Cumitech 7B: Lower Respiratory Tract Infections

7B Lower Respiratory Tract Infections SUSAN E. SHARP, ANN ROBINSON, MICHAEL SAUBOLLE, MICHAEL SANTA CRUZ, KAREN CARROLL, AND VICKIE BASELSKI COORDINATING EDITOR

SUSAN E. SHARP

Cumitech CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY

Cumitech 1B

Blood Cultures III

Cumitech 2B

Laboratory Diagnosis of Urinary Tract Infections

Cumitech 3A

Quality Control and Quality Assurance Practices in Clinical Microbiology

Cumitech 5A

Practical Anaerobic Bacteriology

Cumitech 6A

New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide

Cumitech 7B

Lower Respiratory Tract Infections

Cumitech 12A

Laboratory Diagnosis of Bacterial Diarrhea

Cumitech 13A

Laboratory Diagnosis of Ocular Infections

Cumitech 16A

Laboratory Diagnosis of the Mycobacterioses

Cumitech 18A

Laboratory Diagnosis of Hepatitis Viruses

Cumitech 19A

Laboratory Diagnosis of Chlamydia trachomatis Infections

Cumitech 21

Laboratory Diagnosis of Viral Respiratory Disease

Cumitech 23

Infections of the Skin and Subcutaneous Tissues

Cumitech 24

Rapid Detection of Viruses by Immunofluorescence

Cumitech 25

Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing

Cumitech 26

Laboratory Diagnosis of Viral Infections Producing Enteritis

Cumitech 27

Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals

Cumitech 28

Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals

Cumitech 29

Laboratory Safety in Clinical Microbiology

Cumitech 30A

Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract

Cumitech 31

Verification and Validation of Procedures in the Clinical Microbiology Laboratory

Cumitech 32

Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife

Cumitech 33

Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism

Cumitech 34

Laboratory Diagnosis of Mycoplasmal Infections

Cumitech 35

Postmortem Microbiology

Cumitech 36

Biosafety Considerations for Large-Scale Production of Microorganisms

Cumitech 37

Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, and Wildlife

Cumitech 38

Human Cytomegalovirus

Cumitech 39

Competency Assessment in the Clinical Microbiology Laboratory

Cumitechs should be cited as follows, e.g.: Sharp, S. E., A. Robinson, M. Saubolle, M. Santa Cruz, K. Carroll, and V. Baselski. 2004. Cumitech 7B, Lower Respiratory Tract Infections. Coordinating ed., S. E. Sharp. ASM Press, Washington, D.C. Editorial Board for ASM Cumitechs: Alice S. Weissfeld, Chair ; Maria D. Appleman, Vickie Baselski, B. Kay Buchanan, Mitchell I. Burken, Roberta Carey, Linda Cook, Lynne Garcia, Richard M. Jamison, Karen Krisher, Susan L. Mottice, Michael Saubolle, David L. Sewell, Daniel Shapiro, Susan E. Sharp, James W. Snyder, Allan Truant. Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious use of clinical microbiology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians, laboratorians, and other interested stakeholders to provide a broad overview of various aspects of infectious disease testing. These aspects include a discussion of relevant clinical considerations; collection, transport, processing, and interpretive guidelines; the clinical utility of culture-based and non-culture-based methods and emerging technologies; and issues surrounding coding, medical necessity, frequency limits, and reimbursement. The recommendations in Cumitechs do not represent the official views or policies of any third-party payer. Copyright © 2004 ASM Press American Society for Microbiology 1752 N Street NW Washington, DC 20036-2904 All Rights Reserved

Lower Respiratory Tract Infections Susan E. Sharp Kaiser Permanente, 13705 Airport Way, Portland, OR 97230

Ann Robinson Sacred Heart Medical Center and Pathology Associates Medical Laboratories, 101 W. Eighth Ave., Spokane, WA 99220-2555

Michael Saubolle Laboratory Sciences of Arizona, Good Samaritan Regional Medical Center, 1111 E. McDowell Rd., Phoenix, AZ 85006

Michael Santa Cruz Microbiology Laboratory, Good Samaritan Regional Medical Center, 1111 E. McDowell Rd., Phoenix, AZ 85006

Karen Carroll Division of Medical Microbiology, The Johns Hopkins Hospital, The Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287

Vickie Baselski Department of Pathology, University of Tennessee at Memphis, 899 Madison Ave., Memphis, TN 38163

COORDINATING EDITOR: Susan E. Sharp Kaiser Permanente, 13705 Airport Way, Portland, OR 97230

Introduction ......................................................................................... 2 Clinical Aspects and Pathogenesis ......................................................... 2 Specimen Collection and Transport ........................................................ 3 Specimen Types ........................................................................................................ 3 Clinical Utility of Specimen Types ................................................................................ 5

Specimen Processing ............................................................................ 7 Primary Culture Media .......................................................................... 8 Gram Stain Interpretation and Reporting ................................................ 8 Overview .................................................................................................................. 8 Evidence Supporting the Utility of the Gram Stain ......................................................... 8 Gram Stain Screening Criteria ..................................................................................... 9 Interpretation and Reporting of Organisms in Direct Smears ........................................ 11 Summary ................................................................................................................ 12

Bacterial-Culture Evaluation in Lower Respiratory Tract Infections .......... 12 Protocol 1: Q-Score System ...................................................................................... 13 Protocol 2: Q234 System .......................................................................................... 13 Protocol 3: Association with WBCs Method ............................................................... 14

Implementing Change ......................................................................... 15 Lower Respiratory Tract Infectious Diseases and Agents ........................ 15 Acute Bronchitis ...................................................................................................... Bronchiolitis ............................................................................................................. Acute Pneumonia ..................................................................................................... Chronic Pneumonia .................................................................................................. Respiratory Cultures from CF Patients .......................................................................

15 17 17 21 23

Public Health Issues ............................................................................ 24 Frequency of Testing .......................................................................... 25 Reimbursement and Coding Issues ....................................................... 25 CPT-4 Codes ........................................................................................................... 25 1

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CUMITECH 7B ICD-9-CM Codes ...................................................................................................... 26 DRG Codes ............................................................................................................. 26 Data Review ............................................................................................................ 26

References ......................................................................................... 26

INTRODUCTION The major goal of the clinical microbiology laboratory is to provide information of maximal clinical or epidemiological usefulness as rapidly as is consistent with acceptable accuracy and minimal cost. Jay P. Sanford, M.D. (242) The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen. Raymond C. Bartlett, M.D. (25)

T

hree decades later, the words of Sanford and Bartlett are still true. We continue to develop and apply methods that hopefully will utilize the resources of the microbiology laboratory more effectively and efficiently while endeavoring to determine the relevance of these diagnostic methods. This Cumitech is intended to guide the practice of clinical bacteriology as it applies to the culture of lower respiratory tract secretions. Clinical aspects of diseases associated with the lower respiratory tract, specimen collection, specimen processing, and interpretation of bacterial cultures are presented. The most common organisms associated with infections of the lower respiratory tract are discussed, along with methods for implementing change in the laboratory, guidelines for frequency of testing, public health issues, and reimbursement codes for these specimens. We hope that the reader will find within these pages useful guidance for continuing education in the bacterial analysis of lower respiratory tract specimens with the goal of “providing information of maximal clinical usefulness as rapidly as is consistent with acceptable accuracy and minimal cost.”

CLINICAL ASPECTS AND PATHOGENESIS The human oropharyngeal tract harbors large numbers of aerobic, as well as anaerobic, microorganisms (243). In the gingival crevices, anaerobic organisms may outnumber aerobic organisms 10-fold. The oral flora may reach total bacterial counts of 1010 to 1012 CFU/ml. Sublaryngeal bacterial colonization is minimal in the healthy host but increases in patients with chronic lung illness or those requiring intubation. The dynamics of these organism populations are affected by the patient’s overall health and immediate environment. Differing flora may be seen in patients with

underlying conditions, such as immunosuppression, diabetes mellitus, alcoholism, or chronic lung disease. Patients receiving broad-spectrum antimicrobial therapy or individuals having protracted exposure to others harboring more invasive or antibiotic-resistant organisms may also present with various species of flora. Thus, through continued exposure, the frequency of colonization with Streptococcus pneumoniae and Haemophilus influenzae can increase in parents with children enrolled in day care centers. Although the normal aerobic oropharyngeal flora consists primarily of gram-positive organisms, the prevalence of gram-negative bacilli increases dramatically in acutely ill patients receiving antimicrobial therapy and hospitalized for several days, in chronic alcoholics, in diabetics, and in the institutionalized elderly population (132). Aspiration of colonizing flora into the alveoli is the most common mechanism initiating a pneumonic infection. Prior to aspiration, there is often a change in the flora from benign bacterial species to more invasive or resistant organisms, such as S. pneumoniae or gram-negative bacilli. Such changes in flora may be precipitated by a number of events, including viral infection or antibiotic use. Asymptomatic aspiration is common even in healthy hosts, and the organisms are usually cleared by the mucociliary apparatus. The outcome of any invasion of the pulmonary tree depends on the balance between the virulence and inoculum size of the aspirated species and the status of the patient’s immune system and respiratory function. Inhalation of aerosols is a second, albeit less frequent, mechanism for microorganism access to the lower respiratory tract. In home care situations, poorly cleaned respiratory equipment is often to blame for transmission. Other prime examples of aerosolization-caused pneumonia include Mycobacterium tuberculosis (associated with droplet nuclei), Legionella spp. (associated with shower heads), dimorphic fungi (associated with dust storms and bird droppings), Aspergillus spp., mycoplasmas, chlamydiae, and Coxiella burnetii (243). Hematologic seeding of the lung from a distant focus is a third, but least common, mechanism for initiating an infection in the lower respiratory tract. This type of pneumonia may be seen in patients who are intravenous-drug abusers or who are receiving hemodialysis. The infectious process may involve

CUMITECH 7B

both of the lower lobes of the lungs, since blood flows preferentially to those areas.

SPECIMEN COLLECTION AND TRANSPORT Specimen Types There are a number of specimen types, corresponding to the various inflamed areas of the lower respiratory tract, that may be submitted for microbiological analysis. These samples may be obtained noninvasively or by an invasive bronchoscopic or transthoracic procedure. In addition, for specific bronchial pathogens, it may be appropriate to submit upper respiratory samples (e.g., throat or nasopharyngeal) for detection of the suspect agent. For a select group of infectious agents, urine may be submitted for antigen testing, and in a few situations, serum may be collected to establish a retrospective diagnosis using serologic testing. Noninvasive Samples Upper Respiratory Swabs

In cases of bronchial, bronchiolar, or alveolar infection, throat swabs are collected perorally, taking care to swab the tonsillar area vigorously, for detection of Mycoplasma pneumoniae or Chlamydia pneumoniae (125, 248). Alternatively, nasopharyngeal swabs may be collected, particularly from pediatric patients and preferentially for the detection of Bordetella pertussis (105). Swabs for culture should be placed into an appropriate transport medium to maintain organism integrity for analysis as specified by the method. Specific mycoplasmal and chlamydial transport media are available, including some appropriate for both agents, such as M4 (Remel, Lenexa, Kans.). For B. pertussis, a different transport medium is preferred, such as 1% Casamino Acid solution or Amies or Stuart’s medium with charcoal. For molecular assays, assay-specific transport devices are used to maintain the integrity of the nucleic acid targets (225). Sputa

Sputum contains a composite of microorganisms which may be responsible for inflammation in the lower respiratory tract and host cellular, proteinaceous, and other secretory materials produced in the lungs as a result of the inflammatory response. Sputum is moved upwards from the deep lung spaces into the trachea by coughing and, if unimpaired, by mucociliary activity until it reaches the oral cavity. Sputum may be naturally expectorated and used for analysis, or it may be necessary to nebulize the patient with a warm saline solution to induce coughing. To minimize superficial contamination of the specimen with colonizing flora from the upper respiratory tract and

Lower Respiratory Tract Infections

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the oral cavity, patients should be instructed to remove dentures, if present, and to rinse their mouths with water prior to collection of a sample. Either expectorated or induced sputum may be used to assist in the diagnosis of a lower respiratory tract infection (23). Sputum is most commonly used for stains and cultures, and the sample should be transported to the laboratory as soon as possible and processed immediately upon receipt (49). Several classic studies have demonstrated that when sample delivery is delayed prior to analysis, there is both loss of significant pathogens (191) and overgrowth of pathogens by nonfastidious colonizing flora (187). Although definitive data are unavailable, a 2-h maximum delay between collection and processing of specimens is generally accepted. This goal is particularly problematic in large, consolidated, off-site laboratory settings, and alternative specimen-handling protocols have been developed, including refrigeration of samples during transit and culture medium inoculation on site with subsequent transport of the inoculated medium. Although not rigorously evaluated, these modifications would seem to offer an improved chance for recovery of pathogens. Tracheal Aspirates

Patients with serious pneumonia often require ventilatory support and are intubated. Patients who are intubated for respiratory failure due to another cause are also at risk for developing ventilator-associated pneumonia. In these patients, tracheal or endotracheal suctioning may be used to collect secretions for detection of the etiological agent of a lower respiratory tract infection (176). On occasion, saline may be used to dilute the viscous secretions and facilitate collection. Tracheal secretions should be considered comparable to sputum for analysis by stain and culture, with application of the same requirements for minimizing contamination and expediting transport and processing. Blood Cultures

Blood cultures are generally indicated in serious lower respiratory tract infections requiring hospitalization, such as pneumonia. Sample collection should be in accordance with the published guidelines in Cumitech 1B (Blood Cultures III) (85). Urine

Antigenuria testing detects renally excreted microbial antigens. Tests are currently available for Histoplasma capsulatum, S. pneumoniae, and Legionella pneumophila. Serum

Serum samples may be collected to support a specific diagnosis when the patient presents after the acute phase of the infection or if the etiological agent

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is difficult to detect by direct testing methods. Serologic testing may be performed for M. pneumoniae, C. pneumoniae, and Legionella spp. in particular (125). In addition, serology may be a useful adjunct in the diagnosis of infection caused by organisms with bioterrorism potential, such as Francisella tularensis and Yersinia pestis. Bronchoscopic Samples Bronchoscopy Specimens

Fiber optic bronchoscopy is frequently performed for patients with serious pneumonia requiring hospitalization, particularly for immunocompromised patients (29) and in the setting of ventilator-associated pneumonia (VAP) (30). The procedure provides both a visual analysis of the gross findings in the lower respiratory tract and the opportunity to collect goodquality samples from the actual site of infection. Sampling areas that correspond to areas of radiologically demonstrated inflammation are generally selected, but these areas may be difficult to identify in patients with acute respiratory distress syndrome. There are several technical issues that may compromise the quality of the sample obtained. These factors include, but are not limited to, contamination of the working channel of the bronchoscope during passage of the scope and suctioning, suppression of the growth of some bacteria by topical anesthetic agents, the quantity of fluid instilled into the alveoli and the amount aspirated subsequently, and localization of the affected area of the lung (30). Newer “bedside” modifications of the procedure that are not fiber optically guided but that nonetheless allow collection of similar but lower-volume samples may be performed for critically ill patients. Care should be taken to avoid excessive topical lidocaine use due to possible inhibition of pathogen recovery (290), and the same guidelines described for sputum apply for expedient transport and processing. The types of samples collected include the following (29, 30). Bronchial Washings

Bronchial washings are the secretions aspirated through a bronchoscope channel after instillation of saline into a major bronchus. These washings are considered similar in quality to sputa or endotracheal aspirates. Oropharyngeal contamination is likely, and the utility of bronchial washings is generally limited to the diagnosis of recalcitrant bronchitis (an inflammatory condition of the tracheobronchial tree) and bronchiolitis (an acute viral lower respiratory tract illness occurring during the first 2 years of life) (109, 120, 212). Occasionally, samples also may be used to diagnose pneumonia caused by strictly pathogenic organisms, such as M. tuberculosis ordimorphic fungi, or in situations in which the bronchoalveolar lavage (BAL) aspirated volume is inadequate.

CUMITECH 7B

Bronchial Brushings

Conventional bronchial brushings are collected with a stiff brush designed to obtain samples from large-airway walls for exfoliative cytologic diagnosis of malignancy. The brush is not protected from oropharyngeal contamination during passage through the bronchoscope channel and thus is not generally used for routine cultures. However, the samples may be useful for the detection of strict pathogens by stain or culture, and they are particularly helpful for the detection of cytopathologic changes associated with viral infection of airway epithelial cells. PSB

Protected specimen brushings (PSB) are collected using a small flexible brush located within two telescoping catheters. The outermost catheter is occluded with a wax plug that protects the inner catheter and brush from upper-airway contamination. After the device has passed through the main bronchoscope channel to the desired level of the bronchial tree, generally the third subsegmental bronchus, the inner catheter is pushed to expel the plug, which eventually dissolves, and the brush is extended to collect secretions from the distal bronchioles. Protected specimen brush bristles are designed to collect from 0.001 to 0.01 ml of material. After specimen collection, the sequence is reversed. The tip is drawn into the inner catheter; then the inner catheter passes into the outer catheter, and the device is removed. After the outer and then the inner catheter tips are cleaned with alcohol, the brush is advanced, aseptically cut, and placed in 1 ml of diluent, generally a saline solution. The brush, submerged in diluent, should be taken to the laboratory as soon as possible for processing. BAL

BAL provides one of the largest-volume and most versatile samples for the laboratory diagnosis of lower respiratory tract infections. In this procedure, the bronchoscope channel is wedged tightly into an airway lumen, generally at the level of the third subsegmental bronchus, effectively isolating that segment. A variable volume of saline is injected through the lumen and aspirated out in three or four aliquots. The volume of saline instilled ranges from ⬎160 ml in a typical procedure to ⱕ20 ml in a bedside, nonbronchoscopic “mini-BAL” procedure, using a Metras catheter. The volume returned by aspiration varies with the volume instilled and ranges from 10 to 100 ml. It is estimated that ⬎1 million alveoli are sampled by this technique, with approximately 1 ml of actual lung secretions obtained (168). To avoid excessive bronchial contamination, the initial aliquot is enriched for secretions from the bronchus and is thus similar to bronchial washings. This aliquot is generally discarded prior to performing bacterial cultures.

CUMITECH 7B Table 1.

Lower Respiratory Tract Infections

Suitabilities of bronchoscopically obtained samples for various diseasesa

Sample type

Suggested workupb

Disease

Bronchial washings

Useful only for pneumonia caused by strict pathogens, such as M. tuberculosis, Legionella, endemic fungi

PSB BAL

Useful only for diagnosis of bacterial pneumonia Useful for all tests for opportunistic pathogens

TBB

Useful for diagnosis of malignancy, sarcoid; limited role in pneumonia

a b

5

Plate to selective medium for pathogen of interest. Reasonable requests include mycobacteria; fungal media; stains for fungi and mycobacteria; DFA for Legionella and Pneumocystis. Quantitative bacterial culture and Gram stain Quantitative bacterial culture Mycobacteria stain and culture Fungal stain and culture Viral DFA and culture Pneumocystis DFA Legionella DFA and culture Histopathologic assessment for tumor, noninfectious diseases; possibly helpful with Pneumocystis and M. tuberculosis

Table derived from reference 30. DFA, direct fluorescent-antibody assay.

A protected BAL procedure has been described that uses a balloon device and a distally occluded tip to more effectively segregate the bronchus from the sampled area prior to collection of the aspirates (183). For Legionella culture, concerns about possible saline toxicity have prompted the collection of a water aliquot by some clinicians to enhance organism recovery (88). Table 1 indicates the suitabilities of samples obtained by bronchoscopy for various diseases. TBB

Transbronchial biopsy (TBB) samples are obtained by using forceps designed to snip small pieces of tissue from peribronchial or alveolar areas. These samples are used primarily to document noninfectious disorders, such as sarcoidosis and neoplasms, or to document active tissue invasion by opportunistic organisms, such as fungi and herpesviruses, both through histopathologic examination. These samples also may be used for other microbiological analyses, but the amount of tissue is limited and there is a risk of upper-airway contamination of the specimen. TBNA

Transbronchial needle aspirates (TBNA) are similar to TBB samples and are collected by direct aspiration from a lesion identified radiologically. Their utility and limitations are similar to those of TBB samples. Other Invasive Specimen Types Transtracheal Aspirates

Transtracheal aspirates were popularized in the 1970s as a specimen suitable for anaerobic culture. Samples are collected by direct needle puncture of the trachea, with the use of a small catheter to aspirate secretions from lower-airway spaces. The risk of contamination with flora from the upper airway is lower than for sputum, but the procedure causes discomfort

to the patient and has been largely replaced by bronchoscopy to obtain samples (77). Pleural Fluid

When infection and inflammation occur in the pleural space, aseptic transcutaneous aspiration or drainage of pleural fluid provides a specimen from a normally sterile site that is appropriate for direct stains, culture, and direct antigen or molecular detection of pathogens (including special pathogen groups, such as anaerobes) (282). Transthoracic-Needle Biopsies

Transthoracic-needle biopsies may be performed when a radiologically confirmed lesion is accessible by direct needle puncture. This sample type is similar to TBB and TBNA in quantity, utility, and limitations due to the limited amount of material obtained. Open-Lung Biopsy

Tissue may be obtained following a surgical procedure, particularly when alternative specimen types have failed to yield an etiology in patients for whom the establishment of a diagnosis is critical for management. Samples may be used for virtually any microbiological procedure, and protocols to ensure appropriate handling by surgical pathology and microbiology should be established (65). Clinical Utility of Specimen Types General Concepts With the great diversity of specimen types available for the diagnosis of lower respiratory tract infections, as well as the great diversity of potential etiological agents in several distinctive clinical settings, it is absolutely critical to establish guidelines for specimen collection. The specimen collection guidelines should be in compliance with clinical-care guidelines established for community-acquired (14, 40) and hospital-

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CUMITECH 7B

Table 2.

General guidelines for specimen collection and transport Specimen type

Collection device

Transport conditionsb

Upper respiratory (throat or nasopharyngeal) swabs Sputum Tracheal aspirate Blood cultures Urine Serum Bronchial washings Bronchial brushings

Pathogen-specific transport system Sterile cup Sterile cup or aspiration tube Blood culture bottles Sterile cup or tube Serum tube Sterile cup or tube Sterile cup or tube with several milliliters of saline (use cytology fixative as required)

PSBa BALa TBB

Sterile tube with 1 ml of saline Sterile cup or tube Sterile cup with moistened gauze in bottom (use formalin for histology) Sterile tube Sterile cup Sterile tube (anaerobic if indicated) Sterile cup with moistened gauze in bottom (use formalin for histology) Sterile cup with moistened gauze in bottom (use formalin for histology)

Up to 24 h, RT 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C RT 24 h, 4°C 24 h, RT 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C (cytology fixative, indefinite) 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C (formalin, indefinite) 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C 2 h, RT; ⬎2–24 h, 4°C (formalin, indefinite) 2 h, RT; ⬎2–24 h, 4°C (formalin, indefinite)

TBNA Transtracheal aspirate Pleural fluid Transthoracic needle biopsy Open-lung biopsy a b

For Legionella isolation, a sterile-water lavage may be preferred because of potential saline toxicity. RT, room temperature.

acquired (52) lower respiratory tract infections. Further, these guidelines should be clearly communicated to clinicians and other health care personnel directly caring for patients and clinical laboratory personnel involved in microbiological testing. Tables 1, 2, 3, 4, 5, and 6 are designed to assist in the development of such guidelines for the laboratory diagnosis of lower respiratory tract infections caused by bacteria. Specimen Guidelines General guidelines for appropriate specimen collection devices and transport conditions needed to assure bacterial-pathogen integrity on receipt in the laboraTable 3.

tory are given in Table 2. Note that there may be special requirements for the fastidious organisms referred to in this document. Testing Guidelines According to the laboratory test method, guidelines for specimen selection are outlined in Table 3. Respiratory specimens may be used for a number of diagnostic methods, including cytology or histopathology, direct microscopy for microorganisms, routine or special selective culture, direct antigen detection by immunofluorescence or enzyme immunoassay methods, and direct or amplified probe methodology.

Utilities of respiratory-specimen types according to laboratory method Utility fora

Specimen type

Direct microscopy

Culture

Antigen detectionb,c

Molecular detectionc

Serologyc

Upper respiratory swab Sputum Tracheal aspirates Blood Urine Bronchial washings Bronchial brushings PBB BAL TBB TBNA Transtracheal aspirate Pleural fluid Transthoracic needle biopsy Open-lung biopsy

⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ (whole blood) ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫹ (serum) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫹, useful; ⫺, not useful. Primarily immunofluorescence. c For specific microorganism information, refer to Table 5. a

b

CUMITECH 7B Table 4.

Lower Respiratory Tract Infections

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Utilities of specimen types according to clinical condition Utility forb

Specimen typea

Bronchial infection

Upper respiratory swab Sputum Tracheal aspirates Blood cultures Urine Serum Bronchial washings Bronchial brushings PSB BAL TBB TBNA Transtracheal aspirate Pleural fluid Transthoracic needle biopsy Open-lung biopsy

(⫹) ⫹ NA ⫺ ⫺ (⫹) (⫹) (⫹) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

a b

Community-acquired pneumonia Outpatient

Inpatient

(⫹) ⫹ NA ⫺ (⫹) (⫹) ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

(⫹) ⫹ (⫹) ⫹ (⫹) (⫹) ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ (⫹) ⫺ ⫺

Microorganism guidelines It is also important to define the specimen types that may be most useful in the detection of specific pathogens, particularly those requiring special techniques. Table 5 summarizes the specimen types recommended Table 5.

VAP

Immunocompromised patient

CF patient

⫺ ⫹ NA (⫹) (⫹) (⫹) ⫺ ⫺ ⫹ ⫹ (⫹) (⫹) (⫹) (⫹) (⫹) (⫹)

⫺ ⫹ (⫹) ⫹ (⫹) (⫹) ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ (⫹) (⫹) ⫺ ⫺

⫺ ⫺ (⫹) ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ (⫹) ⫺ ⫺

(⫹) ⫹ ⫺ ⫹ (⫹) (⫹) ⫺ (⫹) (⫹) ⫹ (⫹) (⫹) ⫺ (⫹) (⫹) (⫹)

(⫹) ⫹ ⫺ (⫹) ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

for identification of the pathogens described in this document.

SPECIMEN PROCESSING As always, specimens should be transported in sterile leakproof containers. For BAL samples, the initial bronchial fraction should be discarded. The alveolar samples should be aseptically divided into portions designated for cytologic analysis, chemical and microbiological tests, and cultures. Pieces of tissue collected by TBB should be placed in a sterile container with a small amount of nonbacteriostatic saline.

Specimens commonly obtained to support a specific etiological diagnosis Etiology

B. pertussis M. pneumoniae

C. pneumoniae

Anaerobes Legionella species

Nocardia and related organisms Burkholderia species F. tularensis Y. pestis S. pneumoniae

b

Hospitalacquired pneumonia

For specific microorganism information, refer to Table 5. ⫹, commonly collected if available; ⫺, not typically collected; (⫹), collected in selected clinical settings; NA, not applicable.

Diagnosis Guidelines Guidelines for specimen collection according to clinical condition are outlined in Table 4. The primary distinguishing parameters are the anatomic area of the lower respiratory tract that is believed to be the site of infection and the clinical circumstances surrounding the patient’s illness.

a

Chronic pneumonia

Specimen typesa NP swab (collected in medium such as ReganLowe or Jones-Kindrick) NP swab (collected in mycoplasma transport medium such as M4); bronchoscopic specimen Serum NP swab (collected in transport medium such as M4); bronchoscopic specimen Serum TTA, pleural fluid, PSB, PBAL PSB, BAL Serum Urine Sputum, BAL, TBB, TBNA, TTNA, OLB Sputum, BAL Sputum; bronchoscopic specimen Serum Sputum; bronchoscopic specimen Serum Sputum; bronchoscopic specimen Urine

Use Selective culture; molecular testing Selective culture; molecular testing Serology Selective culture; molecular testing Serology Gram stain and culture Selective culture; DFAb; molecular testing Serology Antigenuria Gram and acid-fast stains; culture Gram stain and culture Gram stain and culture Serology Gram stain and culture Serology Gram stain and culture Antigenuria

NP, nasopharyngeal; TTA, transthoracic aspirate; PBAL, protected BAL; TTNA, transthoracic needle aspirate; OLB, open-lung biopsy. DFA, direct fluorescent-antibody assay.

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Table 1 summarizes the suitability of each of the above-mentioned sample types for the diagnosis of various disease entities. PSB specimens received in diluent should be vortexed and cultured for bacteria using quantitative methods. BAL samples likewise should be vortexed and then cultured quantitatively for bacteria. The remaining specimen should be concentrated by centrifugation at 1,500 to 1,800 ⫻ g for 15 to 20 min to be used for other cultures. An aliquot of the sample should likewise be spun in a cytocentrifuge for preparation of various direct stains, such as the Gram stain (30). For tissues obtained by TBB, touch preparations can be made for direct smears, followed by homogenization of the sample in nonbacteriostatic saline for inoculating cultures. Quantitative bacterial cultures are recommended for specimens obtained by PSB and BAL. The thresholds for significance are 103 CFU/ml for PSB and 104 CFU/ml for BAL. Two approaches for quantitative culture are acceptable: the serial-dilution method and the calibrated-loop method. In the serial-dilution method, two 100-fold dilutions are made, and colony counts are obtained from 0.1-ml amounts of the diluted specimen inoculated onto media. Counts are made from the plate containing between 30 and 300 colonies. The results are expressed as CFU per milliliter (30). For the calibrated-loop method, 0.1 ml of PSB and 0.001 and 0.01 ml of BAL are inoculated onto agar media. The results are expressed as log10 ranges of bacteria. With each approach, all morphotypes should be quantitated and reported. Those organisms whose numbers approach or exceed the threshold for significance should be identified and have susceptibility testing performed. Those bacteria present in smaller quantities should not be completely characterized.

PRIMARY CULTURE MEDIA The majority of the causative bacterial pathogens of lower respiratory tract infections can be detected on common laboratory media. Sputum, endotracheal aspirates, and specimens obtained by bronchoscopy should be inoculated onto 5% sheep blood agar, MacConkey agar, and chocolate agar. The chocolate agar plate is incubated at 35°C in 5% CO2 for optimal recovery of H. influenzae. Since all of these specimens can be heavily contaminated with normal respiratory flora, neither enrichment broth nor anaerobic media are appropriate. Anaerobic cultures may be performed for specimens obtained by percutaneous aspiration (rarely performed) or for PSB samples if specifically requested. Organisms with special growth requirements, such as Legionella spp. and B. pertussis, may also be recovered from the above-mentioned specimen types. How-

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ever, selective media are required for successful isolation. For Legionella culture, both buffered charcoal yeast extract agar and selective buffered charcoal yeast extract plates incubated at 35°C for at least 3 to 5 days are recommended. Regan-Lowe media with and without cephalexin are ideal for B. pertussis. Specimens from patients with cystic fibrosis (CF) require special handling and are discussed in detail elsewhere in this document (see “Respiratory Cultures from CF Patients” below).

GRAM STAIN INTERPRETATION AND REPORTING Overview Gram-stained smears of lower respiratory tract secretions provide rapid presumptive information to guide the initial selection of antimicrobial therapy for patients with acute bacterial pneumonia. Interpretation of Gram-stained smears also offers a method to identify superficially contaminated specimens, enhancing discrimination between samples likely to harbor organisms associated with infection and those containing colonizing oropharyngeal flora. The quantity and diversity of resident colonizing flora in the oropharyngeal cavity and the tracheobronchial tree pose the greatest challenge to the interpretation and reporting of Gram-stained smears. There are numerous reports in the literature that assess the clinical utility of the sputum Gram stain. Unfortunately, the complex variables encompassed in the studies complicate the comparison of diverse conclusions ranging from unequivocal endorsement of the use of Gram-stained smears to identify the etiological agents of bacterial pneumonia to emphatic rejection of the test. Evidence Supporting the Utility of the Gram Stain The majority of the literature supports the clinical usefulness of Gram-stained sputum smears, more so than the utility of culturing lower respiratory tract secretions (102, 119, 135, 136, 157, 166, 214, 258, 287). Valuable information about host cell types and the predominant organism can be obtained. In an evaluation of community-acquired pneumonia caused by Acinetobacter baumannii, Gram-stained sputum smears with ⬎25 leukocytes per field at ⫻100 magnification assisted in the early diagnosis and treatment of the infection (61). A number of studies have shown the sputum Gram stain to be a sensitive and specific indicator of pneumococcal pneumonia, providing useful information to guide therapy (36, 84, 162, 235). A prospective study of community-acquired pneumonia showed that the Gram stain had a sensitivity of 57% and a specificity of 97% for diagnosing S. pneumoniae pneumonia and a sensitivity of 82%

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and a specificity of 99% for H. influenzae pneumonia (235). The authors concluded that the Gram stain was highly specific and beneficial in guiding pathogenspecific antimicrobial therapy, as evidenced by significantly more patients with a predominant morphotype than those who failed to exhibit a predominant morphotype receiving monotherapy. Importantly, if only purulent samples were included in the data analysis, the Gram stain gave a presumptive diagnosis for 80% of the patients (235). Similarly, another study showed that the sputum Gram stain, in conjunction with clinical findings, predicted the etiological agent in 80% of all cases (37). Drew also found the sputum Gram stain to be a helpful, rapid diagnostic tool with a sensitivity of 70% and a specificity of 100% in the case of pneumococcal pneumonia (84). In a military population, the Gram stain was positive for 65% of the patients with a definite diagnosis of pneumococcal pneumonia, and sputum purulence successfully differentiated pneumococcal from mycoplasmal and viral pneumonia (162). Additionally, the sputum Gram stain was useful in the CF patient population, for both assessing the quality of the specimen and directing the workup of potential pathogens in the culture (238). Using a leukocyte-to-squamous epithelial cell ratio of ⬎5, 77% of the specimens were considered acceptable, and the positive predictive value of bacterial morphology by Gram stain compared with culture was 98% for Pseudomonas aeruginosa, 84% for Burkholderia cepacia, 86% for Staphylococcus aureus, and 100% for H. influenzae (238). Recently, however, Nair and colleagues found poor correlation between quantitative sputum Gram stain examination and culture results in patients with CF. It was suggested that subjective sputum evaluation together with bacteriological culture provided results superior to those obtained from specimens which had been microscopically evaluated for quality (200). Several studies concluded that the sputum Gram stain is fraught with problems that compromise its value as a diagnostic test (229). In the case of tracheal aspirates from mechanically ventilated, very low birth weight infants, it was concluded that purulence in the aspirates correlated with prolonged endotracheal intubation, not with the development of respiratory symptoms (68). Likewise, Lentino and Lucks concluded that purulence was a nonspecific indicator of pneumonia (163). These findings are not surprising, since there are a host of conditions, involving both the lower and the upper airways, that can cause a proliferation of leukocytes, including prolonged mechanical ventilation (182). In some reports, a criterion for assessing the value of the sputum Gram stain that had a significant adverse impact on performance of the method was the expectation that the test result could or should diagnose the presence of pneumonia. The

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appropriate use of the test is not to make a clinical diagnosis, but once the clinical diagnosis is determined based on clinical parameters, the Gram stain can be used to determine the probable etiological agent and the therapeutic intervention. Many studies used sputum culture as the reference standard to which the Gram stain was compared, as summarized by Namias et al. and Reed et al. (201, 229). Using this criterion, the sensitivity of the Gram stain ranged from 15 to 100%, and the specificity ranged from 11 to 100% (229). It can be argued that culture is an imperfect standard that is compromised by many limitations that affect its specificity, as well as its sensitivity, in the case of transport delays or prior antimicrobial therapy (119). For this reason, it has been recommended that the direct Gram stain results should be used to guide the selection of potential pathogens in the culture that merit further identification and susceptibility testing (119, 257). Heineman et al. reported that half of the information gleaned from sputum culture is clinically misleading in the absence of correlation with direct Gram stain results (119). Importantly, studies have shown that Gramstained sputum or tracheal-aspirate smears can effectively predict the etiological agent in pneumonias documented by positive blood cultures (105, 217). Gleckman et al. presented data indicating that a physician could select appropriate monotherapy ⬎90% of the time when guided by bacterial morphologies reported from the Gram stain (105). Another study showed that sputa with ⬍25 squamous epithelial cells per 100⫻ field had culture results that correlated with paired transtracheal-aspirate cultures 79% of the time, compared with a correlation of only 27% when specimens had ⬎25 squamous cells per 100⫻ field (102). A potential pathogen isolated from a sputum culture with ⬎25 leukocytes and ⬍10 squamous epithelial cells per 100⫻ field was 94% predictive of growth in the corresponding transtracheal aspirate (102). Gram Stain Screening Criteria Standardized screening criteria are required to achieve consistent smear interpretations among technologists and to establish the quality of the specimen submitted for analysis. Optimal smear preparation and staining are essential precursors to the production of clinically meaningful information, including targeted selection of the grossly mucopurulent portions of the specimen. A number of quantitative criteria have been developed to screen for specimen quality, and fundamental to each scheme is the premise that an abundance of squamous epithelial cells is suggestive of superficial oropharyngeal contamination, requiring specimen recollection (Table 6). Notably, these criteria should not be applied to specimens submitted for culture of

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Table 6.

Criteria for assessing specimen quality by microscopic screening of lower respiratory tract secretions Reference

Methoda

Bartlett (1974)

25

Murray and Washington (1975) Van Scoy (1977) Geckler et al. (1977) Barry (1978)

196 280 102 12

Heineman and Radano (1979) Kalin et al. (1983) Morris et al. (1993)

118 136 189

Zaidi and Reller (1996)

298

Sum of neutrophils/LPF (10 –25, ⫹1; ⬎25, ⫹2), mucus (⫹1), and SEC/LPF (10 –25, ⫺1; ⬎25, ⫺2) Enumerate SEC/LPF Enumerate neutrophils/LPF Enumerate SEC/LPF Sum of neutrophils/LPF (1–75, ⫹1; 76 –150, ⫹2; ⬎150, (5–15, ⫹3) and SEC/LPF (⫺1; 16 –25, ⫺2; ⬎25, ⫺3) Ratio of neutrophils to SEC Ratio of neutrophils to SEC Enumerate SEC/LPF and presence or absence of organisms/OIF Presence or absence of organisms/OIF

Author(s) (yr)

a

Minimum criteria for specimen acceptancea Score of ⬎0 ⬍10 SEC/LPF ⬎25 neutrophils/LPF ⬍25 SEC/LPF Positive summation score

⬎10 neutrophils/SEC ⬎5 neutrophils/SEC ⬍10 SEC/LPF and organisms present Organisms present

LPF, low-power field; SEC, squamous epithelial cells.

Legionella species (128). If desired, Gram stain results indicating a poor-quality specimen can be accompanied by an interpretive comment conveying the suboptimal status of the sample, such as one of the following. “Squamous cells in the specimen indicate the presence of superficial material that may contain contaminating or colonizing bacteria unrelated to infection. Collection of another specimen is suggested, avoiding superficial sources of contamination.” “Not representative of lower respiratory secretions. Please repeat.” However, in some circumstances, antibiotic therapy will already have been initiated prior to requesting the submission of another specimen. Thus, the contributory nature of some repeat samples may be limited. In one observation (M. Saubolle, unpublished data) of 96 consecutive patients whose initial sputum specimens had been rejected and for whom verbal as well as written reports of “resubmit if clinically warranted” had been made, only 48 (52%) sputum specimens were resubmitted. Of those, 18 (38%) were again rejected for inadequacy, while 27 of 48 (56%) were satisfactory by screening but failed to yield a potential pathogen. Only 3 specimens (6% of the resubmitted sputa and 3% of the 96 originally rejected sputa) yielded a potential pathogen on reculture. However, in each case, the patient had already been started on appropriate therapy and the new information had no effect on patient care or outcome. Laboratories may wish to individually evaluate the clinical efficacy and cost-effectiveness of requesting repeat specimens when initial sputa are rejected. Alternatively, it might be more appropriate to add the comment “The collection of another specimen avoiding superficial sources of contamination is suggested only if clinically warranted and if the patient has not received antibiotics.” In 1974, Bartlett was the first to publish criteria for evaluating the quality of lower respiratory tract secretions (26). He reported a quality score system that produced scores ranging from 0 to 3 based on the

observed numbers of squamous epithelial cells (10 to 25 per 10⫻ field, ⫺1; ⬎25, ⫺2), mucus (⫹1), and neutrophils (10 to 25 per 10⫻ field, ⫹1; ⬎25, ⫹2). Specimen recollection was requested when a score of ⱕ0 was obtained. The quality score was subsequently used to determine the extent of work to be performed on the culture; for scores of 1, 2, and 3, a maximum of one, two, and three potential pathogens were identified to the species level and tested for susceptibility, respectively. The following year, Murray and Washington proposed a simpler protocol for evaluating the quality of sputum specimens that involved rejection of specimens with ⬎10 squamous epithelial cells per 10⫻ field (196). This scheme was later modified by Van Scoy to include acceptance of all specimens with ⬎25 leukocytes per low-power field, regardless of the number of squamous epithelial cells present (280). Geckler et al. reported that only specimens with ⬍25 squamous epithelial cells per low-power field were acceptable, regardless of the number of leukocytes present (102). In 1978, Barry proposed a scheme that was based on the ratio between the white blood cells (WBCs) (1 to 75 per 10⫻ field, ⫹1; 76 to 150 per 10⫻ field, ⫹2; ⬎150, ⫹3) and the squamous epithelial cells (5 to 15, ⫺1; 16 to 25, ⫺2; ⬎25, ⫺3) in the specimen, with a positive summation score of the two cell types indicating an acceptable specimen (12). Heineman and Radano described a similar protocol, requiring a ratio of ⬎10 leukocytes per squamous epithelial cell observed (118). These six different criteria were nicely summarized and compared by Wong et al., who determined that all six screening criteria produced the same interpretation for 60% of 391 expectorated sputum specimens (293). Wong et al. contended that inclusion of both leukocytes and squamous epithelial cells in the screening criteria minimizes the impact of variation in the thickness of the smear by providing a more reproducible ratio of one cell type to the other in various areas of the slide (293). A study of community-acquired pneumonia determined that a ratio of ⬎5

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leukocytes to 1 squamous epithelial cell was a good indicator of acceptable specimen quality and that the presence of alveolar macrophages was not (136). Morris et al. developed screening criteria specifically for endotracheal aspirates from adults (189). They recommended the rejection of specimens that showed either no organisms per oil immersion field (OIF) or ⬎10 squamous epithelial cells per low-power field, resulting in the rejection of 41% of the specimens. Upon extending this work to endotracheal aspirates from pediatric patients, it was concluded that squamous epithelial cells were not a useful screening criterion in children, since only 5% of the patients had ⬎10 squamous epithelial cells per low-power field (298). However, the absence of organisms in Gramstained smears was a useful criterion for excluding endotracheal aspirates from pediatric patients for culture, resulting in rejection of 62% of the specimens (298). Similarly, a study of intubated neonates showed that the absence of organisms in the Gram stains of endotracheal aspirates corresponded with a very low rate of recovery of potential pathogens from culture (226). A number of studies have reported the percentage of acceptable versus unacceptable specimens submitted for culture. In one evaluation, good-quality sputum specimens were obtained from 210 (39%) of 533 patients, with 175 (83%) of those samples exhibiting a predominant bacterial morphotype (235). Using the criterion of ⬎25 neutrophils and ⬍10 squamous epithelial cells as a minimal indicator of a good-quality specimen, another report showed that culture of poorquality specimens, which in this study represented 62% of the samples received, did not provide useful information (260). Similarly, an earlier publication showed that 60% of sputum specimens were superficially contaminated (119). Of 266 specimens evaluated by Kalin et al., 76% were considered high-quality samples (136). In one author’s experience, only 40 to 60% of lower respiratory tract specimens warranted processing for culture when criteria for assessing specimen quality were initially implemented (A. Robinson, unpublished data). However, each of the three evaluated institutions achieved and sustained a threshold of ⬎90% acceptable specimens within 1 month of implementing the criteria, as a result of increased awareness of appropriate specimen collection (Robinson, unpublished). In the case of repeated poor-quality specimens from the same patient, respiratory therapists can sometimes obtain higher-quality specimens. A recent study, however, showed that sputum induction using hypertonic saline did not improve the quality of the specimen, as evaluated using a ratio of neutrophils to squamous epithelial cells in Gram-stained smears (63).

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Interpretation and Reporting of Organisms in Direct Smears Criteria for the interpretation and reporting of microorganisms in Gram-stained smears of lower respiratory tract secretions are highly variable, both in terms of quantitation and morphology. This situation is complicated by the normal flora that typically contaminate these types of specimens. It is important to convey to the clinician what, if any, potential pathogen is present to provide an early indication of the cause of the pneumonia for administration of appropriate antimicrobial therapy (27). Bacteroides or Haemophilus species and enteric bacilli can be reliably identified in gram-stained direct smears 95 and 82% of the time, respectively (28). To have an impact on patient care, the laboratory must report clinically useful information, and the designation of the genera of the microorganisms present is more useful to clinicians than a descriptive narrative of organism morphology, such as gram-negative pleomorphic coccobacilli. There is a virtual vacuum in the literature regarding the correlation of reporting criteria for organisms in Gram-stained smears with clinical pneumonia. By elevating the threshold for reporting staphylococci in sputum Gram stains to ⬎50 organisms per OIF, 69% of the patients with staphylococci reported in the Gram stain had clinical evidence of staphylococcal pneumonia versus only 6% if a threshold of 25 organisms per OIF was used (M. Normandin, J. Tetreault, and A. Robinson, Abstr. 97th Gen. Meet. Am. Soc. Microbiol., abstr. C-91, p. 136, 1997). Following adoption of the threshold of ⬎50 staphylococcal organisms per OIF, a chart review of patients with smears exceeding this threshold showed that 93% had clinically defined staphylococcal pneumonia (M. Normandin, J. Tetreault, and A. Robinson, unpublished data). Another study showed that approximately 90% of patients diagnosed with Moraxella catarrhalis pneumonia produced good-quality specimens and exhibited 10 to ⬎50 gram-negative cocci per OIF (296). Although Gram staining of high-quality lower respiratory tract secretions has been recommended as an approach to the diagnosis of lung infections caused by anaerobic bacteria, in conjunction with radiographic and clinical findings (284), there is a paucity of data defining the criteria to use for Gram stain interpretation. Guidelines were developed for reporting a direct Gram stain of lower respiratory tract secretions as “suggestive of an aspiration event” that had a positive predictive value of 79% (A. Robinson, Y. McCarter, M. Normandin, and J. Tetreault, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. C-468, p. 209, 1998). Subsequently, reanalysis of the data showed the criteria to have a positive predictive value of 87% (Robinson, unpublished). The following direct Gram stain smear criteria were correlated with an aspiration

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event: ⱕ10 squamous epithelial cells per low-power field, ⱖ25 neutrophils per low-power field, and an increase in mixed flora to ⱖ50 organisms per OIF with intracellular gram-positive and gram-negative organisms in at least one field. Gram-stained smears of lower respiratory tract secretions provide information that can be useful in the medical management of patients with aspiration pneumonia because bacterial aspiration pneumonia can be difficult to diagnose clinically, is rarely confirmed by culture, and requires antimicrobial therapy targeting both aerobic and anaerobic bacteria. Summary In conclusion, because expectorated sputa and aspirated tracheal secretions are superficially contaminated with upper-airway flora, microscopic screening of specimens reduces the production of potentially misleading information and increases the diagnostic utility of culture methods. Considerable improvement in the provision of clinically meaningful results can be realized through the implementation of a Gram stain screening protocol. Gram-stained-smear results from lower respiratory specimens of an acceptable quality can be provided rapidly to the clinician for patient management and can also guide the interpretation of the culture results as detailed below.

BACTERIAL-CULTURE EVALUATION IN LOWER RESPIRATORY TRACT INFECTIONS The value of microbiological studies in the diagnosis of lower respiratory tract infections is controversial. Many investigators report that cultures from tracheobronchial secretions, such as sputum and tracheal aspirates, have limited value in the management of severe community-acquired pneumonia, pneumonia in primary-care hospitalized patients, or VAP (93, 146, 163, 222, 267, 268, 295). Indeed, many authors have stated that these secretions are commonly contaminated with microorganisms colonizing the upper airways and that culture of these specimens is not a meaningful exercise, resulting in the production of misleading results (163, 236, 267, 271). Data indicate that only 60% of patients with pneumonia actually produce sputum, and of those that do, as many as 50% of the cases have negative sputum cultures (11, 82, 227). Sputum cultures, compared to lung secretions collected by a more invasive technique, grow more bacterial species, including many that are not associated with the disease process. Sputum cultures also fail to correlate with blood cultures and serologic studies (94). On the other hand, collection of sputum is a noninvasive technique, carrying no risk for the patient.

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Additionally, others report that cultures of sputum and endotracheal aspirates can provide important clinical information, and they are considered to be useful specimens and valuable tools in the presumptive diagnosis and treatment of lower respiratory tract infections (2, 42, 44, 67, 135, 240). When goodquality sputum and endotracheal aspirates are collected, oropharyngeal contamination is minimal, and culture results are comparable to those for transtracheal aspirates in terms of the bacteria isolated (196). In addition, some reports suggest that when goodquality specimens are collected and delivered to the laboratory for prompt culture, the value of bacterial culture is improved (237, 269, 270). Although the original fiber optic brush (FB) technique had limited utility due to contamination by oropharyngeal flora, the newer PSB have decreased such contamination (181, 291). Organisms collected from PSB in quantities exceeding 103 to 104 organisms per ml are considered more likely to be true infecting agents. These cultures have been shown to have sensitivities of 70 to 97% and specificities of 95 to 100%. However, for patients receiving antibiotics or with underlying obstructive diseases, FB-collected specimens have not been useful. The FB procedure has been used to aid in the diagnosis of VAP, with sensitivities ranging from 36 to 100%, specificities of 50 to 77%, a positive predictive value of 43 to 74%, and a negative predictive value of 43 to 100% (41, 58, 59, 60, 131, 176, 273, J. A. Martos, M. Ferrer, A. Torres, J. Gonzalez, J. Puig de la Bellacasa, R. Celis, A. Gene, and A. AgustiVidal, abstract from the World Conference on Lung Health, Am. Rev. Respir. Dis. 161:A276, 1990). Quantitative cultures of BAL-collected specimens have been shown to be clinically significant at concentrations of ⬎104 organisms per ml and are best defined in cases of VAP. For BAL cultures, sensitivities of 42 to 95% and specificities of 45 to 100% have been reported, which are comparable to the results obtained with FB specimens (49, 134). It has been reported that the more invasive collection techniques for obtaining lower respiratory tract secretions, such as BAL, FB, and PSB, can be valuable in the management of patients with pneumonia, especially those with community-acquired pneumonia (209, 232). Until definitive studies are performed to determine the appropriate collection method(s) and whether culture analysis of lower respiratory tract secretions should be performed, the microbiology laboratory will be asked to perform bacterial culture analyses of a variety of respiratory samples. As previously mentioned for FB-, PSB-, and BAL-collected specimens, a threshold of ⱖ104 organisms should be used to determine which organisms are most likely pathogens. Routine identification and appropriate antimicrobial susceptibility testing should be limited to one or two

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Lower Respiratory Tract Infections

FIGURE 1.

13

Q-score system chart.

potential pathogenic organisms from these quantitative cultures. For sputa and endotracheal aspirates, routine workup should be limited to two or three potential pathogens. With this objective in mind, two examples of protocols for analyzing these specimens for possible pathogenic bacteria are presented. These protocols are Gram stain directed and are based on the quality of the specimen (as determined from the Gram stain), the presence of organisms in the Gram stain, and the number of potential pathogens present in the culture. These protocols presume that the more superficially contaminated a specimen (as determined by the number of squamous epithelial cells relative to the number of polymorphonuclear cells observed in the initial Gram stain), the greater the likelihood that contaminating organisms will be present in the culture. Thus, the standardized approaches outlined below for respiratory-culture workup will promote consistent practice in the laboratory, beginning with the necessity for properly collected specimens. Protocol 1: Q-Score System The quality (Q) score approach was first introduced by Bartlett in 1974 (25) and was reported again by Sharp in 1999 (254). The protocol is based on the Q-score system described above. In this protocol, if the number of potential pathogens isolated does not exceed the Q score, all of the potential pathogens in the specimen are worked up (Fig. 1). For use in the Q-score system, potential pathogens include S. aureus, S. pneumoniae, Streptococcus pyogenes, H. influenzae, the Enterobacteriaceae, other aerobic and facultatively anaerobic gram-negative bacilli, M. catarrhalis, and Neisseria meningitidis. Yeasts are not considered potential pathogens, unless Cryptococcus neoformans is isolated (195). Specifically, specimens are worked up as follows: Q0, do not process the

specimen; Q1, work up only one potential pathogen; Q2, work up only two potential pathogens; and Q3, work up a maximum of three potential pathogens. If, however, the number of potential pathogens exceeds the Q score, the determination of which organisms to work up is dictated by the organisms seen in the direct specimen Gram stain. If the organisms grown in culture do not correlate with those observed in the Gram stain, then only limited identifications are performed using spot or other rapid tests, and no antibiotic susceptibility testing (AST) is performed. Protocol 2: Q234 System Another respiratory culture protocol that is Gram stain directed is referred to as the Q234 approach (Fig. 2) (S. E. Sharp, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1778, p. 475, 2002). As with the Q-score system, the specimen must be of sufficient quality to preclude rejection for bacterial culture, and a maximum of two potential pathogens are worked up. In this simplified Q234 protocol that standardizes laboratory practices, if two potential pathogens are grown in culture, complete identification and AST are performed, and if four potential pathogens are grown in culture, all isolates are identified based only on spot tests or other rapid tests, with no AST performed. If the culture grows three potential pathogens, the workup is based on the correlation of the organisms grown in culture with the organisms seen in the direct Gram-stained smear. If one or two of the three potential pathogens grown in culture are seen in the Gram stain, they are worked up with identification and AST (if appropriate). If all three of the potential pathogens grown in the culture are observed in the Gram stain, their workup is limited to spot and rapid testing, and no AST is performed. The same protocol is followed with the Q345 System, only

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FIGURE 2.

Q234 (Q345) system flow sheet.

working up one more organism for each criterion. Additionally, potential pathogen workup can be limited by the quantity of normal flora present in the specimen. For example, if there are few or no potential pathogens with a moderate or greater number of normal flora, the potential pathogen workup is limited to spot and rapid tests, with no AST. Although the systems are similar, in the Q234 system, only two potential pathogens are worked up routinely, while in the Q-score system, a maximum of three potential pathogens may be worked up from a good-quality specimen. Both of these culture protocols are Gram stain directed, limit the identification and susceptibility testing of organisms in mixed cultures, and can be used for standardized workup and reporting of respiratory-culture results. For both of these protocols, a statement explaining the limited workup of mixed cultures can be added to the culture results. This statement should convey the following information: “This is a mixed culture of potential pathogens. Correlation of culture results with the Gram-stained direct smear does not help identify any of these isolates as more significant than others. Bacteria may not be related to infection and may represent colonization or contamination.” Identification and susceptibility testing of all organisms from mixed cultures can generate confusion and lead physicians to inappropriate utilization of antimicrobials and unnecessary treatment of patients. One must keep in mind that exceptions to these protocols exist, and a physician may have a valid reason for deviation from these recommendations. All potential pathogens are indicated in the culture results, and a physician can consult with the microbiology laboratory on the necessity to have further identification and/or susceptibility tests performed.

Protocol 3: Association with WBCs Method Success in the evaluation of specimens by simplified microscopic screening depends on strict use of initial appropriate cytological criteria for acceptability of quality sputum specimens in patients with normal or elevated WBC counts (i.e. nonimmunocompromised patients). Cytological acceptability is determined by scanning sputum Gram stains at ⫻100 to 120 magnification (high-dry) for the following three criteria: a. ⬎25 polymorphonuclear cells, ⬍10 epithelial cells b. ⬎25 polymorphonuclear cells, ⬍25 epithelial cells c. ⬎10 polymorphonuclear cells per epithelial cell (ratio, 10:1) The laboratory may choose any one of these criteria to judge the acceptability of sputum specimens, depending on local patient demographics (23, 49). Sputum specimens not meeting any of the acceptability criteria should be rejected. Acceptable specimens are observed under ⫻1,000 to 1,200 magnification (OIF). Gleckman et al. evaluated the following Gram stain interpretations and associated them with the appropriate clinical conditions (105). A. No organisms seen B. Mixed flora; no one morphotype with ⬎10 organisms OIF C. Mixed flora in which there were two or more morphotypes with ⬎10 organisms/OIF D. One morphotype in concentrations of ⬍10 organisms/OIF E. One morphotype in concentrations of ⬎10 organisms/OIF

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The slides should be reviewed for the presence of a predominant bacterial morphotype(s) associated with WBCs and not adhering to the epithelial cells. The presence of single morphotypes in association with WBCs (categories D and E) can be helpful in predicting pulmonary pathogens and should be reported. On rare occasions, a copathogen may be present as a cause of pneumonia, and two morphotypes may be seen in association with WBCs and may also be reported. A lack of organisms can also be helpful in assessing a patient’s situation, since “stealth” organisms (e.g., legionellae, endemic fungi, M. tuberculosis, or other atypical causes of pneumonia) may be the causative agents. The presence of multiple microorganisms consistent with normal flora (often within vacuolated WBCs) in a good-quality specimen may indicate aspiration pneumonia and should be reported as such (see the criteria for aspiration pneumonia in “Gram Stain Interpretation and Reporting” above). Interpretation is difficult if a patient has been started on antibiotics prior to specimen collection; it may be prudent to report disclaimers on cultures from such patients. Quantitation of organisms in smears can be an inconsistent (and therefore inaccurate) assessment and may vary from technologist to technologist and from day to day (277). Variability in specimen sampling is also a problem that can add to inaccurate assessment (199). Thus, the quantitation of organisms in Gram stains should not be relied upon to convey relatedness to infection and should not be reported. Rather, association with WBCs is a much more predictive criterion for relationship as a causative agent, and such associations may be helpful in reports.

IMPLEMENTING CHANGE The implementation of new and potentially controversial procedural changes frequently elicits trepidation on the part of laboratorians, who are concerned about adverse reactions from clinicians. Education within the laboratory, institution, and client base is often essential to the successful conversion to new protocols. Within the laboratory, technologist education can be achieved by inservices that explain how the new procedure will be implemented, the rationale for the change, and the time line for completing the conversion. In all cases, a new or revised procedure manual is required, and in many situations, blind unknowns, such as Gram-stained smear study sets, can facilitate successful technologist training and validation of competency through hands-on experience with the new protocols. Equally important is knowledge of the prevalence of a particular organism or disease within

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the test population or any idiosyncrasies unique to the local organisms. Within an institution, it is important to target and gain consensus from groups or individuals who can be advocates for the laboratory, including infectiousdisease specialists, pulmonologists, the pharmacy and therapeutics committee, the infection control committee, and the quality assurance committee. It is beneficial to build a communication network within an institution for many reasons, including facilitation of the implementation of new procedures. The communication approach may include routine meetings between the microbiology laboratory and infectiousdisease personnel, nursing staff educational services, newsletters, and memoranda. For example, in the case of microscopic screening of specimens, a successful implementation program included laboratory, hospital, and outpatient client components. Initially, hundreds of Gram-stained smears were evaluated to determine the incidence of poor-quality specimens, to assess any unusual aspects of the patient population, and to create a slide study set for technologist training and competency assessment. Technologist training included the review of a standardized procedure manual, educational lectures, and successful completion of a slide study collection containing approximately 40 smears. Additionally, the work flow impact of interpreting smears before inoculating media was assessed. Within the hospital, consensus was gained with the infectious-disease pulmonary and respiratory-therapy sections. The change was communicated by a memorandum to the medical and nursing staff and by a weekly newsletter with a hospitalwide distribution. The essential components of each communication included a brief description of the change, the rationale and literature citations supporting the new method, the mechanism for reporting poor-quality specimen results, and the availability of laboratory resources for discussing specific patient considerations. Communication before, during, and after implementation was essential for the achievement of a successful conversion to the new protocol.

LOWER RESPIRATORY TRACT INFECTIOUS DISEASES AND AGENTS Acute Bronchitis General Aspects Bronchitis is an inflammation of the epithelial lining of the bronchial tubes. The inflammation obstructs airflow, causing shortness of breath and coughing to remove mucus and inflammatory debris. Acute bronchitis may occur as a secondary bacterial infection following a viral upper respiratory tract illness or as a result of a primary infection with one of several specific agents, most notably B. pertussis, M. pneu-

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moniae, and C. pneumoniae. The role of the laboratory in an acute cough illness is twofold. First, the laboratory should exclude etiological agents of epidemiologic significance, particularly if empirical antibiotic therapy has failed. Second, a clinical evaluation should be completed to rule out a more serious illness, particularly community-acquired pneumonia (109). Bronchitis may become chronic, as defined by the presence of a mucus-producing cough for a total of 3 months during each of two successive years. In its most severe form, chronic obstructive pulmonary disease (COPD) may develop as a result of the sustained inflammation. The role of the laboratory in the treatment of chronic bronchitis is limited, as therapy is primarily aimed at reducing bronchial irritation and inflammation. However, specimens may be microbiologically examined in acute exacerbations when empirical therapy has failed and antibiotics are indicated. B. pertussis B. pertussis is a pathogen that causes the highly communicable, vaccine-preventable disease of pertussis, or whooping cough (54, 288). The organism is not considered to be a member of the normal commensal flora, and its presence in a clinical sample is an indication for specific therapy. The organism is a fastidious gram-negative coccobacillus that requires the use of nontoxic swabs, such as Dacron or calcium alginate. The specimen should be collected from the nasopharynx and placed into a specific transport medium, such as 1% Casamino Acids, or directly plated onto a specific primary isolation medium, such as Regan-Lowe or Bordet-Gengou medium, for diagnosis by culture. Direct fluorescent-antibody testing may also be performed but has variable sensitivity. The best diagnostic technique is a PCR test performed on a specimen collected on Dacron swabs obtained from the nasopharynx (64, 249). Serology is not standardized and has limited utility, although it was used recently in an outbreak among adults for whom a diagnosis was sought beyond the period considered acceptable for collection of samples to detect the organism (55). Pertussis classically occurs in unimmunized children in the summer and fall and is characterized by paroxysmal spasms of severe coughing, inspiratory whooping, and posttussive vomiting. However, pertussis has recently been known to cause prolonged illness with cough in adults and adolescents, including individuals who had been previously immunized (252). Whooping cough is primarily toxin mediated and carries a risk of neurological sequelae in ⬍1% of cases. The disease appears to be confined primarily to the ciliated epithelial surfaces, but alveolar macrophage invasion can occur. The disease can be treated effectively with macrolides, trimethoprim-sultamethoxazole, and quinolones. Several erythromycin-re-

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sistant isolates have been documented recently, but concerns about the emergence of widespread erythromycin resistance were not confirmed in a U.S. study (111). The disease is reportable to public health authorities, and close contacts frequently require prophylactic antimicrobial therapy. Notably, other organisms can cause a clinically similar disease, including Bordetella parapertussis, Bordetella bronchiseptica, some respiratory viruses, and C. pneumoniae. However, these agents do not generally have the epidemiologic implications that are associated with B. pertussis. M. pneumoniae M. pneumoniae is a respiratory pathogen representative of a small group of prokaryotic organisms in the class Mollicutes that are unique in that they lack a cell wall. Mycoplasmas are very labile, and specimens must be collected and transported under carefully controlled conditions. The organism, like B. pertussis, is not considered a commensal organism, and its presence in an ill person is an indication for antimicrobial therapy. However, there appears to be a high percentage of asymptomatic infections. Direct stains do not have clinical utility in diagnosis, primarily because of the organism’s small size and the absence of a cell wall. Its fastidious growth characteristics make isolation of the organism in culture difficult. Molecular tests performed on upper respiratory specimens collected on swabs have been reported to have the highest sensitivity for detection of the organism and are available from reference laboratories (83). However, the “gold standard” of clinical diagnosis for this infection is serology. Specific M. pneumoniae serologic tests for the detection of immunoglobulin M or increasing titers of immunoglobulin G to M. pneumoniae are readily available. Tests for the presence of cold agglutinins, previously thought to be a good indicator for mycoplasmal disease, are no longer considered to have adequate sensitivity or specificity to be clinically useful. The primary clinical presentation of M. pneumoniae infection is tracheobronchitis with fever and a nonproductive cough in both children (89, 219, 220) and adults, occurring most commonly during the summer months. The infection progresses to bronchiolitis and pneumonia in only about 10 to 15% of cases. Rarely, there may be significant extrapulmonary manifestations, primarily involving the cardiac and neurological systems, which may be caused by an autoimmune process. In addition, recent data suggest a correlation between early M. pneumoniae infection and the development of asthma (90), and there is evidence for an immunopathologic mechanism for severe lung injury, including acute respiratory distress syndrome (223). Treatment of an acute infection may require antimicrobials, most commonly macrolides or

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tetracyclines. However, management of severe disease may require immunomodulation therapy in addition to antimicrobials. There is also evidence of prolonged carriage of the organism, even following completion of appropriate antibiotic therapy. The consequences of this prolonged carriage are not clear in terms of epidemiologic risk (50). M. pneumoniae infection remains one of the most challenging lower respiratory tract infections to diagnose and manage for a myriad of reasons, including laboratory test limitations, the lack of specificity associated with clinical symptoms, the suspicion of disease frequently only after betalactam treatment failure, varied clinical disease with early direct pathogen effects followed by immunopathologic effects, and limited data on effective outbreak control (8). C. pneumoniae Chlamydia species are obligate intracellular gramnegative bacteria with a predilection for infection of mononuclear cells, primarily columnar epithelial cells and macrophages. Three known species are associated with lower respiratory tract infection. Chlamydia trachomatis is a well-established cause of bronchial infection and pneumonia in infants exposed during birth by an infected mother. Chlamydia psittaci is the etiological agent of psittacosis, which is acquired following exposure to infected psittacine birds, and C. pneumoniae causes a “pertussis-like” bronchial infection, as well as a more chronic bronchial infectious process, both of which can progress to pneumonia. It has been proposed recently that a new genus, Chlamydophila, be established to include C. psittaci and C. pneumoniae (92). If clinically indicated, diagnosis of these infections can be made by cell culture isolation of the organisms followed by direct fluorescent-antibody confirmation of their presence in inclusion bodies or by detection of the extracellular form in lower respiratory tract samples by direct detection. Both test methods require the use of either a genus-specific reagent or a speciesspecific reagent for each suspected species. Like the other fastidious pathogens discussed in this document, molecular detection methods appear to have the greatest sensitivity, and it is anticipated that multiplex formats will be developed commercially in the future (272). Serologic testing is useful following the acute phase of the illness, generally after a failed course of beta-lactam therapy. Again, it is necessary to know the specificities of the reagents employed for each suspected pathogenic species. These organisms are not considered to be commensals, and their presence is an indication for antimicrobial therapy with a macrolide, a tetracycline, or a quinolone. Little is known about the control of outbreaks caused by these organisms or the necessity for surveillance or prophylaxis in a documented situation. There is, in fact, a need for

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accurate diagnostic testing for all of the fastidious atypical pathogens, given the considerable overlap in clinical presentations (266). C. pneumoniae causes bronchitis or pneumonia in both children and adults (185, 219, 220), as well as COPD exacerbations that are generally accompanied by a gradually progressing and persistent cough with little or no fever. The organism may also cause upper respiratory symptoms, including pharyngitis, laryngitis, and sinusitis. Of particular concern is the organism’s association with a severe pertussis-like illness (115), chronic bronchitis (35), and exacerbations in asthmatics (167) and patients with COPD (250). As with M. pneumoniae, there is an association between infection with the organism and the subsequent development of asthma and neurological complications, presumably autoimmune in nature (153). Finally, there are data suggesting a role for C. pneumoniae in atherosclerosis, presumably secondary to an earlier acute respiratory infection (46). There are substantial data to support this role, including evidence for a strong epidemiologic association based primarily on serology, as well as direct detection of the organism in atherosclerotic plaque. What is not clear is whether there is any role for antibiotic prophylaxis or treatment in coronary heart disease or whether this is an exclusive association. Indeed, a role for other pathogens has been suggested, although the data are strongest for C. pneumoniae (206). Infection with C. psittaci is not commonly diagnosed in the United States but occurs sporadically following exposure to secretions from an infected bird. The diseases are treated similarly, but the epidemiologic implications are quite different (52). Therefore, specific diagnosis is desirable. Like C. pneumoniae, C. psittaci can cause severe disease, extrapulmonary complications, and neurologic sequelae (123). Bronchiolitis Bronchiolitis refers to the inflammation of the smaller-diameter bronchiolar epithelial surfaces. This disease most commonly occurs in infants as a result of respiratory syncytial virus infection, but it may occasionally occur as a result of a descending bronchial infection, particularly in infections with M. pneumoniae. Patient management is primarily based on clinical parameters, with the laboratory having a role only in cases that fail to respond to therapy. The same diagnostic issues that apply to the diagnosis of bronchitis due to atypical pathogens also apply to this condition. Acute Pneumonia Pneumonia is a disease of the lungs caused by infection or irritants and characterized by inflammation and consolidation.

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S. pneumoniae S. pneumoniae is the most common bacterial agent of community-acquired pneumonia, most frequently affecting young children and the elderly. There are approximately half a million cases of pneumococcal pneumonia annually in the United States (289). The pneumococcus causes 25 to 35% of the cases of community-acquired pneumonia that result in hospitalization (177), and it is the most common cause of bacterial pneumonia in individuals infected with human immunodeficiency virus (HIV) (141). S. pneumoniae colonizes the upper respiratory tract. Prior to the advent of antibiotics, studies showed that 40% of adults and an even higher percentage of children were positive for nasopharyngeal colonization with S. pneumoniae (197). The average period of carriage is 6 weeks in adults, extending to more than a year in some cases (197). Virtually all children become colonized with the organism at least once during the first 2 years of life (112). More than 80 serotypes of S. pneumoniae can be differentiated based on their capsular polysaccharides (197). The incidence of pneumococcal pneumonia is substantially higher among adults with bronchopulmonary disease or an underlying immunodeficiency (197). Factors that increase the risk of pneumonia following colonization with S. pneumoniae include: smoking, alcoholism, malnutrition, diabetes, chronic bronchitis, COPD, asthma, viral respiratory infections, HIV infection, lung cancer, lymphoma, and chronic liver or renal disease (197). It is believed that pneumococcal infection develops only in those individuals who lack functional humoral immunity to the polysaccharide capsule of the colonizing serotype (197). The pneumonia is characterized by symptoms of cough, prominent sputum production, and fever. Asplenic patients may die precipitously following an illness that lasts only 12 to 18 h (263). S. aureus S. aureus can cause pneumonia following aspiration or hematogenous transmission of the organism. It is associated with 3 to 14% of cases of communityacquired pneumonia (97, 98) and 2 to 33% of nosocomial pneumonia infections (16, 221). Staphylococcal pneumonia can lead to complications, including lung abscesses and pleural empyema. Colonization with S. aureus occurs soon after birth in many neonates, progressing to nasal colonization rates in adults that range from 20 to 40% (149). Approximately 80% of the population is colonized with the organism at some time during life, with 20% of these individuals persistently carrying S. aureus and the other 60% intermittently harboring the organism (149). Health care workers are more likely to have nasopharyngeal colonization than the general population, with S. aureus carrier rates as high as 90%

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reported, depending on the caregiver group (107). Certain patient populations are also more likely to exhibit high colonization rates, including patients with insulin-dependent diabetes, receiving dialysis, using intravenous drugs, exhibiting various dermatologic abnormalities, or infected with HIV (145, 149, 274, 275). Community-acquired S. aureus inhalation pneumonia is commonly a secondary complication of influenza virus infection; in contrast, nosocomial pneumonia often follows aspiration or intubation (169, 228). The pneumonia also can be acquired by hematogenous transmission of the organism from various niduses, including skin infections, septic phlebitis, and endocarditis vegetations (71, 174, 276). Staphylococcal pneumonia can progress rapidly to cavitation, with 10% of patients with pneumonia developing pleural empyema (169, 228). S. aureus is an important etiological agent of pleural empyema, causing onethird of all cases (43). Aspiration Pneumonia Bacterial aspiration pneumonia is a syndrome that can be a challenge to diagnose clinically and that is rarely confirmed bacteriologically. Aspiration of oropharyngeal secretions is a common event that is usually managed effectively by host defense mechanisms. When the normal clearance mechanisms are impaired or overwhelmed by a large amount of aspirated material, pulmonary infection may develop (15). Conditions that predispose an individual to aspiration include altered consciousness, dysphagia, and the mechanical disruption of airway integrity (15, 175, 213). The likelihood that an individual will develop aspiration pneumonia is related to the volume and/or nature of the inoculum and the frequency of aspiration (15, 175). The symptoms of aspiration pneumonia are highly variable and usually develop gradually several days after the aspiration episode (15). The initial phase of the infection is pneumonitis. A common late complication following aspiration is tissue necrosis with abscess formation or empyema (17, 18). Several studies report that aspiration pneumonia represents 5 to 15% of community-acquired pneumonias (175). A study of 178 patients with severe pneumonia showed that 16% had aspiration pneumonia (217). The microorganisms associated with pulmonary infections following aspiration are different, depending on whether the patient aspirates in the community or hospital setting. Anaerobic bacteria primarily cause community-acquired aspiration pneumonia (19). In hospital-acquired aspiration pneumonia, both anaerobic and aerobic bacteria are invariably present, including nosocomial pathogens, such as S. aureus and various aerobic and facultative gramnegative bacilli (19, 171). Anaerobic organisms com-

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monly associated with aspiration pneumonia include Fusobacterium spp., Bacteroides spp., Prevotella spp., Peptostreptococcus spp., and various anaerobic gram-positive bacilli (15). The anaerobic organisms associated with aspiration pneumonia usually are not confirmed by culture because specimens superficially contaminated with oropharyngeal flora are submitted for culture, invalidating the anaerobic-culture results. Specimens collected from below the larynx are considered appropriate for anaerobic culture, including pleural fluid, transtracheal aspirates, and transthoracic lung aspirates (15). In most cases of aspiration pneumonia, an appropriate specimen for anaerobic culture is not collected. Enterobacteriaceae The genera within the family Enterobacteriaceae are ubiquitously distributed in nature and are members of the normal gastrointestinal flora of humans and animals. These organisms are opportunistic agents of respiratory tract infections (165). Entrance into the lungs is primarily gained following aspiration of organisms colonizing the oropharyngeal cavity, with hematogenous transmission serving as a less common route of infection (132). Gram-negative pneumonia is usually a nosocomial rather than a communityacquired infection, because gram-negative bacilli more commonly colonize hospitalized patients than healthy persons, due to altered host cell binding sites for gram-negative bacilli (133). Additionally, gastric colonization with gram-negative bacilli can precede retrograde transfer of the organisms to the oropharynx (244). In contrast, Serratia spp. are more typically transmitted horizontally to hospitalized patients from health care workers (297). Gram-negative bacilli have been reported to cause ⬎50% of nosocomial pneumonias (140), although this group of organisms may have a reduced incidence in non-tertiary-care settings (247). Patients with a history of chronic debilitating disease, such as diabetes or COPD, have an increased risk of pneumonia caused by gram-negative bacilli, regardless of the physical location where the infection was acquired (165). The members of the family Enterobacteriaceae that have been associated with lower respiratory tract infections include Escherichia coli, Klebsiella spp., Enterobacter spp., Serratia spp., Hafnia spp., and Citrobacter spp. E. coli bronchopneumonia results in empyema formation in approximately one-third of patients and is associated with a high mortality rate (165). Klebsiella pneumoniae causes a spectrum of pulmonary diseases, ranging from bronchitis to lobar pneumonia with complications of abscesses and pleural adhesions, that are almost exclusively limited to individuals with underlying diseases (47). Mortality is

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high in the case of lobar pneumonia, and it requires aggressive antimicrobial therapy. M. catarrhalis M. catarrhalis is solely a colonizer and pathogen of humans. The organism can colonize the upper respiratory tract and cause disease in both the upper and lower respiratory tracts. It is estimated that 1 to 5% of healthy adults and a higher percentage of adults with chronic lung diseases are colonized with this organism in the upper respiratory tract (87, 148, 278). There is an active turnover of strains that colonize the respiratory tract in infants and adults (95, 148). Throughout infancy, nasopharyngeal colonization with M. catarrhalis is associated with otitis media. M. catarrhalis is also the third most common cause of bacterial sinusitis, after H. influenzae and S. pneumoniae. It is estimated that M. catarrhalis causes 10% of community-acquired pneumonia in the elderly, most of whom suffer from an underlying illness, such as chronic obstructive lung disease, congestive heart failure, diabetes mellitus, or other chronic diseases (48). N. meningitidis For more than 100 years, it has been known that N. meningitidis can colonize the nasopharynx in healthy adults. These carriers fall into three groups, chronic, intermittent, and transient, as defined many years ago by Rake, with the chronic carrier state lasting for up to 2 years (224). Adult men have the highest N. meningitidis carriage rate. When male carriers introduce the organism into their families, the carrier rates of the women and children increase to the levels observed in men (114). Transmission from carrier to carrier occurs via the respiratory route (224). Treatment of carriers with appropriate antibiotics can eradicate the carrier state rapidly and for prolonged periods (10, 96, 113). Meningococcal disease occurs primarily in persons who have been recently colonized with the organism (86). Meningococcal pneumonia has been recognized for a long time as a clinical disease and is associated with previous colonization with N. meningitidis, with evidence of pharyngitis in the majority of patients with pneumonia. The prognosis is normally favorable. Antecedent viral or mycoplasmal infection may serve as a cofactor that increases the likelihood of developing the carrier state or disease caused by N. meningitidis (188, 208). H. influenzae H. influenzae, indigenous to humans only, is a member of the normal flora of the pharynx and is a less frequent colonizer of the conjunctiva and genital tract (192). Person-to-person transmission occurs by airborne droplets or by direct contact with infected secretions. Colonization of the respiratory tract is often characterized by the acquisition of new strains that

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are subsequently spontaneously eliminated from the respiratory tract (241). Primary pneumonia in children is frequently accompanied by evidence of meningitis, epiglottitis, or otitis (192). H. influenzae is the etiological agent of community-acquired pneumonia primarily in the elderly, those with pulmonary disease, and AIDS patients (215, 246, 261). P. aeruginosa P. aeruginosa is ubiquitously distributed and can be isolated from soil, water, plants, and animals. The organism has a predilection for moist environments. It is a member of the normal microbial flora of humans, exhibiting low rates of colonization of the skin, nasal mucosa, and throat in nonhospitalized persons. In contrast, in hospitalized patients, colonization rates can exceed 50% (190, 245). Hospitalization, extended courses of antimicrobial therapy, and administration of respiratory therapy can promote respiratory disease caused by this pathogen (39, 231, 256). Lower respiratory tract infections with P. aeruginosa occur primarily in persons who are immunocompromised or have altered respiratory mechanisms. Primary pneumonia occurs in patients with chronic lung disease or congestive heart failure as a result of aspiration of the organism from the pharynx and upper respiratory tract. P. aeruginosa contributes to the development of bronchiectasis (a chronic inflammatory or degenerative condition of one or more bronchi or bronchioles marked by dilatation and loss of elasticity of the walls). It is also an etiological agent of bacteremia in patients with lung infections, malignancies, or neutropenia and of lung disease in CF patients (150, 154, 198). Bacteremic P. aeruginosa pneumonia, a fulminant disease causing death in 3 to 4 days, occurs primarily in neutropenic patients following cancer chemotherapy or in persons with AIDS (99, 127, 143, 211, 233). Acinetobacter species Acinetobacter species can be found in many animals and in an array of environmental sources and food products. Acinetobacter has been isolated from cultures of a variety of human specimens, including sputum, feces, and vaginal secretions (122). It colonizes healthy ambulatory adults and infants and is the gram-negative organism that is most commonly carried persistently on the skin of hospital personnel (159). The Centers for Disease Control and Prevention report that this organism causes 4% of the nosocomial pneumonias in sentinel U.S. hospitals (202). The respiratory tract is the most common site for Acinetobacter infection due to its high rate of colonization of this organ system (106, 234). Acinetobacter can cause community-acquired bronchiolitis and tracheobronchitis in healthy children and tracheobronchitis in compromised adults (205). Adult com-

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munity-acquired pneumonia generally occurs in immunocompromised persons (69, 110, 286). Acinetobacter also causes nosocomially acquired pneumonia, particularly in patients receiving ventilator support. Acinetobacter primarily causes nosocomial infections due to aspiration of organisms from the colonized respiratory tract. Nosocomial transmission in the intensive care unit setting has been attributed to ventilator equipment, gloves, colonized nursing and respiratory therapy personnel, and contaminated parenteral nutrition solutions (45, 72, 203, 262). Stenotrophomonas maltophilia S. maltophilia is ubiquitous in the environment and is an opportunistic, primarily nosocomial pathogen that mainly affects immunocompromised patients (31, 103, 158, 207, 251, 264). The organism can be transmitted to patients from environmental sources, such as various pharmacological solutions or hospital water or on the hands of hospital personnel (34, 62, 78, 142, 158, 292). This gram-negative bacillus readily colonizes the respiratory tract (285; K. G. Kerr, C. M. Corps, and P. M. Hawkey, Letter, Rev. Infect. Dis. 13: 762, 1991), which is the most common site for isolation of the organism. The majority of these isolates represent colonization rather than infection. In the case of infection, the organisms reach the lungs by one of two routes: (i) aspiration of contaminated oropharyngeal secretions or direct aerosolization (primary pneumonia) or (ii) hematogenous spread (secondary pneumonia) (281). Pneumonia caused by S. maltophilia is associated with a high mortality rate. L. pneumophila There are ⬎40 species of Legionella, with a total of 64 serogroups (33, 121). The natural habitat of L. pneumophila is environmental water sources, such as rivers, streams, and lakes, where it is found in relatively small numbers. The organisms are chlorine tolerant, and as a result, they can survive in water treatment plants and multiply to high numbers in water distribution systems (155, 194). L. pneumophila is responsible for as much as 90% of the infections caused by this group of organisms, with serogroups 1, 4, and 6 causing the majority of infections. Legionella species are transmitted to humans through direct inhalation, aspiration or instillation of fluids during respiratory tract manipulations, or hematogenous dissemination (7, 38, 81, 139, 173, 178, 186, 193, 294). However, oropharyngeal colonization with the organism has not been demonstrated. Legionellosis may be clinically manifested either as Legionnaire’s disease, pneumonia caused by L. pneumophila, or as Pontiac fever, a flu-like, self-limiting illness without pneumonia caused by L. pneumophila and other Legionella species.

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Chronic Pneumonia Mixed Aerobic-Anaerobic Bacteria Chronic pneumonia is defined as an inflammatory process of the pulmonary parenchyma that may be caused by either an infectious or a noninfectious agent. Chronic pneumonia is normally a condition that is present for weeks to months and is accompanied by abnormal chest radiographic findings and chronic or progressive pulmonary symptoms (80). The infectious agents that typically cause chronic pneumonia include Staphylococcus spp., the Enterobacteriaceae, P. aeruginosa, Actinomyces spp., Nocardia spp., Rhodococcus equi, Burkholderia pseudomallei, and other mixed aerobic and anaerobic bacteria. Elderly debilitated persons have a greater risk of developing chronic necrotizing pneumonia, usually caused by enteric gram-negative bacilli. Chronic pneumonia is especially common in people with AIDS (138), in hospitalized patients, and in individuals with an underlying disease, such as alcoholism, diabetes mellitus, or COPD (20). Due to the multiple causes of chronic pneumonia, no single symptom complex is universal; however, the onset of symptoms often includes fever, chills, and malaise, with a history of progressive anorexia and weight loss (80). Nosocomial Pneumonia Pneumonia is the second most common nosocomial infection in the United States and is associated with substantial morbidity and mortality. Nosocomial pneumonias are frequently polymicrobial, with gramnegative bacilli causing 60% of the infections and representing six of the following seven most frequently identified pathogens: P. aeruginosa, 17%; S. aureus, 16%; Enterobacter spp., 11%; Klebsiella spp., 7%; E. coli, 6%; H. influenzae, 6%; and Serratia marcescens, 5%. In addition, Acinetobacter spp., especially multidrug-resistant strains, have been recognized as significant pathogens in nosocomial pneumonia (138). Gram-positive organisms are also involved in nosocomial pneumonia and consist predominately of S. aureus, S. pneumoniae, and Candida spp. Candida spp. are most commonly seen in cases of nosocomial pneumonia in patients who are immunocompromised (152). Nosocomial pneumonia can be indolent or more fulminant, in the case of more virulent aerobic organisms, with symptoms resembling those of pneumococcal pneumonia (13). The most significant risk factor for nosocomial pneumonia is mechanical ventilation, and often the terms “nosocomial pneumonia” and “VAP” are used interchangeably. Intubation increases the risk of nosocomial pneumonia 6- to 21-fold (151). Most patients exhibit the extremes of age, severe underlying disease, immunosuppression, depressed sensorium, cardiopulmonary disease, or status postthoracoabdominal surgery. Symptoms of

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nosocomial pneumonia include peripheral leukocytosis with fever, purulent sputum with a significant respiratory pathogen predominating on the Gram stain, and a new or persistent radiographic infiltrate (53). Females are at increased risk for adverse outcomes from nosocomial pneumonia, as women are twice as likely as men to die from nosocomial pneumonia (70). In addition, the elderly, who are at higher risk of cerebrovascular accident, are predisposed to an aspiration episode, leading to pneumonia and/or abscess formation (138). Lung Abscess Lung abscesses develop as a result of pulmonary parenchymal necrosis caused by microbial infection. Some authorities use the term “necrotizing pneumonia” or “lung gangrene” to distinguish pulmonary necrosis with multiple small abscesses from a larger cavitary lesion, but these entities represent a continuum of the same process (21, 22). Anaerobic bacteria are the most frequent cause of lung abscess, which is not surprising given the predominantly anaerobic flora of the gingival crevice. The most common organisms involved in lung abscesses are Peptostreptococcus spp., Prevotella spp., Bacteroides spp. (other than the Bacteroides fragilis group), and Fusobacterium spp. (13, 24). If anaerobes are part of the polymicrobial flora of a mixed infection, additional organisms often include at least one other, usually more pathogenic, aerobic bacterial species, such as S. aureus (principally methicillin resistant), K. pneumoniae, other gram-negative bacilli, or S. pyogenes (138). Patients with lung abscesses involving these pathogenic aerobic bacteria are generally more acutely ill. The patient typically is seen during the early pneumonitis phase, which is followed by cavitation when the parenchymal necrosis progresses to the point of communication with the bronchus, producing a characteristic air-fluid level (21). Although mixed organism infections are not uncommon in lung abscesses, several organisms, such as B. pseudomallei (126), H. influenzae type b. Legionella spp. (160), Nocardia spp., and Actinomyces spp., can be exclusive pathogens. Commonly, most patients with lung abscess have indolent symptoms, including cough, fever, dyspnea, and the production of putrid sputum. Accompanying weight loss and anemia are also common features of a more chronic process. Evidence of pulmonary necrosis can be seen on X-ray or computed tomography scans. Propionibacterium propionicum P. propionicum, an agent of actinomycosis, is a member of the endogenous flora of the mucous membranes, including the mouth and the genitourinary tract. P. propionicum is morphologically indistinguishable from Actinomyces israelii. Both organisms

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form pleomorphic, gram-positive “diphtheroidal” rods and long, branched filaments and can grow under microaerophilic or anaerobic conditions, with optimal growth occurring in an anaerobic atmosphere. Although its pathogenicity and morphologic characteristics resemble those of certain species of Actinomyces, P. propionicum differs from Actinomyces spp. by its production of propionic acid as a major metabolic product. Aspiration of the organism from the oropharynx is the usual route of infection for P. propionicum pulmonary disease (144). The elderly have a higher risk of cerebrovascular accident that predisposes them to an aspiration episode. P. propionicum pulmonary disease is an indolent process that affects the pulmonary parenchyma and pleural space (156). Chest pain, fever, weight loss, and hemoptysis are prominent symptoms; a cough, if present, is variably productive. P. propionicum has been associated with cases of actinomycosis that included pulmonary-abscess formation with or without thoracic empyema (151). Nocardia spp. The nocardiae, bacteria belonging to the aerobic actinomycetes, are an important component of the normal soil microflora worldwide. Nocardia asteroides, Nocardia brasiliensis, Nocardia pseudobrasiliensis, Nocardia farcinica, Nocardia otitidiscaviarum, Nocardia nova, Nocardia brevicatena, and Nocardia transvalensis complex cause a variety of diseases, referred to as nocardiosis, in both healthy and immunocompromised humans and animals (32). Two characteristics that distinguish the nocardiae are their ability to disseminate to virtually any organ, particularly the central nervous system, and their tendency to cause relapsing or progressive disease despite appropriate therapy (180). The onset of pulmonary nocardiosis may be acute, subacute, or chronic and is not distinguished by any specific signs or symptoms. Fever, night sweats, fatigue, anorexia, weight loss, dyspnea, cough, hemoptysis, and pleuritic chest pain have been associated with pulmonary nocardiosis (164). A broad spectrum of radiographic findings has been demonstrated in nocardiosis, including single or multiple nodules, lung masses (with or without cavitation), reticulonodular infiltrates, interstitial infiltrates, lobar consolidation, subpleural plaques, and pleural effusions (32). As a result, nocardiosis has been frequently misdiagnosed initially as a lung abscess, tuberculosis, invasive fungal disease, or malignancy (164). Complications, such as empyema, mediastinitis, pericarditis, and superior vena cava syndrome, can arise following contiguous spread of the infection from a lung, pleural, or cutaneous focus (1). Surgical intervention under these circumstances is crucial to achieve a favorable outcome. For example, in cases of

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pulmonary nocardiosis complicated by pericarditis, mortality was 100% in those patients who did not have a pericardial drainage procedure, compared with 0% mortality in patients who received antibiotics and pericardial surgical drainage (1). In contrast, localized pleuropulmonary disease usually does not require surgery; the outcome is generally favorable with appropriate antibiotic therapy if the infection has not disseminated. Nocardiosis is mainly an opportunistic infection, predominantly in patients with lymphoreticular neoplasms as well as chronic pulmonary disorders and almost any condition requiring long-term corticosteroid usage (100). In addition, high-risk individuals also include renal, cardiac, and liver transplant recipients and patients with AIDS (57, 130). R. equi R. equi has been recognized for many years as a veterinary pathogen, as a result of its role in suppurative bronchopneumonia in foals and other animals (218). It is primarily associated with human disease as an opportunistic agent of invasive cavitary pneumonia in immunocompromised patients (255). Golub et al. reported the first case of rhodococcus infection in 1967 (108), and only 12 additional cases were recorded during the next 15 years (279). While still relatively uncommon, R. equi has been isolated from cultures more frequently in recent years, especially as an opportunistic pathogen. Most human infections have been associated with immune system dysfunction, and a dramatic increase in recognized cases has occurred since the recognition of the HIV pandemic (6, 117). R. equi is a soil organism carried in the gut of many herbivores and is widespread in their environment. The highest numbers of organisms have been found in the surface soil on horse farms where disease is endemic. Exposure to soil contaminated with herbivore manure is probably the major route of acquisition for both animal and human infection. Contact with farm animals or manure has been reported in 32 to 50% of cases (6); however, considering that this epidemiologic information is not often sought or reported, the true percentage may be higher. The majority of R. equi infections have occurred in adults, but infection in children as young as 9 months old has been reported. Most cases have been observed in men, which may be partially explained by the predominance of HIV infection in men from North America and Europe (283). Pneumonia is the most common form of human disease caused by R. equi, which suggests that humans acquire infection by inhalation. In one extensive review of 72 cases, pneumonia occurred in 76% of the patients, and the lung was the sole site of infection in 82% (283). A number of local complications can

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occur in R. equi pulmonary infection. Cavitation arises in ⬎50% of cases, and pleural effusion occurs in approximately 20%. Invasion of contiguous chest structures, including the chest wall, pericardium, and mediastinum, and recurrent pneumothoraces are unusual complications. When the underlying host immune deficit cannot be ameliorated, pulmonary infection is usually chronic and progressive. Even though pulmonary infection is significantly associated with immunosuppression, pneumonia also occurs in immunocompetent hosts. It is difficult to assess the prevalence of disease in this group, as the diagnosis may be overlooked in healthy individuals (259). B. pseudomallei B. pseudomallei is a facultative, intracellular, gramnegative bacterium that causes a clinically diverse disease, melioidosis, in humans and animals. The organism is a widely distributed environmental saprophyte in soil and fresh surface water in regions of endemicity in Southeast Asia; melioidosis is “hyperendemic” in northeast Thailand and areas in northern Australia (56, 73). Sporadic human or animal cases and occasional environmental isolates of B. pseudomallei have been described from Africa, the Middle East, the Caribbean, and Central and South America (75, 76). Although most infections are subclinical, local skin or pulmonary infection can occur. The most important risk factors for melioidosis are diabetes, excessive alcohol consumption, and chronic renal disease (161). Diabetics have an increased risk for asymptomatic infection, clinical disease, and bacteremia. Serologic studies suggest that most infections with B. pseudomallei are asymptomatic (137), with severe clinical disease occurring mainly in individuals exhibiting risk factors. Patients with the most severe manifestations of the disease can develop fulminant septic shock. Melioidosis has also been called the great mimicker because of the inconsistent clinical features associated with it (74). The most common clinical sign is an acute pulmonary infection. The chronic pulmonary form often resembles tuberculosis (91). Melioidosis can become a latent infection that later, sometimes decades after the initial exposure, reactivates into symptomatic disease (179). Respiratory Cultures from CF Patients Specimens from patients with CF continue to present a challenge to microbiology laboratories. The epidemiology of infections in this patient population has been well studied, and patients predictably progress from infection with nontypeable H. influenzae and S. aureus to P. aeruginosa infection. According to the Cystic Fibrosis Foundation patient registry annual data report for 2000, approximately 40% of infants sampled during the first year of life have S. aureus in

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23

respiratory secretions (239). The prevalence rises to 52% during adolescence and decreases throughout adulthood. By the age of 18 years, 80% of patients are infected with P. aeruginosa. A small but increasing percentage of patients proceed to infection with unusual organisms, such as B. cepacia complex (⬃3% overall); multidrug-resistant nonfermenters, including S. maltophilia and Achromobacter spp.; nontuberculous Mycobacteria; and moulds, such as Aspergillus spp. CF patients are often infected with more than one strain of P. aeruginosa, which typically have characteristic mucoid phenotypes. Oftentimes, the concentration of organisms exceeds 109 CFU per g of sputum, resulting in overgrowth of more fastidious pathogens. For this reason, specific laboratory protocols for detection of the more common pathogens have been developed. The following are recommended protocols. S. aureus The role of S. aureus as a significant pathogen in CF patients was ascertained primarily from early clinical observations of its presence in the oropharyngeal secretions of children and the concern that this reflected its presence in the lower airways (172). Definitive studies demonstrating improvement in lung function with treatment of S. aureus have not been performed. Nevertheless, the Cystic Fibrosis Foundation recommends selective media for recovery of the pathogen from respiratory secretions of CF patients (239). Mannitol salt agar is the most useful selective medium for staphylococci because it distinguishes between S. aureus and coagulase-negative staphylococci. This agar is particularly useful for detecting thymidine-dependent S. aureus, which may exhibit atypical morphology on routine media and hence may be missed (104). Columbia colistin-nalidixic acid medium is also selective for staphylococci but lacks the advantage of distinguishing S. aureus from other gram-positive cocci. P. aeruginosa Chronic airway infection with P. aeruginosa is the major clinical infectious disease problem for the CF patient. As stated previously, approximately 80% of patients are infected by age 18, most likely from environmental sources, where the organism is ubiquitous. After the establishment of P. aeruginosa airway infection, the clinical course is associated with numerous exacerbations, requiring antimicrobial therapy. The accompanying intense inflammatory response leads to significant morbidity and mortality, resulting from tissue destruction and the eventual loss of lung function. The major hypothesized factor predisposing to organism persistence is the abnormal composition of airway secretions (172). During the course of infec-

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tion, the P. aeruginosa strains often change to a mucoid phenotype, resulting from the production of alginate and mucoid exopolysaccharide. The mucoid phenotype allows the organism to evade host immune responses and correlates with the progression to chronic disease and deterioration of lung function (79). This mucoid phenotype, along with the development of resistance to antimicrobial agents, possibly related to the establishment of biofilms, prevents the eradication of the P. aeruginosa strains. P. aeruginosa grows well on standard laboratory media. However, anaerobically incubated horse blood agar may be used to isolate H. influenzae from CF patients who are heavily colonized with P. aeruginosa. The challenge for clinical microbiologists is recognition of these unusual phenotypes. For example, mucoid strains may lack pigmentation or appear more susceptible to antimicrobial agents than “typical” strains. B. cepacia Complex B. cepacia has been recognized as a significant pathogen among CF patients, and selective media are required to recover these organisms from sputum cultures containing the more rapidly growing staphylococci and Pseudomonas species. In 1987, a laboratory proficiency study, using simulated sputum containing ⱖ105 CFU of B. cepacia, S. aureus, and P. aeruginosa/ml, showed that only 22% of the laboratories not using selective media detected B. cepacia compared to 95% of the laboratories that used B. cepacia-specific media (265). Several different media have been developed. P cepacia agar contains 300 U of polymyxin B/ml and 100 ␮g of ticarcillin/ml. OFPBL agar contains oxidation-fermentation agar supplemented with lactose, 300 U of polymyxin B/ml, and 0.2 U of bacitracin/ml. B. cepacia selective agar contains 1% lactose and 1% sucrose in an enriched base of casein and yeast extract with 600 U of polymyxin B/ml, 10 ␮g of gentamicin/ml, and 2.5 ␮g of vancomycin/ml. Recent data suggest that B. cepacia selective agar is superior to OFPBL and P cepacia agar in time to organism recovery, quality of growth, and suppression of organisms other than B. cepacia complex (124). Commercial systems do not have sufficient sensitivity and specificity to be used as stand-alone tests for the identification of B. cepacia and its distinction from Burkholderia gladioli and members of related genera, such as Ralstonia spp., Alcaligenes spp., and S. maltophilia (147). The consensus is that suspect isolates should be confirmed as B. cepacia based on B. cepacia-specific media and phenotypic tests, such as the presence of lysine and ornithine decarboxylase, oxidation of sucrose and adonitol, presence of oxidase activity, and growth at 42°C (66). These phenotypic tests should allow the distinction of B. cepacia com-

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plex from related Burkholderia species and other genera; however, they may not allow differentiation within the Burkholderia complex to the genomospecies level. Reports indicate that genomospecies III may be associated with the “cepacia complex” that results in rapid deterioration of lung function in about 20% of CF patients (239). PCR-based methods and other molecular tools that have added to our understanding of the taxonomy of this complex group of organisms have been developed, and they may be helpful in difficult cases when growth characteristics and phenotypic-test results are equivocal or when the clinical presentation is suggestive of a more aggressive course (66). The U.S. Cystic Fibrosis Foundation established the B. cepacia Research Laboratory and Repository in 1998 (170). Laboratories participating in the care of CF patients are encouraged to send B. cepacia isolates to this repository for confirmatory identification.

PUBLIC HEALTH ISSUES Infections of the lower respiratory tract, including bronchitis and pneumonia, are common causes of morbidity and mortality and contribute to the economic burden (230, 239). It is difficult to provide precise data as to their incidence because they are not reportable. Nevertheless, pneumonia is the leading infectious-disease cause of death in the United States and the sixth overall cause of death. It is estimated that between 2 million and 4 million cases of community-acquired pneumonia occur each year, with at least 500,000 patients requiring hospitalization (23, 129, 243). In fact, community-acquired pneumonia was responsible for the hospitalization of ⬎1.3 million patients in 1998 and for 3.9 million outpatient visits (216, 253). In terms of economic burden, the cost of a hospitalized patient with community-acquired pneumonia is estimated at $6,000 to $7,000, while that of an outpatient is less than $200 (204). Pneumonia also accounts for 10 to 15% of nosocomial infections, with an overall mortality rate of 15 to 50% (116). A public health concern is the changing epidemiology of pneumonia and the increasing antimicrobial resistance of the causative organisms. The changing characteristics of the population in the United States contribute to this evolving epidemiology. The average age is increasing. Older persons have a higher risk for acquiring pneumonia, and once ill, they have a higher rate of hospitalization. Older patients also have a higher rate of admission to the intensive-care unit than their younger counterparts. At the same time, the number of persons at high risk for infection has increased with the increasing numbers of immunocompromised patients (230). Initiation of appropriate empirical therapy early in the course of pneumonia is paramount for adequate patient outcome. However,

CUMITECH 7B Table 7.

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CPT-4 coding guidance for diagnosis of lower respiratory tract infectionsa

Procedure categoryb Direct microscopy

Cultures

Direct specimen antigen detection

CPT-4 code series 87205 87206 87210 87040 87070 87075 87109 87110 87081

87260 – 87300 87301– 87451 87802– 87899

Direct specimen molecular detection

87470 – 87652

Serology

86602– 86804

Procedurec Gram, Kinyoun, or Ziehl-Neelsen acid-fast; PAS or GMS Auramine-rhodamine acid-fast; calcofluor white Wet mount or KOH Blood Lower respiratory specimen (aerobic) Lower respiratory specimen (anaerobic) Mycoplasma (any source) Chlamydia (any source) Individual specific pathogens (each) (note: add codes for definitive identification and susceptibility testing as appropriate.) Immunofluorescence technique, e.g., 87265 (B. pertussis/ parapertussis) Enzyme immunoassay technique, e.g., 87449 (not otherwise specified for Legionella) Immunoassay by direct optical observation, e.g., 87899 (not otherwise specified for S. pneumoniae antigenuria) Detection by nucleic acid, e.g., 87486 (C. pneumoniae amplified probe) Detection of antibodies to infectious agents, e.g., 86738 (Mycoplasma)

a

American Medical Association CPT-4 book (3) must be reviewed annually for additions, deletions, and descriptor changes. It is expected that more than one code may be used to detect multiple analytes from each specimen from the same date of service. It is necessary to add appropriate modifiers or other indication that a duplicate service has not been performed. c PAS, periodic acid-Schiff stain; GMS, Gomori methenamine silver stain. b

because of long turnaround times and poor predictive values for available diagnostic tools, together with the changing spectrum of resistance patterns of the pathogens, recommendations for therapeutic approaches are frequently in a state of flux. Resistance in microorganisms is driven by overuse of antimicrobial agents. Unfortunately, the public has come to rely on antimicrobial agents for a wide variety of symptoms, often inappropriately, and pressures clinicians for unnecessary prescriptions. The total amount of antimicrobial use in communities has been associated with the appearance and dissemination of resistant organisms (9). The growing concern about antibiotic resistance in etiological agents of pneumonia has prompted a public health campaign to immunize appropriate individuals with pneumococcal and influenza vaccines. The goal is to provide these vaccines to ⱖ90% of those who are 65 years of age or over by 2010. The same vaccines are recommended for health care workers and younger persons who are in groups at high risk for infection (101).

FREQUENCY OF TESTING The frequency of respiratory-specimen collection from patients should be driven by the clinical presentation, suspected diagnosis, and response to therapy, commensurate with established institutional protocols. Respiratory cultures are not indicated for the patient with acute bronchitis. Controversy exists regarding their usefulness in acute exacerbation of

chronic bronchitis and community-acquired pneumonia. If it is decided to submit a clinical sample, a single well-collected specimen obtained prior to antibiotic therapy is sufficient. Multiple samples submitted in the same 24-h period should not be processed. Once an etiological diagnosis is established and the patient has responded to the course of therapy, repeat testing for microbiological cure is not necessary. Repeat testing for patients who do not respond to therapy within 48 to 72 h may be a reasonable approach. In these circumstances changes in organism susceptibility may develop due to the expression of resistance mechanisms in response to antimicrobial therapy, so repeat susceptibility testing may be a reasonable request to detect these and other resistant organisms.

REIMBURSEMENT AND CODING ISSUES CPT-4 Codes Correct selection of Current Procedural Terminology version 4 (CPT-4) codes for lower respiratory tract laboratory diagnostic procedures ensures appropriate reimbursement for testing, as well as capture of the workload for the procedures performed. All regulations and guidelines that govern correct coding should be followed. Code according to the following hierarchy of specificity. Select the specific analyte first, and if it is not available, select according to the specific method. Note that for microbiology, some non-culture-dependent codes address both parameters. If a specific code is not available, then select a generic code, and finally use an unlisted code only if neces-

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sary. It is appropriate to develop reflex and composite code sets when justified by regulatory or accreditation guidelines or by published literature, as long as all compliance conditions are met. Table 7 provides an overview of codes or code series applicable to specific procedures used for the diagnosis of lower respiratory tract infections (3). ICD-9-CM Codes Justification of the medical necessity of lower respiratory tract clinical laboratory diagnostic testing requires that for each CPT-4 code an ICD-9-CM (4) clinical condition code must be provided that meets coding payment criteria in the form of either local medical review policies or national coverage decisions. For lower respiratory tract infections, one may provide a nonspecific sign or symptom code (e.g., fever, cough, and shortness of breath) or one may choose a more specific code (e.g., septicemia or pneumonia) if a higher level of diagnostic specificity has been achieved at the time of the order. One should stay informed regarding all prepay edits that are based on CPT-4 and ICD-9-CM codes. DRG Codes For inpatients, the clinical situation is generally categorized based on final admission diagnosis. The Medicare system classifies according to the diagnosisrelated groups (DRG). The DRG are in turn tied to reimbursement through a prospective payment system. The DRG diagnosis of “pneumonia” has recently been the subject of intense scrutiny by the Office of the Inspector General in evaluation of increasing financial outlays for this DRG, an activity dubbed “operation DRG creep” (5, 210). Data Review Correct coding is critical for utilization review and outcome assessment, which are used to determine the effectiveness of care. For example, a CPT-4 utilization review can be mapped to specific diagnosis codes to assess compliance with clinical-care pathways. DRG can be mapped to expense outlay on all of the procedures to determine the net financial outcome. Finally, the utility of specific procedure codes in clinical outcome for specific diagnosis codes can be determined. As an example of this, it has been shown that the collection of blood cultures is associated with an improved clinical outcome in patients hospitalized with serious pneumonia (184). REFERENCES

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Schur, and V. Brade. 1997. Role in quantitative cultures and microscopic examinations of endotracheal aspirates in the diagnosis of pulmonary infections in ventilated patients. J. Hosp. Infect. 37:25–37. 3. American Medical Association. 2003. Current Procedural Terminology. AMA Press, Chicago, Ill. 4. American Medical Association. 2003. ICD-9-CM for Physicians. AMA Press, Chicago, Ill. 5. Anonymous. 2002. Avoid pneumonia upcoding. Pathol. Lab Coding Alert 3:94 –95. 6. Arlotti, M., G. Zoboli, G. L. Moscatelli, G. Magnani, R. Maserati, V. Borghi, M. Andreoni, M. Libanore, L. Bonazzi, A. Piscina, and R. Ciammarughi. 1996. Rhodococcus equi infection in HIV-positive subjects: a retrospective analysis of 24 cases. Scand. J. Infect. Dis. 28:463– 467. 7. Arnow, P., T. Chou, D. Weil, E. N. Shapiro, and C. Kretzschmar. 1982. Nosocomial legionnaires’ disease caused by aerosolized tap water from respiratory devices. J. Infect. Dis. 146:460 – 467. 8. Atmar, R. 1999. Chlamydia species and Mycoplasma pneumoniae. Curr. Infect. Dis. Rep. 1:73–79. 9. Austin, D. J., K. G. Kristinsson, and R. M. Anderson. 1999. The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance. Proc. Natl. Acad. Sci. USA 96:1152–1156. 10. Aycock, W. L., and J. H. Meuller. 1950. Meningococcus carrier rates and meningitis incidence. Bacteriol. Rev. 14:115–160. 11. Barrett-Connor, E. 1971. The non-value of sputum culture in the diagnosis of pneumococcal pneumonia. Am. Rev. Respir. Dis. 103:845– 848. 12. Barry, A. 1978. Clinical specimens for microbiologic examination, p. 92–96. In P. D. Hoeprich (ed.), Infectious Diseases, 2nd ed. Harper & Row, New York, N.Y. 13. Bartlett, J. G., S. L. Gorbach, F. P. Tally, and S. M. Finegold. 1974. Bacteriology and treatment of primary lung abscess. Am. Rev. Respir. Dis. 109:510 – 518. 14. Bartlett, J. G., R. F. Brieman, L. A. Mandell, and T. M. File, Jr. 1998. Community-acquired pneumonia in adults: guidelines for management. Clin. Infect. Dis. 26:811– 838. 15. Bartlett, J. G. 1989. Respiratory tract and other thoracic infections, p. 311–331. In S. M. Finegold and W. L. George (ed.), Anaerobic Infections in Humans. Academic Press, Inc., San Diego, Calif. 16. Bartlett, J. G. 1996. IDCP guidelines: lower respiratory tract infections. Infect. Dis. Clin. Pract. 5:147– 167.

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vsky, A. Bourdois, P. Mariani-Kurkdjian, J.-P. Cezard, J. Navarro, and J. Elion. 1991. DNA restriction fragment length polymorphism differentiates crossed from independent infections in nosocomial Xanthomonas maltophilia bacteremia. J. Clin. Microbiol. 29:1348 –1350. 35. Blasi, F., S. Damato, R. Cosentini, P. Tarsia, R. Racannelli, S. Centanni, and L. Allegra. 2002. Chlamydia pneumoniae and chronic bronchitis: association with severity and bacterial clearance following treatment. Thorax 57:672– 676. 36. Boerner, D. F., and P. Zwadyk. 1982. The value of the sputum Gram’s stain in community-acquired pneumonia. JAMA 247:642– 645. 37. Bohte, R., J. Hermans, and P. J. van den Broek. 1996. Early recognition of Streptococcus pneumoniae in patients with community-acquired pneumonia. Eur. J. Clin. Microbiol. Infect. Dis. 15:201– 205. 38. Brady, M. 1989. Nosocomial legionnaires’ disease in a children’s hospital. J. Pediatr. 115:46 –50. 39. Brewer, S. C., R. G. Wunderink, C. B. Jones, and K. V. Leeper. 1996. Ventilator-associated pneumonia due to Pseudomonas aeruginosa. Chest 109: 1019 –1029. 40. British Thoracic Society. 2001. British Thoracic Society guidelines for the management of communityacquired pneumonia in adults admitted to hospitals. Thorax 56(Suppl. IV):iv.1–iv.64. 41. Broughton, W. A., R. M. Middleton, M. B. Kirkpatrick, and J. B. Bass, Jr. 1991. Bronchoscopic protected specimen brush and bronchoalveolar lavage in the diagnosis of bacterial pneumonia. Infect. Dis. Clin. N. Am. 5:437– 452. 42. Bryan, C. S. 2001. Acute community-acquired pneumonia: current diagnosis and treatment. J. S. C. Med. Assoc. 97:19 –26. 43. Bryant, R. E., and C. J. Salmon. 1996. Pleural empyema. Clin. Infect. Dis. 22:747–762. 44. Busk, M. F., E. C. Rosenow III, and W. R. Wilson. 1988. Invasive procedures in the diagnosis of pneumonia. Semin. Respir. Infect. 3:113–122. 45. Buxton, A. E., R. L. Anderson, and D. Werdegar. 1978. Nosocomial respiratory tract infections and colonization with Acinetobacter calcoaceticus. Am. J. Med. 65:507–513. 46. Campbell, L., C. Kuo, and J. Grayston. 1998. Chlamydia pneumoniae and cardiovascular disease. Emerg. Infect. Dis. 4:571–579.

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49. Carroll, K. C. 2002. Laboratory diagnosis of lower respiratory tract infections: controversy and conundrums. J. Clin. Microbiol. 40:3115–3120.

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