Postmortem Analysis and the Role of the Clinical Microbiology Laboratory

Abstract

The clinical microbiology laboratory frequently receives specimens for culture collected at autopsy. The results generated from these culture requests can either provide valuable information or produce confusion, depending largely on the selection of sites to sample, collection technique, and interpretation of results in the context of data available from patient history and histology. This mini-review is intended to provide guidance to the pathologist in the best use of microbiology testing at autopsy.

Keywords

Autopsy Infectious disease

Introduction: Clinical Microbiology and the Decline of Autopsy

Throughout the history of medicine, autopsy has played a central role in our understanding of normal anatomy, disease and treatment efficacies or side effects. Despite the instructive value of autopsy, it now plays only an ancillary role in contemporary medical education (1). Medical students who have participated in surveys about autopsies have given responses similar to those of other professional students reflecting perhaps the absence of appropriately specialized fundamental training (2). Similarly, physicians who did not observe autopsies during their training are less likely to request an autopsy during the course of their careers (3). In 1986, Medicare stopped paying directly for autopsies and, as early as 1970, the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) eliminated the requirement for a minimum autopsy rate from its accreditation process (4). A once actively growing body of literature from the mid-1980s to the mid-1990s demonstrated that although most physicians found a final autopsy report useful, many failed to request an autopsy because of the lack of training on how to seek autopsy permission, fear of offending the family of the deceased, fear of malpractice litigation, confidence in contemporary diagnostic technology or the desire to limit costs (3, 5–10). With all of these forces aligned against this service, it is not surprising that autopsy rates have declined in the United States (US) and other Western countries over the past three decades (5). However, the sequelae of this decline include the loss of an established teaching tool along with inability to identify and correct clinical errors and missed diagnoses (5).

Studies that have attempted to quantify and categorize the types of clinical errors that are missed pre-mortem in an era where only 5-10% of deaths in the US are examined by autopsy (4, 5) have been controversial. Although the classification scheme has changed over time, recent analyses categorize the discovery at autopsy of a missed clinical diagnosis affecting outcome as a class I error while class II and III errors have respectively declining impacts on expected prognosis (4, 11). Class I errors were found in 10% of autopsies over three decades at a Boston teaching hospital (12) and major discrepancies (Class I and II errors) were found in 35% of autopsies in another study (11). While these numbers might be inflated by selection bias, further studies have shown that physicians generally are not able to predict which cases will reveal major discrepancies at autopsy (13), an observation that alone suggests the importance of autopsy in education and in revealing missed clinical diagnosis. It has been observed that the three most common missed diagnoses identified at autopsy include aortic dissection (AD), pulmonary embolism (PE) and active tuberculosis (TB) and that calculating the expected prevalence of missed cases among patients not autopsied reduced the rate of detection from 93% to 82%, 97% to 91% and 96% to 83% for each of these respectively (4).

Following the H1N1 pandemic and at a time when consideration of potential bioterrorism is still fresh in the public memory, it is interesting that active tuberculosis is one of the most commonly missed clinical diagnoses in autopsy studies. Clinical microbiology might play less of a role in hospital autopsy than in forensic autopsy since hospital directed autopsies are usually performed on patients with pre-mortem cultures from a variety of sites already in progress and on individuals who might have received antimicrobial therapy prior to death. Since the report of Shojania and Burton (4) was based on hospital-based autopsies where active tuberculosis was one of the most overlooked diagnoses, clinical microbiology testing may be a more important component of hospital autopsies than generally appreciated. Of course, the general decline in autopsy numbers has further limited the impact of post-mortem clinical microbiology. Clinical microbiology may be even more relevant in a forensic setting where the post-mortem interval may be longer and patients more likely carry undiagnosed infections. The high prevalence of immunodeficiency in the setting of HIV, transplant, cancer or aging adds to the relevance of an infectious disease-based argument for reversing the trend of declining post-mortem examinations. This short review attempts to summarize the current literature regarding the use of microbiology sampling techniques and testing at autopsy.

The Decision to Collect Cultures at Autopsy

The pathologist may approach the subject of autopsy microbiology with considerable skepticism given the reports of discrepant findings between organisms isolated from patients before death and those recovered from postmortem sampling (14, 15). We suggest that there is a limited but useful role for autopsy microbiology, but that a high index of suspicion for an infectious agent as a cause or co-factor of death along with careful attention to sample type, collection technique, and interpretation of findings is absolutely critical. Particular symptoms correlate well with infectious disease as a cause of death, and should alert the pathologist that microbiological testing may be indicated. Flu-like symptoms, fever with respiratory symptoms, and encephalopathy or new onset seizures all have high positive predictive values (65%, 72% and 50% respectively) for identification of an infectious etiology (16).

Selection of samples for microbiological testing should be guided by evidence of a local or systemic host response to infection (e.g. abscess, granuloma, purulent fluid, etc) (17). The probability of obtaining meaningful results from such samples is higher than from sites that show no signs of inflammation and from which growth would more likely represent contamination. It should be noted that immunocompromised patients may not exhibit an inflammatory response and would require a different approach to specimen selection and result interpretation. In the event of disseminated infection in the immuno-compromised host it may be necessary to attempt to identify an entry site for infection, such as catheters or skin wounds, leading to more extensive culturing (18).

Historically, blood, CSF and lung tissue are the samples that yield the most useful results for microbiological testing (19). Bacterial growth from normally sterile sites such as blood and CSF can result from several possible scenarios. 1) The organism may have been present during life, either transiently or causing infection. Correlation with signs and symptoms of infection and inflammation are useful in distinguishing between these two possibilities. 2) Organisms can spread to normally sterile sites either at the time of death or shortly thereafter. So-called agonal spread at the time of death is thought to result from the loss of integrity at mucosal surfaces due to ischemia and/or as a result of physical trauma associated with resuscitation attempts. However, there is little evidence to support that this process actually occurs, and translocation of organisms via migration from mucosal surfaces following death is less likely with appropriate storage. 3) Growth in cultures may result from contamination introduced at the time of autopsy specimen collection.

An extensive review of the available literature on postmortem bacteriology from blood and CSF was conducted by Morris et al. (20), finding that when careful precautions were taken to avoid contamination at sample collection approximately two-thirds of blood cultures are negative for bacterial growth after appropriate incubation periods. Among those that are positive for growth, two-thirds are monomicrobic whereas the balance demonstrates recovery of mixed microorganisms. In one particularly large study of 2033 autopsies with an age range of 0-90, only 7% yielded mixed cultures (21), a rate only slightly higher than blood culture contamination rates from living patients in many settings. These findings suggest that agonal or immediate postmortem spread of normal flora from mucosal surfaces is unlikely given that mixed blood cultures make up a clear minority of results. Additionally, the time to collection after death (within 48 hours) does not change these findings if the body is stored appropriately at 4^°C (19, 22).

It should be noted that far higher rates of positive autopsy blood cultures have been reported in other studies (14, 22), and that those reports typically show a preponderance of mixed cultures rather than pure cultures of a single organism, even with appropriate storage. Rather than supporting the notion that translocation of polymicrobial flora takes place in the setting of some studies and not others, these findings suggest that many samples are contaminated during the collection process and that attention must be paid to sterile collection technique in order to obtain meaningful results. Sterilization of the collection site before specimen collection and use of sterile instruments, as might be expected, yields a lower rate of culture positivity than does collection with non-sterile instruments and subsequent surface sterilization of the tissue section upon plating (12.5% vs. 43% positive culture rate for spleen culture); (23–25). While this point would seem intuitive, much of the reputation that autopsy microbiology has attained for producing poor quality results could potentially be alleviated by careful attention to sterile technique in the autopsy suite. Details of techniques for avoiding contamination in sample collection and handling are beyond the scope of this review, and are described elsewhere (18, 26).

The same is true of CSF cultures, as the blood-brain barrier is maintained for large molecules after death (20, 27), indicating that growth is likely due to either presence of organisms in CSF prior to death, or introduction of contamination during the autopsy. The WBC count does rise in the CSF following death, but this pleocytosis is represented by lymphocytes and monocytes rather than neutrophils, unless inflammation is also present (28, 29).

Specimen Collection

Blood culture: The decision to collect blood for culture should be based upon clinical suspicion of sepsis. Patients who die in a hospital setting may already have positive blood cultures obtained prior to death. In this case it is unnecessary to attempt to reproduce those results postmortem (17). Signs and symptoms of sepsis in the person who dies outside the hospital setting, and presumably prior to antibiotic treatment or blood culture collection, may yield more meaningful results. Blood cultures should be collected before removal or manipulation of the bowel and may be drawn from the right atrium, inferior vena cava, or aorta, following searing of the area to sterilize the site (17). If a femoral draw is done the skin should first be sterilized with povidone-iodine (17). In a patient with a suspicion for sepsis, Reznicek and Koontz recommend collection of heart blood and spleen, as identical isolates from both sites support a diagnosis of sepsis (18).

CSF: For suspected cases of meningitis, CSF collection can be done by cisternal tap following skin disinfection with iodine and then alcohol, or by inserting a needle into the subarachnoid space after removal of the calvarium. If a brain abscess is suspected the surface should be sterilized by searing and the abscess aspirated with a needle (17).

Lung cultures: The lungs should be observed for evidence of infection (e.g. parenchymal consolidation or fibrinous pleuritis), and if a site is identified that suggests infection the surface should be sterilized by searing and a small cube of lung tissue removed with a sterile blade and forceps (17). Low levels of bacteria may be present in healthy tissues, and quantitative culture may assist with interpretation. Knapp and Kent demonstrated that less than 10⁵ organisms/ml in lung tissue is indicative of histological absence of disease, while greater than 10⁵ organisms/ml correlates with pneumonia (30).

Other specimen types may present themselves during the course of autopsy, such as abscesses or valvular vegetations. As with the previously mentioned cultures it is important to collect the sample in a sterile manner to avoid contamination. Certain sample types, such as a perforated bowel, should not be cultured as the culture will obviously yield mixed fecal flora and will not add meaningful information to the report.

Interpretation of Culture Results

Determining the significance of a positive blood culture is not always a straightforward task. The type of organism identified is an independent correlate of true bacteremia (31), and the presence of particular organisms in the blood of living patients is nearly always (>90%) indicative of a “true positive”, including Staphylococcus aureus, E. coli and other Enterobacteriaceae, Streptococcus pneumoniae, Pseudomonas aeruginosa and Candida albicans (32, 33). Additionally, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Bacteroides fragilis group organisms, Cryptococcus neoformans, and Candida spp. other than albicans nearly always represent true infection, while organisms such as Corynebacterium spp., Bacillus spp. other than anthracis, and Propionibacterium acnes are frequent contaminants (32, 33). Some findings, such as growth of coagulase negative Staphylococcus spp. and viridans group streptococci can be difficult to interpret, making corroborating information of signs and symptoms and histology particularly important (32, 33).

The most meaningful interpretation of results will take into account clinical history, microbiology reports, and histopathology. Positive blood cultures should be interpreted on the basis of what organism is growing (likely pathogen vs. likely contaminant), whether it is a pure culture, whether it was isolated from other sites as well, and whether the result is consistent with what is known of the patient's clinical course (18). The relevance of positive cultures from body fluids or tissues is supported by demonstration of organisms on gram stain or in concomitant histology samples, particularly along with evidence of inflammation and isolation of an organism that is suggestive of infection at that site (i.e. Streptococcus pneumoniae from lung tissue). Culture results may be supported by immunohistochemistry (IHC). While not frequently available locally, IHC can be performed on tissue samples for some viruses, bacteria, and parasites through the CDC or reference labs, assisting in the diagnosis of difficult infectious disease cases (34). Coordinated efforts between anatomic pathology and clinical microbiology are invaluable to this effort. The quality of culture results is largely related to the skill of the pathologist in selecting and collecting the appropriate samples to be tested, and in their subsequent ability to correlate these results with history and histology.

Molecular Testing in Autopsy

The development of molecular diagnostics has brought about a major change in medical research, clinical diagnostics and the modern day autopsy. No longer are laboratories limited to the detection of microorganisms that can be cultured in vitro or visualized microscopically (35). Molecular technology encompasses a myriad of applications which employ the use of genetic material (RNA and DNA), antigen-antibody reactions, or specific protein signatures as targets (35). These techniques allow for the rapid detection and identification of infectious agents found many times only at the time of autopsy such as tuberculosis, hepatitis viruses, bacterial pneumonia, legionnaire's disease, Creutzfeldt-Jakob disease, Toxic Shock Syndrome, and HIV/AIDS (36).

Molecular technology has gained much prominence in the clinical laboratory over the past decade. This is also true for postmortem evaluations. Polymerase chain reaction (PCR), in situ hybridization, nanotechnology, microarrays, mass spectrometry, proteomics, and other advanced technologies are used to detect and characterize infectious agents that once were impossible to identify by conventional methods. Standard microbiological methods such as culture and microscopy are important in the clinical and postmortem evaluation; however, these techniques have limitations that molecular techniques can surpass. For instance, not all microorganisms can be cultivated in an artificial environment or can be visualized without the use of electron microscopy - as is the case with many viruses. Therefore, molecular methodologies are a perfect supplement to standard microbiology techniques used in a postmortem investigation (37).

One of the more familiar molecular technologies used in clinical and postmortem evaluations is polymerase chain reaction or PCR. It is used to selectively amplify the nucleic acid of specific targets present in very low concentrations to detectable levels (39). Both DNA and RNA amplification can be done providing a greater range and sensitivity of organism detection than culture and in less time. As already mentioned this technology is especially useful in cases where agents such as viruses are not easily cultivated (37). There are automated platforms that allow multiple samples to be processed, amplified, and analyzed simultaneously in hours versus days. This technology can be used to quickly identify communicable diseases making it an optimal tool to assist in early Public Health intervention (37). Finding a suitable nucleic acid isolation system is important for molecular studies. Successful analysis begins with proper collection, and processing of samples. After obtaining high quality nucleic acid proper storage is important as this provides samples for additional tests. The basics of nucleic acid isolation are disruption of the cellular structure, separation of the nucleic acid from insoluble material and purification of the nucleic acid of choice from proteins and other nucleic acids (38). There are many kits on the market that make this process quick and easy but space doesn't permit a complete review of the commercially available extraction kits. These products, whether manual or automated, provide for the isolation of amplifiable nucleic acid in as little as an hour with high purity by using chaotropic salt solutions, detergents, silica beads, filtration matrices or magnetic capture and elution buffers. Different types of nucleic acids require different measures for isolation. DNA is very stable because of its double stranded configuration while RNA is less stable because of its single stranded configuration. There are various kits available that address these differences and are designed to selectively isolate the nucleic acid of choice. There are also kits that permit the isolation of DNA and RNA simultaneously from the same sample. Once the nucleic acid is purified it can be stored for hours or up to several years depending upon the type and storage temperature and/or conditions. RNA can be stored for a year at -80C in RNAse free water with EDTA to chelate metals that can cause non-specific cleavage of RNA. DNA can be stored for much longer periods of time (thousands of years in amber for example).

Fresh frozen tissue is the best source sample to use for future nucleic acid analysis but access to equipment for cryopreservation may not always be an option. Formalin fixed and paraffin-embedded is often used as a low cost method for long term storage of tissue. However, extraction of useful nucleic acid has been problematic due especially to the damaging effects of formalin (40). DNA is relatively stable in a slightly acidic environment but, at pH level below six protonation of guanine and adenine bases occurs creating targets for hydrolysis (41), and making DNA more susceptible to single strand breaks (42). Yet another issue when formalin fixation is used is the cross-linkage between proteins and nucleic acids causing fragmentation of the nucleic acid as it is extracted using methods for fresh frozen tissue (43). There have been breakthroughs on this front, however, from techniques that allow routine amplification of DNA from formalin-fixed and paraffin embedded tissue to the use of other non-cryogenic techniques for tissue preservation that improve preservation of nucleic acids (40). According to studies conducted by Kilpatrick and colleagues, ethanol and DMSO can be used to preserve tissue and still maintain the integrity of DNA for up to two years (44). Some degradation does occur with the use of ethanol but this is mainly during the extraction process and can be mitigated by soaking the tissue in lysis buffer for a couple of hours prior to extraction. In addition to direct amplification of infectious agents from fresh/frozen tissue, whole blood or stabilized blood (i.e., EDTA or ACS), specific methods have been successfully employed for amplification of microorganisms from formalin-fixed, paraffin-embedded tissue (45, 46). While the permutations of the extraction process are limitless, a number of commercial kits developed specifically for embedded tissue have been compared for recovery efficiency and would likely work sufficiently well for all molecular targets including fungi (47).

Transplant and Autopsy

As the number of individuals who receive cadaveric organ transplants continues to grow, an increasing number of safeguards must be established to protect the future health of the recipients. Transplanted organs and tissue must be tested to ensure their safety and in case of non-heart-beating donors testing is performed postmortem. Accreditation agents such as JCAHO, CAP, and ASCP provide guidelines for the handling and testing of cadaveric transplant organs and tissue. According to JCAHO one of the new uses of the autopsy is as a reservoir of tissue and organs for transplant and research (ASCP autopsy policy 91-01, (www.ascp.org/pdf/Autopsy.aspx)). There are also special categories for which an autopsy should be given special consideration such as the death of an organ or tissue transplant recipient who received a cadaveric related transplant within sixty days prior to death and in cases where death results from high risk infectious and contagious diseases, to include AIDS (www.ascp.org/pdf/Autopsy.aspx). Viruses such as HIV, HBV, HCV and a host of other infectious agents such as prion diseases, Babesiosis, Chagas disease, and tuberculosis have been transmitted via tissue transplantation in the past due to the limitations of the testing employed. Immunoassays used to screen blood donors have also been used to test deceased donors (48). The downside of this method of testing is the difficulty in detecting an infectious period during the viremic stage before antibodies can be detected (48). It is believed that the risk of a tissue or organ donor being in the “infectious window period” is even higher than that for blood donors. Molecular testing has been added to supplement antibody testing for specimens from cadaveric non-heart-beating donors: http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Tissue/ucm091374.pdf. Testing for a battery of infectious agents is performed on cadaveric donors (brain-dead or non-heart-beating) depending upon the tissue or organ retrieved for transplant (HCV, HIV, CJD, fungal, bacterial, and parasitic agents). These tests are performed after the removal of organs in the non-heart-beat donor due to the time constraints to ensure organ viability (49). It is vital to rule out the presence of infectious etiology prior to transplantation and molecular technology plays a pivotal role in that process. Similar to the success achieved with blood transfusion, nucleic acid testing is being used to decrease the risk associated with cadaveric tissue transplants (48).

Bioterrorism and Autopsy

Since the events of 9-11-2001 and the infamous anthrax threat, there has been a heightened awareness of the potential for biowarfare agents being used against an unsuspecting community. The postmortem evaluation unfortunately may be the first opportunity for the medical system to identify and confirm the presence of a biological “select agent”. Therefore autopsies play a critical role in the process of specimen collection as evidentiary material for analysis and dissemination of timely and accurate information. In a bioterroist attack the autopsy accomplishes three very important goals according to the CDC, medical examiner, coroners, and biological terrorism guidebook. 1) establish the identity of the etiologic agent and the disease process, 2) verify that the agent causing disease is also the cause of death, and 3) rule out the possibility that there may be a competing cause of death (50).

Autopsy and Public Health

According to Nolte (51), anywhere from 20-30% of patient who die in the hospital have an infectious disease that goes undiagnosed until postmortem evaluation. A retrospective study was conducted by Bonds (52) to determine if clinical and postmortem diagnoses of infectious diseases were comparable and to determine the discrepancy rate. The study showed that for adults 43.1% of the patients with infectious diseases were diagnosed at autopsy with infections that had not been previously recognized. The findings were higher for neonates and fetuses with 58% of those with an infectious disease being diagnosed only at autopsy. Clinical microbiology has also been used to study pathogenesis and transmission of infectious agents of public health concern to prevent or curtail transmission (51). Schwartz and Herman in 1996 spoke to the value of autopsy for the early diagnosis of outbreaks caused by Hantavirus, Ebola virus, leptospirosis, Legionella, and Lassa hemorrhagic fever (53). The significance of these findings suggests that microbiology testing conducted from autopsy investigations plays an invaluable role in public health surveillance. Medical examiners and coroners often encounter infectious processes as a cause of death with public health implications (54). In a retrospective study of undisclosed infection first detected in autopsies performed between 1950 and 1988, Hill and Anderson (36) identified 87 diseases that were not previously identified prior to death, a small proportion of which were infectious in nature. Over the past two decades many advances have been made and more diseases have been uncovered through the use of postmortem microbiology analysis and molecular techniques.

Summary: Autopsy and the Relevance of Microbiology Testing

According to Hill and Anderson (36) virtually all of the knowledge of modern medicine is derived from the study of autopsies which were greatly influenced by microbiology, as well as physiology and physical diagnosis. However, in recent years there has been much debate as to the relevance of an autopsy in current medicine and even more debate on the usefulness that microbiology plays in this setting. One recurring theme however is that when autopsies are used in a targeted approach such as investigating the sudden death of a formerly healthy young adult, or for obtaining timely information in an outbreak be it natural or man-made the value increases dramatically (37). This is especially so in today's environment of emerging and remerging pathogens, and bioterroism. In conclusion, microbiology can add valuable information to the autopsy report with the careful selection and collection of samples and interpretation of results.

References

Horowitz

R.E.

, Naritoku

W.Y.

. The autopsy as a performance measure and teaching tool. Hum Pathol 2007; 38(5): 688–695.

Mende

, Laubach

, Friedrich

. Attitudes of students of medicine, sociology and jurisprudence towards autopsy. Dtsch Med Wochenschr 2001; 126(43): 1191–6.

Stolman

C.J.

. Attitudes of pediatricians and pediatric residents toward obtaining permission for autopsy. Arch Pediatr Adolesc Med 1994; 148(8): 843–7.

Shojania

K.G.

, Burton

E.C.

. The vanishing nonforensic autopsy. N Engl J Med 2008; 358(9): 873–5.

Xiao

. The impact of declining clinical autopsy: need for revised healthcare policy. Am J Med Sci 2009; 337(1): 41–6.

Peacock

S.J.

. The autopsy: a useful tool or an old relic? J Pathol 1988; 156(1): 9–14.

Cottreau

, McIntyre

, Favara

B.E.

. Professional attitudes toward the autopsy. A survey of clinicians and pathologists. Am J Clin Pathol 1989; 92(5): 673–6.

Karunaratne

, Benbow

E.W.

. A survey of general practitioners’ views on autopsy reports. J Clin Pathol 1997; 50(7): 548–52.

Start

R.D.

. Attitudes of senior pathologists towards the autopsy. J Pathol 1994; 172(1): 81–4.

10.

Grunberg

S.M.

. Analysis of physician attitudes concerning requests for autopsy. Cancer Invest 1994; 12(5): 463–8.

11.

Gibson

T.N.

. Discrepancies between clinical and postmortem diagnoses in Jamaica: a study from the University Hospital of the West Indies. J Clin Pathol 2004; 57(9): 980–5.

12.

Goldman

. The value of the autopsy in three medical eras. N Engl J Med 1983; 308(17): 1000–5.

13.

Shojania

K.G.

. Changes in rates of autopsy-detected diagnostic errors over time: a systematic review. JAMA 2003; 289(21): 2849–56.

14.

Wilson

S.J.

, Wilson

M.L.

, Relier

L.B.

. Diagnostic utility of postmortem blood cultures. Arch Pathol Lab Med 1993; 117(10): 986–8.

15.

Koneman

E.W.

, Davis

M.A.

. Postmortem bacteriology. 3. Clinical significance of microorganisms recovered at autopsy. Am J Clin Pathol 1974; 61(1): 28–40.

16.

Nolte

K.B.

. “Med-X”: a medical examiner surveillance model for bioterrorism and infectious disease mortality. Hum Pathol 2007; 38(5): 718–25.

17.

Waters

B.L.

. Handbook of Autopsy Practice. 4 ed. Humana Press; 2009.

18.

Reznicek

M.J.

, Koontz

F.P.

. Autopsy Microbiology. In Clinical Laboratory Medicine. Lippincott Williams & Wilkins; 2002.

19.

Lobmaier

I.V.

. Bacteriological investigation–significance of time lapse after death. Eur J Clin Microbiol Infect Dis 2009; 28(10): 1191–8.

20.

Morris

J.A.

, Harrison

L.M.

, Partridge

S.M.

. Practical and theoretical aspects of postmortem bacteriology. Current Diagnostic Pathology 2007; 13: 65–74.

21.

Carpenter

H.M.

, Wilkins

R.M.

. Autopsy Bacteriology: Review of 2,033 Cases. Arch Pathol 1964; 77: 73–81.

22.

Weber

M.A.

. Post-mortem interval and bacteriological culture yield in sudden unexpected death in infancy (SUDI). Forensic Sci Int 2010; 198(1-3): 121–5.

23.

Dolan

C.T.

, Brown

A.L.

Jr. , Ritts

R.E.

Jr. . Microbiological examination of postmortem tissues. Arch Pathol 1971; 92(3): 206–11.

24.

Wise

. The ‘septic spleen'–a critical evaluation. J Clin Pathol 1976; 29(3): 228–30.

25.

Morris

J.A.

, Harrison

L.M.

, Partridge

S.M.

. Postmortem bacteriology: a re-evaluation. J Clin Pathol 2006; 59(1): 1–9.

26.

de Jongh

D.S.

. Postmortem bacteriology: a practical method for routine use. Am J Clin Pathol 1968; 49(3): 424–8.

27.

Coe

J.I.

. Postmortem chemistry update. Emphasis on forensic application. Am J Forensic Med Pathol 1993; 14(2): 91–117.

28.

Platt

M.S.

. Postmortem cerebrospinal fluid pleocytosis. Am J Forensic Med Pathol 1989; 10(3): 209–12.

29.

Wyler

, Marty

, Bar

. Correlation between the post-mortem cell content of cerebrospinal fluid and time of death. Int J Legal Med 1994. 106(4): 194–9.

30.

Knapp

B.E.

, Kent

T.H.

, Postmortem lung cultures. Arch Pathol 1968; 85(2): 200–3.

31.

Bates

D.W.

, Lee

T.H.

. Rapid classification of positive blood cultures. Prospective validation of a multivariate algorithm. JAMA 1992; 267(14): 1962–6.

32.

Weinstein

M.P.

. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis 1997; 24(4): 584–602.

33.

Weinstein

M.P.

. Blood culture contamination: persisting problems and partial progress. J Clin Microbiol 2003; 41(6): 2275–8.

34.

Guarner

. Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis. Hum Pathol 2007; 38(9): 1412–9.

35.

Talal

A.H.

, Weiss

L.M.

, Vanderhorst

. Molecular diagnosis of gastrointestinal infections associated with HIV infection. Gastroenterol Clin North Am 1997; 26(2): 417–44.

36.

Hill

R.B.

, Anderson

R.E.

. The Autopsy - Medical Practice and Public Policy. Boston: Butterworths; 1988.

37.

Mazuchowski

2nd , Meier

P.A.

. The modern autopsy: what to do if infection is suspected. Arch Med Res 2005; 36(6): 713–23.

38.

Ivarsson

, Carlson

. Extraction, quantitation, and evaluation of function DNA from various sample types. Methods Mol Biol 2011; 675: 261–77.

39.

Pfaller

M.A.

. Molecular approaches to diagnosing and managing infectious diseases: practicality and costs. Emerg Infect Dis 2001; 7(2): 312–8.

40.

Klopfleisch

, Weiss

A.T.

, Gruber

A.D.

. Excavation of a buried treasure – DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues. Histol Histopathol 2011; 26(In Press).

41.

McGhee

J.D.

, von Hippel

P.H.

. Formaldehyde as a probe of DNA structure. 3. Equilibrium denaturation of DNA and synthetic polynucleotides. Biochemistry 1977; 16(15): 3267–76.

42.

Akalu

, Reichardt

J.K.

. A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue. Genet Anal 1999; 15(6): 229–33.

43.

Lehmann

, Kreipe

. Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 2001; 25(4): 409–18.

44.

Kilpatrick

C.W.

. Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods. Biochem Genet 2002; 40(1-2): 53–62.

45.

Odenthal

. Detection of opportunistic infections by low-density microarrays: a diagnostic approach for granulomatous lymphadenitis. Diagn Mol Pathol 2007; 16(1): 18–26.

46.

Paterson

P.J.

. Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections. Mol Pathol 2003; 56(6): 368–70.

47.

Munoz-Cadavid

. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol 2010; 48(6): 2147–53.

48.

Strong

D.M.

. Preventing disease transmission by deceased tissue donors by testing blood for viral nucleic acid. Cell Tissue Bank 2005; 6(4): 255–62.

49.

Brockbank

K.G.M.

, Battjes Siler

D.J.

. Disinfection, Sterilization and Preservation. Fifth ed, ed. Block

S.S.

. Philadelphia: Lippincott Williams & Wilkins; 2001.

50.

Nolte

K.D.

. Medical examiners, coroners, and biologic terrorism: a guidebook for surveillance and case management. MMWR Recomm Rep 2004; 53(RR-8): 1–27.

51.

Nolte

K.B.

. Infectious disease pathology and the autopsy. Clin Infect Dis 2002; 34(1): 130–1.

52.

Bonds

L.A.

. Infectious diseases detected at autopsy at an urban public hospital, 1996-2001. Am J Clin Pathol 2003; 119(6): 866–72.

53.

Schwartz

D.A.

, Herman

C.J.

. The importance of the autopsy in emerging and reemerging infectious diseases. Clin Infect Dis 1996; 23(2): 248–54.

54.

Nolte

K.B.

. Guidelines to implement medical examiner/coroner-based surveillance for fatal infectious diseases and bioterrorism (“Med-X”). Am J Forensic Med Pathol 2010; 31(4): 308–12.