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Current Diagnostic Pathology (2007) 13, 6574

www.elsevier.com/locate/cdip

REVIEW

Practical and theoretical aspects of postmortem bacteriology

J.A. Morrisa,, Linda M. Harrisona, Susan M. Partridgeb
a

Department of Pathology, Royal Lancaster Inrmary, Lancaster LA1 4RP, UK Department of Pathology, Furness General Hospital, Barrow in Furness LA14 4LF, UK

KEYWORDS
Blood cultures; Cerebrospinal uid; Neutrophils; Postmortem bacteriology

Summary A positive blood culture or culture of cerebrospinal uid (CSF) obtained postmortem could be due to a genuine positive, to agonal spread, to postmortem translocation or to contamination. A review of published evidence indicates that, if specimens are taken with care, the majority is sterile. Mixed growth occurs in less than 10% and is mainly due to contamination. Postmortem translocation is rarely a problem if the body is refrigerated promptly after death. Agonal change is less common than often assumed. A single isolate of a potential pathogen in perinatal cases and in adult practice is likely to be a genuine positive (PPV450%); in sudden unexpected death in infancy (SUDI), however, a single isolate should be regarded as only a possible cause of death (PPVo50%) in the absence of corroboration. If bacterial invasion causes or contributes to rapid death, there might not be time for the histological changes of inammation to develop and more subtle signs should be sought; including cell counts and protein estimations of CSF. Sampling upper-airways secretions shortly after death and before refrigeration provides useful information if infection is suspected. The results can be compared with community controls in epidemiological studies. In the future, genomic and proteomic techniques need to be added to standard methods to determine the role of bacteria in sudden death. The key message is that if specimens are taken with care, and interpreted with care, then useful information can be obtained. & 2006 Elsevier Ltd. All rights reserved.

Introduction
Bacterial infection causes and contributes to death at all ages and those who perform postmortem examinations must be able to take the appropriate
Corresponding author. Tel.: +44 1524 583 794;

fax: +44 1524 583 798. E-mail address: Jim.A.Morris@rli.mbht.nhs.uk (J.A. Morris).

specimens and interpret the results.1 It is a standard practice to obtain specimens for bacteriological examination in perinatal cases, in cases of sudden unexpected death in infancy (SUDI) and also in young children and young adults whose death is sudden and unexpected; the great majority of adult autopsies, however, are undertaken without bacteriological investigation. This might change in the future as antibiotic resistance in common hospital

0968-6053/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cdip.2006.07.005

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66 and community pathogens becomes more of a problem, both medically and legally. Interpretation of bacteriological specimens is straightforward if a signicant pathogen is isolated in association with clinical and pathological evidence of inammation; it is much more difcult if a common organism is found in a case where death was rapid and inammation has not developed. This applies with particular force in SUDI. In general, it is best to obtain specimens for bacteriological examination prior to autopsy, so as to keep contamination to a minimum. If, however, during the course of an autopsy it becomes apparent that cultures are required, they should still be takenwith as much care as possiblealthough it is inevitable that interpretation in this situation will be difcult. This review is concerned with bacteriology, not virology. Its primary focus is the interpretation of the signicance of isolates of potential pathogens in cases where supporting evidence of infection is not clear-cut. It is emphasized that a condent diagnosis can rarely be made on the basis of bacteriology alone, and corroborative evidence of infection must be sought, although this might be subtle. Recent changes in the law and in Coroners rules have put hurdles in the way of conducting ancillary investigations at autopsy. These changes might prevent the introduction of some of the recommendations in this review. For instance, our suggestion that, in cases of SUDI, specimens should be obtained by paediatricians in A&E rather than by pathologists at autopsy is based on sound pathological principles but might fall foul of local rules and procedures. In this review it is our intention to concentrate on the pathology and leave the law to others. J.A. Morris et al.

Box 1 Causes of a positive postmortem blood culture 1. 2. 3. 4. Invasion in life Agonal spread Postmortem translocation Contamination

period that the circulation is articially maintained by resuscitation, there is relative ischaemia/hypoxia of mucosal surfaces and bacteria can invade. As there are very many different types of bacteria on mucosal surfaces, the growthin theoryshould be mixed. 3. Postmortem translocation: bacteria can grow on the mucosal surface after death and after the circulation has ceased. These bacteria can then invade the body and the blood. This is the basis of putrefaction, which occurs normally after death if the body is not stored at a low temperature. The growth is likely to be mixed rather than a single isolate. 4. Contamination: bacteria were not present in the blood at the time of autopsy but were introduced when the sample was obtained. Once again, this is likely to lead to a mixed growth of organisms. Agonal spread and postmortem translocation are artefacts specic to the postmortem period. Contamination is a problem in life and in death.

Assessment in life
Bacteria isolated from blood cultures in life are genuine positives or are due to contamination. Standards issued by the American Society of Microbiology indicate that the rate of blood culture contamination should not exceed 3%, although, in practice, in most hospitals the contamination rate is closer to 46%.2 A pure growth of a potential pathogen is likely to be a genuine positive; a mixed growth of commensal organisms is likely to be a result of contamination. Weinstein3 used a predictive model with multiple variables to assess the results of blood cultures obtained in life. Microorganism identity was an independent predictor (Box 2). Organisms that nearly always (490%) represent true bacteraemia or fungaemia include not only Neisseria meningitidis, Neisseria gonorrhoea, Haemophilus inuenzae, Salmonella species and Candida albicans but also more common pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Escherichia

General considerations
Invasion of the bloodstream
The isolation of bacteria from blood postmortem can arise in four ways (Box 1): 1. Genuine positive: the bacteria invaded the blood in life and were present at the time of death. Bacteraemia can occur in life without causing symptoms or signicant disease but if bacteraemia is followed by death then one cannot exclude the possibility that it is a contributing factor to death. 2. Agonal spread: this is a theoretical concept. The idea is that during the agonal process, or in the

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Practical and theoretical aspects of postmortem bacteriology 67

Box 2 Postmortem blood isolates that are likely to be true positives

       

Neisseria meningitidis Haemophilus inuenzae Streptococcus pneumoniae Candida albicans Salmonella species Staphylococcus aureus E. coli Other Enterobacteriaceae

coli and other members of the Enterobacteriaceae. By contrast, Corynebacterium species, Bacillus species and Propionibacterium acnes rarely represent true bacteraemia.

Corroboration
The assessment of the signicance of a bacterial isolate in life and in death depends on the specic isolate and the clinical context. The clinical history and investigations before death might support a diagnosis of infection. The pathologist will also seek macroscopic and microscopic evidence of infection at autopsy. The diagnosis of inammation at autopsy, however, is not straightforward and this applies in particular if death is sudden. One of the cardinal features of inammation is a neutrophilic inltrate in tissues, but this takes time to develop. This review will emphasize the importance of:

   

The nature of the bacterial isolate: the type of organism and whether or not it is in pure growth. The clinical context: does the history support a diagnosis of infection? The correct assessment of microscopic evidence of inammation. The potential of new techniques, both genomics and proteomics, to provide corroboration of infection.

days. The output of neutrophils from the bone marrow is 1.5 109 kg body weight per 24 h. The neutrophils circulate in the blood for approximately 10 h and then enter the tissues. They then survive for a further 14 days.6,7 In tissue sections, there is approximately one neutrophil per high-power eld and the vast majority are present within blood vessels. It is unusual to see extravascular neutrophils in tissue in the absence of inammation, other than on mucosal surfaces. As they live for 14 days, spend only 10 h in the bloodstream and are not normally seen in tissue, they must quickly exit onto mucosal surfaces. Their function on the mucosal surface is presumably to control the commensal bacterial ora and purge the surface of potential pathogens. The intravascular pool of neutrophils is 0.7 109 cells/kg body weight and in the bone marrow there is a reserve of 8.8 109 neutrophils/ kg body weight. In the intravascular pool, 0.4 109 neutrophils/kg body weight are marginated and 0.3 109 are circulating. The bone marrow reserve allows for up to a ten-fold increase in circulating neutrophils during bacterial infection. This, however, takes time to develop. During bacterial infection, neutrophils can show toxic granulation. This is due to an increased number of primary granules but, once again, takes time to develop. Neutrophils derive energy from glycolysis and can survive on the mucosal surface and phagocytose bacteria in anaerobic conditions. But following phagocytosis in tissues there is a respiratory burst in which oxygen is used to generate oxygen radicals for optimum bacterial killing. This is impaired in the absence of oxygen.8

Blood cultures
A careful aseptic technique is essential to minimize contamination. One approach is to obtain blood from a peripheral vein before the body is dissected. The skin over the vein is wiped clean with spirits and then the blood sample is collected and dispensed into blood-culture bottles. An alternative approach is to open the body in the standard way, heat-sterilize the surface of the heart or pulmonary artery and then pass a needle through the sterilized area to collect the sample of blood. With a careful technique, contamination can be kept below 10%. Carpenter and Wilkins9 reviewed 2033 autopsy cases in which blood samples had been obtained. Only 7% yielded two or more organisms; the rest were either sterile (68%) or

Neutrophil kinetics
The primary function of neutrophil polymorphonuclear leukocytes is the phagocytosis and destruction of microorganisms.4,5 These cells are produced in the bone marrow from myeloblasts. Myeloblasts produce promyelocytes, myelocytes, band cells and then segmented neutrophils by a process of cell division and then maturation The cell generation time in the marrow is 24 h and the transit time from myeloblast to mature segmented neutrophil is 9

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68 yielded a single organism (25%). Wise10 studied 192 autopsy cases aged 3191 years. He sterilized the spleen surface and then passed a swab into the spleen to obtain tissue for culture; 87.5% were sterile, 11% yielded a single organism and only 1.5% had a mixed growth. Pryse-Davies and Hurley11 reviewed blood cultures in 797 infants in the perinatal period; mixed growth occurred in only 4%. If the body is promptly stored at 4 1C after death, postmortem translocation is rarely a problem. Thus, although Carpenter and Wilkins9 examined their cases within 118 h of death, many of Wises cases10 were stored for 24 or 48 h before autopsy. In the series reported by Pryse-Davies and Hurley,11 one-third of the samples were obtained within 24 h but one-third were obtained over 48 h after death. In a review of over 5000 autopsy cases, comprising mainly adults but including 1108 perinatal cases and 468 cases of SUDI, approximately two-thirds of blood cultures were sterile, over two-ninths yielded a single organism, and less than one-ninth had a mixed growth.1 It is likely that most of the mixed growth was due to contamination. Thus agonal spread of organisms is much less of a problem than is often assumed. In two-thirds of the cases there was no agonal spread because the cultures were sterile. Furthermore, in over ninetenths, the cultures were sterile or yielded only a single organism. Thus the concept that the mucosa is compromised during the agonal phase, leading to invasion of a range of bacterial organisms, is incorrect. If agonal spread occurs then in most cases only a single species invades. This invasion must reect a property of the organism, such as its invasive potential or concentration, or something peculiar to its location. Few autopsy studies allow an estimation of the sensitivity and specicity of blood culture. An exception is a study carried out in 1916 by Fredette,12 who obtained blood cultures from 119 cases within 30 min of death. The age range was 4 months to 84 years but most were adults. Blood was obtained from the antecubital veins using a careful aseptic technique. This study was carried out in the preantibiotic era and many of the cases were young adults with lobar pneumonia. The growth of Strep. pneumoniae as a marker for lobar pneumonia had a sensitivity of 47%, a specicity of 98%, a positive predictive value (PPV) of 78% and a negative predictive value (NPV) of 92%. This is a single example but the indication is that the sensitivity is low but the specicity is reasonably high. Predicitve values depend on the sensitivity and specicity of the test and also on the prevalence or a priori probability of the condition under consideration. If infection is likely, as determined by J.A. Morris et al. the clinical context, and the specicity of blood culture is reasonably high, then the PPV will be well in excess of 50%. But if infection is highly unlikely then the PPV can be o50% even if the specicity is high. A good working rule, therefore, is to regard a pure growth of a pathogen as a possible genuine positive even in the absence of clinical or pathological corroboration (low PPV) but to accept it as a likely genuine positive (high PPV) if there is supporting evidence from the clinical context. In most adult autopsies, and in most perinatal cases, a pure growth of a potential pathogen, such as Staph. aureus or E. coli, in blood culture is likely to occur in association with supportive evidence and the PPV will be over 50%. But in cases of SUDI, the situation is more complicated. There is evidence that infection is a cause of SUDI and therefore it is not difcult to accept that bacteraemia caused by organisms such as Staph. aureus and E. coli could lead to toxaemia and sudden death.13 But this sequence is not universally accepted and therefore a prudent approach is to regard a pure growth of a potential pathogen in blood culture as no more than a possible explanation for death in a case of SUDI that is otherwise unascertained. The diagnosis, however, moves from possible to probable if there is supporting pathological evidence, i.e. evidence of inammation in major organs such as the lungs or cerebrospinal uid (CSF).14

Cerebrospinal uid
A specimen of CSF can be obtained by lumbar puncture or by cisternal puncture. The specimen should be taken using a careful aseptic technique. It is best to obtain the specimen prior to autopsy and follow the procedure set out for use on the hospital wards. If a careful technique is used, the rate of contamination can be kept to well below 10%. Adelson and Kinney15 had no growth in 38 consecutive CSF cultures in cases of SUDI; a contamination rate of 0% [95% condence interval (CI) 08%]. This study also indicates that agonal spread of bacteria from mucosal surfaces through the blood to the CSF is extremely rare and probably does not occur. Pryse-Davis and Hurley11 examined 479 specimens of CSF from perinatal cases. They found that 88% were sterile, 10% had a single isolate and only 2% had a mixed growth. They judged most single isolates to be genuine positives and therefore the rate of contamination was very low.

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Practical and theoretical aspects of postmortem bacteriology If a body is refrigerated promptly after death it is unlikely that a positive CSF specimen is due to postmortem translocation or agonal spread. Thus a positive CSF obtained by careful technique is likely to be a genuine positive. There is evidence that bacteraemia can occur in life without symptoms or signs of disease, but there is no evidence that bacteria can enter the CSF in life other than as part of a serious and life threatening condition. A pure growth of a potential pathogen, such as E. coli or Staph. aureus obtained from CSF postmortem must be regarded as at a least a possible explanation for death. A condent diagnosis of serious disease is easy if a bacterial isolate from CSF is associated with evidence of meningitis. Difculty arises, however, in cases of sudden death, particularly sudden death in infancy, in which there is a pure growth of a potential pathogen in the CSF but no histological conrmation of meningitis. The majority of cases of SUDI die rapidly. In the CESDI SUDI study,14 of those infants who died in the daytime, the interval between last seen and found dead was less than 1 h 10 min in 50% of the cases; in 25% it was less than 25 min. All these infants appeared to be well when last seen or had only a very minor illness. Bacteraemia can be present in infants who appear to be well. As the potential doubling time of bacteria is 20 min, clearance mechanisms must be capable of removing the bacterial load in less than 20 min or the battle will be lost. If bacteria release toxins when cleared there is a chance of toxic shock and rapid death. In this situation there will not be enough time for the histological signs of meningitis to become apparent. A cubic millimetre of tissue cut into 5 mm slices produces 200 parallel histological sections. Four high-power elds covers an area of approximately 1 mm2 (it depends on the microscope but this is the average). There is approximately one polymorph per high-power eld in non-inamed tissue, which is present almost exclusively in vascular channels. In the entire cubic millimetre of tissue there will be approximately 800 polymorphs. The acute onset of inammation causes a movement of polymorphs from blood vessels into tissues. It normally takes 10 h for all the polymorphs in the blood to move into tissues and, even if this is greatly speeded up in inammation, only a small percentage will move in 2060 min. Thus, in a short period of time there might be only one extravascular polymorph per ten high-power elds, which is not regarded as sufcient for a condent diagnosis of inammation. However, one polymorph per ten high-power elds is equivalent to 80 polymorphs per cubic millimetre 69

of tissue; 80 polymorphs per cubic millimetre of CSF would be regarded as diagnostic of meningitis.16 Thus, counting polymorphs in the CSF is likely to be a much more sensitive way of diagnosing meningitis than examining tissue sections. The difculty with counting polymorphs in CSF is that they have a half-life of only 24 h.16 It is important, therefore, to obtain the specimen as soon after death as possible. In cases of SUDI, most are now admitted to A and E departments and a specimen of CSF should be obtained by paediatricians and submitted for urgent microbiological investigation prior to autopsy. The specimen should be examined as in life: with a cell count, a differential cell count and protein estimation as well as culture. The other advantage of this approach is that clinicians are familiar with obtaining CSF specimens aseptically so that contamination rates should be low. A normal specimen of CSF contains no more than four mononuclear cells per cubic millimetre and no polymorphs. After death, however, the CSF cell count rises. Platt et al.17 found CSF counts between 37 and 3250 cells per cubic millimetre in 26 cases of SUDI in which there was no evidence of meningitis. The postmortem intervals were 228 h. The typeable cells were mononuclear, 6070% lymphocytes and 2040% monocytes; there were no polymorphs. Following death, therefore, a raised cell count alone does not support a diagnosis of meningitis. These studies, however, indicate that polymorphs do not enter the CSF normally after death and their presence, as in life, indicates inammation. Brain capillaries form a barrier to the movement of proteins from blood into the CSF. This barrier is reduced in inammation and proteins enter the CSF by diffusion between endothelial cell junctions and by active transport across the endothelial cell cytoplasm. Following death, the bloodbrain barrier is preserved, at least initially, and protein measurements in CSF are of value.18,19 The precise rate at which the barrier breaks down following death at different ages, however, has not be dened with precision and therefore more work needs to be done to evaluate CSF protein as a marker of inammation postmortem (Box 3).

Upper respiratory tract ora

Samples of secretions from the anterior nares (nasal swab), from the nasopharynx (pernasal swab) and from the throat (throat swab) can be obtained prior to the autopsy. If the swabs are obtained soon after death and before the body is

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70 J.A. Morris et al. early morning secretions of infants sleeping prone and suffering from an upper respiratory viral infection.22 The explanation is that secretions pool in the upper airways in infants sleeping in the prone position; this is most marked in infants who have a viral respiratory infection. In this situation, organisms from the environment, such as bedding, can be inhaled and can grow in the secretions. When the infant moves to the supine or upright position the secretions drain into the oesophagus, are swallowed and the Gram-negative ora is lost. In studies of the nasopharyngeal ora in SUDI carried out in the 1980s, when most infants slept prone, E. coli and other Enterobacteriaceae were found in 45%.24 This was a highly signicant difference compared with community controls. In this study the isolation rate of Enterobacteriaceae fell with delay in obtaining the sample so that it was probably not explained by postmortem growth. In a study of SUDI in Avon, UK,25 carried out between 1987 and 1989, throat swabs were obtained on 95 cases and 190 healthy age- and season-matched controls from the community. The bacteriological results obtained from the throat swabs were as follows: 1. Staph. aureus: 24/95 (25%) in cases versus 12/ 190 (6%) in controls, odds ratio 5 (2.410). 2. Coliforms: 15/95 (16%) in cases versus 2/190 (1%) in controls, odds ratio 29 (7.5111). 3. Strep. pneumoniae: 10/95 (11%) in cases versus 2/190 (1%) in controls, odds ratio 10 (2.934). 4. Group B streptococcus: 6/95 (6%) in cases versus 2/190 (1%) in controls, odds ratio 11 (1.963). The authors of this study were reticent about attaching too much weight to these results because of the possibility of postmortem growth. But the throat swabs were obtained in A and E shortly after death was detected and prior to autopsy. It is therefore likely that the ora detected in cases reects the ora at the time of death. The odds ratios in this study are impressive (Box 4) and provide support for the idea that bacteria from the upper respiratory tract have a role in the pathogenesis of SUDI. Box 4 Throat swabs: odds ratios for SUDI versus controls Staphylococcus aureus Coliforms Streptococcus pneumoniae Group B streptococcus 5 29 10 11

Box 3

Postmortem cerebrospinal uid

   

Agonal spread of bacteria into the CSF is rare Mononuclear cells are found in the CSF after death but neutrophils are not in the absence of meningitis or bleeding The balance of evidence is that protein levels in the CSF reect those at the time of death for up to 24 h after death A CSF neutrophil count, obtained shortly after death, is a more sensitive indicator of meningitis than is histological examination of the meninges

refrigerated then the bacterial ora should reect that present at death. Following death, however, some bacteria will outgrow others; further, following refrigeration some bacteria will die more quickly than others. The composition of the ora will therefore change with time. The composition of the upper respiratory tract ora is particularly relevant in cases of SUDI. There is a body of evidence supporting the hypothesis that toxins produced by common bacteria such as Staph. aureus and E. coli are a cause of some cases of SUDI.20 The best single piece of evidence is that over 50% of the cases of SUDI have detectable staphylococcal toxins (toxic shock syndrome toxin and staphylococcal enterotoxin C) in the brainstem.21 Nasopharyngeal carriage of Staph. aureus is over 50% in the rst 3 months of life and then falls to around 30% from 3 to 12 months.22,23 One-third of isolates carry the genes for one or more of the pyrogenic enterotoxins, such as TSST or SEC, but signicant toxin production only occurs when the mucosal surface temperature rises above 37 1C. If Staph. aureus is isolated from a pernasal swab, and if the isolate is shown to have the genes for pyrogenic toxins and these genes are switched on, then this is at least a possible explanation for death in SUDI. Further conrmation would require demonstrating the bacteria and or the toxins in blood and or CSF. Assays for staphylococcal pyrogenic toxins in blood or CSF are not routinely available at the time of writing but are likely to become available in the near future. The tests could be based on immunoassays for the toxins or measurements of the RNA transcript of the toxin gene. E. coli and other Enterobacteriacea are not normally found in the nasopharynx of infants; in one community study the isolation rate was below 4%.22 These organisms are found, however, in the

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Practical and theoretical aspects of postmortem bacteriology In cases of SUDI it is best to obtain both pernasal and throat swabs. Bacteria are more likely to be found in the nasopharynx but the odds ratios are more impressive in throat swabs. The likely explanation is that if there are only a few organisms present these will be found in the nasopharynx but not the throat, whereas if there are many organismsa more dangerous situationthey are likely to be found in both sites. 71

Lower respiratory tract

There is no normal resident bacterial ora in the lower respiratory tract. Bacteria from the upper airways will be inhaled with each breath but they are deposited on the mucociliary escalator of the bronchi and trachea and cleared into the oesophagus. At the time of death there might be a few bacteria present in the bronchial secretions and these could grow in number after death. Otherwise the lung will be sterile in the absence of infection. Sampling the lower respiratory tract at autopsy can be done by passing a swab down the trachea and bronchi after careful dissection and elevation of the tongue and larynx. An alternative is to inject saline down the trachea and then aspirate and collect the washings. A tissue sample can be obtained by opening the chest and taking a piece of lung using a sterile forceps and a sterile blade. As always, careful technique is essential. The evaluation of the bacterial ora follows the same principles as above. A pure, heavy growth of a potential respiratory pathogen such as Strep. pneumoniae points to genuine infection. A scanty growth of mixed organisms of the type commonly seen as commensals in the upper airways points to postmortem aspiration. If there is a pure growth of a potential pathogen and histological evidence of bronchopneumonia, the diagnosis is easy. However, if death is sudden and a potential pathogen is found in the absence of evidence of pneumonia, assessment is much more difcult. Once again, this particular problem of a possibly signicant isolate in the absence of inammation occurs in SUDI. If death follows dissemination of bacteria from the upper airways the process can occur too quickly for a signicant neutrophil response in the lungs. There are 25 million alveoli at birth and the adult level of 300 million alveoli is reached by 8 years.26 The intravascular pool of neutrophils is 700 million/ kg body weight. If all the neutrophils in the circulation enter the lung there will be 100200 per alveolus. However, if disseminated infection

causes disseminated inammation and sudden death, only a tiny percentage of the circulating neutrophils will enter the lung. If there are fewer than 20 per alveolus there will be less than one per alveolar section. Examination of tracheal and bronchial secretions by cytology could be useful. If bacteria are growing in the upper airways then they would have been entering the lung for some time prior to death and there could be a signicant number of neutrophils in bronchial secretions. The cells are anaerobic and will survive for a few hours. The disadvantage of examining bronchial secretions compared with nasopharyngeal secretions is that they cannot be obtained till the autopsy is done, usually many hours after death, and there are no controls.

The intestinal ora

The number of bacteria in the intestinal tract is ten times greater than the total number of human cells in the body; there are many different species and many of them cannot be grown on conventional media. The role of the intestinal ora in a whole range of diseases is receiving increasing attention. It has long been thought that the normal bowel ora could have a pathogenic role in inammatory bowel disease; but more recently it has been realized that exposure to bacterial antigens in the gut could precipitate a range of autoimmune and allergic mechanisms, and that this could be implicated in the onset of type 1 diabetes mellitus, asthma, eczema, multiple sclerosis and rheumatoid arthritis to name but a few.27,28 In view of this, study of the intestinal ora at autopsy could become increasingly important in the future. Its current role, however, is much more limited. If there is evidence from the clinical history or from ndings at autopsy of bowel pathology that could have an infective cause then a sample of faeces should be obtained. Careful technique is less important as the microbiological approach is to try to identify specic pathogens from a background of commensals. Thus if Salmonella, Shigella or Campylobacter spp. are present in association with intestinal inammation a condent diagnosis can be made. Equally, recognition of toxins of toxigenic E. coli or Clostridia in the appropriate clinical setting will conrm a diagnosis of enterocolitis or pseudomembranous colitis. Occasionally, specic infections such as tuberculosis can be conrmed. Murrell et al.29 in Australia have investigated the large-bowel bacterial ora in SUDI. They found

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72 evidence of increased carriage of Clostridium perfringens in cases compared with controls. This has been conrmed by others.30,31 C. perfringens is toxigenic and it is possible, in theory, that toxins from this organism could cause sudden death in infants around 23 months of age when protective antibody levels are low. This organism is commonly carried in the proximal colon in adults; it is in competition with other members of the gut ora and is usually present in low numbers. If the bowel ora is disturbed then overgrowth can lead to necrotising colitis and septicaemia with local and systemic gas production. This full-blown picture, however, is rarely seen in SUDI. C. perfringens is occasionally found in blood at autopsy, with gas in tissues and as a heavy growth of organisms in the proximal colon. In most cases this is due to postmortem translocation in bodies that have not been refrigerated following death. A number of organisms will grow in the gut after death and invade tissues as part of the process of putrefaction. C. perfringens grows more quickly than other bacteria; the standard doubling time for bacteria is 20 min but C. perfringens can double in 8 min under optimum conditions.32 Thus this organism comes to dominate the cultures and appears to be in pure growth. But occasionally C. perfringens is found in pure growth in the blood in association with gas in tissues shortly after death or in bodies appropriately stored following death. In some case reports this appears to be genuine infection.33,34 A review of published literature1 shows that agonal spread of C. perfringens is extremely rare, therefore if the organism is found shortly after death in pure growth this should not be dismissed too lightly. If there is also evidence of damage to the proximal colon then it is at least a possible explanation of death (Box 5). J.A. Morris et al. the body is appropriately stored prior to autopsy. Agonal spread leading to a mixed growth is rare. Agonal spread might lead to a single isolate but, if so, one still needs to ask Why this organism? Spread must be related to the amount of the organism on the mucosal surface or to the invasive potential of the organism, or perhaps to something specic to the particular local site. In perinatal cases, contamination of blood cultures and specimens of CSF should be less than 10%. In this setting, if either yields a single isolate of a potential pathogen then it is more likely to be a genuine positive than a postmortem artefact. The interpretation of postmortem cultures is most difcult in SUDI. A simple rule is that a single isolate of a potential pathogen in blood or CSF, in a case that is otherwise unascertained at autopsy, gives a possible explanation for death. If, however, the isolate is associated with some corroborative evidence of inammation then it becomes a probable explanation for death. To gain corroboration it is necessary to obtain samples, particularly a sample of CSF, shortly after death. A differential cell count and protein estimation should be performed on the CSF specimen. In the near future it is likely that assays will be available to measure specic bacterial toxins in blood and CSF. Correlation of blood and CSF ndings with the bacterial ora of the upper airways is also useful in SUDI. If Staph. aureus is present in blood or CSF, and is also found in the nasopharynx and/or throat, then further studies are indicated; if the organism carries the genes for pyrogenic toxins, if the genes are shown to be switched on (RNA transcript present) and if there is a neutrophilic inltrate in the upper airways then this all supports an infective explanation for death. Equally, if E. coli is present in blood or CSF and is also found in the upper airways in association with neutrophils, then this too supports the infection hypothesis. Postmortem bacteriology is less commonly carried out in adult autopsies but a great deal of Coroners practice concerns sudden unexpected death and, even if ischaemic heart disease or other degenerative disease is present, the question of what precipitated the acute event is left unanswered. Episodes of bacteraemia occur throughout life and could have a role in the acute events of later life. We can only know if we take the specimens. The progressive rise of antibiotic resistance in common hospital and community pathogens might create a legal climate in which postmortem bacteriology in adult autopsies needs to be done. If so, the general principles set out in this review will apply; take the samples with care and interpret the results with care.

Discussion
Postmortem bacteriology can provide useful information if the specimens are taken and interpreted with care. The major problem is contamination. Postmortem translocation is not a problem if specimens are obtained shortly after death or if

Box 5 Agonal spread of C. perfringens is extremely rare therefore if the organism is found shortly after death in pure growth it should not be dismissed too lightly.

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Practical and theoretical aspects of postmortem bacteriology 73

Research direction

What is the rate of change of CSF protein with postmortem interval at different ages? In fact, relatively little information is currently available, particularly in the rst year of life, but it is important to know if CSF protein levels are a reliable marker of inammation. The idea that a raised mononuclear cell count in the CSF after death does not indicate CNS disease should be challenged. A rst step would be to characterize the cells using the array of CD markers now available. Do the mononuclear cells enter from the blood or are they from the lining cells of the meninges? The chemical composition of the CSF in SUDI should be dened. Are there bacterial toxins, are there other bacterial antigens, is bacterial DNA or RNA present? This will require the techniques of proteomics and genomics; it is big science for the future but that future is not far off. A particular advantage of careful assessment of the upper respiratory ora and secretions in SUDI is that the ndings can be compared with the secretions of matched control infants from the community. This was shown to good effect in the Avon study25 but needs to be done on an even larger scale. As in the case of the upper respiratory tract ora, it is possible to obtain appropriately matched specimens of faecal ora for comparative studies. Autopsy investigation is bedevilled by lack of controls but this is one area where large-scale controlled investigations are possible.

References
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