This emerging disease was originally described in the Guangdong province of China in 2002. Even when the global outbreak of SARS was under control in 2003, new infections were reported in persons who had contacts with animals in 2003 and 2004 [38]. The typical SARS-CoV-like virus is not transmitted from animals to humans. However, under certain conditions the virus can evolve into the early human SARS-CoV, which has the ability to be transmitted from animals to humans or even humans to humans, thereby leading to localized outbreaks of mild disease. The early human SARS-CoV, under selective pressure in humans, may further evolve into the late human SARS-CoV, which can cause local or global outbreaks of typical SARS [39]. SARS can be easily grown in cell cultures [38]. Thus, there is an urgent need for vaccines, not only to prevent naturally occurring epidemic outbreaks, but also as a tool against the threat of biological weapons. Several structural proteins are expressed by SARS-CoV, including nucleocapsid, envelope and spike (S) proteins [38]. The latter is a type I trans-membrane glycoprotein, which is responsible for virus binding, fusion and entry, and being the major target of neutralizing antibodies [38, 40]. The extracelullar domain of the S protein consists of two subunits, S1 and S2 [40]. The S1 subunit possess a receptor-binding domain (RBD), which is responsible for viral binding to one of its receptors [41, 42]. Vector-based vaccines expressing the S protein, as well as DNA vaccines encoding full-length S protein have been assessed in preclinical studies [43, 44]. When modified vaccinia virus Ankara (MVA) coding for full-length S protein was administered by either intranasal or intramuscular route, neutralizing antibodies were elicited [45]. However, vaccination of ferrets resulted in liver damage after challenge, raising some concerns about the safety of this approach [46]. Vaccines formulated using different synthetic peptides encompassing linear B cell epitopes from the S protein, which were identified using sera from convalescent patients, stimulated high antibody titres. Nevertheless, none of them triggered the elicitation of neutralizing activity. On the other hand, some studies demonstrated that although antibodies against S protein of the late SARS-CoV (Urbani strain) exhibit neutralizing activity, they can also enhance infection by an early human SARS-CoV isolate (GD03T0013) and the civet SARS-CoV-like viruses. A derivative of the S protein with a truncation at amino acid (aa) 1153 fails to cause antibody dependent enhancement of infection, but retains the ability to induce neutralizing antibodies. These findings suggest that the elimination of the putative heptad repeated 2 (HR2, aa 1153-1194), which is implicated in viral fusion, might abrogate the stimulation of virus infection-enhancing antibodies [47, 48]. The use of the nucleoprotein of the coronavirus in a DNA vaccination protocol also led to the stimulation of a protective response [49]. In contrast, protection

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was not achieved when a recombinant PIV-3 expressing the nucleoprotein alone or together with the matrix protein was used [50]. This demonstrates that the selection of the delivery system and immunization strategy play a critical role in vaccine efficacy.

Vaccines against adenovirus The human adenoviruses are divided into six subgroups (A–F). The adenovirus can cause large-scale epidemics of acute respiratory disease, and dissemination is especially favoured under conditions in which persons are housed communally. The subgroup A viruses, such as Ad31, have been associated with pneumonia in immunocompromised patients. Neutralizing antibodies directed against the capsid (hexon and fiber proteins) seems to be the main effector mechanism to prevent re-infections by adenovirus [3]. Until 1998, military recruits in USA were administered enteric-coated capsules containing live viruses from the serotypes 4 and 7. The virus, which was not attenuated if delivered by respiratory route, was able to replicate in the gastrointestinal tract without causing disease, thereby stimulating protective responses in the respiratory tract [51]. When the vaccine went out of production, outbreaks of respiratory diseases caused by adenovirus reemerged among the military recruits [3]. Since serotypes 1, 2, 3 and 5 cause the 80% of adenovirus associated respiratory disease in young children, the development of a tetravalent vaccine similar to the above mentioned might solve the problem in children [52]. However, the implementation of a vaccine (live or attenuated) against adenovirus should be carefully evaluated, since recombinant adenoviruses are proposed both as vaccine vectors and as tools for the transfer of foreign genes in gene therapy protocols.

Vaccines against Streptococcus pneumoniae Polysaccharide-based vaccines against S. pneumoniae In 1945, MacLeod et al. [53] reported the protective efficacy of a capsular polysaccharide (PS) vaccine in military personnel during an outbreak of pneumococcal pneumonia. The immunization with purified PS showed a drastically reduced reactogenicity, in comparison with the previously used inactivated whole cell vaccines. This was a major breakthrough, not only in terms of safety, but also because it demonstrated that a specific virulence factor can be purified and effectively implemented for the prevention of an infectious disease, thereby paving the road for modern non toxoid-based subunit vaccines. Although the serological correlates of immunity are poorly defined, type-specific anti-capsular antibodies are responsible for protective immu-

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nity. However, immunity is serotype specific, rendering extremely difficult the development of a universal vaccine. This is in part due to the elevate number of serotypes, the regional variations in dominant serotypes and the lack of updated sero-prevalence data for certain regions. These problems have been partially solved by the use of PS-based polyvalent vaccines. The currently licensed formulations contain 23 serotypes of S. pneumoniae, which cover approximately 90% of serious pneumococcal disease, but only in Western industrialized countries. Relatively good antibody responses (60–70%) are elicited in healthy adults 2–3 weeks after a single intramuscular or subcutaneous immunization [54]. Unfortunately, they are poorly immunogenic in children aged less than 2 years, in immune compromised individuals (e.g., AIDS patients) and in elderly people with concomitant disease, and they do not induce good immunological memory. Randomized controlled trials in healthy elderly and young men also failed to show a beneficial effect against pneumonia [55]. However, vaccination is recommended for healthy people over 65 years of age to confer protection against invasive disease [54]. PS-based vaccines can be also used in pregnant women to stimulate the production of antibodies, which are transferred to the foetus via the placenta or to the newborns by breast-feeding. However, it is still a matter of controversy whether maternal vaccination can indeed protect newborns against pneumococcal infections [56].

Conjugate vaccines against S. pneumoniae The second generation of PS-based conjugate vaccines stimulates stronger antibody responses, even in infants, young children and immune deficient individuals, as well as immunological memory. These vaccines also suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community leading to herd immunity, which adds considerable value to their implementation. The introduction of these vaccines in USA in 2000 resulted in a dramatic decline in the rates of invasive pneumococcal disease [1, 57, 58]. A significant reduction in the incidence rates among non vaccinated individuals was also observed as a result of herd immunity [59, 60]. However, the licensed seven-valent vaccine does not contain some of serotypes that cause severe disease in developing countries (i.e., serotypes 1 and 5). New conjugate vaccines including more serotypes, such as the ninevalent vaccine (Wyeth) and two 11-valent vaccines (GlaxoSmithKline and Sanofi-Pasteur), should provide better serotype coverage.

Protein-based subunit vaccines against S. pneumoniae New approaches to develop protein-based subunit vaccines against S. pneumoniae are currently being pursued by different research groups. This is

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expected to enable the generation of a universal vaccine conferring protective immunity against a large number of serotypes, as well as to avoid the complexity of manufacturing a conjugate vaccine [61]. There are different pneumococcal candidate antigens, such as the pneumolysin, neuraminidase, autolysin, pneumococcal surface protein A (PspA) and adhesin A (PsaA), which are in an early phase of clinical development [1]. In addition, several promising candidates have been identified, which are currently being tested in pre-clinical experimental models [1]. Among them, the two iron uptake ABC transporters of S. pneumoniae (PiaA and PiuA), which trigger protective immunity against invasive pneumococcal disease in mice. Through the screening of S. pneumoniae genomic expression libraries with sera from convalescent patients, bacterial surface proteins were identified (e.g., BVH3 and BVH-11) that promote the elicitation of protective anti-pneumococcal antibodies in mice [1]. A recombinant hybrid protein, BVH3/11V, has successfully been tested in toddlers and elderly volunteers. This candidate vaccine should be able to trigger serotype-independent responses, since the BVH3 and BVH11 antigens are common to all serotypes of S. pneumoniae.

Vaccines against typeable and non typeable Haemophilus influenzae Conjugated Haemophilus influenzae type b vaccines The major obstacle for developing an effective vaccine against H. influenzae capsular PS was related to the inherently poor immunogenicity of this T-cell-independent antigen. Antibody responses against PS are age-related, with extremely poor immunogenicity in infants during the first 18 months of life. Unfortunately, this age group exhibits the highest risk for invasive infections caused by H. influenzae. A PS-based vaccine against the H. influenzae type b (Hib) was licensed in the United States in 1985, for children more than 18 months old [62, 63]. The protective efficacy after licensure studies showed the inefficacy of this vaccine not only in infants, but also in older children [64]. This problem was solved by the generation of a conjugate Hib vaccine. To this end, the Hib PS (i.e., polyribosylribitol phosphate; PRP) was covalently linked to an immunogenic carrier protein, thereby leading to T-cell-dependent responses against the PS. Different conjugate Hib vaccines currently exist. These vaccines are HbOC, PRP-T and PRP-OMP, which make use of the mutant diphtheria toxin CRM197, the tetanus toxoid and the outer membrane protein from group B N. meningitidis as carriers, respectively. All of them trigger similar immune responses at the recommended doses. However, the dynamic of the elicited response may vary for each of them [65, 66]. Efficacy studies of these vaccines showed that they confer protection not only against meningitis, but also against pneumonia [67–69]. Although

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Hib vaccines are highly effective, their cost is still prohibitive for the world’s poorest nations. However, with the establishment of the Global Alliance for Vaccines and Immunization (GAVI), we have moved consistently ahead in making them also available for developing countries. GAVI has approved the establishment of a Hib initiative to support countries wishing to sustain Hib vaccination, as well as those exploring whether their introduction could be considered a priority in the near future.

H. influenzae typeable and non typeable: vaccination perspectives Although the introduction of conjugated PS vaccines has significantly decreased the prevalence of invasive Hib disease, paediatric infections due to non typeable H. influenzae (NTHi) are still highly prevalent. NTHi is most often associated with otitis media, sinusitis and bronchitis. In addition, NTHi is an important cause of lower respiratory infection in adults with chronic obstructive pulmonary disease (COPD). Thus, the development of a vaccine against NTHi is considered an important goal in public health. In contrast to Hib, vaccines against the non-encapsulated NTHi strains must be directed against alternative virulence factors. The lipoproteins D and P6 are widely distributed and antigenically conserved among H. influenzae strains, and also trigger the elicitation of protective immunity in animals vaccinated by mucosal route [70–73]. Thus, their incorporation in vaccine candidates might facilitate the generation of a universal vaccine against all typeable and non typeable H. influenzae.

Vaccines against Bordetella pertussis Even in the age of vaccine availability, B. pertussis continues to be a major cause of childhood morbidity and mortality (i.e., approximately 50 million cases and 300,000 deaths occur annually worldwide). Since the late 1940s, the incidence of whooping cough has dramatically decreased in most developed countries, as a result of widespread immunization. The first vaccine formulations, which are still in use, consist of preparations based on killed B. pertussis. The frequent incidence of minor adverse effects (e.g., fever, protracted crying and local erythematous reactions), as well as concerns raised by reports of serious neurological side-effects, resulted in a decline in vaccine acceptance and use [74]. This in turn led to a re-emergence of whooping cough and its complications. This serious problem prompted the development of a new generation of acellular vaccines. In 1981 Japan was the first country to successfully introduce acellular vaccines against whooping cough in its immunisation programme [75], leading to a consistent reduction in the reported side-effects. In the mid 1980‘s a major phase III trial of acellular vaccines was undertaken in Sweden, at a

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time when the banning of the whole cell vaccine had resulted in a pertussis epidemic in that country [76]. The first vaccine trials contained chemically detoxified pertussis toxin (PT) and filamentous haemagglutinin (FHA), or detoxified PT alone. The results of these trials showed that whilst producing good antibody responses, the vaccines failed to give an adequate level of protection in infants. The mono-component vaccine conferred no protection against infection, whereas the use of the two component candidate only gave incomplete protection against infection [77]. The results obtained in Japan and Sweden stimulated vaccine companies in the USA and Europe to establish vigorous research programmes aimed at the development of a new generation of acellular vaccines with higher efficacy. Currently available vaccines have incorporated chemically or genetically inactivated PT and additional virulence factors, such as FHA, the outer membrane protein pertactin (PRN) and fimbrial proteins (FIMs). The efficacy studies of this second generation of acellular vaccines have demonstrated that they confer levels of protection equivalent to the whole cell vaccines. The advent of improved techniques for antigenic characterisation and the introduction of acellular vaccines containing genetically defined components also resulted in a reduction of lot-to-lot variation in comparison with conventional whole cell vaccines and the acellular formulation originally introduced in Japan. However, despite the wide implementation of vaccination campaigns in infants and children, the disease continues to be endemic. In addition, in countries with high vaccine coverage we are now observing a consistent increment in the cases of pertussis in adolescents and adults [78–80]. These patients can then transmit the disease to infants, thereby now representing a primary reservoir for bacterial transmission and cycling in the community. The above-mentioned observations can be explained by one or more of the following factors: (i) improved detection techniques, (ii) major awareness on the possibility that bacteria may affect these age groups, (iii) vaccinedriven antigenic changes in circulating isolates, and (iv) reduction in vaccine efficacy over time. In this context, concerns have been raised about genetic variation between the strains used for vaccine preparation and circulating isolates. This seems to be true, since the currently used whole cell and acellular vaccines are prepared with strains that were isolated before mass vaccine introduction and show clear mismatches with respect to circulating strains. There is a steady tendency to decrease diversity in recent isolates, together with clonal expansion during epidemic outbreaks [81, 82]. Over time, at least two surface proteins (PT and PRN) may have changed sufficiently to allow for an increase in the incidence of disease. Unfortunately, our global information on antigenic variation and disease in adults and adolescent is extremely limited. Thus, despite widespread introduction of pertussis vaccines, it is essential to continue surveillance studies and collection of circulating strains. The present view is that successful control of pertussis in the community may require routine immunization of adolescents and adults

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with the new acellular vaccines, perhaps in combination with the diphtheria and tetanus toxoids (DTaP). This intervention might help in turn to reduce the burden of disease and transmission to infants.

Vaccines against Chlamydia pneumoniae Chlamydia pneumonia is an intracellular bacterium transmitted person-toperson via respiratory droplets. This pathogen is a common cause of pneumonia, with infections usually being oligosymptomatic or asymptomatic in young age groups. However, the rate of asymptomatic carriage in the normal population is unknown. There is also a tremendous gap in our understanding of host response to infections caused by C. pneumoniae. Most of the studies have been focused on the development of efficient diagnostic methods. However, less work has been done on vaccine development, and there is a paucity of knowledge on the microbial components which may serve as target antigens. In fact, at present there are no licensed vaccines against C. pneumoniae. However, the potential of different antigens, such as the major OMP2 [83] have been assessed in experimental animal models. Nevertheless, mice vaccinated with OMP2 using a protocol based on priming with DNA and boosting with recombinant VLP showed only partial protection [84]. Recent studies also suggested that CTL responses play a role in protection and clearance [85]. Animals immunized with a mini-gene encoding seven H-2(b)-restricted CTL epitopes fused to a endothelial reticulum-translocation signal showed protection following intranasal challenge with a virulent C. pneumoniae [85]. The current view is that multi-component vaccine will be required in order to induce a protective response [86]. Using the promising approach of reverse vaccinology combined with proteomics (see section “Reverse vaccinology”), the whole-genome of C. pneumoniae was screened searching for vaccine candidate antigens among exposed and immune accessible surface proteins [87]. The selected candidates were then expressed in a heterologous system and used in immunization studies. Approximately 53 proteins were able to trigger the elicitation of C. pneumoniae-binding antibodies. When tested in secondary screenings, six of them were also able to neutralize bacteria in vitro, and four inhibited systemic dissemination of C. pneumoniae in a hamster model [86].

Vaccines against Moraxella catarrhalis Moraxella catarrhalis is the third most common bacterial etiologic agent of otitis media in children. Furthermore, M. catarrhalis is an important cause of respiratory infections in patients with COPD. Thus, different studies have been carried out to characterize potential protective antigens. In

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this context, two major OMP (CD and E) have been identified, which are considered prime candidate antigens for vaccine development. These proteins are expressed on the surface and show a high degree of conservation among circulating strains. Both OMP triggered the elicitation of bactericidal antibodies and protective immunity in preclinical models [88]. Additional candidates are the UspA1 protein [89], which seems to be required for bacterial colonization of the human upper respiratory tract, the iron-induced OMP B1 and LBP, and the iron-repressed OMP B2 [90]. A conjugate vaccine based on detoxified lipo-oligosaccharide was also tested in mice by intranasal route with encouraging results [91, 92]. Some of these candidates are planned to be tested in clinical studies soon [90].

Vaccines against Mycoplasma pneumoniae Mycoplasmas are commensal microorganisms, as well as opportunistic pathogens. Mycoplasma pneumoniae is one of the causative agents of acute and chronic human respiratory diseases and the main responsible for primary atypical pneumonia, accounting for approximately 20–30% of all community-acquired pneumonia [93]. There is a considerable underreporting for M. pneumoniae-associated diseases. This is in part due to the wide diversity of clinical manifestations, the difficulties associated with its cultivation from clinical specimens and the lack of adequate diagnostic tools. No vaccines are currently available against this pathogen. However, studies conducted in human volunteers in the late 1960s demonstrated that a formalin-inactivated whole cell vaccine and an acellular extract were able to confer moderately protective immunity against M. pneumoniae [94]. Unfortunately, immune pathological reactions were observed following challenge with live organisms. Therefore, studies are still needed to understand the underlying mechanisms to the observed autoimmune responses [95]. More specifically, we need to elucidate the specific role played by humoral and cellular response in protection against M. pneumoniae. M. pneumoniae is one of the smallest self-replicating prokaryotic pathogens (approximately 800 kb). The complete genome sequence is now available. This is expected to expand our knowledge on the physiological and virulence properties of this agent, as well as new hints for vaccine development.

Vaccines against Legionella pneumophila A previously unrecognized bacterium was isolated after the outbreak of Legionnaires disease in 1976, which was designated Legionella pneumophila [96, 97]. The spreading of L. pneumophila is increasing due to the use of air-conditioners and humidifiers, since infections can occur by inhalation of aerosolized contaminated water sources. Several approaches have

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been developed in the fight against this facultative intracellular pathogen. Infection and immunization induce a rapid increase of antibody titres. However, antibodies do not seem to play a significant role in host resistance, particularly after aerosol challenge [98–100]. Some authors also suggested that these antibodies can promote bacterial phagocytosis, thereby favouring invasion and subsequent intracellular replication [101]. In contrast, cellular responses appear to be important for protection. Different vaccine candidates were tested in the past. Heat-, acetone- and formalin-killed L. pneumophila vaccines were not able to confer protective immunity in guinea pigs, whereas animals immunized with L. pneumophila membranes survive an aerosol challenge with virulent bacteria [98, 99]. Additional work demonstrated that also purified antigens, such as the major secretory protein [98], the major cytoplasmatic membrane protein [102], the peptidoglycan-associated lipoprotein [103], OmpS [104] and flagella [100] can confer protection against challenge with virulent L. pneumophila. Finally, different live attenuated mutants of L. pneumophila were used in animal infection models with promising results [105].

Vaccines against Pseudomonas aeruginosa Cystic fibrosis (CF) patients are particularly susceptible to severe bacterial infections of the lung, being Pseudomonas aeruginosa one of the most prominent etiologic agents. Thus, significant efforts have been invested to develop a vaccine against this pathogen. Surface PS are among the antigens that were most intensively assessed. Berna Biotech have developed an octavalent vaccine against the eight most prevalent serotypes based on O-PS conjugated with the exotoxin A [106–113]. A consistent reduction in the number of CF patients with chronic P. aeruginosa lung infection was observed in a cohort receiving the basic immunization protocol, followed by yearly boosters over a period of 10 years [112, 113]. The conjugate vaccine induced the production of specific IgG antibodies and increased the number of IgG memory B cells. It is still unclear if cellular responses might contribute to the overall protection conferred by this vaccine. However, strong proliferative responses of lymphocytes with a Th1 phenotype were observed in vaccinated individuals in response to the carrier exotoxin A protein [113]. Alternative vaccination strategies are currently being tested in clinical trials. Among them, formulations based on a fusion protein between the outer membrane proteins F and I, which have been administered by parenteral and mucosal routes [114, 115]. These formulations were demonstrated to be safe in volunteers and conferred increased protection against P. aeruginosa in CF patients. Cell-surface alginate, flagella, components of the type III secretion system, inactivated toxins and proteases are other proposed target antigens [116]. Some of them are already in clinical trials alone or in combination [116].

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New advances in vaccinology When Pasteur returned from his summer holidays in 1881 to continue with his studies on chicken cholera, he inoculated chickens with an old culture of Pasteurella multocida, which was left during the whole summer on his bench. The animals that received the preparation were protected against a challenge performed with a fresh isolate. Thus, Pasteur developed the hypothesis that pathogens could be attenuated by exposure to environmental insults (e.g., high temperature, oxygen and chemicals) [117]. The strategy was then successfully extrapolated for developing anthrax vaccines in livestock in the 1880s, with significant economic benefits. This was followed by the generation of attenuated vaccines against rabies and other important pathogens towards the end of the nineteenth century. Pasteur’s approach for “attenuating” or “inactivating” a pathogenic organisms still constitutes a cornerstone in vaccine technology [117]. This exemplifies that until recently the major achievements in vaccinology have been facilitated by technological (e.g., adjuvants, delivery systems, reverse vaccinology, genetic engineering) rather than immunological advances [117–119]. However, it is expected that the impressive knowledge accumulated in recent years in the fields of immunology, immune pathology and microbial pathogenesis will pave the road to a new golden era in vaccinology, in which knowledge and technology will enable rational vaccine design.

New technologies and approaches for vaccine development Reverse vaccinology In the 20th century, pertussis vaccines progressed from crude bacterial preparations to the highly purified antigens used for acellular vaccines. A similar quantum jump in technology allowed the development of subunit vaccines against influenza, Hib and S. pneumoniae, as well as the production of antigens by recombinant DNA techniques (e.g., genetically inactivated PT). Despite the fact that these techniques enable the production of almost any foreseeable antigen, the identification of suitable targets still remained as a main bottleneck for vaccine development [120]. The advent of genomics and its exploitation in the vaccinology field have rendered possible the implementation of a systematic and holistic approach for the screening, identification and prioritisation of candidate antigens. This new approach, called “reverse vaccinology” [121], does not require cultivation of the original pathogen, thereby being amenable for highlypathogenic or non culturable micro-organisms. It is possible to predict and select the most promising candidates by the analysis of genomic sequences in silico, which will then be cloned and expressed in heterologous systems. The resulting proteins are then used to perform immunological and/or

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functional studies to select the most promising candidates (e.g., able to induce the production of microbicidal or neutralizing antibodies, capacity to confer protective immunity). Flanking studies are usually carried out, such as molecular epidemiological analysis to assess their degree of conservation among circulating strains, or transcriptional profiling to evaluate their expression during natural infections [122]. The time-consuming process in which highly expressed components of an in vitro cultivable organism are identified (one at a time) and separated (different components between them) is one of the disadvantages that reverse vaccinology has solved. The conventional method usually requires 15–20 years to arrive to a clinical trial, whereas reverse vaccinology reduces the process to approximately 5 years. Reverse vaccinology also allows the identification of hundreds of potential candidates in a few days, in comparison with the small number of antigens that conventional approaches have provided after decades of research. Moreover, reverse vaccinology offers the possibility to select potential candidates independent of their expression levels or purification easiness. The reverse vaccinology approach has proved its usefulness in the field for both viral and bacterial pathogens (e.g. hepatitis C virus, Group B meningococci, group B streptococci) [123, 124]. Reverse vaccinology has also become an essential tool for several vaccine development projects against agents causing community-acquired pneumonia (e.g., C. pneumoniae, streptococci). The potential and speed of genomic-based approaches was also shown when the nucleotide sequence of the coronavirus causing SARS was made available in less than one month. In addition, the increasing number of available genomes from bacteria and viruses would allow comparative genomic studies, thereby providing hints on conserved protein families and/or functional domains. This would facilitate the generation of vaccines using immunogens covering multiple micro-organisms [125]. Despite the incredible potential of reverse vaccinology, this approach also has some important limitations (Tab. 1). Among them is the fact that it is not be possible to identify non-protein antigens (e.g., PS, glycolipids), which are the cornerstone for many successful vaccines (e.g., pneumococcal and Hib vaccines).

Reverse genetics Currently available influenza vaccines (see above) are based on inactivated viruses, and, more recently, attenuated ca viruses and virosomes. All these vaccines exploit the same starting material (wild-type virus), which is inactivated or attenuated. The last approach consists in the co-infection of chicken eggs with the new isolate and a master attenuated strain, and subsequent selection for re-assorted viruses with the desired genotype/phenotype. However, the virulence of certain virus strains, such as the H5N1,

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Table 1. Classical vaccine development versus reverse vaccinology Reverse vaccinology

Classical approach

Time required to reach the clinical phase

~ 5 years

~ 20 years

Type of organisms

Culturable and nonculturable

Only culturable

Antigens

Only proteins

Proteins, lipoproteins, polysaccharides and glycolipids

Genome

Necessary

Unnecessary

Target genes

All

Mainly in vitro expressed

Exclusion of known antigens

Yes

No

Need to handle microorganisms (e.g., highly pathogenic)

No

Yes

Surface and structural antigens

Yes (only proteins)

Yes

Internal antigens

Yes (only proteins)

Rarely

Antigens with low expression levels

Yes

Rarely selected

Number of candidate antigens

Many (more than hundred)

Few

Selection of antigens

Poorly or highly immunogenic

Mainly highly immunogenic

Antigens identification and separation

Easy

Could be difficult

Need for genetic tools during the initial discovery process

Not necessary

Usually essential (e.g., to create and complement mutants)

Need for genetic tools for antigen expression

Necessary for the initial phase of development

Only in a late phase of development

renders difficult the implementation of this traditional strategy. The use of reverse genetics represents a valid alternative for the generation of vaccines against RNA respiratory viruses, such as the influenza virus, PIV and RSV. It consists in the production of the virus from cloned DNA [126], thereby allowing the development of vaccines against any pandemic viral strain. In some cases (e.g., avian H5N1) an additional mutagenesis step would be required to attenuate its virulence [127]. Then, the new HA and NA segments would be transferred into an appropriate influenza A virus master strain adapted to grow in a cell line. The final re-assorted virus will have the antigenic specificity of the pandemic strain and the growth characteristics of the master strain [128, 129]. This technology would also allow production of the influenza vaccine in cells that are co-transfected with plasmids encoding for different frag-

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ments of the virus [130]. Therefore, the complete genome is inside the cell and virus can be produced and assembled. One of the main advantages is that a plasmid encoding for HA and NA can be easily replaced. Therefore, re-assortment and selection become unnecessary. This method would considerably reduce the time for vaccine production, from many months to only a few weeks. Another advantage would be the simple manipulation of the genome (contained in plasmids), which would enable detoxification of specific virulence factors. Similar approaches can be implemented for other viruses, such as RSV, PIV and SARS-CoV. However, intellectual property and liability issues are still obstacles for the industrial development of reverse-genetics-based vaccines [131]. Furthermore, since the resulting viruses are considered genetically modified organisms, additional problems may arise from the regulatory stand point [131].

Mucosal delivery systems Most of the infective agents are either limited to the mucosal membranes, or need to transit across them in order to cause disease. Therefore, it is highly desirable to elicit an efficient immune response at the local site in which the first line of defence is laid. The stimulation of a pathogen-specific response at the portal of entry is expected to impair infection (i.e. colonization), thereby reducing the risk of transmission to susceptible hosts. Parenterally administered vaccines mainly stimulate systemic responses, whereas vaccines given by the mucosal route mimic natural infections, thereby leading to efficient mucosal and systemic responses. Thus, there is a considerable interest in the development of mucosal vaccines. However, antigens administered by this route are usually poorly immunogenic. Different strategies are being pursued to overcome this bottleneck, among them can be cited the use of (i) advanced synthetic delivery systems, (ii) live attenuated bacterial or viral vectors, (iii) bacterial ghosts, (iv) pseudoviruses and (v) mucosal adjuvants [132–135]. Advanced synthetic mucosal delivery systems Particulate antigens are more immunogenic than those in solution, due to their vulnerability to degradation by enzymes and extreme pH. Thus, it would be helpful to incorporate them into a protective vehicle. Often, these vehicles do not serve only to protect them, but can also enhance their uptake, promote targeting to antigen presenting cells and serve as adjuvants [136]. The most commonly exploited delivery systems are: (i) gelatine capsules, which are dissolved at alkaline pH in the intestine but not in the stomach, (ii) muco-adhesive polymers that are highly viscous inert PS, (iii) eldexomer and carboxymethyl cellulose, which have been used for oral, nasal and vaginal delivery, (iv) lipid-based structures with

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entrapped antigens, such as immune stimulating complexes (ISCOMs) and liposomes, and (v) biodegradable micro/nano-spheres based on biocompatible materials such as starch, copolymers of lactic or glycolic acid [137, 138]. Some of these approaches are currently being explored to develop vaccines against agents causing community-acquired pneumonia. Encouraging results have been obtained, among others, using surface antigens from S. pneumoniae encapsulated in micro-spheres [139] and a ISCOM-adjuvanted vaccine obtained by reverse genetics against the influenza virus, in preclinical models [140]. Live attenuated bacterial or viral vectors Attenuated viruses and bacteria can be used not only as vaccine candidates per se, but also as delivery systems for heterologous antigens. Thus, many attenuated microorganisms have been exploited as a scaffold for the development of subunit vaccines against other agents, under the premise that the expression of the recombinant antigen(s) does not increase their pathogenic potential for humans or animals. The most frequently exploited bacterial vectors are attenuated derivatives of Salmonella enterica and Shigella spp., and the Bacille Calmette-Guérin (BCG). For example, vaccination with an attenuated Salmonella expressing the OprF-OprI was also shown to be able to confer protection against P. aeruginosa in a murine experimental infection model [141]. In addition, it was also demonstrated that a recombinant BCGbased vaccine expressing the PspA confers protection against S. pneumoniae in an infection animal model [142]. The use of commensals represents an alternative to attenuated organisms (e.g., lactobacilli). In this context it was demonstrated that oral administration of Lactobacillus expressing proteins from coronavirus can protect against a gastric infection [143]. Thus, this approach has been also proposed to combat SARS. Promising results were also obtained using x Chlamydia psittaci [144]. On the other hand, different attenuated viruses, such as MVA, bovine or attenuated hPIV-3 and adenovirus can be used as delivery systems for heterologous antigens [25, 145]. In fact, MVA has recently been exploited for antigens of the SARS associated coronavirus [146]. Bacterial ghosts An alternative approach to the use of live attenuated carriers is given by the use of bacterial ghosts. Ghosts are generated by the conditional expression of the lethal lysis gene E from bacteriophage PhiX174 in Gram-negative bacteria [147–151]. This leads to the formation of a trans-membrane tunnel through the bacterial cellular envelope [147]. Due to the high internal osmotic pressure, the cytoplasm content is expelled through the tunnel, thereby leading to an empty bacterial cell envelope [152]. The presence of envelope components in the ghosts provides a strong danger signal through the activation of pattern recognition receptors [153]. In addition, bacterial

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ghosts are efficiently taken up by antigen-presenting cells, stimulating their maturation and activation [154]. Bacterial ghosts retain all morphological, structural, and antigenic features of the cell wall and can be used as vaccine candidates per se. Ghosts can also be externally loaded with purified antigens. Alternatively, ghosts can be generated from recombinant bacteria expressing heterologous antigens, hence avoiding the difficulties associated with the purification steps. This technology also offers the possibility to manipulate the topology of the recombinant antigen (e.g., the antigen can be bound to the inner membrane, secreted into the periplasmic space or associated to the surface). Encouraging results has been obtained in preclinical models using ghosts expressing chlamydial antigens [135, 155]. Pseudoviruses as antigen delivery systems Promising results have been reported using different types of pseudoviruses, such as virosomes and virus-like particles (VLP), which are non-replicating viral-like structures. Virosomes are based on the principle of reconstituting empty viral envelopes through integration of viral envelope proteins in liposomes. They offer the versatility of liposomes in terms of lipid composition, with the advantage of including viral membrane proteins. Virosomes are produced by disassembling the viral membrane envelope with detergents. Then, the viral nucleocapsid is removed by ultracentrifugation before reconstitution (Fig. 1). In contrast, VLP exploit the capacity of recombinant viral coat proteins to spontaneously self-assemble, thereby mimicking at structural level the viral capsid. VLP can be isolated after protein expression in eukaryotic cells or by in vitro assemblage from subunits produced in an heterologous system [156]. Their main advantages are the lack the viral genetic material with an “intact” envelope, and the fact that they are significantly more immunogenic than soluble proteins. They can be used as vaccines per se, as well as a delivery system for protein- or nucleic acid based vaccines, or as carriers for small molecules. Foreign antigens can be expressed on their surface, or can be simply encapsulated. In addition, amphiphilic adjuvants can be incorporated into their membranes, thereby offering the advantage of combining an adjuvant and the antigen in one entity without a covalent attachment. Pseudoviruses are especially attractive for mucosal vaccination protocols, since they offer the opportunity to use the natural route of transmission of the agents. Induction of serum antibodies, secretory IgA, T helper and CTL responses, and protection against mucosal pathogen challenge has been reported from studies in animals and humans [157–159]. The virosomes generated using the influenza virus retain membrane fusion properties very similar to the naïve virus. Therefore, they are able to deliver material to the cytosol of target cells, offering the possibility to access the MHC class I-restricted pathway of antigen presentation to prime CTL activity [160–162].

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Figure 1. Virosomes are reconstituted viral envelopes, which incorporate the cell binding and fusion proteins of native virus without its viral genetic material. (a) Virosomes are produced by disassembling the viral membrane envelope with detergents. (b) The viral nucleocapsid is then removed by ultracentrifugation, and (c) they are reconstituted by removing the detergent with or without addition of lipids. (d) Electron-microscopy of an influenza virosome kindly provided by Etna Biotech.

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Mucosal adjuvants Bacterial toxins and their derivatives are among the first molecules that have been used as mucosal adjuvants. They are characterized by the presence of an A moiety with enzymatic activity, and a B moiety that mediates toxin binding to the target cells. Cholera toxin and the closely related Escherichia coli heat-labile toxin showed potent adjuvant activity when co-administrated with different antigens by the mucosal route [163–165]. However, their use in humans is hampered by their intrinsic toxicity. Thus, mutated derivatives were developed, in which the A subunit was modified to remove the ADP-ribosylating activity. The resulting polypeptides retain their adjuvanticity, in the absence of detectable toxicity [166–168]. However, additional studies have demonstrated that even these derivatives can lead to potential severe side-effects, such as retrograde homing of adjuvant and antigen to neural tissues [169]. This might explain, at least in part, the side-effects observed after intranasal vaccination against influenza with a virosomes-based formulation containing heat-labile toxin (i.e., Bell’s palsy), which in turn led to its retraction from the market. However, chimeric derivatives lacking the targeting moiety for neural tissues (i.e., B subunit) are now available [170]. They might allow the exploitation of the high potential of these molecules for the development of vaccines against respiratory pathogens. In fact, preclinical studies provided the proof-of-concept for the usefulness of derivatives of bacterial toxins in the generation of acellular vaccines against microorganisms, such as S. pneumonia and H. influenzae [171, 172]. Other bacterial components were also explored for their activity as adjuvants. The monophosphoryl lipid A retains much of the immune stimulatory properties of LPS, without the inherent toxicity [165]. On the other hand, extracellular matrix binding proteins, such as the fibronectin binding protein I of Streptococcus pyogenes, also exhibit adjuvant activity [173]. This offers the possibility of using them as dual antigen/adjuvant moieties in the same formulation. Recent reports also demonstrate that vaccine formulations containing adamantylamide dipeptide, a non-toxic compound obtained by linking the L-alanine-D-isoglutamine residue of the muramyl dipeptide to the antiviral drug amantadine, confer protection against non typeable H. influenzae in preclinical models [73]. The innate immune system plays a critical early role in host defence against pathogenic microorganisms through the recognition of pathogenassociated molecular patterns [174]. This is achieved through the stimulation of pattern-recognition receptors (PRR) that sense a broad range of exogenous and endogenous danger signals [153, 174]. Toll-like receptors (TLR) represent the best-characterized family of PRR. Natural and synthetic TLR agonists are being used as immune modulators to optimize responses after vaccination. Since the identification of the TLR4, many mammalian TLR homologues have been identified (i.e., 10 in humans

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Table 2. Toll-like receptors (TLR) and their ligands TLR

Ligands

TLR1 (with TLR2)

Mycobacterial lipoprotein (LP), triacylated lipopeptides, lipoteichoic acid (LTA)

TLR2

LPS (P. gingivalis), fungal products (zymosan), peptidogycan (PGN), LP, GPI anchors (T. cruzi) , lipoarabinomannan, muramyl dipeptide

TLR3

Viral dsRNA, synthetic Poly (I:C)

TLR4

Gram-negative bacterial products, LPS, respiratory syncytial virus, synthetic lipid A, E5564, plant products, saturated and unsaturated fatty acids, murine ß-defensin 2, BCG

TLR5

Flagellin

TLR6 (with TLR2)

Mycoplasma LP, LTA, PGN, diacylated LP

TLR7

GU-rich ssRNA, resiquimod, imiquimod

TLR8

GU-rich ssRNA, resiquimod, imiquimod

TLR9

Bacterial and viral DNA, unmethylated CpG-ODN

TLR10

Unknown

TLR11 (in mice)

Components from uropathogenic E. coli, and profiling-like from Toxoplasma gondii

and 13 in mice) [175]. Each TLR member binds specifically to different ligands (Tab. 2), alone or in combinations (e.g., heterodimers formed by TLR2 with either TLR1 or TLR6). An example of TLR agonist is bacterial DNA, but not vertebrate DNA, and synthetic oligodeoxynucleotides containing unmethylated CpG motifs. They act on TLR9, thereby inducing a strong Th1 responses by activation of dendritic cells [176, 177]. CpG motifs have been successfully used as adjuvants in preclinical studies of different candidate vaccines against agents causing community-acquired pneumonia [178-180]. Another important adjuvant with TLR-binding capacities is the Mycoplasma-derived macrophage-activating lipopeptide MALP-2, which act a the level of the TLR heterodimer 2/6 [181, 182]. MALP-2 promotes a global activation of cells from the innate and adaptive immune system [183, 184], such as macrophages, DC, T- and B-lymphocytes [183, 185]. When coadministered with an antigen by either the parenteral or the mucosal route, MALP-2 promotes the elicitation of humoral and cellular responses at systemic and mucosal level [186]. Preclinical studies suggested that MALP2 could be exploited in vaccine formulations against the SARS-associated coronavirus, M. catarrhalis and influenza virus, among others (unpublished data).

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DNA vaccines DNA vaccination offers some advantage over the normal antigen vaccination, such as the fact that it is not necessary to express any antigen. In contrast, it is the biosynthetic machinery present in the cells of the vaccinees that takes care of this work. Furthermore, since eukaryotic cells are in charge of protein synthesis, their glycosylation and folding are optimal. However, the large-scale purification of DNA might be associated with high costs. This can be solved by the use of attenuated or inactivated bacteria or viruses as delivery systems [187]. This approach can also lead to an enhanced induction of antibodies, which is otherwise poor using conventional naked DNA vaccines. We have recently demonstrated that bacterial ghosts can be also exploited as a delivery system for DNA vaccines for both in vivo and ex vivo applications [188]. The potential of this approach is demonstrated by the fact that it is possible to optimize performance by a broad range of manipulations, such as (i) choice of optimal promoters, (ii) use of codon optimized genes for expression in mammalian cells, (iii) addition of nuclear localization signals or ubiquitination signals to improve expression and processing, and (iv) co-delivery of DNA constructs coding for immune modulatory molecules [189]. In addition, by the presence of immune stimulatory CpG motifs, the DNA vaccine constructs has built-in adjuvant properties. This vaccination approach is particularly suited for the stimulation of cellular immune responses [190]. Interestingly, several reports suggest that DNA vaccines may represent a valid alternative to prime the neonatal immune system, even in the presence of passive transferred maternal antibodies [191, 192]. In fact, promising results were also obtained in preclinical models of community-acquired pneumonia, such as influenza [193] and S. pneumoniae [194]. Furthermore, DNA coding for vaccine antigens appears to induce excellent immunological memory, which can be reawakened by later immunization or exposure to the pathogen.

New immunological concepts that need to be addressed to optimise vaccine design The knowledge generated in several basic disciplines, such as immunology and microbial pathogenesis, has allowed the identification of critical bottlenecks for establishing a successful vaccination strategy. It is expected that in the coming years we will develop customized approaches to address each of them, in order to stimulate efficient protection against infective agents under specific clinical settings (i.e., newborns, aging individuals, immunocompromised patients).

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The importance of immunological memory B lymphocytes that have differentiated into plasma cells are the producers of antigen-specific IgG antibodies. Bone-marrow (BM) plasma cells have a short life, therefore, the BM reservoir needs to be replenished by the stimulation of memory B cells [195, 196]. The maximal life span of BM plasma cells is still debated. Only few factors have been identified that control the differentiation of antigen-specific B cells toward short- or long-life plasma cells or to memory B cells [119]. Beside the requirement of CD4 + T cells, the nature of the antigen [197] and the dose are also important. Higher antigen doses, as well as rapid vaccination schedules (closely spaced vaccine doses) tend to favour the rapid induction of short-term effectors, whereas lower doses of antigens preferentially support the induction of immune memory [198-201]. It was demonstrated that neonatal vaccination (priming) and infant boosting might be effective even when pathogen exposure occurs very early in life. In children in whom vaccine-induced Hib antibody titres have fallen to undetectable levels, memory is readily demonstrated [202]. However, immune memory per se is not enough to protect against pathogens that required high levels of neutralizing antibodies. The delay between memory B-cell reactivation and differentiation may limit the ability to interrupt pathogen invasion. Therefore, it is important to establish vaccination protocols in which the population is boosted at different ages in order to maintain the required levels of antibodies. This is particularly important in diseases in which antibodies play a central role in microbial clearance or toxin neutralization. In the particular case of community-acquired pneumonia, we should consider that aging individuals are neglected in many vaccination programs. However, the strategies proposed for elderly would be different from those used for small children, since the main factors affecting vaccine efficacy are immune senescence and immaturity, respectively. The attempts to give a rational solution to this issue are discussed in the next sections.

The immune system in children and elderly The immune system in children Immune responses to bacterial and viral antigens usually increase with age in a stepwise manner [203]. Prompt immunization after birth is required to induce active immunity against diseases that may occur early in life. Unfortunately, this strategy is limited by the relative immaturity of the neonatal and infant immune system. Some factors implicated in this poor response are the limited switch from IgM to IgG2 antibodies, impaired complement-mediated reactions and deficient organization of the splenic marginal zone. Vaccination studies performed in newborn mice suggested that limited germinal centre reactions may results from the delayed devel-

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opment of follicular DC and limit plasma cell differentiation [204]. It was also showed that the neonatal BM has a limited capacity to support the establishment of long-life antibody-secreting plasma cells [205]. Thus, the responses to glyco-conjugates and to most T-cell-dependent antigens are usually affected [119]. Therefore, only few and highly immunogenic vaccines show significant protective efficacy after a single dose in infants. The limited IgG responses are extended all over the first year of life. In addition, the immune responses, particularly antibodies, elicited in the first year of life after vaccination rapidly decline [203]. However, the problem observed in infants in terms of magnitude and duration of immune response does not seem to affect efficient priming. In fact, the immune memory generated in neonates may be recalled later in life [119]. Nevertheless, strategies to generate strong and long-lasting protective responses in infants are still needed. This is in part due to the presence of maternal antibodies, which inactivate and clear the vaccine antigens, thereby rendering difficult the stimulation of an immature immune system [203]. In addition, the effects of adjuvants reported in adults cannot be extrapolated to neonates [206]. A potential strategy to overcome these problems would be to implement vaccination during pregnancy, to provide the required antibodies by placenta and later by maternal feeding [30, 207–209]. This could be complemented with an early priming of the “immature” immune system of the newborn by DNA vaccination, followed by a boost during the second half of the first year or later in life [203]. The immune system in the elderly Poly-pathology and multiple organ failure is the rule rather than the exception in aging individuals. Thus, many systems are affected (e.g., endocrine, cardiovascular), and the immune system is not an exception. The mechanisms involved in the immune senescence process, which in turn may lead to poor response to vaccination, are not fully understood. However, it is clear that responses against certain vaccines are more affected by immune senescence than others (e.g., PS-based vaccines against S. pneumoniae) [210]. In contrast, the responses to a boost dose of the anti-tetanus vaccine are hardly affected by age [211]. A rapid decline of antibody responses, together with a relative restriction of the T-cell repertoire is characteristic of the immune senescence process. This restriction and the reduction in the pool of naïve cells can explain the poor CD4 + T cell responses against antigens that are cross-reacting with proteins which were seen earlier in life. In contrast, T-cells responses of healthy elderly individuals to new antigens are often unaffected. Nevertheless, the overall response to vaccination in the elderly is less efficient than in young adults, making more vigorous approaches necessary (Fig. 2). In the case of influenza, the actual strategy is annual re-vaccination. However, there are concerns regarding the capacity to increase antibodies with proper specificity against re-assorted viruses in aging adults

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Figure 2. Factors affecting the responses in young adults and aging individuals after vaccination. The process of immune senescence impairs host response to both infection and vaccination. This critical issue needs to be considered during vaccine design and will require the development of special approaches.

who have been repeatedly infected or immunized. After exposure to a new, but cross-reacting antigenic variant, such individuals may respond by producing antibodies. However, these antibodies could be primarily directed against influenza strains, which were encountered earlier in life.

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For example, individuals previously exposed to the “old” H1N1 influenza strain (i.e., 50 years ago), may respond differently from naïve adults who are vaccinated with a “new” H1N1 strain which have accumulated different mutations. The former might produce antibodies against the HA of the “old” H1N1 strain rather than to the cross-reacting epitopes of the new strain [212]. This is phenomenon is the so-called “original antigenic sin” [119]. On the basis of this observations, it was proposed that variations in vaccine efficacy might be due to differences in the antigenic distance between the vaccine strains and the epidemic strains responsible for influenza outbreaks [213]. However, this hypothesis was not confirmed by epidemiologic studies [214]. Even more, individuals aged 65 years or older who were annually vaccinated showed a significantly reduced mortality risk. Therefore, until now, it seems that the antigenic sin does not represent a major practical obstacle in influenza vaccination and additional strategies may not be required.

Concluding remarks Despite the broad availability of vaccines against agents causing community-acquired pneumonia, they still represent an important cause of death, human suffering and economic losses. However, we have dramatically expanded our knowledge on the pathophysiology of diseases caused by respiratory pathogens, their virulence factors and the effector mechanisms responsible for their clearance. It is becoming clearer which microbial components are attractive as vaccine targets, as well as the type of immune response needed to confer protection against disease. Thus, it is now possible to address vaccine development using rational rather than empiric approaches. This is facilitated by powerful bioinformatics tools for the accurate prediction of epitopes and proteasome trimming [215–217], as well as by the availability of a broad palette of immune modulators and delivery systems. Therefore, we can predict that new and improved vaccines against the etiologic agents of community-acquired pneumonia will considerably reduce the global impact of this disease in the coming years.

Acknowledgments This work was supported in part by grants from the DFG (GU482/2-3) and the BMBF (“PathoGenoMik” – Competence Center for Genome Research of Pathogenic Bacteria “Pathogenomik”, 031U213B) to CAG. We are particularly grateful to D. Felnerova from Etna Biotech, who provided us with a micrograph from a transmission electron microscopy of a virosome, and to M. Höfle for critical reading of the manuscript.

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Index adaptive immunity 183, 188, 190 adenovirus, vaccines 209 alternative activation pathway 155 alveolar macrophage 150 amantadine 82 amantadine-resistant virus strain 82 antibiotics 7 ff penicillins 8 macrolides 8 fluorquinolones 9 resistance 5 antigen, lipid and protein 194 antigenic drift 76 antigenic shift 76 antiviral compounds 82 antiviral therapy 31, 33, 36, 45 asthma, and M. pneumoniae 192 autoimmune reaction 191 bacterial ghosts 221 biological characteristics 184 bird flu virus 77 blood cultures 6 blood-brain barrier (BBB) 149 Bordetella pertussis, vaccines 212 bronchiectasis 191 bronchitis 79, 80 C. pneumoniae see Chlamydia pneumoniae C3 degrading activity 154 CAPNETZ 16 capsular polysaccharide 142 capsule regulation 153 CD4+ 79 CD8+ T-cell responses to influenza 79 cell wall hydrolase 144 cellular activation 188 chest x-ray 6 Chlamydia pneumoniae 15, 84, 86, 89–95 detection by PCR 86 differential diagnosis 84 immunity 89–93

reinfection 84 seroprevalence 84 virulence factors 95 vaccines 212 Chlamydia, nomenclature 84 Chlamydophila, nomenclature 84 choline-binding proteins (CBP) 144 chronic obstructive pulmonary disease (COPD) 80, 193 classical pathway of complement activation 154 clathrin-coated vesicle 150 clone, international (pneumococcal) 61 clone, pneumococcal 68 co-infection with microorganisms (influenzavirus) 78 combined bacterial and viral pneumonia 80 complement factor H 155 complement fixation (CF) 194 CRB-65 score 5 C-reactive protein 154 cytokine production 189 differential fluorescence induction (DIF) 151 DNA vaccines 226 Dot/Icm type IV secretion system 119 efflux, PmrA 65 elderly, immune system 228 encephalitis 81 encephalopathy 80 enolase 148 enzyme immunoassay (EIA) 194 extrapulmonary complication 190 extrapulmonary syndrome 191 factor H (complement) 155 failure, treatment 67 fluoroquinolone 196 fowl plague 77

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GAPDH 148 gene target 195 gene, mosaic 60 ghosts (bacterial) 221 G-protein pathway 150 Guillain-Barré syndrome 81, 191 Haemophilus influenzae 3, 15, 78, 80 non typeable, vaccines 212 type b (Hib), conjugated vaccines 212 vaccines 211 healthcare associated pneumonia 2 hemagglutinin (HA) 74–76 hemolytic anemia 191 hypogammaglobulinemia 189 hypotension 80 IFN-alpha, in influenza 79 Immunoglobulin subclasses, and diagnosis 194 immunologic hypersensitivity 189 immunological memory 227 influenza infection of neonates 80 influenza vaccines 202–204 influenza virus 73 adaptive immune response to 79 co-infection with microorganisms 78 influenza, laboratory diagnosis 204 innate immunity 79, 183, 188 interferon antagonism 78 interleukin (IL)-6 79 interleukin (IL)-8 79 ketolide, resistance 65 Legionella 15 dot/icm genes 112, 120 dot/icm mutants 117 egress 123 entry in host cells 117 growth phase 122 immunity 125 in free-living amoebae 113 Naip proteins 117 persistence 114 Toll-like receptors 125 transient association with amoebae 112 vesicles 118 Legionella pneumophila, vaccines 215 Legionnaires’ disease, outbreak 111 leukocyte recruitment 150 linezolid, resistance 66 lipoteichoic acid (LTA) 156

Index

local (mucosal) immune response 189 LPxTG sequence 144 lung abscesses 191 lymphocyte activation 189 lymphoid infiltration 189 macrolide 196 macrolide resistance 196 macrolide treatment 193 macrophage receptor, MARCO 161 manipulation of vesicle traffic 118 mast cell degranulation 193 microimmuno-fluorescence (MIF) 22 Moraxella catarrhalis, vaccines 212 mouse infection model 146 mucosal adjuvants 224 mucosal delivery systems 220 multi-locus sequence typing (MLST) 61 MurM protein 63 Mycoplasma pneumoniae 3, 15, 183, 184 adaptive immune response 190 adherence to epithelium 186 biological characteristics 184 cell biology 183 epidemiology 185 genome 184 pathogenesis 183 vaccines 215 myocarditis 80 myositis 80 naip5 117 natural reservoir of influenza-A-virus 76 neuraminidase (NA) 74, 76 neuraminidase (NanA, NanB, NanC) 145 neuraminidase inhibitors 82 neuraminidase subtype 76 neutralizing antibody directed against the HA 79 nucleic acid amplification assay 15, 20, 195 opsonophagocytosis 153 orthomyxoviridae 74 oseltamivir 82 otitis media 80 P1 adhesin 185 pandemic (influenza) 76 parainfluenza virus (PIV), vaccines 205 pathogen-associated molecular patterns (PAMPs) 156 Pce 150 PCR assay 15, 20, 195 penicillin-binding protein (PBP) 62

Index

pericarditis 80 pharmacodynamics 66 phase variation 141 phospholipase C 150 phosphorylcholine (PCho) 144 phosphorylcholine esterase 150 photophobia 80 PK/PD 67 plasmin(ogen) 148 platelet-activating factor (PAF) 148 pleural effusion 191 pneumococcal clone 61, 68 pneumococcal colonization 141 pneumococcal adherence and virulence factor A (PavA protein) 147, 148 pneumococcal pneumonia 142 pneumococcal serotype 140 pneumococcal surface adhesin A (PsaA) 146 pneumococcal surface protein A (PspA) 151 pneumococcal surface protein C (PspC, SpsA, CbpA) 147 pneumococcal molecular epidemiology network (PMEN) 61 pneumolysin 142 polymeric immunoglobulin receptor 147 primary atypical pneumonia (PAP), diagnosis 190 primary viral pneumonia 79, 80 progressive pulmonary failure 193 Pseudomonas aeruginosa 3 Pseudomonas aeruginosa, vaccines216 pseudoviruses 222 pulmonary fibrosis 191 pulmonary infiltrates 80 quinolone resistance-determining region (QRDR) 64 real-time PCR assay 195 receptor binding 74 recombination 60, 65 requirement for sterols 184 resistance, acquired 60 resistance, fluoroquinolone 64 resistance, macrolides 63 resistance, tetracycline 64 resistant virus variants 33, 35, 36, 39, 40 respiratory syncytial virus (RSV), vaccines 206, 207 respiratory viruses 28, 29, 42–44, 48 reverse genetics 218 reverse vaccinology 217, 219

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Reye’s syndrome 81 ribosomal protein 64 rimantadine 82 risk stratification 5, 6 secondary bacterial pneumonia 79, 80 secretory component (SC) 147 senescence, immune 229 seroconversion 194 severe acute respiratory syndrome (SARS), vaccines 208 signature-tagged mutagenesis (STM) 151 Spanish influenza 74, 76 Staphylococcus aureus 78, 80 streptococci, viridans 60, 63 Streptococcus pneumoniae 3, 5, 15, 78, 80, 209, 210 vaccines 140, 209, 210 Streptococcus, erm gene 64 Streptococcus, mef gene 63 streptogramin, resistance 66 telithromycin 196 telithromycin, resistance 65 terminal sialic acid 146 tetracycline 196 Toll-like receptor 156 transmission 185 transverse myelitis 81 treatment 7ff two-component regulatory systems (TCS) 152 urinary antigen 6, 20 vaccine 140, 202–216 adenovirus 209 B. pertussis 212 C. pneumoniae 214 H. influenzae 211, 212 influenza 202-204 L. pneumophila 215 M. catarrhalis 214 M. pneumoniae 215 P. aeruginosa 216 parainfluenza virus (PIV) 205 respiratory syncytial virus (RSV) 206, 207 S. pneumoniae 209, 210 severe acute respiratory syndrome (SARS) 208 vaccine design 226 vaccine, pneumococcal 69 vectors, live attenuated bacterial 221

250

vectors, live attenuated viral 221 vesicle traffic, manipulation 118 viral pneumonia 27, 32, 33, 37, 41, 42, 47 virosomes 222, 223 virosomes 223 virulence 183 virus detection 42-48 virus-like particles (VLP) 222 wheezing attacks in asthmatics 80 zanamivir 82 zinc metalloprotease 146

Index

The BAID-Series Birkhäuser Advances in Infectious Diseases Infectious diseases remain a substantial drain on human well-being and economies despite the availability of modern drugs. New pathogens emerge and known pathogens change their geographical distribution and their susceptibility to the available drugs. An understanding of the structure and function of infectious disease pathogens is a major scientific challenge with important potential applications. This new cross-disciplinary monograph series will provide up-to-date information on the latest developments in infectious disease research. The multi-authored volumes will cover basic biology and biochemistry of pathogens as well as applied medical aspects and implications for public health and policy. The contributions are written by leading infectious disease researchers and pharmaceutical scientists with a wide range of expertise. The envisaged readership includes academic and industrial researchers in medicine and infectious diseases as well as clinicians and others involved in diagnostics and drug development.

Forthcoming volumes: Pediatric Infectious Diseases Revisited, H. Schroten, S. Wirth (Editors), 2007 Available volumes: Coronaviruses with Special Emphasis on First Insights Concerning SARS, A. Schmidt, M.H. Wolff, O. Weber (Editors), 2005 The Grand Challenge for the Future. Vaccines for Poverty-Related Diseases from Bench to Field, S.H.E. Kaufmann and P.-H. Lambert (Editors), 2005 Poxviruses, A. Mercer, A. Schmidt, O. Weber (Editors), 2007