Cumitech 35: Postmortem Microbiology

Postmortem Microbiology MICHAEL KOONTZ COORDINATING

BRENDA

J. CAPLAN

EDITOR

AND FRANKLIN

P.

-

W. McCURDY

Gumitech CUMULATIVE

TECHNIQUES

AND PROCEDURES

IN CLINICAL

MICROBIOLOGY

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1B 2B 3A 4A

Blood Cultures III Laboratory Diagnosis of Urinary Tract Infections Quality Control and Quality Assurance Practices in Clinical Laboratory Diagnosis of Gonorrhea

Microbiology

Cumitech SA Cumitech GA Cumitech 7A Cumitech 12A Cumitech 13A Cumitech 14A

Practical Anaerobic Bacteriology New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide Laboratory Diagnosis of Lower Respiratory Tract Infections Laboratory Diagnosis of Bacterial Diarrhea Laboratory Diagnosis of Ocular Infections Laboratory Diagnosis of Central Nervous System Infections

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I.aboratory Laboratory Laboratory Laboratory

Diagnosis Diagnosis Diagnosis Diagnosis

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19A 20 21

Laboratory Therapeutic Laboratory

Diagnosis of Chlumydia truchomatis Infections Drug Monitoring: Antimicrobial Agents Diagnosis of Viral Respiratory Disease

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22 23 24 25

Immunoserology of Staphylococcal Disease Infections of the Skin and Subcutaneous Tissues Rapid Detection of Viruses by Immunofluorescence Current Concepts and Approaches to Antimicrobial

of of of of

Viral Infections the Mycobacterioses Female Genital Tract Infections Hepatitis Viruses

Agent Susceptibility

Testing

Cumitech 26 Cumitech 27

Laboratory Diagnosis of Viral Infections Producing Enteritis Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections oratory Animals

Cumitech 28

Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals Laboratory Safety in Clinical Microbiology Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract Verification and Validation of Procedures in the Clinical Microbiology Laboratory Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife

Cumitech 29 Cumitech 30 Cumitech Cumitech

31 32

Cumitech Cumitech Cumitech

33 34 35

Cumltechs Coordlnatlng

Laboratory Safety, Management, and Diagnosis of Biological Laboratory Diagnosis of Mycoplasmal Infections Postmortem Microbiology

should be cited as follows, e g ed , B W McCurdy ASM Press,

Caplan, M WashIngton,

Editorial Board for ASM Cumitechs: AlIce S Welssfeld, Burken, Roberta Carey, Linda Cook, Lynne Garcia, Richard Sewell, Daniel Shapiro. James W Snyder, Allan Truant

J , and D C

F

P

Koontz

Obtained

from Companion

and Lab-

Agents Associated with Bioterrorism

2001

Char, Mana D Appleman, M Jamlson, Karen Krlsher,

Cumltech

Vickle Susan

35,

Postmorem

mIcrobIology

Baselskl, B Kay Buchanan, Mltchell L Motttce, Michael Saubolle. David

I L

Effective as of January 2000, the purpose of the CumItech series IS to provide consensus recommendations regarding the @clous use of clinical microbIology and Immunology laboratories and their role In patient care Each Cum/tech IS written by a team of cllnlclans, laboratonans, and other interested stakeholders to provide a broad overvlew of various aspects of infectious collectlon, transport, processing. and disease testing These aspects Include a dIscussIon of relevant cllnical conslderatlons, interpretive guidellnes, the cllnlcal utlllty of culture-based and non-culture-based methods and emerging technologies, and issues surrounding coding, medical necessity, frequency Ilmlts, and reimbursement The recommendations in Cum/tech do not represent the official views or pollcles of any third-party payer Copyright 0 2001 ASM Press American Society for MIcrobIology 1752 N Street NW Washington, DC 20036-2904 All Rights

Reserved

Postmortem

Microbiology Michael J. Caplan

State of Delaware

Ofice of the Chef Medical Examiner, Wilmington, Delaware 19801

Franklin P. Koontz Department

of Pathology,

University of Iowa Hospitals, Iowa City, Iowa 52242

COORDINATING EDITOR: Brenda W. McCurdy Department

of Pathology and Laboratory

Medicine,

VA Medical Center, Detroit, MI 48201

The Autopsy as a Quality Assurance Tool ............................................... The Microbiology of Postmortem Tissues ............................................... ................................. The Conceptual Scheme for Postmortem Culturing

1 2 3

The Clinical Database ................................................................................................. Indications for Postmortem Cultures ............................................................................ Institutional Responses to the Questionnaire: Question 1 .............................................. Review of Responses to Question 1 ............................................................................ Extent of Postmortem Culturing: Sites and Types ......................................................... Institutional Responses to the Questionnaire: Question 2 .............................................. Review of Responses to Question 2 ............................................................................

3 4 5 5 6 7 7

Specimen

Collection,

Transport,

and Processing

.....................................

9

Contamination in the Autopsy Room ............................................................................ Methods and Techniques for Procuring Samples ......................................................... .......................................................................................... Surface Decontamination Further Recommendations for Sample Acquisition ...................................................... Specimen Transport ................................................................................................. Institutional Responses to the Questionnaire: Question 3 ........... ................................. Review of Responses to Question 3 .......................................................................... Alternative Modes of Specimen Processing for Postmortem Microbiology ..................... .............................................. Communication between Pathologist and Microbiologist

9 IO 11 12 12 12 12 13 14

Interpretation of Postmortem Culture Results and Correlation with the Clinicopathologic Database ............................................................

14

General and Specific Guidelines for the Interpretation of Postmortem Cultures .............. The University of Michigan Hospital Database ............................................................ Review of the Database and Patterns of Correlation with Case Examples ..................... Institutional Responses to the Questionnaire: Question 4 ............................................ Review of Responses to Question 4 ..........................................................................

14 16 16 20 20

Conclusion ......................................................................................... Appendix 1: Institutional Questionnaire: Individual Responses ................ Appendix 2: The University of Michigan Hospital Database .................... Appendix 3: Histochemical Stains Used To Demonstrate Microorganisms in Tissues .................................................................. References .........................................................................................

21 22 28 36 36

from both pathology house officers rotating on the hospital autopsy service and clinical microbiologists. From the house officer’s perspective, postmortem cultures signify just another laborious activity to further impede an already tedious enterprise-the autopsygenerating results that are not worth the effort it took to acquire them. For the microbiologist, postmortem

THE AUTOPSY AS A QUALITY ASSURANCE TOOL The subject of postmortem microbiology is historitally a problematic and troublesome one in the field of autopsy pathology. The mere mention of “postmortern cultures” is likely to elicit equally loud groans 1

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Caplan and Koontz

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cultures mean homogenizing multiple tissue samples; setting up growth environments for aerobic and anaerobic bacteria, fungi, and occasionally mycobacteria; and trying to make sense of the often dazzling array of organisms which flourish on the culture media but which may play absolutely no contributory role in the decedent’s death. Simply stated, the majority of pathologists and microbiologists do not feel that postmortem cultures are worth the time, effort, or expense that they require because the results are at best ambiguous and at worst uninterpretable, so that no truly meaningful information is gleaned regarding the role of infection in the individual’s death. Perhaps, to some degree, the negative sentiments regarding postmortem cultures are a microcosm of the prevailing attitudes on the part of pathologists regarding autopsies in general. Many pathologists feel that autopsies do not yield significant data that were not known antemortem, are not an efficient or effective diagnostic tool, and should be avoided if at all possible. The saddest part of all is that such attitudes, which are passed on to each new group of pathology residents rotating on the autopsy service, are based on false assumptions. If this Cumitech were reduced to communicating one major point, it would be that that postmortem microbiology can be a useful adjunct to the autopsy if placed in its proper context-as one component of the total database generated by the autopsy, the pathologic database. However, the strength of the pathologic database, in turn, is directly related to the quality of the clinical database, and the primary determinant of a solid clinical database is sound communication between the pathologist and the clinician. Analogously, communication between the pathologist and the microbiologist is essential for obtaining the greatest yield from postmortem culture studies. It is equally astounding and disturbing that people are so ready to write off postmortem examinations simply because they have forgotten how to use the telephone. Facetiousness aside, however, the establishment of the clinician-pathologist-microbiologist communication chain is the most potent instrument that can be created to ensure that the value of postmortem microbiology is not lost upon the current generation of pathologists and that the autopsy retains its usefulness as a quality assurance tool.

THE MICROBIOLOGY TISSUES

OF POSTMORTEM

The major problem in autopsy microbiology is not the collection or processing of the specimen but rather the interpretation of the culture results. Were the resultant isolates significant or nonsignificant ancillary organisms? The opinions on the validity of autopsy culture vary almost as much as the organisms isolated. Was the isolate(s) the cause of death or the result? Was its appearance a late-breaking event that had no

35

effect on the subsequent death, or was it an organism that arose during the interval between demise and dissection? If autopsy material is unsuitable for culture, the two most common supportive theories are those of agonal inv .asion and postmortem invasion. The former is based on the concept that the dying body has such diminished vitality that it is predisposed to disseminated infection from localized infection (e.g., lung to blood or gut to blood or viscera) or from normal flora of the naso-oropharynx to lung or blood or gut flora to the blood or viscera. These are resultant culture findings, not the causes of death, and there is very little medical science can do to prevent agonal invasion. Conversely, we may be able to deter or perhaps prevent the latter problem of postmortem invasion, which theorizes that bacteria multiply and migrate throughout the body after death. This theory could hold true if the incidence of positive autopsy cultures increased in direct relation to the length of the postmortem interval (PMI). Simply phrased, the longer the time between death and autopsy, the greater the percentage of positive autopsy cultures. This does not seem to be the case. Several studies have shown there is no correlation between the length of the PM1 and the number of positive autopsy cultures (8, 12, 16, 18, 20, 23, 27). These studies had PMIs ranging from 2 to 4 days at 4 to S°C refrigeration. Although the preponderance of data indicated lack of postmortem invasion, one retrospective study by Carpenter and Wilkins (5) of 2,033 autopsies reported a rise in positive postmortem cultures as the PM1 increased up to 18 h. This paper did not report the distribution of cases between short and long delay intervals, and thus, the conclusions espoused by these authors are open to debate. One study of 41 autopsies bY Sulavik et al. (2 5) demonstrated that the number of positive cultures did not simply increase with the PMI: 53% of cultures were positive at a PM1 of 0 to 6 h, 50% were positive at a PMI of 6 to 12 h, 71% were positive at a PM1 of 12 to 18 h, and 59% were positive at a PM1 of >24 h. Thus, while the theories of agonal and postmortem invasion remain suspect but not completely disproven, the preponderance of the published data suggests that, if the body is promptly refrigerated after death, the autopsy is performed within a reasonable PM1 (within 48 h), and the specimens for culture are procured early in the autopsy before manipulation of the gastrointestinal (GI) tract, neither agonal nor postmortem invasion-if either exists - should produce falsepositive cultures (2 1). There is little doubt that manipulation of the cadaver prior to obtaining heart blood cultures could lead to the physical migration of organisms from the lung to the heart. It is also possible that manipulation could lead to positive cultures from the visceral organs. Thus, concern or controversy surrounds the

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harvesting of cadaver organs. Are they sterile, or is the microbial content sufficiently low that infection will not occur in the organ recipient? The data that originally sparked this concern were in a report by Minckler et al. (18), which showed that 28 % of 148 surgical biopsy specimens (excluding skin and GI tract) were culture positive. Obviously, agonal or postmortem invasion could not occur in a surgical biopsy, asthe patient is alive! Culture positivity in those instances could have been due to transient bacteremia related to another procedure (dental, urologic, gynecologic, etc.), as many papers on diagnostic blood culture isolates have shown. Therefore, one must correlate autopsy culture results with the clinical history and histologic findings. As with diagnostic blood cultures, an isolate can be real or valid without being of any clinical significance or consequence in the death of the patient. Knapp and Kent (12) addressedthis problem over 30 years ago by utilizing quantitative cultures in their study of autopsies at the University of Iowa. They concluded that low levels of organisms may be present in noninfected organs at autopsy. They showed that culture results of 100,000 organisms per ml correlated with both clinical and pathologic (gross and histologic) evidence of pulmonary disease. It is absolutely essential that proper culture techniques be utilized in obtaining specimens at autopsy, as the previous discussion has hopefully illustrated. However, Wilson et al. (26) challenge the utility of postmortem blood cultures evenwhen obtained via the standard technique of Kurtin (16), which is over 40 years old but is still the standard protocol method. Wilson et al. reported that more than half of 111 autopsy cultures were positive despite a causeof death not related to an infectious cause.Although there were 20 autopsieswith previous positive antemortem blood cultures, half of these yielded multiple organisms considered to be contaminants while only one-third had the sameorganism isolated antemortem and postmortem. This study is discussedfurther below (see“The Conceptual Schemefor Postmortem Culturing”), but the bottom line of the study is that postmortem cultures performed without a specificendpoint in mind are costly and of questionable diagnostic utility. Thus, autopsy microbiology must be performed via standardized collection methods. However, if it is performed routinely without total understanding of the patient’s clinical history and pathologic findings, its clinical or quality assurancevalue will still be open to question.

THE CONCEPTUAL SCHEME FOR POSTMORTEM CULTURING As Reznicek and Koontz (21) have appropriately emphasized in their chapter on the subject, the value of

Postmortem

Microbiology

3

the autopsy “is directly proportional to the preparation by the prosector.” The following scenario is greatly exaggerated for the purpose of making a point. A pathologist cannot expect to walk into an autopsy room, armed with a samurai sword but with no knowledge of the clinical course of the deceasedpatient or of the questions that clinicians hope to be answered or at least addressed by the autopsy, and expect to acquire meaningful information simply by hacking away mindlessly at tissues, plopping them into a stock jar, and saying, “All done! ” Unfortunately, such a caricature is not necessarily always far from the truth. The Clinical

Database

The preautopsy preparation and review form the processthat allows the pathologist to establish the clinical database. The process consists of the following steps: (i) review of the decedent’s hospital chart, which broadly defines the clinical questions that may need to be addressed and answered at the autopsy; (ii) review of pertinent diagnostic and imaging studies, which may help to target suspected sites of infection to be examined during the autopsy; and (iii) perhaps most importantly, consultation with the clinical service in order to establish firsthand the specific clinical questions to be pursued at autopsy as well as how postmortem microbiology can assist in resolving them. Direct communication between the pathologist and clinician is the single most critical and essential component of a meaningful clinicopathologic correlation; with regard to postmortem microbiology, this will be referred to as the “clinician-pathologist arm” of the communication loop, the other being that between the pathologist and microbiologist. A dialogue between the pathologist and clinician provides the “betweenthe-lines” information which may not be appropriate for documentation in the patient’s chart but which includes the clinical hunches, biases, and thought processesthat allow the optimal information gathering to be derived at the autopsy. Furthermore, especially if the patient experienced a rapid, precipitous clinical decline, there may very well be issues and questions that were never recorded in the patient’s chart. Finally, charts on some clinical services are laconic to the point of providing no meaningful information, which is exacerbated by scribbled or illegible handwriting. In our experience, the quality of information gleaned from a simple phone conversation is exponentially more valuable than that obtained solely from review of the hospital chart. One last point with regard to this subject is that it is impor tant to acquire this information . before the autopsy, so as to avoid the undesirable situation of “Oh, by the way, we wanted you to check for. . . .” From the perspective of postmortem microbiology, postautopsy cultures obviously would be fraught with abundant potential con-

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CUMITECH

taminants that could render any results meaningless or uninterpretable. Indications

for Postmortem

Cultures

The particular scheme for postmortem culturing in a given case is most likely to be dictated by the clinical questions or considerations surrounding the autopsy. The following general scenarios encompass the majority of indications for postmortem microbiology. Indication Class 1 The purpose of this type of indication is to confirm the presence of an infection that was suspected clinically but could not be proven antemortem. This is the most straightforward, unequivocal indication for postmortem microbiology, becausethe site of potential infection has already been targeted. Therefore, the autopsy serves to confirm the existence of infection through complementary techniques, which include gross (naked eye), histopathologic (microscopic), and microbiologic examination. A frequent argument made against postmortem cultures is that they are superfluous for the reason that if an infection is indeed present, it will manifest itself unequivocally in the tissues. However, this position ignores the fact that, often, the inflammatory reaction may obscure the nature of the offending organism; furthermore, even with tissue Gram stains,the identification of microorganisms solely by histologic means is rarely etiologically specific. Granted, in some situations, it may not really matter which gram-positive or gram-negative bacterium is responsible for a decedent’s pneumonia or peritonitis, but in an age when resistant organisms are ubiquitous, the precise identification of the causative organism is frequently a pertinent clinical question. To summarize, it is prudent in this first scenario to examine grossly and histologically, and to culture, any clinically suspected site of infection, especially one that has not been confirmed antemortem. A corollary to the above indication relates to the finding of an unexpected focus of infection at autopsy. The particular location and gross appearance of the infectious focus, as well as the underlying clinical and immune status of the patient, will determine the spectrum of microorganisms to be investigated. In this situation, the site of potential infection is targeted pathologically, at autopsy, rather than clinically, but the same schemeof analysis is employed, as postmortem microbiology serves as an adjunct to gross and histopathologic examination. Indication Class 2 The purpose of this type of indication is to detect or uncover a clinically unsuspected infection when the cause of death is unknown (sudden, unexpected, or unexplained death). In this scenario, infection constitutes one entity in the differential diagnosis of the

35

death. For example, a 39-year-old man is found dead one morning after complaining of fever and malaise the night before. Additional investigation reveals that he underwent a splenectomy for a lacerated spleen sustained in a motorcycle act ident approximately 5 years earlier; his pneumococcal vaccine status is unknown. He is also known to have used recreational drugs in the past. In this circumstance, postsplenectomy sepsisshould be considered in the differential diagnosis of the cause of death, and postmortem microbology should be included in the ancillary studies performed at autopsy, even though many other possibilities for the cause of death exist. However, even if cultures are not performed, microbiological data can be acquired through alternative means if the appropriate specimens are obtained (for example, latex agglutination Ml for Streptococcus pneumoniue or serologies for viral titers in a case of unsuspected myocarditis). Although most pathologists would agreethat postmortem microbiology is indicated in the first scenario (indication class 1, clinically suspectedinfection or infectious focus identified at autopsy), many would not consider culturing in the present situation, where infection may not be such an obvious consideration. It is for this reason that the alternative techniques assumeimportance. Indication Class 3 The purpose of this type of indication is to evaluate the efficacy of antimicrobial therapy in eradicating an infection-the indication for performing postmortem cultures in the face of prior antibiotic treatment. This is clearly the most controversial and equivocal indication. Many autopsy pathologists feel that postmortem microbiology in this setting is a fruitless enterprise. However, it must be acknowledged that the presence of antibiotics does not necessarily obviate the possibility of positive cultures. Particularly when a closed or isolated infection does not permit accessor penetration of antibiotics, or if the antibiotic is inappropriate for the infection or ineffective due to a resistant organism, a positive culture might provide a clue to one of these possibilities. Also, resin bottles can be utilized to remove antibiotics from a matrix, allowing a greater yield of positive microbial cultures. Therefore, in certain situations, it is indeed justified to perform postmortem cultures, even with a history of previous antibiotic therapy. Suffice it to say that prior antimicrobial therapy does not necessarily or automatically preclude the acquisition of postmortem cultures, which may serve as a monitor for the effectivenessof such treatment. The available literature is ambivalent regarding the efficacy of postmortem microbiology in the setting of prior antibiotic therapy. For example, Klastersky et al. (11) attributed negative postmortem cultures in 5 of 23 patients (22%) with culture-proven antemortem bacteremia to high-dose antibiotic therapy (as two cases of improperly treated or untreated bacteremia

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Postmortem

35

yielded positive cultures of blood, right and left lung, and spleen at autopsy) and suggested that antibiotic therapy “may influence the results of postmortem bacteriological cultures”; they acknowledged that the relationship between antimicrobial therapy and postmortem microbiology merited further investigation. In contrast, Carpenter and Wilkins’ (5) study of 2,033 autopsies demonstrated no convincing correlation between prior antibiotic therapy and postmortem heart blood and lung culture results. Furthermore, Wood et al. (29) succeeded in recovering bacteria from the heart blood of many patients treated with antimicrobials. Koneman et al. ( 15), in their review of 91 consecutive unselected autopsies, demonstrated no significant difference in the percentage of positive postmortem cultures between patients who received prior antimicrobial therapy (37 of 41, or 90%) and those who did not (42 of 50, or 84%), concluding that antibiotic use “has little effect on the frequency with which bacteria can be cultured postmortem” and debunking the popular perception that prior antibiotic therapy “invalidates autopsy culture results.” Sulavik et al. (25), in their study of 41 autopsies, reported that 7 of 17, or 41%, of antibiotic-treated patients had significant postmortem cultures. The authors concluded that “antibiotic use did not preclude valid sampling.” Finally, in 1998, Aranda et al. (2), in a study of 92 patients, compared the diagnostic utility of postmortem cultures performed by fine-needle aspiration puncture in the immediate postmortem period with the utility of those obtained by conventional autopsy methods. They observed that antimicrobial therapy was not significantly linked to the percentage of positive cultures in any of the diagnostic groups. Clearly, the issue of prior antibiotic therapy and its influence upon postmortem microbiology is one which begs for further investigation and study. Institutional Responses Question 1

to the Questionnaire:

In an attempt to acquire a broad, general sense of how the subject of postmortem microbiology is approached and handled in current practice, we developed a short four-item questionnaire and contacted autopsy service directors or their representatives from 25 academic pathology programs throughout the United States; institutions offering residency training programs were chosen specifically in order to gain insight as to how the subject of postmortem microbiology was being taught to pathology house officers (pathologists-in-training) in anatomic pathology. The individual institutional responses are presented in tables in appendix 1, while the summary reviews of the responses to all of the questions are retained in the text. The first question addresses the indications and/or rationale for performing postmortem cultures:

Microbiology

‘(In which cases do you perform tures - what are the indications?” Review

of Responses

to Question

postmortem

5

cul-

1

While review of the above-mentioned institutional responses may not result in a resounding consensus regarding the appropriate indications for postmortem cultures, certain definite trends emerge from the responses, 1. Infection suspected clinically but not corroborated by antemortem microbial cultures remains the most common and consistent indication for obtaining postmortem cultures (indication class 1). In fact, 15 of the 25 institutions (60%) used this criterion as an absolute indication for obtaining postmortem cultures (this figure does not include the six institutions that perform postmortem cultures routinely in all cases). The discovery of a potentially infectious lesion at autopsy is another common indication for pursuing postmortem microbiology. Ten of the 25 responding institutions (40 % ) specifically identified the finding of a localized infectious process at autopsy (one institution referred exclusively to fungal lesions) as an additional indication for postmortem cultures. V.

Another trend, particularly among the tertiary care centers which treat complex and advanced forms of cancer and complications of human immunodeficiency virus (HIV) infection and perform bone marrow and/or solid organ transplantation, is the practice of obtaining microbial cultures for nonbacterial organisms - viruses, mycobacteria, fungi, and protozoa. Eleven of the 25 institutions (44%) acknowledged the importance of obtaining nonbacterial postmortem cultures in the appropriate clinical setting (posttransplant, HIV-infected, postchemotherapy, or other immunoincompetent state). Two of the responding institutions reported obtaining fungal cultures for the purpose of determining the species of rare or unusual fungi in immunocompromised hosts, and one center, whose autopsy population is comprised predominantly (60 to 70%) of posttransplant patients, admitted acquiescing to the specific requests of clinicians who, besieged with a “zoo” of microorganisms, attempt to make sense of them by identifying them through culture. One institution goes so far as to exclude identification of bacteria from postmortem cultures and reserve the use of cultures for isolation of nonbacterial microbes. Nonetheless, it is clear that the terms postmortem “microbiology” and “bacteriology” are no longer synonymous as they may have been when this subject was discussed 30 years ago (11); rather, the latter discipline is a now a mere subset of the former.

6

Caplan and Koontz

4 . Opinions vary regarding the utility of postmortem cultures (viral as well as bacterial) in fetal and perinatal deaths. Of the four institutions which specifically addressed these types of deaths, two reported routinely obtaining cultures (one viral, the other unspecified) while the other two stated that they did not routinely take cultures in perinatal deaths becauseof low yields or difficulties in interpreting equivocal results. 5 . Two institutions referred to extended PMIs (exceeding 20 h) as contraindications to culturing. 6 . Only one of the institutions acknowledged evaluating the efficacy of antibiotic treatment as a legitimate indication for culture (indication class 3). Four other centers stated that a history of previous antimicrobial therapy rendered interpretation of the postmortem culture results significantly more problematic; that is, cultures were “less” or “least” helpful in resolving questions relating to infectious diseasewhen the patient had a history of prior antibiotic treatment. 7 . While 6 of the 25 institutions (24%) continue to obtain postmortem cultures routinely (in every case), citing as reasons the educational value for pathology house staff and usefulness in the unanticipated “failure to diagnose” infections (i.e&,the potential harm resulting from failure to obtain cultures when they are indicated or may be helpful), many centers advocate adopting an individualized approach toward the acquisition of postmortem cultures. Nine of the 25 institutions (36%) emphasized the importance of performing cultures in the context of a focused investigation into the cause of death. They used such descriptors as “individualized,” “selective,” “not routine,” “not standard,” and “not always,” in an effort to free themselvesfrom a blind, nonthinking approach to postmortem microbiology. Furthermore, two institutions admitted a preconceived bias against the acquisition of postmortem (particularly bacterial) cultures, primarily because the results are unreliable, and two additional centers acknowledged that their interpretation was frequently confounded by postmortem contamination. 8. Three of the 25 institutions (12%) stated that they performed postmortem cultures infrequently (lessthan 10% of cases),rarely (a few in over 500 cases), or not at all, citing most of the reasons mentioned in the previous paragraph. 9 . Only one of the 25 responding institutions alluded to indication class 2 (the inclusion of infectious diseasein the differential diagnosis of a sudden, unexplained death) and did so tangentially at most, referring to the performance of routine lung

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cultures in the event of clinically occult sepsis;this practice is not altogether different from the traditional practice of obtaining routine postmortem cultures of heart blood. None of the centers advocated performing cultures of multiple anatomic sites to investigate the possibility of undiagnosed sepsis.Thus, it is apparent that the scenario of the sudden, unexplained death is not generally recognized as an indication for postmortem cultures. However, since these deaths fall more frequently under the domain of the medical examiner rather than the hospital pathologist, this particular indication for postmortem microbiology may not be as applicable to academic pathology centers. 10 . Finally, 3 of 25 centers (12%) have composed

their own written guidelines to assist pathology house officers in approaching the subject of postmortem microbiology: (i) G. M. Hutchins, Utjl~xation of Microbiology irt Autopsy, written guidelines, Division of Autopsy Pathology, Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Md., 1998; (ii) A. A. League, Guidelines for 0 btaining Post-Mortem M;crobiological Cultures, written guidelines, Department of

Anatomic Pathology, Emory University Hospital, Atlanta, Ga., 1996; (iii) L. A. Perez-Jaffeand I. Nachamkin, Autopsy Microbiology Protocol, written guidelines, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine and Hospital of the University of Pennsylvania, Philadelphia, 1998). All of these were made available to us. Extent of Postmortem

Culturing:

Sites and Types

The extent of postmortem culturing, that is, the number and choice of sites, is logically related to the indication for postmortem microbiology in a particular case. In casesof clinically suspected infection, the target site or sites would be cultured. Where infection is one component of the differential diagnosis, either the target organ would be cultured or random sites would be sampled, depending upon the particular clinical manifestation of the illness. For example, in a sudden unexplained death, the approach would necessarily be random (nonselective), but if the death is preceded by clinical heart failure, a more directed approach (culturing the heart) might be more efficacious. What then, is the ideal random site(s) to be cultured? This question has never been addressed comprehensively, let alone resolved, in the autopsy literature. Traditionally, heart blood has been the standard site for culture. Reznicek and Koontz (21) advocate culturing heart blood in tandem with spleen to corroborate a clinical diagnosis of sepsis, the rationale being that isolation of the same organism from both sites will provide supportive evidence for sepsis.

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recomLungs, liver, and kidneys are additional mended sites for random cultures. Regardless of the Particular organ chosen, however, we maintain that a culture from only one anatomic site invites results that are potentially equivocal and interpretations which mav be frau ght with error. Probably the study that provides the most compelling evidence in support of this view is that of Wilson et al. (26) (cited above), which compared the positive postmortem blood culture results for patients from 111 autopsies with the results of their antemortem blood cultures. A remarkable 60 of the 111 patients (54%) had positive postmortem blood cultures despite a cause of death that was entirely unrelated to an infectious etiology! Furthermore, of the 20 patients with documented positive antemortem cultures, only 7 (35%) had postmortem blood culture isolates that matched the antemortem growths; 10 cultures (50%) featured contaminants in addition to the antemortem isolates, and 3 cultures (15%) grew entirely different bacteria! Of the 91 patients who had either no, negative, or contaminated antemortem cultures (57,29, and 5, respectively), 69 (76 % ) had contaminated postmortem blood cultures and 22 cultures (24%) produced organisms that were indeterminate for sepsis. The authors concluded that isolated postmortem blood cultures were rarely useful or efficacious because they provided minimal diagnostic information. These authors also agreed that the spleen might serve as a desirable correlative site with the heart blood. The reasons for the low diagnostic yield centered on the high contamination rate for postmortem blood cultures, which has never been satisfactorily explained. Clemmensen et al. (7) recorded a similar percenta ge of positive cultures of heart blood (51% ) in 100 consecutive au topsies and further noted that the incidence of positive cultures in cases with clinically suspected infection, negative control cases (patients dying from acute cardiovascular failure), and the remainder of the cases was not significantly different! The available literature strongly suggests that the indications for and usefulness of an isolated postmortem blood culture are highly suspect, and the unreliability of this practice has been exposed. Types of cultures as well as number and sites of cultures are also important considerations in postmortem microbiology. With the emergence of HIV infection and developments and advances in organ transplantation, conditions setting the stage for immunoincompetent hosts, the scope of postmortem microbiology has extended beyond the traditional bacteriological horizons and now involves viral, fungal, mycobacterial, and even protozoa1 cultures. This trend is evident from the institutional responses to question 1 and is discussed in detail in the postresponse review of trends (question 3). The adoption of a more compulsive approach for immunoincompetent

Postmortem

Microbiology

7

patients also includes those with widely metastatic neoplasms, primary immunodeficiencies, severe injuries (i.e., burns), and extremes of age (elderly patients and premature neonates and infants). Institutional Question 2

Responses

to the Questionnaire:

The second question directed to the academic institutions specifically addresses the extent of postmortem culturing: “Which sites do you culture?” Review

of Responses

to Question

2

The institutional responses to the second question span the spectrum from the traditional routine postmortem blood culture to sampling of multiple anatomic sites. However, as with question 1, the following clear-cut trends are observable. 1. Only 9 of the 25 responding institutions (36%) sampled sites routinely for microbial culture. Of these institutions, heart blood alone was sampled routinely at three (12%); lung alone was sampled routinely at one (4%); and blood and lung were routinely sampled at five (20%). 2. In contrast, 19 of the 25 responding institutions (76%) chose to culture other anatomic sites, either in addition to the traditional (“routine”) sites or exclusive of them. The particular sites were selected on the basis of either the clinical scenario (6 of 25, or 24%), the discovery of clinically unsuspected foci of infection at autopsy (7 of 25, or 28%), or a combination of both clinical and autopsy considerations (10 of 25, or 40%). Since some institutions within the group of 19 also sampled certain sites routinely, a small overlap exists between the groups of 9 and 19, and the total number of responses exceeds the list of responding institutions (28 versus 25). 3 Excerpts from two institutions’ written guidelines effectively illustrate the trend away from routine postmortem cultures toward a more case-specific approach. The guidelines at Emory University state, “Until recently, it has been standard to obtain cultures of blood, lung, and spleen regardless of the suspected cause of death. . .[but] numerous studies have indicated that routine post-mortem microbiological cultures are usually unnecessary. . . [and] when obtained. . .rarely contribute to the postmortem diagnosis. . . .” The guidelines at the University of Pennsylvania state, “In order to maximize the information obtained from post-mortem microbiology, cultures should not be a routine procedure but rather done to answer specific questions regarding the cause of death and factors contributing to it.” The above quotes also relate to postresponse trend 7 under “Review of Responses to Question 1.” 4. Only 9 of the 25 responding institutions (36%)

8

Caplan and Koontz

designated preferable sites for clinically suspected sepsis. Blood, lung, spleen, and a “sterile site” alone were chosen in one institution each (4%); blood and 1ung were chosen at four institutions (16%); and blood and spleen were chosen at one institution (4%). Curiously, none of the institutions sampled multiple sites (defined here as more than two traditional sites, i.e., blood, lung, or spleen) to confirm a clinical suspicion of sepsis. This is a disturbing observation, since the diagnosis of sepsis depends upon the hematogenous dissemination of infectious microorganisms throughout the body, and thus, the demonstration of the same organism (or organisms) would seem to be a logical prerequisite for establishing such a diagnosis postmortem. Koneman et al. (15) cautioned in their study that “a single spot culture” is entirely insufficient to evaluate for the presence of infection at autopsy, and cultures from multiple sites are obligatory in order to obtain a complete and comprehensive picture of the microbial status of the case under study. This point has been amply reinforced in the study of Wilson et al. (26) (discussed above) as well as in that of Klastersky et al. (11). In the latter study, which involved postmortem microbiologic examination of 143 consecutive autopsies, only 9 of 3 1 positive blood cultures (29%) were supported by clinical or pathologic evidence of infection (sepsis), but when both blood and lung contained the same organism (13 cases), clinicopathologic evidence of infection was present in 6 cases (46%), and when blood and spleen harbored the same isolates, 18 of 22 cases (8 1%) confirmed sepsis as the cause of death. Furthermore, both Koneman et al. (15) and Klastersky et al. (11) report the unreliability of the lung as a single anatomic site for postmortem confirmation of infection. In the study by Koneman et al. of postmortem cultures of heart blood, lungs, liver, and kidney from 91 consecutive unselected hospital autopsies, 17 patients had antemortem sputum samples available for comparison with postmortem lung samples. Of the 16 antemortem samples that grew bacteria on culture, 4 (25%) had complete identity (bacterial species from sputum and lungs identical), 4 had partial identity (presence of additional nonidentical species in one of the samples), and 9 (56%) had no identity (species completely different) (15). In the study by Klastersky et al., positive bacterial isolates were obtained from the lungs from 16 of the decedents, but only 6 of the 16 (37%) had clinical and pathological evidence of respiratory tract infection (11). Because colonization of the respiratory tract is common in hospitalized patients and does not necessarily correlate with clinical infection, and because the rapid

CUMITECH Table 1.

Institutional

preferences

Organism sought

for autopsy

35

specimens

Preferred specimen(s) (no. of institutions)

Viral . ..*...*..............*.........*..** Fungal ,...*........................*..*

Brain (2), lung (2), liver (2), CSF (1) Liver (3), lung (21, bone marrow (I), spleen (I) TBa . ..“*....~.......~...........~*....** Lung (4) Mycobacterial (non-TB) . . . . . . . Liver (3), lung (I ), spleen (I), bone marrow (1) Legionella *..*....*.......*..*..**.*.. Serum (I), urine (I), lung (I)

a TB, Mycobactet-ium

tuberculosis.

postmortem migration of microorganisms from the oral cavity to the lungs has been amply documented (22), the practice of sampling lung as a solitary anatomic site should be performed with caution (with the exception of an abscess or other clinically or anatomically suspicious nidus of infection). Furthermore, Larsen et al. have stressed the importance of “isolating the same organism from several organs” in supporting a diagnosis of sepsis attributable to an isolated organism (17), and Reznicek and Koontz, echoing the previous authors’ sentiments, point out that “additional cultures” from alternative anatomic sites (they use as examples lung and kidney) “will more accurately reflect the spectrum of bacteria affecting the patient” (21). 5. Institutions whose autopsy populations included substantial numbers of immunoincompetent patients (HIV positive, posttransplant, postchemotherapy, etc.), as well as those which cultured commonly for particularly fastidious bacteria (e.g., Legionella pneumophila) and nonbacterial organisms, had their own preferences for anatomic sites, as is illustrated in Table 1. A few additional comments from the individual reporting institutions are worthy of mention: (i) the importance of collecting a sufficient volume of cerebrospinal fluid (CSF) for viral culture (the recommended volume is at least 10 ml); (ii) the usefulness of serology, which can be performed postmortem, as an adjunct method in the diagnosis of viral infections; (iii) the high suspicion for herpesviruses in immunoincompetent patients afflicted with viral disease; and (iv) the preference for and high yield of lung in the detection of fungal lesions in immunocompromised hosts. Although HIV-positive patients are at risk for both tuberculous and nontuberculous mycobacterial lesions, lung lesions in HIV-negative patients with the appropriate clinical scenario must always be considered for the possibility of harboring Mycobacterium tuberculosis. Although individual practices will continue to vary, it is reasonable at this point to propose at least a minimum standard for the selection of anatomic sites for postmortem microbiology when performed in the two most common following settings:

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Postmortem Microbiology

35

1. For postmortem confirmation of clinically suspectedbut unconfirmed sepsis:multiple (at least two, but preferably more) sites;Reznicek and Koontz (21) prefer heart blood and spleen,but we advocate sampling heart blood, lung, liver, and spleen in order to establish an unequivocal profile of multilocus growth (recovery of the same organism at multiple anatomic sites). 2. For a localized infectious process (abscess,pneumonia, etc.), the target organ or tissue must be cultured in conjunction with histopathologic examination of the suspectedinfectious focus, which preferably would include special histochemical or immunohistochemical stains for the demonstration of the offending virus, bacterium, mycobacterium, fungus, or protozoon. To summarize this section, a pragmatic and realistic view of postmortem microbiology begins with the realization that its usefulness does not exist in a vacuum; that is, it must be integrated with the clinical and pathologic database in order to provide meaningful information. Stated alternatively, the limitations of postmortem cultures must be understood and respected, as is the case with any diagnostic test; however, the existence of such limitations need and should not preclude their use in the autopsy room. SPECIMEN COLLECTION, AND PROCESSING

TRANSPORT,

One of the most powerful and recurrent indictments against postmortem microbiology has been the perception that any potential usefulness of its results is underm ined by the inadequacy of techniques employed to procure the specimens at the autopsy table or the inability to achieve sterility within the autopsy room. Authors writing on this subject cite postmortem contamination as one of the two most important reasons for difficulty in correlating microbiologic with clinical and other pathologic data (the other being the theory of postmortem transmigration). Although most pathologists and microbiologists would agree that the particular techniques utilized in the acquisition of postmortem cultures “clearly affect the usefulness of the results” (2l), Wilson et al. go so far as to suggestthat inadequate technique “is the only reasonable one [explanation] to explain the majority of postmortem blood culture results” and that postmortem cultures are “rarely, if ever, performed with the same attention to aseptic technique as that given to antemortem cultures” (26). This view appears to be somewhat extreme and harsh, for although the autopsy room should ideally approach the sterility of the surgical operating room, is a truly sterile autopsy room- one achieving strict sterile operating condi-

9

tions -necessary for the attainment of reliable, interpretable, and uncontaminated postmortem cultures? Contamination

in the Autopsy

Room

Studies addressing the preceding question are woefully scant in the literature, but one small seriesgave it at least an admirable try. Babb et al. (3) reported their experience with 30 hospital and coroners’ postmortem rooms over the course of 2 years, in order to assessthe role of the ambient postmortem room environment in the dissemination of infection. Although studies were performed on sampling of the room air; surfaces of instruments, autopsy tables, and cutting boards; and protective clothing and hands and gloves of staff (including pathologists and morgue technicians), the data most relevant and applicable to the issue of contamination in postmortem microbiology are that regarding cadaver sampling. Contact plate samples were taken from the skin of thoracic, abdominal, and thigh regions of the deceasedpatients both before and after autopsy and after the body was sewn up and washed down. The bacterial organisms recovered from the decedents’ bodies were similar to those present on the normal healthy skin of living persons: coagulase-negative Staphylococcus spp. (primarily Staphylococcus epidermidis) and diphtheroids. During the course of the autopsy, however, the number of gram-negative rods increased rapidly; while only 1 of the 66 contact plate samples (1.5%) taken from the skin before autopsy contained counts of gram-negative rods greater than 100 CFU/25 cm2, 15% (approximately 10) of the 66 samples acquired during the autopsy yielded counts exceeding 100 CFW25 cm2. Furthermore, gram-negative rods splashed onto “settle” plates that were placed around the decedents before the autopsy to monitor splashing and dispersal of bacteria over small distances. The speciesof gramnegative rods isolated from cadaveric samples were as follows: Escherichia coli (32%), Klebsiella spp. (17%), Acinetobacter calcoaceticus (14%), Proteus spp. (11%)) Enterobacterspp. (lo%), Citrobacterspp. (9%), Pseudomonas aeruginosa (2% ), Serratia marcescens (1%), and Flauobacterium (Chryseobacterium) spp. (1%).

Also, as would be expected, the incidences of recovery of gram-negative rods on the hands of staff before autopsy and on their gloves after autopsy were dramatically different (6 of 64, or 9%, before and 50 of 64, or 78%, after, with 44 of the 50 equaling or exceeding counts of 10 CFU/sample). The investigators concluded that although bacterial counts in the air were low (related more to the number of people present in thg autopsy room than to the air exchange frequency or to the particular procedure being performed), splashing did indeed occur on settle plates adjacent to the decedents’bodies and on the Reuter Biotest Centrifugal sampler (which can be held close to the site of

10

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Caplan and Koontz

investigation) during washing of the intestines; gloves became heavily contaminated with gram-negative rods during the autopsy; and cadaveric skin was also frequently contaminated with gram-negative rods during autopsy (the numbers of which were not significantly decreased following cleaning with disinfectants). The study, while providing some very illuminating observations, is nevertheless subject to a number of pitfalls and criticisms. Obviously, the larger the sample size is, the more vigorous will be the conclusions that can be drawn. Probably the most important issue omitted with regard to postmortem microbiology is that although room air sampling, with the recording of numbers of recovered gram-negative rods, was performed at different intervals during the autopsy according to the particular procedure being performed (i.e., removal of bowel, removal of skull, or washing out of intestines), cadaveric sampling was not. Such a practice applied to the body itself (for example, cadaveric sampling before the autopsy and following removal of the chest plate; intestines; and stomach, duodenum, and pancreas) could provide valuable information regarding the optimal point during autopsy at which to obtain samples for culture. Acknowledging the limitations of this study with respect to the particular issue of postmortem contamination (the authors were addressing primarily the role of the autopsy room environment in the risk of infection to staff), it nevertheless appears feasible to perform postmortem microbiology in a nonsterile environment with the following precautions: 1 . The number of people in the autopsy room proper should be limited. 2 . Sterile gloves should be worn at the time of sampling. 3 . Samples should be procured before removal or washing of intestines and before significant manipulation of solid organs. 4 . Samples should not be allowed to sit in the autopsy room, but rather should be transported immediately to the microbiology laboratory. 5 . Clean disposable aprons ideally should be worn during sampling. 6 . Sterile scalpel blades should be used for each sampling site. Methods Samples

and Techniques

for Procuring

Exploring further the issue of autopsy room sterility, de Jongh et al. (8) made an important contribution to this area by debunking the myth that postmortem microbiology offers meaningful results only when performed under “surgically sterile” conditions. The authors argued that while such stringent methods were of considerable research value, their practical application for routine use in the autopsy room was restric-

35

tive and limited. Their concerns have been validated by subsequent reports which cite the impracticality of applying the “elaborate and sterile technique” (19) to the autopsy room; in fact, it is the strong opinion of many pathologists and microbiologists that the practical difficulty in adhering to the sterile autopsy technique has been a potent demotivating force with regard to performing postmortem cultures (11). The greatest advantage of the method of de Jongh et al. (8) is that it makes postmortem microbiology practical and applicable for routine use by the autopsy pathologist without a statistically significant difference in culture results (and without significantly prolonging the autopsy). In their study of 100 consecutive autopsies, patients were divided into two series of 50 each in order to compare two different methods of obtaining samples. The first method used a sterile scalpel blade to incise the center of an area previously seared by a round metal plate which was attached to the tip of an electric solder gun; a sterile cotton-tipped swab was inserted into the incised parenchyma, and the swab was placed into a thioglycolate culture tube. Lung was the only routinely sampled solid organ in this method. In the second method, a thin steel spatula was substituted for the solder gun to sear the surface of the organ and l-cm3 blocks of tissue (lung and kidney) were retrieved with a sterile forceps and scissors from the seared area. Separate sets of sterile instruments were used for each sampling site. The samples were then placed into sterile petri dishes and transported to the microbiology laboratory. The results were revealing: in the series of 50 patients for which the first method (swab) was employed, only 37 of 195, or 19%, of cultures were negative (sterile); in the second group (tissue block with separate sterile instruments for each organ), 103 of 197, or 52%, were sterile. Furthermore, the 158 positive cultures in the first series yielded a total of 267 isolates (196 bacterial and 71 fungal), while the 94 positive cultures in the second group produced only 164 isolates (113 bacterial and 51 fungal), a discrepancy which the authors interpreted as a “substantial increase in sterility” (8). The authors concluded with the following recommendations: 1 . Avoid sectioning large vessels before obtaining samples. 2 . Apply a large area of cauterization to the organ surface in order to provide ample room for sampling without contamination. 3 . Use dry gloves while obtaining samples to avoid possibility of dripping contaminated fluid. 4 . Use sterile instruments for each separate anatomic site. 5 . Process all specimens with minimal delay or refrigerate them until processing (transport ).

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35

Several subsequent studies (9, 11, 15, 19) have adopted the method of de Jongh et al. (S), and Reznicek and Koontz (21) have made the following modifications, including guidelines for obtaining specimens of CSF and abscesses or granulomas: 1. Inspect body cavities for fluid accumulations. 2. A sterile blade, instead of scissors, may be used with forceps to excise the l-cm3 block of tissue. 3. Instead of 20 ml, 5 ml of heart blood is aspirated for aerobic and anaerobic blood cultures; also, resin-containing culture bottles are recommended in the setting of prior antibiotic therapy, and centrifugation lysis tubes may be used for clinically suspected fungemia. 4. CSF can be aspirated percutaneously by cisterna magna puncture or transcallosally (through the corpus callosum) into the lateral ventricle following removal of the calvarium. 5. Both the central cavities and the walls of abscesses and granulomas should be sampled, as the density of organisms may vary at different sites. 6. Specimens should be divided for bacteriology, mycobacteriology, mycology, and virology analyses if indicated. Reznicek and Koontz (21) also emphasized the importance of three main elements in obtaining cultures that may provide useful microbiologic information: (i) careful sampling, (ii) surface decontamination, and (iii) careful handling of tissues. Surface

Decontamination

While the contributions by de Jongh et al. (8) to practical culture procurement methods have been substantial, there have been no significant modifications of or improvements in these techniques. The first, and probably most important, issue meriting further evaluation and discussion is that of surface decontamination. The traditionally accepted method of sterilizing surfaces of tissues and organs has been the application of a metal (iron or steel) spatula, heated with a flame, to the organ surface, thus searing the surface and theoretically decontaminating it from any microorganisms originally present on the surface. In fact, Wilson et al. (26) noted that the technique currently employed for obtaining heart blood for culture is essentially the same as that described in the early 1900s. Unfortunately, this time-honored method is not necessarily “time tested”; that is, to our knowledge, no studies in the second half of the 20th century have ever evaluated the effectiveness of surface decontamination with the heated spatula. Furthermore, the sterility of the instrument itself may be at issue: do we really know for sure that a hot flame effectively decontaminates repeated applications of the spatula to different surfaces? What is the mini-

Postmortem

Microbiology

11

mum duration of exposure to the flame to ensure effective decontamination? What is the critical microbicidal temperature? The answers to these questions are unknown but should be pursued vigorously if pathologists and microbiologists are ever to arrive at an understanding of the role of postmortem contamination in confounding the data derived from postmortem microbiological studies. Wilson et al. (26) discussed the possibility of alternative methods for surface disinfection, which may not only provide more reliable results but may be safer to perform, mentioning specifically the use of iodophor compounds. Huston et al. (lo), in their study of percutaneous needle autopsy sampling, used a Betadine surgical scrub pad (povidine-iodine, 7.5%) for 3 min to decontaminate the skin surface before percutaneously inserting a sterile 15gauge needle into the target site. Aranda et al. (2), comparing the diagnostic utility of postmortem microbial cultures obtained by fine-needle aspiration puncture with that of samples collected at conventional autopsy, employed a similar technique with the same antiseptic (povidine-iodine wash) to acquire samples for culture with fine-needle aspiration puncture. Curiously, sampling of anatomic sites during the traditional autopsy procedure was accomplished with a sterile scalpel and pair of tweezers but without the application of povidine-iodine. The only study of which we are aware that made use of iodophor compounds to disinfect surfaces internally is that of Larsen et al. (V), which used 5% iodine-ethanol both to disinfect the skin at the site of the initial autopsy incision and to decontaminate capsular or serous surfaces. To our knowledge, no other data exist regarding the decontaminant or disinfectant potency of iodophor compounds or other antiseptics within the body, either by themselves or in comparison with heated spatulas. The current literature involving preparation of the patient’s skin for surgery provides one source from which to extrapolate data regarding the relative efficacy of antiseptics in decontamination and disinfection ( 1). An ideal surgical scrub preparation is one that is “effective and persistent in reducing the numbers of a broad spectrum of microorganisms without irritating intact skin.” The five commonly utilized classes of agents recognized to have a significant microbicidal effect include alcohols, iodophors, chlorhexidine gluconate, hexachlorophene, and benzalkonium. Hexachloraphene and benzalkonium are too unstable, susceptible to contamination, or potentially toxic for routine use, and the volatility and flammability of alcoholic preparations have discouraged their application. In assessing the results of numerous studies on the subject, the consensus of the literature appears to indicate that the most-effective antiseptic agents are chlorhexidine gluconate, povidone-iodine,

12

Caplan and Koontz

CUMITECH

and alcoholic preparations of chlorhexidine or povidone-iodine. While differences in their ability to reduce resident and transient floras have been documented, the differences have been neutralized by the results of other studies, and their overall efficacies are considered to be similar (1). If the current knowledge regarding decontamination of skin surfaces were applied to internal organs, useful data might be obtained by experimenting with different antiseptic agents on surfaces of internal organs and comparing their levels of individual microbicidal and decontaminant effectiveness with each other, as well as their general disinfectant efficacies with that of a heated metal spatula. It is perplexing that in this day of technologically advanced, complex medicine, some of the simplest, most basic, yet most decisive and meaningful studies are yet to be performed. Until they are, this section remains necessarily truncated. Further Recommendations Acquisition

for Sample

The physical procurement of the tissue or organ sample is one more component of the specimen collection process that deserves mention. While sterile cottontipped swabs were popular in the older literature, most current practices advocate obtaining blocks or cubes of solid tissue which are subsequently homogenized with tissue grinders (or their equivalent) and processed according to standard microbiologic techniques. Swabs are agreed to be generally unsatisfactory specimens because of their ability to retain only small amounts of tissue on their surfaces, a feature which may significantly compromise the recovery of fastidious microorganisms. In addition, they are susceptible to desiccation, a process which may also sacrifice organisms (i.e., gram-negative rods, which die upon drying). Solid tissue, because it is the specimen that most accurately reflects the microbiology of the infectious process in vivo, should always be the preferred sample (4). The particular choice of tool to procure the specimen, whether a sterile forceps, surgical clamp, or scissors, is not crucial to the collection process as long as it remains sterile prior to the procedure and can be handled and manipulated effectively to obtain the sample. One practice potentially facilitating this process would be the assembly of premade sterilized postmortem culture “trays” or “kits,” analogous to surgical trays. Such kits would include setsof sterile instruments (sufficient to allow a different one for each site sampled), antiseptics, and specimen collection containers or petri dishes, as well as blood culture bottles, culture resin-containing broth bottles, lysis centrifugation tubes, a surface for dividing tissue samples for different types of cultures (i.e., bacterial, mycobacterial, viral, and fungal), specimen labels, a

35

marking pen, and microbiology laboratory requisitions. Specimen

Transport

With regard to the transport of specimens, the general guiding principle is that they should be delivered as promptly as possible following collection to the microbiology laboratory in order to maximize the recovery of any microorganisms. While this makes good common sense,a logical follow-up question is “How promptly?” Anyone who has performed autopsies which require the collection of specimens for microbiology knows how inconvenient it is to interrupt the autopsy, take off the protective clothing, label and gather the specimens, fill out the (multiple) requisitions, and leave the autopsy room to bring them to the microbiology laboratory. Consequently, they are often left unattended on a countertop for several hours before they are noticed and brought to the laboratory. Once again, the answer to this question is not known because the appropriate comparative studies (immediate versus delayed transport of identical specimens and their relative microbial yields) have not been performed or, at least, have not been reported; all that we can advise is that sooner rather than later is preferred in order to promote the highest recovery of organisms. Special transport conditions for specimensare warranted in certain situations. For anaerobic bacteria, recovery is promoted best with the use of anaerobic transport vials; the specimen should be placed at the interface of the gel and air to permit the survival of both aerobes and anaerobes within the tissue. Specimens for viral culture also require transport media (usually a combination of protein with saline buffer, often containing antibiotics to minimize bacterial overgrowth) and should be maintained at 4OC if any delay in transport to the virology laboratory is expected; for longer delays, snap-freezing in liquid nitrogen is recommended,sinceslow freezing may be lethal to unstable viruses. Additionally, the recovery of viruses may be enhanced if excreted samples (urine and feces) are collected in addition to the target organ(s) (21). Institutional Question 3

Responses

to the Questionnaire:

The third question presented to the responding institutions inquires about the specific methods or techniques used to obtain samples for culture: “What techniques do you use to culture- specimen collection, transport, processing. etc. 3” Review

of Responses

to Question

3

The above practices are summarized as follows. 1. All of the institutions obtained heart blood in a similar fashion, by inserting a sterile needle into the wall of the right atrium, inferior vena cava, or

CUMITECH

Postmortem

35

ascending aorta, with or without searing the epicardial or adventitial surface with a heated spatula. 2. As would be exnected. the most nonular method of decontaminating organ surfaces lwas by searing them with a heated spatula or similar instrument (blade, putty knife, or iron); 21 of 25 institutions (84%) used this technique. The perpetuation of this practice for sterilizing surfaces is probably less an endorsement of the scientific validity of the procedure than simple habit, not much unlike the routine measurements obtained during the autopsy- more rote than thought. Five institutions (20%) used antiseptics (alcohol or povidone-iodine) to disinfect surfaces, with one institution employing both spatulas and antiseptics, depending upon the pathologist performing the autopsy. 3 An encouraging statistic is the very high percentage with which blocks of tissue were procured for culture (23 of 2.5 institutions, or 92%). However, this should be qualified with the observation that 2 of the 23 centers used swabs for bacterial culture and tissue for fungal and Mycobacterium tuberculosis culture and for specialized infectious foci (i.e., heart valve vegetation or abscess wall). Five institutions (20%) obtained both swabs and tissue blocks, but only two used swabs alone. As discussed above, the yield from homogenized tissue is substantially better than that of a swab, and the former practice should be standard for tissue samples. It is apparent that old practices and habits are difficult to break regarding the acquisition of specimens; thus, it is likely that only the generation of meaningful data (regarding the relative efficacy of antiseptics versus spatulas, etc.) will effect significant changes in the currently employed methods. Alternative Modes of Specimen Postmortem Microbiology

Processing

for

Additional methods of processing tissues can be used to supplement the database provided by traditional microbial cultures. Print cultures are a technique by which cut surfaces of tissues are applied directly to culture media, in contrast to the traditional homogenization of tissue blocks and inoculation onto the media. The theoretical advantage of this technique over the tissue homogenate inoculum method is that, in addition to being easy and quick and providing similar recovery rates of bacterial isolates (i.e., similar to those from homogenized tissue), it facilitates the localization of microorganisms within the tissues and can correlate that with a tissue inflammatory response, thus helping to separate surface contamination from true infection (21). Fung et al. (9) modified and simplified the print culture technique by using fresh unfrozen tissues whose surfaces were exposed

Microbiology

13

with sterile scalpel blades and by imprinting them onto agar surfaces, as well as streaking one imprint; the method as originally described involved frozen tissues that were sectioned with a microtome and either pressing the cut surfaces or laying them onto culture media. The modification obviated the requirement for a microtome with sterile blades, which could pose logistical problems for a pathology department. The results were encouraging: (i) the imprints (spleen and lung) demonstrated virtually the same growth as the tissue homogenate inocula (types of organisms as well as quantities), and (ii) 18 of 33 lung specimens with growth (54.5%) revealed inflammation on histopathologic sections, but only 2 of the 33 lung specimens without growth (6%) demonstrated pneumonia histologically. The authors interpreted the results as indicative of the generally low specificity of postmortem cultures, noting the similarity of their findings to those of Koneman (13), who reported a 30 to 50% lack of correlation between antemortem and postmortem cultures, but also observed the excellent correlation between negative growth and absence of inflammation (“. . .a negative postmortem culture result is a reliable indicator of no bacterial infection”) (9). It is puzzling that this technique is not used more frequently, as it appears to be relatively simple and easy to apply and allows a more precise histopathologicmicrobiologic correlation than the traditional homogenate inoculum method. Also, it could be applied to fungal and mycobacterial organisms as well. Nevertheless, the print culture technique offers some significant advantages, the most important being its use in interpreting culture results based upon location of the colonies relative to sites of inflammation (for example, the selective localization of contaminating bacteria at the edges of the imprint) (3 1). Techniques employing molecular biology are generally reserved for the identification of organisms which have a low yield with routine culture (i.e., coxsackievirus B in cases of viral myocarditis). Unfortunately, very few laboratories in the country currently are performing such studies, and those that are do so only on a research basis, which is not practical for the demands of a diagnostic autopsy service. The final adjunct method to be mentioned is the use of histopathology, in conjunction with microbial stains, to diagnose infectious disease at autopsy. Because different types of infections elicit broad classes of tissue reactions (host responses), the type of response within the tissue can guide the pathologist toward a more specific etiology with the use of microbial stains designed to demonstrate the morphology of that organism within the tissues. The reader is referred to the excellent discussion of this subject and associated tables and photomicrographs in the chapter entitled “Autopsy Microbiol-

14

Caplan and Koontz

CUMITECH

ogy” by Reznicek and Koontz in Clinical Laboratory Medicine (21); in addition, tables in appendixes 2 and 3 provide integration of postmortem microbiology with autopsy histopathology as well as a more detailed outline of histopathological stains. Communication Microbiologist

between

Pathologist

and

Finally, a discussion of specimen processing cannot be considered complete withou t acknowledging the importance of the interaction between the pathologist and microbiologist; such an interaction is nothing less than crucial in order to optimize the successful isolation of significa nt orga nisms. This is the pathologist-microbiologist arm of the commu .nication loop. Examples of such communication begin when the pathologist brings the specimens to the microbiology laboratory and relates the clinical history, along with the specimens that he or she is submitting to the laboratory. The issue may be as simple as a recommendation from the microbiologist that more specimen be obtained to allow for adequate workup for different microorganisms (i.e., fungal and mycobacterial as well as bacterial cultures). Also, if the clinical history or autopsy findings warrant particular suspicion for a certain microorganism, this information should be communicated to the microbiologist to allow for the greatest chance for recovery of the suspected organism. The death of a person with sickle-cell disease or mixed sickle hemoglobinopathy, for example, would direct the microbiological workup toward a focus upon encapsulated organisms. Gram staining or LA might be a useful adjunct procedure in this circumstance. Reznicek and Koontz also advocate touch preparations in the autopsy room, which are simple to perform and which may allow rapid identification of classes of organisms, thereby guiding the microbiologic workup (21). For example, the identification of broad, pleomorphic hyphae with haphazard right angle branching and occasional septa from the paranasal sinuses of a poorly controlled diabetic obviously should prompt a procedure for the isolation of zygomycetes. As a practical matter, certain microorganisms have special growth requirements. The only reliable way to ensure that the specimens are properly treated so that the suspected organisms are appropriately pursued is to communicate this information to the microbiologist by a legible, clearly specified requisition and, preferably, by reiteration of the request through a short discussion. We accept the risk of being redundant and maintain that the importance of communication cannot be overemphasized. Poor communication is probably the single most common reason for insufficient or inadequate specimens that are submitted to the microbiology lab and for inefficient use of the microbiologist’s time when the micro-

35

biologist is faced with polymicrobial growth on a plate but cannot discontinue the workup of the culture because of inability to contact the pathologist. Although it may not always be practical, we recommend that the pathologist submit the specimens directly to the microbiology laboratory. Subsequent communications can be by phone, provided that the pathologist has indicated his or her beeper number on the requisition. The reason for this suggestion is as much interpersonal as it is a plea for optimal information gathering. The pathologist needs to remember that the microbiologist also has a strong interest in infectious disease and that his or her extent of involvement with the case may influence the ultimate quality of the results. Thus, if the microbiologist is provided with background information regarding the case directly by the pathologist, there will be that much stronger a motivation to assist the pathologist in resolving the pertinent questions.

INTERPRETATION OF POSTMORTEM CULTURE RESULTS AND CORRELATION WITH THE CLINICOPATHOLOGIC DATABASE Of the many reasons offered to explain why the subject of postmortem microbiology takes such a “bad rap” from pathologists and clinicians, perhaps the most compelling is the disquieting uncertainty surrounding the interpretation of the results. In the ideal scenario, postmortem cultures would yield pure unimicrobial isolates, which would correlate perfectly with the clinical hypothesis and the autopsy findings. Unfortunately, the real world does not often permit such neat clinical-pathologic-microbiologic correlation, and consequently, the burden is often placed upon the pathologist to integrate seemingly disconnected and incongruent clinical, pathologic, and microbiologic data. Unfortunately, the pathologist, who tries to make sense of the formidable list of microorganisms, discovers quickly that the names of these organisms may leave a more profound impression than their clinical significance! In order to make optimal use of postmortem microbial cultures and to minimize the frustration involved with the interpretation of the culture results, it is necessary to embrace a few basic yet essential guidelines. General and Specific Guidelines for the Interpretation of Postmortem Cultures 1. A positive culture is not synonymous with infection for the same reason that a single laboratory test is virtually never pathognomonic of a clinical condition or state. The diagnosis of infection generally requires both the invasion of a pathogenic microorganism into the tissues or body fluids of the

CUMITECH

35

host and the elicitation of a host immunologic and/or inflammatory response, with variable degrees of tissue injury. In the majority of immunocompetent hosts, a legitimate response is achieved. However, in certain situations, infection may occur in the absenceof an effective host response: (i) the virulence of the offending microorganism may be so great as to overwhelm the host and nullify the generation of a host response (e.g., Bacillus anthracis); (ii) the organism may elaborate a toxin, which may serve as the mediator of tissue injury in the absence of direct tissue invasion and which may diminish the host response (necrotizing inflammation); and (iii) certain immunoincompetent states may blunt or attenuate the host response (e.g., HIV infection, primary immunodeficiencies, and posttransplant or postchemotherapy states), which may obscure the presence of infection. 2. Organisms are capable of contaminating, colonizing, or invading host tissues. Contamination is the presence within a tissue of microorganisms not typically pathogenic or saprophytic for that tissue and not accompanied by an inflammatory response. Colonization is the presence of microorganisms within a tissue (typically on a surface membrane) which do not elicit an inflammatory host response or produce tissue damage. Infection is the presenceof microorganisms within (recovery from) a tissue, due to penetration from a mucosal or skin surface and invasion into a normally sterile tissue or body fluid with subsequent replication, which both elicits an inflammatory host response and produces tissue injury (2, 30). Therefore, a positive postmortem culture may signify contamination, colonization, or infection. 3. Distinguishing between the above possibilities cannot be accomplished on the basis of microbiologic data alone but requires correlation with the clinicopathologic database. Stated alternatively, the interpretation of positive postmortem cultures must include an attempt to support the culture results with the sum total of information acquired regarding the patient’s clinical course and autopsy findings. This “common sense” approach to the evaluation of autopsy microbiology (2 1) absolutely demands such integration, which is derived only from sound clinical judgement. 4. As illustrated above, but nevertheless applicable and appropriate for reinforcement in this section, there exists an imperfect correlation between antemortem and postmortem microbiology. Two published studies by Koneman et al. and Koneman and Davis (14,lS) and studies by Wood et al. (29) and Klastersky et al. (11) all illustrate the substantial disparity between antemortem and postmortem cultures. Reznicek and Koontz have identified the

Postmortem

Microbiology

15

following factors in determining the strength of the antemortem-postmortem microbiologic congruence: (i) gross (naked eye) observational skills at autopsy (i.e., the ability of the prosector to identify and select the appropriate areas to sample for culture); (ii) use of proper sterile culture technique in the procurement of the samples and adequate tissue sampling; (iii) the administration and duration of antimicrobial therapy; and (iv) contamination by normal flora, thus producing a potential mix of clinically significant and insignificant microorganisms, a situation requiring sound clinical j udgement for their discrimination (2 1) . Practical corollaries to the preceding principles are as follows: 1 . The most consistent and reliable way by which to

interpret postmortem cultures within the proper context of the clinicopathologic database is to ask the following question: is the recovered microbial isolate(s) compatible with the antemortem clinical diagnosis and autopsy findings? One must be careful never to evaluate postmortem cultures in a vacuum. A common example of this phenomenon occurs when a positive isolate results from previous exposure to an organism rather than infection by it, as with prior immunization or vaccination. The fact remains that there exists no convenient formula to substitute for clinical judgment. Thoughtful and meaningful correlation is the best defense against, and will minimize, the misinterpretation of postmortem culture results. 2 . The pathologist must maintain a healthy degree of skepticism in the interpretation of postmortem cultures and should always at least comider the possibility that the isolated organism represents a contaminant rather than an actual pathogen. Familiarity with common environmental (autopsyroom) contaminants and skin colonizers is critical, as the passive transfer of these microorganisms from contaminated gloves,instruments,or surfacesto the specimen container can result in spurious instances of positive isolates.Common examplesof suchorganisms include, but are not necessarilylimited to (depending upon the particular ecosystemsof individual autopsy rooms and populations), coagulase-negativestaphylococci (S. epidermidis), Corynebacterium species (diphtheroids), certain streptococci, and someFlavobacterium species.Clues to the presenceof contaminants include the following observations: (i) a prolonged latency period between inoculation of the sample onto media and subsequentgrowth (typically 5 to 7 days before the appearance of colonies on a plate or turbidity in a blood culture bottle) and (ii) the recovery of multiple nondominant organisms from a particular anatomic site. The latter situation, which

16

Caplan and Koontz

Table 2.

The majority

of postmortem

CUMITECH microbial

isolates

are contaminants

35

or colonizers

Anatomic site

No. of sam ples wi th isolates/ total no. of sam ples (%I

No. of samples with significant isolates/ no . of samples with growth (%)

No. of samples with insignificant isolatesa/ no. of samples with growth (%I

Heart blood Lung Liver Spleen CSF

19/23 (83) 27/29 (93) 9/24 (37.5) IO/29 (34) 6118 (33)

2/19 (II) 5127 (19) II9 (11) 3110 (30) l/6 (17)

19/l 9 (100) 26/27 (96) 819 (8% 8/10 (80)

516(83)

a Colonizing or contaminating organisms.

3.

4.

5.

6.

occurs commonly in the hospital autopsy setting, requires communication between the microbiologist and pathologist, as workup and identification of clinically insignificant, contaminating microorganisms are clearly neither time- nor cost-effective and not only will impose an undue burden on the microbiology laboratory but will, in addition, threaten to quash what little enthusiasm exists for postmortem cultures! Such communication entails reciprocal responsibility: while it is the duty of the microbiologist to inform the pathologist of the presence of probable contaminants, the pathologist must in turn communicate back to the microbiologist and apprise the latter of the likelihood that a particular microorganism has potential clinical significance. The extent of any additional workup is dependent upon that communication, which constitutes the microbiologist-pathologist arm of the communication loop. Isolation of the same organism(s) from multiple anatomic sites constitutes supportive evidence for disseminated infection (sepsis). A compatible clinical picture of fever, hypotension, and oliguria, which occurs typically in a rapid or fulminant fashion, ideally supports a postmortem diagnosis of sepsis. The demonstration of organisms within tissue, on either histologic sections or Gram stains of homogenized tissue, substantially corroborates a diagnosis of infection based upon positive culture results. Conversely, because of the low sensitivity of histologic demonstration of microorganisms (that is, the high number of organisms required to demonstrate only one per high-power histologic field), the absence of such tissue demonstration does not rule out the possibility of infection but indicates that a sufficiently large burden of organisms is necessary for detection in histologic stains under a highpower field and has not been achieved. The particular host response should be compatible with the nature of the presumed offending organism. A complete absence of an inflammatory host response indicates postmortem colonization or agonal invasion rather than true antemortem infection. It is not uncommon to see aggregates of cocci within pulmonary alveoli or bronchioles, often accompanied by squamous cells or aspirated food, but entirely without an inflammatory reaction, in sec-

tions of lung obtained at autopsy. A host response that differs in type or intensity from the expected reaction requires a pathophysiologic explanation; specifically, blunted or diminished immune responses to infection, in the form of ill-defined granulomas, necrosis, or amorphous fibrinous exudates, may reflect an altered immune status on the part of the host (21). The University

of Michigan

Hospital

Database

The above guidelines are enumerated in order to provide a reasonable conceptual framework within which to interpret postmortem cultures. However, the practical value of such guidelines can be assessed only by utilizing the guidelines in the context of an actual database of autopsies for which clinical, pathologic, and microbiologic data are all retrievable. Postmortem culture results were compiled from 36 autopsies performed at the University of Michigan Hospital between September 1993 and March 1997; the finding are displayed in appendix 2. The compilation of cases presented in appendix 2 clearly underscores the importance of recognizing the previously delineated guidelines and their associated corollaries. Table 2 illustrates, in a more compact fashion, the danger of interpreting postmortem cultures outside their proper context. As Table 2 illustrates, the overwhelming majority of microbial isolates, especially those recovered from heart blood or lung, were contaminants or colonizers, once again corroborating the data from Wilson et al. (26), Koneman et al. (15), and Roberts (22) summarized above and highlighting the point made in corollary 6 in this section. Fortunately, meaningful pathologic-microbiologic correlation does exist in some cases; furthermore, postmortem cultures in the cases which lack such correlation can still provide important negative information. The following discussion addresses specifically the process of interpretation of postmortem culture results by integrating them with the clinicopathologic database. Review of the Database and Patterns Correlation with Case Examples

of

A review of the preceding cases demonstrates three broad patterns of correlation among postmortem culture results, autopsy findings, and clinical history. Before we present these patterns, some introductory definitions are necessary.

CUMITECH

35

1. “Pathologic-microbiologic

concordance” exists when the postmortem culture results are in agreement with the autopsy findings with regard to the presenceor absenceof infectious disease. “Positive pathologic-microbiologic concordance” is present either when there is identity of microorganisms at multiple anatomic sites and there is pathologic evidence of both the presence and replication of organisms and of an inflammatory host response within those tissues or when an organism isolated from a target site is congruent with the host response to that particular organism within the site. “Positive clinical-pathologic-microbiologic concordance” occurs when there is a clinical history suggestive of sepsis or a localized infection in addition to the pathologic-microbiologic correlation. “Negative clinical-pathologic-microbiologic concordance,” on the other hand, characterizes the scenario in which organisms are not recovered from any anatomic sites and there is no autopsy evidence of either presence of organisms within tissues or generation of a host response. 2. “Pathologic-microbiologic discordance” is present when a discrepancy exists between the postmortem culture results and the autopsy findings. The most common situation demonstrating this pattern occurs when different organisms are isolated from different anatomic sites (nonidentity or partial identity). While there may be autopsy evidence of organisms at a particular site (as is observed most frequently in situations of postmortem bacterial overgrowth with colonizing or contaminating organisms), there is an unequivocal absence of an inflammatory host response to the organism(s). In reality, the overwhelming majority of casesdemonstrate “partial (incomplete ) Path.ologic-microbiologic concordance, ” in which some agreement exists between the postmortem culture results and the autopsy findings. This may occur either in the form of “partial positive concordance, 99where some but not all isolates are accompanied by an appropriate inflammatory host response, or as “partial negative concordance,” in which cultures from somebut not all sitesare sterile and there is no autopsy evidenceof infection. Applying these definitions to the cases from the University of Michigan database produces the following groups (categories): (i) pathologic-microbiologic pure or partial positive concordance (9 cases [3 pure and 6 partial]), (ii) pathologic-microbiologic pure negative concordance (2 cases), and (iii) pathologicmicrobiologic discordance (including partial negative concordance) (3 1 cases). Although it is understandably not feasible within the limits of this Cumitech to elaborate on all of the casesoutlined in the tables, it is instructive to discuss a few casesdemonstrative of the particular categories

Postmortem

Microbiology

17

in greater detail. Two such illustrative examples are provided from group i. Case 94AE170 The decedent was a 30-year-old woman who, at approximately midnight on the day of her death, had given birth at 3T1/2weeks of gestation to a healthy infant via spontaneous vaginal delivery. At around 10:00 that morning, she complained of “backbone and uterus pain”; she was hypothermic, with a temperature of 94”F, marginally hypotensive (blood pressure, 100/60 mm Hg), and cyanotic, and the skin of her abdomen and lower extremities was noted to be mottled. When she was checked at 12:00 noon, she was unresponsive and apneic, and neither pulse nor blood pressure was recordable. A code was called, but despite aggressive resuscitative efforts, she could not be revived and was pronounced dead at 2:00 p.m. An autopsy was performed the following morning. On external examination, the decedent appeared to be in at least a moderately developed stage of putrefaction (typically requiring a PM1 of 2 to 4 days at moderate ambient temperature), with green discoloration surrounding a subclavian venipuncture site, marbling of superficial veins on upper and lower extremities, and skin slippage involving the surfaces of the face, neck, and trunk. This was curious and not readily explainable, as the decedent’s body had been appropriately refrigerated between the time of death and the beginning of the autopsy except for an approximately 3O-min interval during which the body was transferred from a local community hospital to the University of Michigan Hospital Morgue. Subsequent internal examination, however, provided valuable clues to the mystery of the accelerated putrefactive changes. The uterus, ovaries, fallopian tubes, and uterine ligaments demonstrated confluent hemorrhagic necrosis, with marked softening and black discoloration and formation of firm, dark red thrombi which distended the lumina of ovarian vessels. Approximately 8.50 ml of serosanguineous fluid and clotted blood was present within the peritoneal cavity. The remaining organs were soft, fluctuant, and amorphous. Histologic examination of the uterus, ovaries, and fallopian tubes and surrounding retroperitoneal and pelvic soft tissues revealed extensive necrosis with partially dissolved connective tissue and skeletal muscle; there were sheets of cocci within the necrotic zones, and numerous clusters of largely karyorrhectic neutrophils (fragmented nuclei) lay adjacent to the necrotic regions. The remaining organs displayed variable degreesof necrosis, most pronounced within the myocardium, but without a discernible inflammatory response, and abundant collections of cocci were present within the lumina of small vessels. Cultures of lung, liver, spleen, CSF, peritoneal fluid, and endometrium all grew group A beta-hemolytic

18

Caplan and Koontz

in addition, pyrogenic streptococcal exotoxin A was demonstrated within the CSF. The cause of death was certified as group A beta-hemolytic streptococcal puerperal sepsis (invasive group A betahemolytic streptococcal disease).

Streptococcus;

Case 96AE140 The decedent, a 42-year-old man with a history of morbid obesity and chronic obstructive pulmonary disease(COPD), was a heavy smoker (at least 11/2packs per day); the remainder of his history was obscure, as he had moved recently from Texas to Michigan, but was suspicious for alcohol dependency. He lived with a roommate and had complained to the roommate of a productive cough and fever (up to 103.9”F) but apparently had not sought medical attention. However, the illness forced him to miss severaldays of work at his job as a bus driver for the local public transportation company. He was found unresponsive, kneeling over a closed toilet bowl, in the bathroom of his residence by his roommate; the toilet seat cover and the surrounding bathroom floor wereextensivelystainedwith dark brown fluid, and several tissues were filled with the same brown, viscous, mucoid fluid. Autopsy demonstrated a cirrhotic liver and congestive splenomegaly, strengthening the suspicion of chronic alcoholism, and diffuse consolidation of the lower lobe of the left lung (lobar pneumonia); the parenchyma1cut surface of the affected lobe had a gray, meaty appearance, and the overlying visceral pleura was lined by a fibrinous exudate. Patchy, focally confluent consolidation involved all of the remaining lobes with the exception of the right middle lobe. Histologic examination of the left lower lobe confirmed the gross impression of lobar pneumonia, revealing a diffuse intraalveolar exudate composed of abundant neutrophils and fibrin with viable alveolar walls, along with hyaline membrane formation (acute, exudative, diffuse alveolar damage, the pathologic correlate to clinical adult respiratory distress syndrome [ARDS]). S. pneumoniae was recovered from the lower lobe of the left lung, heart blood, and spleen; Haemophilus influenzae was also isolated from the left lower lobe. Discussion of Cases94AE170 and 96AE140 The first case(94AE170) offers a textbook example of clinical-pathologic-microbiologic concordance. In retrospect, the decedent was exhibiting signs of profound septic shock and exotoxemia with hypothermia, hypotension, and diversion of blood from the skin to vital organs, accounting for the mottling. The accelerated putrefactive changes,which, by definition, require the presence of bacteria within the circulatory system, may be explained by the presence of an antemortem bacteremia which expedited the postmortem intravascular dissemination of organisms by shortening the interval usually necessaryfor bacteria to cross

CUMITECH

35

the blood-intestinal barrier. The relative paucity of a systemic, inflammatory host response is also understandable given that the group A beta-hemolytic streptococci elaborated a pyrogenic exotoxin (exotoxin A), which mediated the eventsaccountable for septic shock. As this was not a suppurative inflammatory reaction, but rather one dominated by necrosis, one would not have expected to observe an exuberant inflammatory host response.The host response,therefore, was entirely concordant with the mechanism of microbial injury (24, 28). In summary, this caseachieves clinical-pathologic-microbiologic concordance in the setting of bacterial sepsis and exotoxin-mediated septic shock. The second case (96AE140) also provides an excellent illustration of concordance. The clinical history of COPD placed the decedent at increased risk for pulmonary infection; furthermore, the autopsy findings of cirrhosis and congestive splenomegaly may have also interfered with the decedent’s ability to opsonize and clear encapsulated organisms from the bloodstream. A target organ (the left lung) was identified as suspicious for infection, and the inflammatory host response, an intra-alveolar exudate with intact alveolar walls and early diffuse alveolar damage, was compatible with that expected from S. plzeumoniae. The recovery of S. pneztmoniae from the heart blood and spleen also suggests pneumococcal sepsis, probably a complication of the pneumonia . In conclusion, this caseillustrates clinical-pathologic microbiologic concordance in the form of a localized infection progressing to probable secondary bacteremia and sepsis. Category iii includes all of the cases certified as sudden infant death syndrome (SIDS). Given that SIDS is a diagnosis of exclusion, and that one category of exclusion is the presence of any infectious diseases either causing or contributing to the death, it is logical that postmortem cultures be included as part of the ancillary studies enumerated in standardized protocols for the workup of sudden infant deaths. In addition, infant deaths which are attributable to known noninfectious causes are included in this category, as are unexplained deaths of apparently healthy adults or children, in which postmortem cultures are obtained in order to rule out an infectious etiology for the death. The nearly ubiquitous phenomenon of positive cultures (with the exception of the two cases with sterile cultures comprising category ii) also serves to emphasize the critical importance of pathologic-microbiologic correlation: despite one’s best efforts at obtaining sterile cultures, this rarely occurs, especially in the case of heart blood and lung, and pathologists must resist the powerful temptation to ascribe significance to seemingly pathogenic microorganisms which are really only contaminants or colonizers of a particular anatomic site. The following caseexample illustrates such an instance.

CUMITECH

Postmortem

35

Case 94AE356 The decedent was a 100month-old infant with a history of bronchial asthma who had been hospitalized for 3 days at W’2 months of age for an acute exacerbation. The asthma was apparently well controlled since that time, without additional asthma exacerbations or other illnesses. The child’s mother put her to bed, uneventfully, on the evening before her death, and found her the next morning, cold and unresponsive. She immediately called 911. Paramedics arrived to find the child without vital signs, and she was transported to a local community hospital, where resuscitative efforts continued for approximately 20 min but subsequently stopped due to the inability to generate vital signs or viable cardiac rhythm. A n autopsy performed on the afternoon of her death was entirely negative. Subsequent histologic examination revealed changes of chronic asthma, including focal hyaline thickening of bronchial mucosal basement membranes, goblet cell hyperplasia, and infiltration of bronchial wall submucosa by eosinophils; neither mucous plugs nor degranulated eosinophils were discernible within bronchial or bronchiolar lumina. There were no intraalveolar exudates or interstitial infiltrates suggestive of an acute interstitial pneumonia or bronchopneumonia. Samples of heart blood, lung, liver, and spleen were submitted for culture; both heart blood and lung grew S. pneumoniae, with the latter site growing it in large quantities. At first glance, the presence of S. pneumoniae in both heart and lung, with recovery of numerous organisms from the latter site, would seem to prompt a suspicion of pneumococcal pneumonia with accompanying sepsis. S. pneumoniae is generally perceived by pathologists as a pathogen with virulence induced by its antiphagocytic polysaccharide mucoid capsule; therefore, this organism seemingly should be regarded as significant. However, the organism was not recovered from the liver or spleen, thus weakening the prospect of sepsis. Even more disturbing was the complete absence of the inflammatory response that would be expected within the lungs of an individual reacting to S. pneumoniae, namely, an exuberant intraalveolar exudate of neutrophils and fibrin with intact alveolar walls. The solution to this apparent conundrum lies in recognizing the misconception of S. pneumoniae as a pathogen; rather, in this setting, it is more correctly viewed as a colonizing organism. S. pneumoniae is part of the known indigenous flora of the mouth and oropharynx. It is also a member of the normal flora of the upper respiratory tract in approximately 25 to 50% of preschool children and almost 20% of adults, and individuals who harbor the organism in their upper respiratory tract are referred to as “carriers” (28). Given this background information, in combination with the absence of pneumonia, clinical signs or symptoms suspi-

Microbiology

19

cious for sepsis, and isolation of S. pneumoniae from the liver or spleen, it becomes evident that a case for pneumococcal pneumonia or sepsis cannot be made. One case in the University of Michigan series, while fitting generally into category iii, is unique in that the concordance that exists is not between postmortem culture results and autopsy findings but between antemortem and postmortem latex agglutination tests for S. pneumoniae antigen.

Case 93A303 The decedent was a 2-year-old African-American boy who was diagnosed as a neonate with sickle-cell disease. He had been placed prophylactically on penicillin VK but had not received the pneumococcal vaccine. He also had not experienced any sickle-cell crises. For unknown reasons, he had not been given his penicillin during the last 1 to 2 days preceding his admission to the hospital. He presented to a community hospital with a 24-h history of high fever (103 to 104°F); there was no accompanying pain, cough, or other symptom. Blood cultures were drawn, a dose of cefuroxime was administered intravenously, and the child was subsequently transferred to the Pediatric Hematology-Oncology Unit at the Mott Children’s Hospital of -the University of Michigan. Upon arrival, the child was in reasonably stable condition; 3 h later, however, a nurse found him to be unresponsive, pulseless, and apneic. A code was called immediately, but after 45 to 50 min of unsuccessful resuscitative efforts, resuscitation was discontinued and the child was pronounced dead. The community hospital had notified the Pediatric Hematology-Oncology floor that a Gram stain of the blood cultures had demonstrated grampositive diplococci in pairs and immunoreactivity to S. pneumoniae capsular antigen by LA. The autopsy was unremarkable except for splenomegaly (74.4 g; expected weight for body weight, -35 g). Sickled cells with distinctly pointed ends were readily observed histologically within blood vessels and sinusoids, and the bone marrow exhibited erythroid hyperplasia. Abundant deposition of hemosiderin, excess storage iron formed as a result of hemorrhage or red cell breakdown, was observed within the cytoplasm of renal tubular cells, a finding suggestive of chronic hemolysis. There was neither gross nor histologic evidence of pneumonia, and the autopsy failed to uncover any other localized site of infection. Although samples of heart blood, lung, liver, and CSF were all submitted for culture, S. pneumoniae could not be isolated from any of the above-mentioned anatomic sites. LA tests for S. pneumoniae capsular antigen were negative with both CSF and urine; however, postmortem serum did demonstrate immunoreactivity for the pneumococcal antigen. To our knowledge, resin-containing blood culture bottles were not used. In this case, in addition to the concordance be-

20

Caplan and Koontz

Table 3.

Summary

of responses

CUMITECH to question

35

4 of the questionnaire Usefulnessa

No. (%)

Useful (most) Clinically suspected but unconfirmed sepsis or infection ...................................................................................................................... Anatomic focus of infection (target site) discovered at autopsy (i.e., TB, abscess, or meningitis) ...................................................... lmmunocompromised pts. (primarily in Dx of fungal, mycobacterial, and viral infections) ................................................................... Dx of invasive fungal infections .............................................................................................................................................................. When performed to answer specific question regarding cause of death ............................................................................................. Known (established) antemortem infection, Rx with antibiotics ............................................................................................................ Educational value for residents ............................................................................................................................................................... Dx of viral infections ............................................................................................................................................................................... Recovery of viruses in perinatal cases ................................................................................................................................................... Indigent (non-HIV-infected) pts. with pulmonary lesions ....................................................................................................................... Dx of mycobacterial infection ................................................................................................................................................................. When selective ....................................................................................................................................................................................... As a negative result ................................................................................................................................................................................

10 (40) 8 (32) 7 (28)

2 (8) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4) 1 (4)

Not (least) useful Routine (standard/nonselective) PM Cx ................................................................................................................................................. 9 (36) When contaminants are present .......................................................................... ................................................................................. 8 (32) History of antibiotic Rx ......................................................................................... ................................................................................. 8 (32) Prolonged postmortem interval ............................................................................ ................................................................................. 5 (25) Clinical-pathologic-microbiologic correlation not achieved (interpretation of resu ts not meaningful) ................................................... 5 (25) Dx of bacterial infections (bacterial Cx) ................................................................ ................................................................................. 3 (12) Perinatal and neonatal autopsies .......................................................................... ............................................~.................................... 2 03) Clinical Hx and/or autopsy findings establish noninfectious cause of death.. ..... ................................................................................. 2 (8) Organism already isolated (identified) in antemortem Cx .................................... ................................................................................. 2 (8) Viral Cx (less frequently useful than fungal or mycobacterial Cx for immunoincompetent pts.) .......................................................... 2 (8) In setting of multisystem organ failure .................................................................................................................................................. 1 (4) Isolated PM blood Cx ............................................................................................................................................................................. 1 (4) Repeated antemortem surveillance Cx .................................................................................................................................................. 1 (4) Cx for fastidious bacteria and fungi ........................................................................................................................................................ 1 (4) a Abbreviations:

Cx, cultures;

Dx, diagnosis;

Hx, history; PM, postmortem;

tureen antemortem and postmortem LA tests for S. pneumoniae antigen, there was strong clinical-patho-

logic concordance. The autopsy signs of sickle-cell disease (i.e., the generalized sickling of red cells, erythroid hyperplasia within bone marrow, and hemosiderin deposition within renal tubular cells) strongly supported the existenceof a condition with an inherently increased susceptibility to infectious diseasescaused by pneumococci and other encapsulated organisms by virtue of its attendant splenic hypofunction. The inability to culture S. pneumoniae is most likely the result of aggressiveintravenous antibiotic treatment (cefuroxime, 600 mg). In this case,although conventional postmortem microbiology was negative, the positive LA result for the S. pneumoniae capsular antigen in postmortem serum helped to confirm pneumococcal sepsis as the cause of death- even in the face of an essentially negative autopsy and a clinical picture comprising solely fever without localizing signs and with a positive blood culture and LA test for S. pneumoniae. For reasonspresentedabove, the positive LA result in isolation would be of questionable significance; in light of supporting clinical and pathologic data, however, it does achievesignificance. Thus, while ancillary immunologic studies are certainly not advocated as replacements for postmortem cultures, they can be valuable when antemortem manipulations prevent the recovery of a suspected organism. Hopefully, the above case examples provide some

pts., patients;

Rx, treatment;

TB, tuberculosis.

insight into the frequently mystifying process of interpreting - and making senseof -postmortem cultures. Institutional Question 4

Responses

to the Questionnaire:

This section concludes with the last of the four questions on the questionnaire sent to the 25 pathology teaching institutions. It is our opinion that the responsesto this last question most faithfully and accurately reflect the current prevailing attitudes among pathologists toward postmortem cultures (or at least among those who consider obtaining postmortem cultures!) with regard to their overall usefulness and place in autopsy pathology. Question 4 is, “In which cases have you found postmortem cultures to be (most) useful? Not (least) useful?” The responsesto the last question can be ranked in order of decreasing frequency (Table 3). Review

of Responses

to Question

4

1. There is a decisive trend away from routine, non-

selective postmortem bacteriology and toward selective (case-based) postmortem virology, mycology, and mycobacteriology. This trend clearly illustrates the evolving importance of diagnosing infectious diseasesin the ever-expanding population of immunoincompetent patients. 2. Institutions are reluctant to pursue postmortem

Recommended

criteria

for acquisition

Clinical condition (suspected diagnosis) I. Sepsis

(bacterial

Recommended

or fungal)

2. Localized infection pneumonia)

cultures specimen(s)

Heart blood, lung, liver, spleen

(e.g., abscess,

3. Tuberculosis

Target

tissue

or organ

Lung

4. Disseminated

fungal

of postmortem

mycobacterial

or

Lung, liver, spleen,

bone marrow

infection

5. Viral infection

Target tissue (lung, liver, brain, CSF in immunoincompetent patients)

6. lmmunoincompetent host (HIV infected, posttransplant, postchemotherapy, leukemic or other neoplasm)

Heart blood, lung, liver, spleen (for bacterial or fungal sepsis); target tissue or organ for infectious focus discovered at autopsy; specimens as indicated above for disseminated viral, fungal, and mycobacterial infection Target tissue if localized infection; heart blood, lung, liver, spleen if sepsis; use resin-containing culture bottles

7. Evaluation therapy

of efficacy

of antimicrobial

cultures (and especially bacterial cultures) in the setting of prior antibiotic therapy, despite data from some studies suggesting that antibiotic therapy does not automatically negate the value of postmortem cultures (studies by Carpenter and Wilkins [6], Wood et al. [29], Koneman et al. [15], and Aranda et al. [2], cited above under “Indications for Postmortem Cultures”). Only additional investigation and research will provide the impetus to modify current thinking with regard to this issue. 3. A prolonged PM1 is another relative contraindication to performing postmortem microbiology, although once again, such a practice contradicts the available data regarding this subject (see “The Microbiology of Postmortem Tissues” above). 4. The nearly ubiquitous presence of contaminants in postmortem bacterial cultures is probably the single most important reason for the aversion of institutions to routine (nonselective) bacterial cultures and is also most commonly responsible for the failure to achieve meaningful clinical-pathologicmicrobiologic correlation following the autopsy. 5. Pathologists are most receptive to obtaining postmortem cultures when they are guided by a positive anatomic finding at autopsy, either at an unexpected site discovered at autopsy or at a clinically suspected focus of infection confirmed at autopsy. They are probably less receptive to obtaining random cultures from sites at which there is no pathologic evidence of infection but when a clinical diagnosis of sepsis has been entertained and the cultures are performed at the request of the clinicians (that is, to satisfy a clinical question rather

Recommended

ancillary

study(ies)

Histopathologic examination; modified tissue Gram stains for bacteria; fungal stains Histopathologic examination; special microbial histochemical or immunohistochemical stains; tissue-based molecular methods (in situ hybridization and PCR) Histopathologic examination; acid-fast stain; immunohistochemistry; molecular method Histopathologic examination; acid-fast stain; fungal stain (see Table A3.1); immunohistochemistry; molecular method Histopathologic examination (routine hematoxylin-and-eosin stain as well as special viral inclusion body stains); immunohistochemistry; electron microscopy; molecular methods All of the above, including Gomori’s methenamine silver, Giemsa stain, immunohistochemistry, or electron microscopy for Pneumot ys tis carinii Histopathologic flammatory

examination response

to evaluate

in-

than to resolve a pathologic issue). Although this observation may be somewhat conjectural, it does suggest that preautopsy communication between pathologist and clinician might be valuable not only in directing the focus of the autopsy but also possibly in eliminating unnecessary cultures or streamlining the scope of the microbiologic studies. Some recommended criteria for acquisition of postmortem cultures are given in Table 4.

CONCLUSION This Cumitech has attempted to provide a comprehensive overview of the present state of postmortem microbiology as it exists in major path01 ogy teaching institutions in the United States. It is hoped that discussion of some of the issues related to postmortem microbiology will, at the very least, stimulate some interest in a field which has been unfortunately dormant for too long. These include the usefulness of postmortem microbiology in the setting of prior antibiotic therapy; relative effectiveness of methods for surface decontamination; variable transport times of identical specimens and their relative microbial yields; the use of fine-needle aspiration to obtain postmortem cultures; and alternative and/or ancillary methods in postmortem microbiology in addition to conventional postmortem culturing, such as immunologic or molecular pathologic techniques. It is also our conviction that as long as infectious diseases cause or contribute to patients’ deaths, postmortem microbiological studies will at least be potentidly useful and, thus, will be assured a place in autopsy pathology.

22

Caplan and Koontz

APPENDIX 1 INSTITUTIONAL Table AM.

QUESTIONNAIRE:

INDIVIDUAL RESPONSES

Ques$ion 1: “In which cases do you pgrform postmortem

institution University of Pennsylvania

University of Cincinnati Cleveland Clinic

University of AlabamaBirmingham

University of Nebraska

University of Wisconsin Northwestern

CUMITECH 35

University

University of Kentucky

Cedars-Sinai Medical Center Emory University

West Los Angeles VA Medical Center Duke University

Penrose/St. Francis Health Center-C

Indications’

cultures--what

are the indications?”

Referenceb

1. If patient chart indicates possibility of sepsis Autopsy Microbiology Protocol (Perez-Jaffe and AM Cx obtained; not when PMI > 20 h and Nachamkin) unless looking for Legionella, TB, or fungus 2. If AM Cx have been obtained: a. to evaluate efficacy of antibiotic Rx b. to obtain information on a different, unsuspected infectious lesion Suspected infection but not confirmed AM 1. Viral Cx in perinatal deaths, unknown COD 2. Fungal Cx in: a. patients with no AM Ox of fungal infection but positive autopsy findings b. immunoincompetent and Txp patient (posttransplant) population (Cx for Aspergillus in order to determine species) Perform cultures in all cases: 1. Aerobic and anaerobic Cx on blood 2. Cx an area of lung that appears abnormal by palpation (indurated or consolidated) PM Cx tend to be clinician driven 60-70% of autopsy population comprised of Txp patients (liver, bone marrow) (immunoincompetent hosts) If lung demonstrates consolidation If H/O sepsis, take blood plus lung Possibility of infection; clinical conditions which support Ox of infection Standard practice is to Cx in all cases (rationale: educational value for pathology house officers) Also useful in “failure to diagnose” cases Most commonly when clinical questions involve potentially infectious etiology 1. Strongest indication for PM Cx: clinically suspected but previously undiagnosed or unconfirmed sepsis or infection; if AM Cx are positive, PM Cx may be unnecessary 2. Additional indication: discovery of unanticipated or unsuspected potential source of infection at autopsy (e.g., abscess) Suspected endocarditis or sepsis (if AM Cx have not been obtained) Occasionally viral Cx in HIV-infected patients PM Cx performed in 40% of autopsies Before performing autopsy, pathologist looks at patient’s computerized database and consults with clinicians: 1. If sepsis or infection is an important or relevant issue, PM Cx are performed 2. In case of immunoincompetent patients, equivocal AM Cx are followed up with PM Cx 1. Question of previous infectious disease 2. Unexplained fever 3. Unexpected finding at autopsy: abscess, pneumonia and/or consolidation

University of Washington

Routine lung Cx performed for potential source of sepsis Local infectious process-abscess, empyema Perinatal and fetal deaths-lung and placenta (stillbirths), R/O chorioamnionitis

Columbia University

PM Cx performed or attempted in a// cases; not always successful

Comment “Cultures should not be a routine procedure but should answer specific questions regarding the cause of death and factors contributing to it.” -Perez-Jaffe and Nachamkin

Exceptions: 1. Do not Cx for perinatal deaths (macerated fetus). 2. Not on embalmed bodies.

Preferable not to take blood Cx alone (contamination) Not performed routinely on all patients. Do not Cx for perinatal cases routinely.

No rigid criteria Guidelines for Obtaining Post-Mortem Microbiological Cultures (League)

Underlying principle: routine postmortem microbiological cultures are usually unnecessary and rarely contribute to autopsy diagnoses

Guiding philosophy: performing PM Cx selectively rather than routinely (in every case) will enhance the value of the results and render them more meaningful. Evolution: initially, PM blood Cx performed routinely Do not routine/y perform viral or fungal Cx; unusual fungi are identified by mycology laboratory, PM Cx are not always performed,

(Table clantinues)

CUMITECH

Postmortem

35

Microbiology

23

Table Al .l. Continued Institution University of California at San Francisco Cornell University

Johns Hopkins University

Indication9

Performed tn every case TB and/or mycobacterial Cx for immunoincompetent or HIV-infected patients Contraindications for bacterial Cx include H/O antibiotic administration, repeated AM surveillance Cx, and extended PMI For immunoincompetent patients (HIV infected, post-Txp, -chemotherapy): viral and mycobacterial Cx or viral and fungal (chemotherapy, Txp) Cx of lung and liver Strong clinical suspicion of infectious disease; no AM Cx

Harvard/Massachusetts General Hospital

Absolute indications (PM Cx always performed): abscess, clinical suspicion or pathologic impression of TB or other mycobacteriosis, suspected endocarditis or meningitis Relative indications (PM Cx usually performed): suspected pneumonia (if important clinical question); pyelonephritis Fairly liberal indications; PM Cx performed in approx 50% of cases Absolute indications (always performed): septicemia, unexplained fever Routine cultures performed in almost all cases

Harvard/Brigham and Women’s Hospital Stanford University

University of Chicago University of Kansas

Comment Most potent disincentive: PM contamination.

Sole indication: isolated infection (abscess)

Washington University/ Barnes Hospital

Mayo Clinic

Referenceb

Utilization of Microbialogy in Autopsy (Hutchins)

Evolution: at one time, PM Cx of heart blood were performed routinely. Presently, routine PM bacterial Cx are not performed; there must be a positive anatomic finding at autopsy or the clinical history must be strongly suggestive of infection in order to proceed with PM bacterial Cx. PM Cx are performed uncommonly (a few in >500 autopsies); reliance is on histopathology for Dx of infectious disease.

1. Any case that lacks a definitive, well-established organism by AM Cx 2. lmmunoincompetent patients Clinical suspicion but no confirmation of sepsis Policy: strong bias against bacterial Cx because of little confidence in their results. PM Cx are reserved for isolation of viruses and fungi.

‘Abbreviations: Cx, cultures; Txp, transplant; AM, antemortem; Dx, diagnosis; PM, postmortem; chemo, chemotherapy; H/O, history of; Rx, treatment; R/O, rule out; COD, cause of death; TB, Mycobacterium tuberculosis. 6 See text for camp lete information on references. c Colorado Springs, Cola.

24

CUMITECH

Caplan and Koontz

Table Al.2

Question

2: “Which

Institution

sites do you culture?“a Site cultured X

X

X

University of Crncinnati Cleveland Clinic

x x

x x

X

University of AlabamaBirmingham

x

x

X

University of Nebraska

X

University of Wisconsin

x

Northwestern Unrversrty University of Kentucky

X

Cedars-Sinai Medical Center

x

x

X

x

X

x

X

X X

Penrose/St. Francis Health Center University of Washington

x

Columbia University

x

Mayo Clinic Harvard/Brigham and Women’s Hospital Stanford University University of Chicago University of Kansas

X X

X

West Los Angeles VA Medical Center Duke University

Washington University/ Barnes Hospital Harvard/Massachusetts General Hospital

X X

Emory University

University of California at San Francisco Cornell University Johns Hopkins University

Comment9

Blood Lung Spleen Other site(s)

University of Pennsylvania

X

x

X X

x

x X

X

X

x

x

X

x x

x x

X

X

x X

35

x X

See written guidelines (Perez-Jaffe and Nachamkin) Spleen for sepsis workup (blood discouraged as it is “rapidly contaminated postmortem by intestinal flora“) Lung for suspected TB Bone marrow, spleen, and liver for disseminated fungal or mycobacterial infection Tissue (i.e., lung and brain) and fluids (i.e., CSF) for clinically suspected viral disease Lung, urine, and serum for suspected L. pneu~~p~j/a Serous cavity and abscess fluids when indicated or dictated by clinical suspicion or autopsy findings Most frequent sites; dependent upon clinical scenario Perinatal autopsies: blood, sometimes lung For fungal infections: suspected organ of involvement, usually lung Do not routinely submit blood or lung for bacterial Cx Blood (aerobic and anaerobic) and lung (aerobic) in all cases Any other site that appears to be involved by infection, including fluids and abscesses Lung is most common site (Txp population); suspected intra-abdominal sites of sepsis Blood and lung for suspected sepsis Lung if consolidated Serous fluids if cloudy Case variable; depends upon presumed source of infection Blood Cx not performed in isolation (contamination issue) Discrete infectious focus (i.e., suspected infectious lesion within lung) Blood and lung routinely (all cases) Vascular catheter (i.e, central venous line) if present Other focal lesion suspicious for infection Blood sampled in almost all cases (including suspected sepsis) Otherwise, site-specific: target organ plus blood See written gui‘delines (League) Traditionally (until recently): blood, lung, and spleen regardless of suspected cause of death Currently: sterile site (blood, lung, or spleen) for clrnically suspected but unconfirmed sepsis (no AM Cx) Clinically unsuspected infectious focus (e.g., abscess) Lung is most frequently cultured site (i.e., abscess, suspected TB) Heart valve (vegetation) in suspected endocarditis CSF (via cisternal tap) for suspected meningitis Liver for suspected viral or fungal infection Target organ for suspected infectious focus Originally: routine blood Cx Currently: routine lung Cx for sepsis Sites determined by clinical Hx and gross autopsy findings (e.g., CSF for suspected meningitis; lung and placenta for stillbirths) Target site for known or suspected infection Blood and lung for unknown infection (screen) in almost all cases (routinely) Suspected infectious focus (e.g., paravertebral abscess) Sites cultured routinely (every case) See written guidelines (Hutchins) Traditionally: routine blood Cx Currently: case determined; based upon clinrcal Hx and autopsy findings-no routine Cx Lung and liver for mycobacterial and viral Cx in HIV-positive patients and for viral and fungal Cx in postchemotherapy and post-Txp (BMT) patients Particular site(s) depends upon circumstances of case Heart blood routinely and with clinical suspicion of sepsis (but do not rely exclusively upon this result; blood and lung) Other suspected sites of infection: abscess; lung (TB); heart valve (endocarditis); CSF (meningitis) Lung (for pneumonia), kidney (for pyelonephritis) if important clinical question or issue Any sites suggestive of or suspicious for infection Sites cultured routinely (all cases) One lung Heart blood cultured routinely (all cases) Sites reserved for detection of viruses and fungi

a See Table Al .I footnotes for additional details. b Abbreviations: Hx, history; BMT, bone marrow transplant; TB, tuberculosis.

CUMITECH

Table A1.3.

35

Question

Postmortem

3: “What

Institution

techniques

Surface decontamination Spatula

University of Pennsylvania

X

University of Cincinnati Cleveland Clinic University of AlabamaBirmingham University of Nebraska University of Wisconsin Northwestern University University of Kentucky Cedars-Sinai Medical Center Emory University West Los Angeles VA Medical Center Duke University Penrose/St. Francis Health Center University of Washington Columbia University University of California at San Francisco Cornell University Johns Hopkins University Washington University/Barnes Hospital Harvard/Massachusetts General Hospital Mayo Clinic Harvard/Brigham and Women’s Hospital Stanford University University of Chicago University of Kansas

X X8 Xb

a Blade. b Putty knife. = Alcohol. d Iodine. e Alcohol swab. f Iron. Q Betadine or alcohol.

do you use to culture-specimen

X X

Antiseptic

transport,

Type of sample Swab

processing,

25

etc.?”

Comment(s)

Tissue X

X XC

X X X X

collection,

Microbiology

See written guidelines (Perez-Jaffe and Nachamkin); swabs uniformly discouraged; “generous piece of tissue”

Forceps to grasp tissue dipped in alcohol

I- to 2-cm3 block of tissue X X

Swab for bacterial Cx; tissue block for fungal and TB

X X

l-cm3 wedge of tissue X

Do not sear or disinfect organ surfaces; rarely use swab

Xd X8

X Xf X

X X X

X

X

X X

X X

X X X

X X X

XQ

Swabs for organs (lung); tissue for heart valve vegetation, abscess wall

Wedge of tissue Method of sterilizing organ surface varies within department

26

Caplan and Koontz

Table Al .4.

Question

CUMITECH

4: “In which

cases have you found

postmortem

cultures

Institution

(Most) useful

University of Pennsylvania

When performed to answer a specific question regarding the COD Clinical suspicion of mycobacterial infection (M. tuberculosis or atypical mycobacteria) When they confirm an AM Dx

University of Cincin nati Cleveland Clinic

lmmunoincompetent patients (species of unusual bacteria and fungi can be determined) (Occasionally) recovery of viruses in perinatal autopsies

University of AlabamaBirmingham University of Nebraska

May be useful as a negative result (sterile Cx)

University of Wi sconsin

Northwestern

U niversity

University of Kentucky

Cedars-Sinai Medical Center Emory University

West Los Angeles VA Medical Center Duke University

Penrose/St. Francis Health Center

University of Washington

Columbia University University of California at San Francisco Cornell University

lmmunoincompetent patients (i.e., HIV infection; post-Txp patients on immunosuppressive Rx) Example: thrombosis of liver allograft with subsequent graft failure; Cx of thrombus and necrotic liver grew Candida albicans (candidal vasculitis) When a positive anatomic finding at autopsy (i.e., consolidation within a lung) correlates with a positive Cx from the same anatomic site lmmunocompromised patients (e.g., HIV infection, post-Txp)

Suspected but unconfirmed AM Dx of sepsis and PM (blood and lung) Cx correspond to anatomic findings at autopsy Known (established) AM infection, treated with antibiotics and PM Cx of involved anatomic site(s) negative (indicates effective antibiotic Rx) Educational value for pathology house staff Unrecognized infection discovered at autopsy (e.g., endocarditis) Cx of target organ/site at autopsy, often with clinical Dx or suspicion of infection and positive AM Cx (PM Cx of target tissue supports AM result) Unexpected site of possible infection at autopsy (target organ; e.g., abscess)

Dx of endocarditis, tuberculosis, fungal infections Occasionally, Dx of viral infections in HIV-infected patients Patients with immunodeficiency syndromes Inability to establish Dx or specific microbial etiology of infection AM PM confirmation of clinical suspicion of infection (e.g., subphrenic abscess; anaerobic pleuropulmonary infection) Selective PM Cx: enhances value of results Anatomic focus of infection (target site); histopathologic findings can be correlated with PM Cx results Example: apparent diffuse alveolar damage (ARDS); sample of lung placed on BCYE agar grew L. pneumophila, and pleomorphic GNR were demonstrated on silver impregnation stain of lung tissue Example: tissue Gram stains to make Dx of Clostridium perfringens sepsis (with severe intravascular hemolysis) Sepsis suspected but not proven (documented) AM Unusual case with pathologic-microbiologic correlation (e.g., paravertebral abscess) Few cases

to be (most)

useful?

Not (least)

35

useful?“a

Not (least) useful When performed as a routine procedure When numerous contaminants are present When PMI > 20 h When contaminants are present Prolonged PM I “Garden-variety” patients (i.e., cardiovascular, pulmonary noninfectious COD) In setting of multiple system organ failure In setting of antibiotic Rx When performed as a routine procedure In setting of antibiotic Rx When numerous contaminants are present Majority of cases (PM Cx do not usually contribute significant information that AM Cx have not already provided)

When antibiotics were administered

Perinatal autopsies Prolonged PMI (X-3 days) Routine (“blind”) Cx (no clinical evidence of infection) Prolonged PMI contamination (i.e., positive PM Cx but no correlation with autopsy findings)

Routine Cx (of blood and lung) When pathologic-microbiologic correlation not achieved (creates problems in interpretation) When an organism has already been isolated AM from a sterile site (PM Cx unnecessary) Extensive use of antibiotics Clinical suspicion of infection (e.g., pneumonia) but no anatomic evidence of infection of target organ at autopsy Standard (routine) Cx usually unnecessary “because they rarely contribute to the PM diagnosis” Dx of bacterial infections (PM Cx not useful due to presence of contaminants) Random (routine) Cx of neonates and infants (where sepsis is part of DDx of illness) Presence of contaminants Uncertain clinical picture followed up by isolated PM blood Cx Nonselective (routine) PM Cx Patient receiving antibiotic Rx Routine Cx

COD is known (clear) Prospect of contamination is major obstacle to performing PM Cx Most cases (difficulties in interpretation of results) (Table continues)

CUMITECH

Postmortem

35

27

Microbiology

Table Al .4. Con hued Not (least) useful

Institution

(Most) useful

Johns Hopkins University

lmmunoincompetent patients (HIV or AIDS, BMT, postchemotherapy); PM fungal and mycobacterial Cx are most useful; viral Cx occasionally useful (especially in cases of suspected herpesvirus infections) Indigent patients with pulmonary lesions: PM mycobacterial Cx most useful lmmunocompetent patients if clinical Hx and gross autopsy findings indicate (are supportive of localized infection or sepsis) Rare cases (clinically suspected viral or fungal infections)

Washington University/ Barnes Hospital

Harvard/Massachusetts General Hospital

Mayo Clinic

Harvard/Brigham and Women’s Hospital

Stanford University University of Chicago

University of Kansas

Clinical or autopsy evidence of localized infection (e.g., abscess, TB, meningitis) When pneumonia or pyelonephritis is an important clinical issue Clinical setting suspicious for sepsis or localized infection (i.e., unexplained fever); AM Cx indeterminate or not obtained lmmunoincompetent patients (post-Txp; leukemic): PM Cx “very helpful,” especially in Dx of fungal infections (morphology difficult in tissue); viral Cx sometimes helpful Clinical sepsis, microbial etiology (organism) unknown Unexplained death in immunoincompetent patient Clinical signs and symptoms of sepsis (shock, multiple system organ failure; sepsis part of DDx) in combination with positive unimicrobial blood Cx In Dx of invasive fungal infections (i.e., aspergillosis, zygomycosis [mucormycosisl)

BSee Table Al .I footnotes for additional details. Abbreviations: gram-negative rods; BMT, bone marrow transplant.

DDx, differential

diagnosis;

Bacterial Cx: seldom useful, especially when accompanied by Hx of antibiotic Rx, repeated surveillance Cx, long PMI Routine PM Cx rarely useful Viral Cx less frequently useful than fungal and mycobacterial Cx in immunosuppressed patients Long PMI Rx with broad-spectrum antibiotics Fastidious bacteria and fungi are difficult to culture (questionable yield) Positive PM Cx, negative autopsy findings (no autopsy evidence of infection)-creates problems in interpretation of results Obvious contaminants (anaerobes; gram-negative bacteria, especially below diaphragm) Routine Cx (primary noninfectious tw

Dx): noncontribu-

Organism already identified in AM Cx When patient is on antibiotics: difficult to interpret

In Dx of bacterial infections: Viral Cx: interpretation of results often confusing BCYE, buffered

charcoal yeast extract;

Hx, history;

GNR,

2

2% mo

11 yr

93A3 17

93A379

M

F

M

2 Yr

93A303

Sex

F

Age

Summary

7 Yr

A2.1.

93A285

no.

Case

Table

THE UNIVERSITY

APPENDIX

NG

blood

University

Anaerobic: betastrep., not Gps A to D, F, G

Possible GNR (2 species [4 CFUI ml and 1 CFU/ ml]); possible staph. species (1 CFU/ml)

Ag

LA: positive for S. pneumoniae

Aerobic:

Heart

of the

Lung

of Michigan

Aerobic: possible betastrep. (numerous); possible Enterococcus sp. (numerous); possible Pseudomonas spp. (2 species; numerous); skin flora Anaerobic: NG Fungal: NG

Fungal: NG Aerobic: coagulase-positive staph. (rare), oral flora Anaerobic: Peptostreptococcus micros (few); Wolinella sp. (rare); Strep toccus in termedius (miileri group) (rare); Actinomyces sp., not A. israeiii (rare); Veillonella sp. (rare) Fungal: NG Aerobic: oral flora Anaerobic: Veil/one/la sp. (rare); Prevotella (Bacteroides) melaninogenica (rare)

Liver

Finding(s)

databasea

DATABASE

Aerobic: NG Anaerobic: NG Fungal: NG

NA

Fungal: NG Aerobic: NG Anaerobic: NG

Aerobic: Enterobacter cloacae (rare; 2 morphotypes) Anaerobic: NG Fungal: NG

hospital

HOSPITAL

Aerobic: coagulase-negative staph. (rare); Aspergillus flaws

OF MICHIGAN

not

Aerobic: NG Anaerobic: NG Fungal: NG

Fungal: NG Aerobic: coagulasepositive staph. (rare) Anaerobic: NG

Fungal: NG Aerobic: NG Anaerobic: NG

NA (sample obtained)

Spleen

NA

CSF: possible GNR (2 species; rare); Enterococcus sp. (possible; rare); yeast (rare); possible staph. (2 species; few); possible strep. sp. (rare)

CSF: NG LA: negative for H. influenzae type B Ag; S. pneumoniae Ag; N. meningitidis Gp A, C, Y, W135, B Ag Feces: LA: negative for H. influenzae type B Ag; S. pneumoniae Ag; N. meningitidis Gp A, C, Y, WI 35, B Ag feces) Viral: NG Coagulase-positive staph. (numerous) Fecal flora CSF: GNR (2 species; rare), y-streptococcus (rare)

Other

Postural asphyxia due to cerebral palsy with spastic quadriplegia and seizure disorder due to perinatal asphyxia due to maternal placenta previa

SIDS

S. pneumoniae sepsis due to sickle-cell disease

(followaddiand of cirsudden

of death

Undetermined ing autopsy, tional studies, investigation cumstances; death)

Cause

history

Possible apneic episodes, worked up with twochannel pneumogram (negative; did not qualify for apnea); resuscitation following 35-40 min of cardiac arrest (survived -18 h) s/p fundoplication for GE reflux w/ gastrostomy tube placement; found suspended from couch in head-down position

Recent high fever (103104°F); child had not taken penicillin in last couple of days and had not received pneumococcal vaccine (Pneumovax); antemortem blood Cx positive for S. pneumoniae (gram-positive diplococci on Gram stain); LA positive for S. pneumoniae Ag

Recent vomiting and diarrhea (gastroenteritis)

Clinical

findings

Autopsy: neuronal loss and gliosis w/ atrophy of corticospinal tracts; organizing pneumonia; bilateral cryptorchidism; contractures of extremities

Autopsy: petechiae of epicardium and visceral pleura; hypoxic encephalopathy; pneumonia

Erythroid hyperplasia of bone marrow; splenomegaly (74 g) with marked congestion of red pulp; generalized congestion and sickling of red cells within major organs; hemosiderin deposition within renal tubular cells

Histology: few interstitial myocardial inflammatory infiltrates in multiple sections (>15) but insufficient for Dx of myocarditis

Autopsy

f XI =i

z? 2 G

0 E

60 yr

27 yr

94AEl88

94AA204

2% mo

30 yr

94AEl70

94AE260

3 mo

48 yr

94AE069

94AEl57

65 yr

93A393

F

M

M

F

F

M

F

6 group (rare)

B

oxyoral (rare)

Aerobic. NG Legronella screen. no Leglonella isolated

NA

Aerobic: pstrep. Gp A (numerous); possible GPC (no further workup [ID]) Anaerobic: Clostrid/um butyricum-C. beijerinckrl (rare); Prevotella sp. (rare) (6-14 days)

Aerobic: strep.

NA

RUL: Aerobic: Enterobacter cloacae (few); Flavobacterium indologenes (few); Enterobacter aerogenes (fare) Anaerobic: NG Fungal: NG

Aerobic: S. pneumoniae Aerobic: Klebs/ella pool D (types 9, 11, toca (numerous); 16, 36, 37; type 9 flora; C. alblcans ruled out); coagulasenegative staph. Anaerobic: S. pneumonjae pool D (types 9, 11, 16, 36, 37; type 9 ruled out)

Aerobic: NG Anaerobic: NG

NA

NA

Aerobic: coagulasepositive staph., coagulase-negative staph. (2 species)

NA

NA

NA

Aerobic

NA

NG

Aerobic: /?-strep. (numerous) Anaerobic: NG

NA

NA

NA

Gp A

@strep

NG

Gp A

NG

Aerobic: coagulasenegative staph. (rare); Pseudomonas fluorescens (rare)

Aerobic.

NA

days)

(numerous) Anaerobic: Clostndium butyncum-C. beoer/rick/i (rare) (7-l 1

Aerobic:

Aerobic:

NA

N4

CSF: NG LA: negative for H. influenzae type b Ag; S. pneumonlae Ag; N. meningitidis Gp A, C, Y, W135, B Ag

NA

merous); positive for streptococcal pyrogenic exotoxrn A Endometrium: Aerobic: kstrep. Gp A (numerous) Anaerobic: NG Peritoneal fluid: @strep. Gp A (numerous); intestinal flora Peritoneal fluid: Enterobacter cloacae (numerous)

GP B Ag Feces: fecal flora (negative for Salmonella, Shigella, Yersinla, Aeromonas, Pleslomonas, Vibrio [not V. cholerae], and Campylobacten CSF: p-strep. Gp A (nu-

CSF: NG LA: negative for H. influenzae type b Ag; strep.

Urine: Klebsrella pneumoniae (2 strains; >I OO,OOO/ml), E. co// (>I OO,OOO/ml)

NA

sclerosis

SIDS

Complications of cirrhosis of liver due to chronic alcoholism (Including spontaneous bacterial peritonitis) Complications of DKA, including ARDS

Group A beta-hemolytrc streptococcal puerperal sepsis

SIDS

Multrple

Complications of dexamethasone treatment of glioblastoma multiforme

alcoholism

Uncomplicated prenatal course; slept in bed w/ parents; able to “roll over”; found unresponsive in bed after being put down for nap in late morning

New-onset diabetes mellitus w/ DKA, complicated by ARDS; obesity

Chronic

s/p spontaneous vaginal delivery; hypothermia, shock, DIC

Neurogenrc bladder w/ suprapu bit vesicostomy site and placement of left ureterovesical stent for urinary obstruction; wheelchair bound (required live-tn nurse’s aide) Recent onset of diarrhea (gastroenteritis); found unresponsive rn crib

(Table con tinuesl

Diffuse alveolar damage (acute or exudative phase) with early bronchopneumonia; fatty change of liver; atherosclerotic CAD Petechiae of vlsceral pleura; milky, curdlike material in stomach

Massive serous ascites (20.5 liters); right pleural effusion (I ,250 mL); renal cell CA

Gangrenous (hemorrhagic) necrosis of uterus, adnexae, and pelvic soft tissues; petechlae of skin, eplcardium, and myocardium; accelerated postmortem putrefactive changes

Epicardial petechlae; pulmonary edema; patchy mononuclear inflammatory infiltrates within pulmonary interstitium

Chronic pyelonephntis; urolithiasis; early bronchopneumonra; flexion contractures and atrophy of lower extremities

Confluent bronchopneumonra (RUL), pulmonary aspergillosis (LUL), pulmonary parenchymal hemorrhage (RUL) during and after bronchoscopy

f 3z =i m

10 mo

21 yr

7wk

2% mo

39 yr

94AE481

94AE501

95AE119

95AEl62

Age

94AE356

no.

Case

M

F

M

F

F

Sex

Heart

blood Lung

Aerobic: possible GNR (2 species); alpha-hemolytic strep. (2 species) Enterococcus sp.; S. loneumoniae

Aerobic: possible GNR; possrble staph. sp.; possible Enterococcus sp. Anaerobic: alpha-hemolytic strep.; possible GNR (2 species); possible staph. sp.; posstble Enterococcus sp. Aerobic: S. pneumoniae Anaerobic: S. pneumonrae

H. influenzae alpha-hemolytic (rare)

LLL: Aerobic: S. pneumoniae (mod.); H. influenzae (mod.); beta-strep., not GD A (rare): oral flora

Aerobic: E. cloacae (numerous); E. co/i (few); drphtherord or lactobacillus (numerous); alpha-hemolytic strep. (numerous); Enterococcus sp. (few)

Aerobic: (rare); strep.

Aerobic: S. pneumoniae; Aerobic: Klebsiella pneualpha-hemolytic strep.; moniae (rare); H. influcoagulase-negative enzae (numerous); staph.; H. influenzae, S. pneumonlae (numernot typeable with b an0~s); oral flora trsera Anaerobic: alpha-hemolytic strep.; H. infhenzae, not typeable with b antisera Aerobic: E. co//; diphtheViral: NG roid or lactobacillus Anaerobic: E. co//; Clostridium ramosum

Table AZ.1. Continued

NA

Aerobic:

Aerobic:

NA

Aerobic:

NG

NG

NG

Liver

Finding(s)

NA

Aerobic:

Aerobic:

Aerobic:

Aerobic: (rare)

NG

NG

NG

possible

Spleen GPC

B

Bronchial

Cause asthma

aerobic:

NG

Urine: LA: negative for S. pneumonlae Ag

Thymus:

Pneumococcal monia

SIDS

pneu-

of death

Viral myocarditis Heart muscle: viral: NG Serum: Coxsackie B virus Ab by complement frxation: type 1, 1:8; type 6, I:1 6 (infection undetermined time); types 2 to 5, cl:8 (Ab not detected); Coxsackie A virus Ab by complement fixation: types 2, 4, 7, 9, 10, 16,
AC4

dis Gp A, C, Y, W135,

CSF: NG LA: negative for H. Influenzae type b Ag; S. pneumoniae Ag; N. meningiti-

Other

history

prenatal course; full-term delivery w/ difficult presentation, req. resuscitation; did well subsequently; found prone on bed (on top of quilt), unresponsive and apneic by babysitter; resuscitation unsuccessful Chronic alcoholism

Unremarkable

Found unresponsive, prone in bed after being put down for afternoon nap

md 4-day H/O upper respiratory tract symptoms, nausea, vomiting, fever (to 101.5”F), and fatigue; treated in ER for dehydration w/ i.v. fluids; suffered ventricular tachycardia arrest, deftbrillated; biventricular congestive heart failure; subsequent cllnical course complrcated by ventricular tachyarrhythmias and cardiogenic shock, refractory to transvenous pacing, intraaortic balloon pump placement, and pressers

Found lying supine in bed by mother in morning, unresponsive and apnerc; last seen alive previous evening; could not be resuscitated; H/O asthma requiring hospitalization and NMT treatments for acute exacerbation (at 8

Clinrcal

findings

Lobar pneumonia (LLL); fatty liver; hypertrophic heart with patchy myocardial fibrosis; atherosclerotrc CAD, moderate

Epicardial and thymic petechiae; bilateral pneumothoraces (artifacts of mechanical ventilation)

Petechrae of thymus and eprcardrum; pulmonary edema (slight)

Soft, flabby, pale myocardium, extensive interstitial lymphocytrc inflammation and myocardral necrosis; pulmonary edema with dilatation of subpleural lymphatics; serous effusions of pleural, pericardial, and peritoneal cavities

Histologic changes of asthma; no gross or hrstologrc signs of pneumonia; limbal dermoid

Autopsy

z

G

2 z =i

z;;]

39 yr

4% mo

2 days

6 mo

95AE227

95AE316

95AE363

95AE442

NA

NA

F

Aerobic: NG

Aerobic: coagulase-posi- Aerobic: diphtheroids tive staph. (rare); oral (rare) flora

Aerobic: E. cloacae (rare); possible GNR

Aerobic: alpha-hemoAerobic: K. pneumoniae Aerobic: NG lytic strep.; coagu(numerous); C&c+ lase-negative staph.; batter ffeundii (nugamma-strep. merous); E. cloacae Anaerobic: alpha-hemo(numerous); gammalytic strep.; coagustrep. (numerous); lase-negative staph. alpha-hemolytic strep. (numerous); diphtheroid or lactobacillus (numerous); yeast (few)

M

M

M

Anaerobic: possible Anaerobic: Fusobactestaph. sp.; possible rium nffcleatum (rare); GNR (2 species); EnActinomyces sp. (not terococcus sp.; alpha- A. israelii 2 strains; hemolytic strep. (no rare) mixed aerobic further ID on isolates and anaerobic oral at 3 days) flora LUL: AFB: smear negative Fungal: Candida albicans; Penicihm sp. NA RUL: Aerobic: S. pneu- NA moniae (numerous); beta-strep. Gp G (numerous); Enterobacter aerogenes (moderate); E. cofi (rare); Pseudomonas putida (rare)

Aerobic: NG

Aerobic: NG

Aerobic: NG

NA

CSF: Aerobic: NG LA: negative for IV. meningitidis Gp A, C, Y, W135 Ag, Gp B Ag

NA

CSF: coagulase-negative staph. (2 species; rare)

Peritoneal fluid: S. pneumoniae (numerous); beta-strep. Gp G (modcrate)

flab/e can tinues)

Diverticulosis of sigmoid Bacterial peritonitis and Sustained partial-thickness colon with spontaneous burn at workplace, treatbronchopneumonia of perforation, inflammaright lung complicating ed with Tylenol #3, c/o constipation, switched to tion, and early repair; sigmoid diverticulosis acute generalized periVicodin; c/o “stomach with spontaneous perupset, ” took Advil; contonitis; confluent bronforation and diverticulistipation continued, ao chopneumonia of upper tis and following adk&e of right lung with ministration of Fleet’s companied by vomiting fibrinous pleuritis enema for constipation Uncomplicated prenatal Petechiae of thymus and SIDS course with spontaneous visceral pleura; artifacts of resuscitation vaginal delivery at 39 wk; (contusions and defino antecedent illnesses; brillator burns of anteplaced supine on twin rior chest wall) bed (thin satin sheets) for nap by mother; found infant approx 1 h later prone, unresponsive, and cyanotic; called 911; transported to ER but resuscitation unsuccessful Marked hypoplasia of left Poor feeding and cyanosis Hypoplastic left heart ventricle with slitlike syndrome on 3rd day of life; admitted to ER w/bradycardia inflow and outflow and lactic acidosis; cartracts; mitral and aortic valvuiar atresia; right diomegaly on CXR; poor left ventricular function ventricular hypertrophy; w/ probable hypoplastic medial hypertrophy of small pulmonary artery left heart syndrome on ECHO branches; complications of hypoperfusion and shock (GI hemorrhage; adrenal hemorrhage) Anomalous origin of right Full-term infant; viral illness Artifacts of resuscitation at 3 wk, but otherwise (pulmonary fat emboli) coronary artery (from left sinus of Valsalva) healthy; infant fell asleep on carpeted floor; babysitter moved infant to floor bedroom (placed her prone on blankets); baby checked 1 h later and 50 min later; 2nd time, infant was apneic; babysitter attempted CPR, called 911; transported to ER, resuscitation unsuccessful

7 mo

3 mo

96AE039

68 yr

2% mo

Age

96AE009

95AE565

95AE492

no.

Case

M

M

F

M

Sex

Heart

blood

Aerobic coagulase-positive staph.; Enterococcus sp.; multiple morphs of lactose-positive GNR Anaerobic: multiple morphs of lactose-positive GN R; coagulase-posrtive staph.; Enterococcus sp. (2)

Aerobic: K. pneumoniae; Serra t/a marcescens; beta-strep. Gp B; coagulase-posrtive staph.; M/crococcus sp.) Anaerobic: P. aeruginosa; beta-strep. Gp B; coagulase-positive staph.

NA

Aerobic: S. sanguis Anaerobtc: S. sangurs

Table A2.1. Continued

Aerobic:

Aerobic: (rare)

NA

E. cloacae

P. aeruginosa

Aerobic: E. co/i (few), S. pneumonlae (rare); Neisseria sp., not N. gonorrhoeae or N. menlngitidis (few); gamma-strep. (few)

Lung

Aerobic:

NG

ID)

Aerobic: E. cloacae (moderate); Cx yields 4 or more organisms at 2 days (no further

Aerobic: S. marcescens (rare); K. pneumoniae (rare); Enterococcus sp. (rare) beta-strep. Gp B (rare)

NG

Aerobic:

NG

NA

Aerobic:

Spleen

NA

Aerobic: coagulasenegative staph. (rare)

Liver

Finding(s)

CSF

NG

NA

SIDS

H. influenzae bronchitis and bronchrolitis with early bronchopneumonia Contributory: chronic obstructrve pulmonary disease and atherosclerotic cardiovascular disease SIDS

Tracheobronchial tree: H. influenzae (numerous); oral flora

of death

SIDS

Cause CSF: coagulase-negative staph. (2 spp.; rare)

Other

history

findings

Pulmonary emphysema; bronchitis and bronchlolitis w/ extensive mu-

Petechrae of thymus and visceral pleura; lipogranuloma of lung (probable aspiration)

Autopsy

pine); found resuscitation

unresponsrve; unsuccessful

komalacra and developmental delay; on day of death, inadvertently administered ferrous sulfate and metolazone but aspirated back by nurse; fed, placed supine for nap, found unresponsive Placed for nap on couch (su-

lntrathoracic

petechrae

cous (mucopurulent) plugging and patchy consolrdatron (bronchopneumonia); atherosclerosis of coronary arteries and aorta Chronic interstitial nephriMaternal cocaine use w/ intrautefine exposure; no pretis with cytomegalovrrus inclusions natal care: premature birth (at 25 wk of gestation), complicated by partial placental abruption, cocaine HISwithdrawal, respiratory tress syndrome, and intraventricular hemorrhage; long-term mechanical ventilation w/ tracheostomy placement and bronchopneumonra; fundoplication w/ tube placement for poor feeding; periventricular leu-

Premature birth (at 34 wk of gestation); recent H/O nasat congestion; baby put to bed at night by mother, found next morning by father, prone in bassinet (face in soft pillow), unresponsive and apneic; resuscitatron unsuccessful Smoker (1% ppd for -40 yr; constant cough; recent onset of weakness and fatigue

Clinical

N

f z

2 CL

3

6 wk

lY2 mo

42 yr

35 yr

96AE091

96AE 103

96AE140

96AA167

M

NA

Aerobic S. pneumonlae; E. CO/I; Enter+ coccus sp

Aerobic: alpha-hemolytic strep.; K. pneumoniae (2 specres)

M

M

NA

M

NG

NA

Aerobic: S pneumoniae (numerous); H mfluenzae (moderate) AFB: smear negative; Cx negative Fungal: NG

Aerobic: K. pneumonlae (few); S. pneumonrae (moderate)

Aerobic:

NG

NA

NA

Aerobrc: K. pneumonlae (rare)

Aerobic:

NG

NG

Aerobic: P. aeruginosa (moderate); P aeruginosa (2nd strain; rare) Fungal: NG

Aerobic: S. pneumonlae (numerous) AFB: smear negative, Cx negative Fungal: NG

Aerobic:

Aerobic:

NA

NA

CSF: NG

CSF: Aerobic:

NG

Neutropenrc colrtrs (typhlitis) complicating induction chemotherapy for ALL

Pneumococcal 6. pneumoniae) pneumonia

SIDS

Carbon monoxide intoxrcation (decedent was exposed to kerosene heater In rear of converted mobile home (bus)

spleno-

0360 g); hyper-

(Table

continues)

trophy and dilatation of heart (moderate; 520 g) Bilateral adrenal hemorPancytopenia (absolute solrhages; petechiae of ute neutrophri count lOO/ eprcardrum, pencarmm3), right lower quadrant abdominal pain, diarrhea, drum, and renal caguaiac-positive stools; thrcklyceal mucosa; subenening of walls of cecum and docardial hemorrhage; right colon by abdominal CT, ileocolic anastomosis; exploratory laparotomy serosanguineous asshowed acute rnflammatron cites and pleural effuand necrosis of cecum, apsrons; hypoplastrc bone pendix, and right colon with marrow perforation of cecum, req. right hemrcolectomy; postoperatively developed resprratory distress, hypotension, bradycardia, and asystole; antemortem blood Cx positive for Pseudomonas and possibly En terococcus

megah/

er; congestive

Hypoxrc-ischemrc brain Parents were evicted from injury with brain swellresidence, lrvrng In Greying, early bronchopneuhound bus, heating it w/ monia; bilateral adrenal kerosene heater, infant hemorrhages; hepatic sleeping on bed between parenchymal iron depoparents; strong kerosene sitron with Involvement smell noted by parents, of Kupffer cells; postInfant unresponsive; called 911; survived several hours mortem COHb saturaIn hospital, treated w/ tion: 1.2% 100% oxygen (0,); antemortem COHb saturation: 5.8% Prenatal course, delivery Petechrae of visceral w/o complrcatrons; found pleura, artifacts of CPR unresponsive and apneic In (intra-alveolar pulmonary hemorrhage) stroller; 1-yr-old brother In stroller, not on top of infant; bottle In Infant’s mouth; resuscitation unsuccessful Heavy smoker, COPD; susLobar pneumonia (LLL); petted chronic alcoholrsm; bilateral confluent bronmorbid obesity (body mass chopneumonia of left Index: 45 6 kg/m21 upper lobe and right upper and lower lobes; diffuse alveolar damage, acute and exudative

8

%

G

=i

c3

F

F

2 mo

3%m0

97AE116

97AE117

F

M

7%wk

24 yr

Sex

Age

96AE310

96AE273

no.

Case

Table A2.1. Continued

blood

possible

staph.

days)

sp.; alpha-hemolytic strep. (2 spp.); gamma-strep.; diphtheroidjactobacillus, Neissefia sp. (no further ID approved at 2

Aerobic:

Aerobic: alpha-hemolytic strep.; E. cloacae; coagulasepositive staph.

NA

indologenes; Stenotrophomonas (Xan thomonas) maltophilia Anaerobic: Vei//one//a sp.

Aerobic: Chryseobacterium (Flavobacterium)

Heart

Bacillus

sp., not B. anthraas (few)

Aerobic:

Aerobic: alpha-hemolytic strep.

Aerobic: E. cloacae (rare); E. cloacae (moderate) i2nd specresl); E. cloacae (rare [3rd speciesl); P. putida (rare); Entefobacter agglomerans (rare); diphtheroids (few) NA

Lung NG

Aerobic:

NG

Aerobic: K. pneumoniae (rare) ; possible Pseudomonas sp. (few); Enterococcus sp. (rare); coagulase-positive staph. (rare); diphtheroids (rare) (no further workup at 3 days) Aerobic: NG

Aerobic:

Liver

Finding(s)

NG

Aerobic:

Aerobic:

days)

NG

NG

Possible GNR (rare 12 species]; possible GNR (few [3 species]) (no further workup at 3

Aerobic:

Spleen

CSF: NG

CSF: NG

Amniotic fluid: f. co/i (rare); diphtheroid and lactobacillus (rare); possible Bacillus sp. (rare) (no further workup at 3 days)

CSF: NG

Other

of death

Bronchial

asthma

Undetermined

Undetermined

Cardiac arrhythmia due to right ventricular myocardial dysplasia of undetermrned etiology

Cause

history

Intrauterine cocaine exposure (meconium positive for cocaine metabolite); born at 38 wk gestation; upper respiratory infection at 2 wk of age; cough, congestion, rhonchi at 1 mo, diagnosed w/ asthma (“reactive airways disease”); treated w/ albuterol; exacerbations at 2 and 2V’2 mo, treated w/ antibiotics, bronchodilators, steroids, nebulizers (NMT); infant fed and placed prone in crib by mother at midnight; found next morning unresponsive; not resuscitatable

successful

found unresponsive in car seat during drive; resuscitation un-

Prenatal course complicated by preeclampsia, requiring emergent C-section at 31 wk of gestation; transient tachypnea of newborn, treated with mechanical ventilation and theophylline;

Hypoplastic right ventricle shown on 28wk fetal echocardiogram (confirmed with postnatal ECHO [multiple] also showing left ventricular hypertrophy and dilatation and biatrial enlargement at 6 wk); hypoxemia, requiring supplemental 02, resolved or discontinued at approx 2% wk of age; cardiac arrest Pregnant (late 2nd trimester [-25 wkl); four previous pregnancies, all miscarriages, latest a molar pregnancy (gestational trophoblastic neoplasia), Rx w/ chemotherapy; mild asthma; recent abdominal cramping, nausea, and vomiting; found unresponsive on floor next to couch

Ctinical

right ventricle and right atrium; endocar-

findings

petechiae

of visceral pleura; diffuse pulmonary interstitial mononuclear inflammatory infiltrates (lymphocytes, macrophages, and plasma cells; no viral inclusions seen) with intraalveolar foam cells and fibrin and organizing inflammation along alveolar walls; lobular hepatitis (lymphocytic) Petechiae of visceral pleura; histologic changes of chronic asthma

Autopsy:

Changes of chronic bronchial asthma; patchy but widespread syncytiotrophoblasts within pulmonary alveolar capillaries (trophoblastic microemboli)

dial fibrosis of right and left ventricular outflow tracts

Hypoplastic enlarged

Autopsy

8 3 i;;f

2

c) G!i a, 2 Q)

F

F

F

M

NA

NA

Aerobic: alpha-hemolytic strep.; Clostridiurn sordellii Anaerobic: alpha-hemolytic strep.; Clostridium sordellii

Aerobic: NG Anaerobic: NG

Aerobic: coagulasenegative staph. (rare) Viral: NG

NA

Aerobic: coagulasepositive staph. (few); oral flora Fungal: NG

Aerobtc: H. influenzae (numerous); saccharolytic Acisp. netobacter A. baumanii complex) (rare); oral flora Viral: sample sub mitted for RSV Cx; result not reported

Fungal:

Aerobic: C. sorde//i (unable to quantify) Fungal: NG Aerobic: coagulase-negative staph. (rare)

NG NG

Aerobic:

Aerobic: Micrococcus sp. (rare)

NG

tifv) Fungal: NG Aerobic: NG

Aerobic: C. sordellii (unable to quan-

Aerobic: NG Fungal: NG

Aerobic:

CSF: E. co/i (moderate); P. aeruginosa (moderate); Enterococcus spp. (2 species; few) (no further ID approved at 3 days) Feces: fecal flora; negative for Salmonella, Shigella, Vibrio Gp (not V. cholerae), Yersinla, Aeromonas, Campylobacter, Plesiomonas Viral: NG larynx swab Vtral: NG trachea swab

NA

NA

CSF: NG

Complicatrons of acute laryngotracheobronchitis (croup) of probable viral etiology

Atherosclerotic heart disease

asphyxia

chopneumonia, due to perinatal

Complications of seizure disorder, including bilateral confluent bron-

etiology

H. rnfluenzae pneumonia complicating bronchiolitis of undetermined ratory distress, seizure activity, and pulmonary hemorrhage at 5 wk of age; dyspnea (presumed bronchiolitis) at 5 mo, progressing to lethargy, respiratory distress, and LLL pneumonia, req. hospitalrzation; cardiopulmonary arrest with unsuccessful resuscitation at 6% mo Perinatal asphyxia complicated by mental retardation, developmental delay, and seizure disorder; s/p PEG tube placement w/ intrapentoneal displacement, abdominal wall abscess, and S. aureus bacteremia; tooth abscess (left lower first molar) status epilepticus (4 mo before death); found unresponsive on bed by mother, in full cardiac arrest; resuscitation unsuccessful Juvenile rheumatoid arthritis and Sjogren’s syndrome; obesity; found unresponsive on floor of residence; not resuscitatable Came to United States from Lebanon 2 days before death; coughing and gagging for -48 h; placed on couch by mother, who returned a few minutes later to find child cyanotic with seizure-like activity; called 911; child transported to community hospital ER in cardiac arrest; resuscitated and intubated; transferred to University of Michigan Hospital; profound acidosis (pH 6.9) and hypotension, req. pressor support; pupils fixed and dilated; no cornea1 or gag reflexes; failed apnea test; clinical brain death confirmed by ce rebral perfusion study (no blood flow), child declared brain dead

Prenatal course complicated by maternal suicide attempt with overdose of acetaminophen and ethanol at 27 wk of gestation; premature birth at 33 wk; respi-

Atherosclerosis of coronary arteries w/ stenosis; organizing myocardial infarct; subgaleal (scalp) contusion Acute ulcer Involving mucosa of true vocal cord, subglottic larynx, and trachea with necrosis of respiratory epithelium, edema, and neutrophilic infiltration of lamina propria, accompanied by mixed inflammatory infiltrate; thymic stress involution with loss of cortical lymphocytes and “starry-sky” appearance; lipid depletion of zona fasciculata of adrenal glands with patchy cortical hemorrhage

cerebellar (Purkinje cell) atrophy w/ Bergmann gliosis and early neuronal ischemic injury; elevated phenytoin concentration

Bilateral confluent acute bronchopneumonia w/ older, organized foci (probable recurrent aspiration); laceration of tongue w/ subjacent hemorrhage (bite);

Acute and chronic bronchiolitis w/ early bronchopneumonia; thymic involution; monunuclear infiltrate within subarachnoid space

a M, male; F, female; Ab, antibody; Ag, antigen; H/O, history of; ALL, acute lymphoid leukemia; Hx, history; ID, identification; i.v. intravenous; C-section, cesarean section, K., Klebsiella; CA, carcinoma; CAD, coronary artery disease; LLL, left lower lobe; c/o, complaint(s) of; LUL, left upper lobe; COD, cause of death; NG, no growth; NA, not applicable COHb, carboxyhemoglobin; CPR, cardiopulmonary resuscitation; CT, computerized tomography; PEG, percutaneous gastrostomy; CXR, chest X-ray; ppd, packs per day; DIC, disseminated intravascular coagulation; RLL, right lower lobe; DKA, diabetic ketoacidosis; RSV, respiratory syncytial virus; Dx, diagnosis; RUL, right upper lobe; Rx, treatment; ECHO, echocardiogram; ER, emergency department; GE, gastroesophageal; s/p, status-post; GNR, gram-negative rods; staph., staphylococcus; GPC, gram-positive cocci; strep., streptococcus; w/, with; w/o, without; req. requiring; Gp, group; Cx, cultures; NMT, nebulized mist treatment.

1% yr

38 yr

97AE401

WME97.489

31 yr

6% mo

97AE390

97AE127

36

Caplan

APPENDIX

and Koontz

Category Routine:

35

3

HISTOCHEMICAL Table A3.1.

CUMITECH

STAINS USED TO DEMONSTRATE

Histochemical

stains

used to demonstrate

microorganisms

or procedure and stain

MICROORGANISMS in tissuesa

Diagnostic

H&E . . . . . . . . . . . . . . . . . . . . . . . ..‘.............

applications

Best stain to demonstrate overall tissue reaction; demonstrates viral rnclusion bodies, protozoans, some bacteria and fungi, and Splendore-Hoeppli material that may surround certain microorganisms and granules of actinomycosis, botryomycosis, and mycetoma, needed to detect phaeoid (naturally pigmented) fungi

Viral inclusion body stains . . . . . . . . . . . . . . . . . . . . None is superior to H&E for detecting inclusion bodies and for studying Lendrum’s phloxine-tartrazine trichrome is especially useful for staining inclusion bodies of CMV Attwood’s phloxrne-tartrazine Shorr’s Masson’s trichrome Bosch’s and Schleifstein’s methods for Negri bodies Silver impregnation Steiner Dieterle Warthrn-Starry

procedures

IN TISSUES

their Internal

details;

Masson’s

. . . . . . . . . ..Blacken all bacteria; required to detect non-Gram-stain-reactive bacteria; e.g., spirochetes, Bartonella spp., Calymmatobacterium granulomatis; useful as a sensitive screen for weakly gram-negative bacteria, e.g. Legionella spp., Francisella tularensis, Burkholderia pseudomallei, and Helicobacter pylori

Modified Gram stains for bacteria Brown and Brenn Brown-Hopps MacCallum-Goodpasture

. . . . . ..Drfferentrate most gram-positive and gram-negative bacteria and stain actinomycetes and microsporidians gram positive; Brown-Hopps is best for gram-negative bacteria and rickettsiae; some fungi, especially Candida glabrata, blastoconidia of Candida spp., and conrdia of Aspergillus spp., are gram-positive

Acid-fast stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Demonstrate Mycobacterium spp.; Truant’s has highest sensitivity and is therefore useful for screening; Ziehl-Neelsen Coates-Fite, Fite-Feraco, or other modified procedures are required for Mycobacterium leprae, NocarCoates-Fite dia spp., Rhodococcus equi, and Legionella micdadei, all of which are weakly acid fast and not alcohol are acid fast Fite-Feraco fast; some spores of Nosema spp. (microsporidians), Truant’s auramine rhodamrne Fungal stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Useful for detecting all fungi and for studying their morphology in detail; GMS IS best for screening and also stains actinomycetes, cyst walls of P. carinii and free-living amoebae, certain encapsulated bacteGMS ria, most mycobacteria, intracytoplasmic granular inclusron bodies of CMV, and spores of certain miPAS Gridley fungus crosporidians; PAS stains the cytoplasm of Entamoeba histolytica trophozoites, the cyst walls of freeliving amoebae, the polar granule of most microspondians, and most mycobacteria Mucin stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Demonstrate mucord capsule of Cryptococcus neoformans, thus differentiating this pleomorphic yeastMayer’s mucicarmine form fungus from others of similar size and shape; also may stain cell walls of Blastomyces dermatitiSouthgate’s mucicarmine dis, Lacazia (Loboa) loboi, Paracoccidioides brasiliensis and Rhinosporidium seeberi Alcian blue Colloidal iron Melanin stain: Modified acFontana-Masson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Useful for staining the cell wall of Cryptococcus neoformans, which contarns melanoid substances; centuates and confirms the presence of melanin in weakly pigmented agents of phaeohyphomycosis Giemsa stains . . . . . . . . . . . . ..-....................... May-Grunwald Wolbach’s Gaff ney’s

Demonstrate

most

protozoa,

bacteria,

and nckettsrae;

trophozoites

and intracystic

mocystis carinii; and the intracytoplasmic krnetoplast and nucleus of lershmania of Tvpanosoma cruzi; Wolbach’s gives greater depth of staining

sporozoites of Pneuand amastrgote stage

Connective tissue stains . . . . . . . . . . . . . . . . . . . . . .Wilder’s stains kinetoplast of leishmania and amastigote stage of Trypanosoma cruzi; Masson’s useful Wilder’s reticulum for demonstrating amoebae but offers little or no advantage if organisms cannot be initially seen in Masson’s trichrome H&E-stained sections; Weber’s trichrome useful for detecting microsporidial spores; Russell-Movat Weber’s modified trichrome pentachrome excellent for studying morphology of nematodes, cestodes, and trematodes Russell-Movat pentachrome =This appendix is modified from reference 6 with the permission of the author. lovirus; GMS, Gomon’s methenamrne silver; PAS, periodic acid-Schiff stain.

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Abbreviations:

H&E, hematoxylin

and eosin;

CMV,

cytomega-

3. Babb, J. R., A. J. Hall, R. Marlin, and G. A. Jayliffe. 1989. Bacteriological sampling of postmortem rooms. J. Clin. Pathol. 42:682-688. 4. Bjornson, H. S. 1988. Microbiology: microbiologic diagnosis of infection in the surgical patient, p. l-2. In Scientific American Surgery. Scientific American Medicine, New York, N.Y. 5. Carpenter, H. M., and R. M. Wilkins.

1964. Autopsy

CUMITECH

35

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review

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Pathol.

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14. Koneman, E. W., and M. A. Davis. 1974. Postmortem bacteriology. III. Clinical significance of microorganisms recovered at autopsy. Am. J. Clin. Pathol. 61: 28-40. 15. Koneman E. W., T. M. Minckler, D. B. Shires, and D. S. de Jongh. 1971. Postmortem bacteriology. II. Selection of cases for culture. Am. J. Clin. Pathol. 55: 17-23. 16. Kurtin, J. J. 1958. Studies in autopsy Am. J. Clin. Pathol. 30:239-243.

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Microbiology

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