Microorganisms have always been one of the great challenges of humankind, being responsible for both high morbidity and mortality throughout history. In a forensic setting microbiological information will always be difficult to interpret due to lack of antemortem information and changes in flora postmortem. With this study we aim to review the use of microbiological procedures at our forensic institute. In a retrospective study including 42 autopsies performed at our Institute, where microbiological test had been applied, analyses were made with regard to: type of microbiological tests performed, microorganisms found, histological findings, antemortem information, C-reactive protein measurement and cause of death. Fiftyone different microorganisms were found distributed among 37 cases, bacteria being the most abundant. Nineteen of the cases were classified as having a microbiological related cause of death. Creactive protein levels were raised in 14 cases of the 19 cases, histological findings either supported or were a decisive factor for the classification of microbiologically related cause of death in 14 cases. As a multitude of abundant microorganisms are able to cause infection under the right circumstances, all findings should be compared to anamnestic antemortem information, before conclusions are drawn. A definite list of true pathogens is nearly impossible to compile.
ß 2015 Elsevier Ireland Ltd. All rights reserved. and/or swaps are collected once suspicion is raised by gross pathology findings during the autopsy. Tissue samples were analyzed at the Department of Clinical Microbiology, University Hospital of Odense. Samples are routinely analyzed for bacteria and fungi, using agar culturing. PCR analysis for viruses is performed per request. CRP was measured in full blood using ''QuickRead CRP'' produced by Orion Diagnostica. The ''QuickRead CRP'' supplies results in the range of 8-160 mg/L, results below or above this interval will be noted as <8 and >160 respectively. Though an exact cut off value for CRP on postmortem material is not easy established [6] , values above 10 mg/L are considered raised.
The age ranged from 2½ months to 84 years, 8 cases were children under the age of 2, one was between 2 and 15 years old, 23 were between 16 and 60, and 10 were older than 60 years. Twenty-eight were male and 14 were female.
In five cases, no microorganisms were found. Bacteria were found in 36 (85.7%) of the cases, amounting to 247 of 354 (69.7%) samples taken. Viruses were found in 3 of 22 cases (1.4%). Fungi were found in eight cases (19.0%), amounting to 21 of 354 (5.9%) samples taken. No other named fungi than Candida albicans was found. In one case C. albicans was the only microbiological finding. Bacteria were found in 16 cases where they were not related to COD. Virus was only found in one case which was not classified as an infectious COD.
In the 42 autopsies COD was classified as infectious in 18 cases (42.8%), distributed among: three cases of viral pneumonia (16.7%), three cases of bacterial pneumonia (16.7%), four cases of sepsis (22.2%), two cases of meningitis (11.1%), two cases of peritonitis, one due to accidental perforation of the intestine during surgery and one due to perforated appendicitis (11.1%), one case of pericarditis (5.6%) and three cases of combined opiate poisoning and infection (16.7%), Table 1 .
The COD of the remaining 24 cases, which were not related to microbiological findings, are listed in Table 2 . In all, 52 different microorganisms were found including one report of ''normal flora'', Table 3 shows the distribution.
Testing in one case, classified as viral pneumonia COD, did not yield any positive viral findings. However the histological findings were consistent with viral pneumonia (interstitial pneumonitis), and the person had been treated with antiviral antibiotics before death which could cause the negative viral findings [7] . CRP was measured in 33 cases. In nine cases CRP was below 8 mg/L. Regarding the 18 cases classified as infectious COD, CRP values were below 8 mg/L in two cases, 13 cases had elevated CRP values (p < 0.05), and in three cases no measurement was performed.
Of the 18 cases with an infectious COD, histological examination was of importance in 14 cases (77.8%). In two of the 14 cases no microorganisms were found but the histological changes, CRP values and other information lead to the conclusion (one case of viral pneumonia and one case of neutrophilic pneumonia in an opiate poisoning case). In four cases, histological findings did not show inflammation to support the microbiological findings, however CRP levels above 160 mg/L and the bacteriological results were sufficient to verify the microbiologically related COD.
Culturing bacteria on agar plates is an old invention whereas polymerase chain reaction (PCR) used for viral detection is a fairly new technique and is still costly compared to agar cultures. Hence bacteria and their colonization of the body is the most thoroughly investigated group of pathogens. A single study has used PCR for bacterial detection with excellent results [8] . Though this might be the way to proceed in the future, the skill and equipment required for PCR, compared to using agar plates, could mean that it will not be the investigation of choice in the near future on a worldwide basis.
The key point in interpreting postmortem microbiological results is to determine whether detected microorganisms arise from postmortem spread or are the result of true antemortem infection. Larsen et al. suggest that to be sure a bacteria was present in the bloodstream before death, it must be cultured from at least two different sites [9] . However authors do not agree on the most reliable sites for sampling, blood, lung and spleen are generally mentioned, as well as cerebrospinal fluid [10, 11] . Tuomisto et al. [8] suggest liver and pericardial sampling as the most reliable within the first five days after death. Preferably, cultures should be verified by gross pathologic or histologic findings [7, 9] .
When bacteria are cultured postmortem, one must consider several mechanisms which could lead to bacteria in the samples. Four lines of spread have been suggested [7, 12] .
A microorganism has genuinely invaded the bloodstream or target organ during life leading to bacteriaemia/ infection/sepsis, the group of cases which could be classified with a microbiological COD. 2. Agonal spread: According to this theory the ischemia in the agonal period or during resuscitation lead to mucosal damage allowing bacteria to invade through the surface in question. 3. Postmortem translocation: The movement of naturally present bacteria across the mucosal barriers, into the surrounding tissue, bloodstream and body. One should expect a mixed growth and not a single isolate bacteria [13] . 4. Contamination: Microorganisms are introduced into the tissue at time of sampling, and were not present in life. The theory of agonal spread is according to a review by Morris et al., considered unlikely [13] . Several papers find that elapsed time from death to sampling point does not have a significant influence on false positive bacterial results provided that bodies are kept refrigerated [11, [13] [14] [15] . However animal experiments performed by Heimesat et al. [16] do show a rather large increase in bacteria in extra-intestinal tissue especially up to 72 h after death. Perhaps even more interesting, they observe a rise in extraintestinal bacteria already five minutes after death. Maujean et al. report an interesting case in which Neisseria meningitidis was detected in a putrefied body after 10 days using molecular analyses [17] . Pryce et al. compared antemortem vs. postmortem sampling and concluded that postmortem sampling is as valuable as antemortem, when related to an infectious COD [18] . The risk of contamination from the autopsy room seems to be small [19] . Table 3 shows the vast diversity of microorganisms found in our cases, and illustrates the large number of possible pathogens. The majority of bacteria could be considered postmortem translocation, however as many of the microorganisms contributing to our normal flora can become pathogens under the right circumstances, all findings must be carefully considered.
In our study bacteria were found in 36 cases, 16 of these cases were not classified as having infectious COD, adding to the presumption of a significant postmortem spread. The vast majority of the microorganisms found in these cases are part of our natural flora, and are to be considered opportunistic pathogens, several being able to cause both pneumonia and bacteremia. In six of these 15 cases no clear COD was found, four were SIDS and two unexplained under the age of two (Table 4 ). Histology did not show inflammation in any of the samples, and none of the cases had elevated CRP values, though in two cases CRP measurement was not performed.
Not surprisingly, these problematic cases are all infants. As several of the bacteria are possible pathogens, infection cannot be completely ruled out due to the fact that a not fully developed immune system could be responsible for the lack of both inflammatory response and rise in CRP values.
Several more or less commonly encountered microorganisms, such as N. meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Salmonella species, Staphylococcus aureus, Streptococcus pneumoniae, b-hemolytic streptococci, Klebsiella species, Escherichia coli, Mycobacterium tuberculosis, members of the Enterobacteriaceae and C. albicans should, according to some authors, always be considered pathogens [10, 11, 13] . Several of the above are however part of our natural flora, and may be considered opportunistic pathogens [20] , only causing disease in persons with an underlying illness, immune deficiency, or are perhaps only known to cause disease in children etc. [21] . Generally all cases should be correlated to anamnestic information, if any present, both medical and socio-economic [22] before a microorganism is classified as a true infection. Hence a definite list of true pathogens is impossible to comprise.
Considering that microbiological testing amounts to only 6.3% of the autopsies in the investigative period versus the yield of cases which were classified as microbiologically related COD (42.8%), this inexpensive testing seems to be rather well founded.
Perhaps microbiological testing should be applied in even more cases. In Denmark around 5% of autopsies do not yield a viable cause of death, and though microbiological testing would be performed on most, the possibility remains that a few of these deaths could be due to infections/sepsis. In this study 13 of 18 cases with a microbiologically related COD had elevated CRP values (p < 0.05). Considering that result and the literature on the subject [6, 23] , one would suggest that CRP measurement is carried out at the beginning of the autopsy. This would give an early indication of a possible infection in cases where antemortem information does not suggest such, allowing for microbiological sampling before significant handling of the body and a hence reduced risk of mechanical contamination, both by direct transfer of bacteria and by displacement of bowel-near blood in arteries and veins.
Besides the possibility of improved sampling methods we also need to consider other methods to improve the microbiological testing in relation to autopsy. PCR analyses have been proved useful [24] , and although it seems to be excellent with regards to virus investigations, it does not bypass the biggest pitfall in bacterial analysis, the postmortem spread. Since it is even more sensitive than culturing [8] it might possibly yield an even higher number of positive results. PCR analysis targeting specific bacterial toxins or the toxin-genes might be of some use [25] , investigations into PM bacterial toxin production could be an interesting area for future work.
Microbiological sampling remains an important part of the autopsy yielding the cause of death in 42.8% of the cases in which it was performed. Optimized storage of the body and precautions to avoid contamination greatly enhance the value of the results.
The decision to classify a COD as microbiological rests on a combination of factors, including CRP levels, histology and an analysis of the microbiological agents.
A raised CRP level is correlated with microbiologically related COD and if performed at the beginning of the autopsy could be used as a guideline to initiate a microbiological autopsy, before mechanical manipulation leads to contamination.
Histology is an important part of the microbiological investigation in almost 2/3 of the cases with a microbiologically related COD, verifying inflammation in the cultured material, and in a few cases being the decisive factor, though supported by other information, in the diagnosis where the microbiological results were negative. Therefor all sites sampled for microbiological testing should also be histologically examined.
In very few of our cases only one single microbiological agent was found or an agent which is without doubt the sole cause of death. Several of the microorganisms contributing to our natural flora, colonizing human skin, respiratory tract or other inner surfaces or which are found in abundance in our surroundings can lead to life-threatening infections under the right circumstances, i.e. persons with underlying illness, immune suppression etc. Conversely widely found microorganisms known to cause sepsis could be the result of postmortem spread, considering their established virulence, target age group and point of infection. Investigators should always take into account all findings and anamnestic information in each case compared to the detected microorganisms before making any conclusions. Hence a definite list of ''true pathogens'' is nearly impossible to compile, and we suggest consulting microbiological literature on each microorganism, once it has been deemed of interest due to the above factors.