Introduction

Post-mortem microbiology (PMM) is an important tool in forensic pathology, assisting to determine the cause and manner of death. This is relevant in unexpected deaths, as a microbiological invasion may cause or contribute to rapid death with minimal or no histological inflammation [1, 2]. PMM also aids in the identification of emergent pathogens, novel presentations of known pathogens, drug resistance or bioterrorism agents [3]. Standardized guidelines for microbiological sampling in different post-mortem scenarios are of public health interest [4]: they assist in the identification of microorganisms causing death, guarantee the allografts’ safety for the recipient of cell/tissue transplants and help in tracking the hazard and identifying bioterrorist pathogens.

Historically, PMM has long been a subject of controversy. Several theories intend to explain false positive post-mortem culture results [2, 5]. One of the difficulties in the interpretation of PMM is the growth of microorganisms that are not necessarily pathogenic. These include microorganisms that are part of the commensal flora, related to bacteremia around the time of death, secondary to post-mortem invasion or translocation from oropharyngeal and gastro-intestinal colonized mucosa, and/or contaminants due to inadequate sampling during autopsy [2].

The difficulties around PMM interpretation have relegated its use as a secondary and almost forgotten strategy in forensic autopsy. However, the implementation of specific sterile sampling techniques at autopsy, the use of molecular techniques, the development of laboratory interpretative criteria and the interconnection between microbiologists, pathologists and medical examiners have demonstrated that PMM has a prominent role in forensic medicine [3, 6–8].

Tissue and cells can only be accepted for transplantation after they have demonstrated to be microbiologically safe [9]. PMM is also a useful resource during the investigation of skeletal remains and mummified bodies [10]. Although there are some PMM sampling protocols in use throughout Europe [5, 7 and http://www.seimc.org], there is still lack of standardization.

The aims of our study are: (1) to present recommendations for microbiological sampling at the most frequent post-mortem scenarios faced by forensic and clinical pathologists, cell and tissue bankers, archeologists and anthropologists and (2) to issue general biosafety recommendations when dealing with these specimens.

Methods

A network of forensic microbiologists, forensic pathologists and forensic physicians from Spain, England, Belgium, Italy and Turkey, all working in the judicial system, was initially set up in 2013. During a 15-month period, the network discussed by email, video conferences and face-to-face sessions. One of the meetings was sponsored by a European Twinning Project. This was a collaboration project between the European Union and Turkey aimed at improving the skills of forensic experts (TR/2008/IB/KH/01). This venture included forensic microbiology activities around the need to standardize the PMM sampling. A literature search and the multi-disciplinary experience helped in shaping a flexible protocol. We herein present the consensus on forensic and post-mortem microbiology reached by the expert panel. The clinical scenarios considered were: (A) sudden death (SD), (B) bioterrorism, (C) cell and tissue transplantation, (D) paleomicrobiology.

Results

In all the scenarios described except for cell and tissue transplantation, the staff should wear protective clothing, disposable gloves, head covering and in certain cases an FFP2 mask. Sterile instruments including disposable scalpels and forceps should be used to collect samples. It is essential that a new kit of instruments is used for each sample [11].

  1. A)

    Sudden death

    General recommendations

    The following precautions should be taken in each (forensic or clinical) autopsy:

    • The body should be placed in a sealed body bag at 4 °C as soon as possible until the autopsy starts.

    • The autopsy should preferably be conducted within 24 hours following death, since microbiological samples should be taken as soon as possible after death.

    • The skin can be disinfected with a water-based antiseptic such as chlorhexidine 0.05 % with cetrimonium bromide 0.5 % in water. In case isopropyl alcohol is used, toxicologists should be notified, since this compound can be employed as an internal control in toxicological analyses. Organs’ surfaces are seared with a red-hot spatula or soldering iron before sampling [1].

    • Blood samples, body fluids and nasopharyngeal exudates are best taken at the beginning of the autopsy. Tissue specimens should be obtained with the organs being in situ, prior to evisceration [11].

    • Tubes with heparin or oxalate (anticoagulant) or fluoride (preservative) should be avoided, as they are toxic to many microorganisms.

    • Depending on the amount of the exudate, this can be collected using a syringe, or polyester or other synthetic (flocked) swabs, avoiding cotton or calcium alginate.

    • As a general principle, retrieved samples should arrive at the laboratory within 2 hours when stored at room temperature and within 48 hours between 2 and 8 °C when stored in adequate transport media [12].

    Based on most frequent case scenarios of SD in which PMM is requested, we envisioned four practical sub-groups according to the age of the deceased person and the circumstances at autopsy. For each sub-group, a minimum number of samples were agreed on. Group 1 includes SD cases in infancy and childhood (0–16 years) without clinical symptoms (Table 1); group 2 corresponds to SD in the young (17–35 years) without clinical symptoms (Table 2); group 3 corresponds to SD at any age with clinical symptoms (Table 3); and group 4 addresses traumatic or iatrogenic deaths (Table 4). Tables 1, 2, 3 and 4 present the recommended microbiology sampling sites at autopsy, the quantity of material, the type of containers and a general recommendation for microbiological analyses.

    Table 1 Sudden death: in infancy and childhood (0–16 years) without clinical symptoms
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    Table 2 Sudden death in the young (17–35 years old) without clinical symptoms
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    Table 3 Sudden-unexpected death with clinical symptoms at any age. Recommended and complementary specimens according to suspected infections
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    Table 4 Post-traumatic (either admitted to the hospital or not) or iatrogenic death at any age
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    Specimens taken during a forensic autopsy are usually considered proof of evidence and a chain of custody must be maintained at all times [13]. This requires documentation of each step in the handling of the evidence, i.e. from the moment of procuring the sample, to its final storage or disposal. The process should be able to demonstrate that the evidence has not been tampered with. This implies the use of tracking forms, both written and in electronic format. Each member of the staff handling the evidence (mortuary, transporters, administrative and laboratory staff) must sign, time and date the type of transaction or task performed. These registers should guaranty the traceability of all the aliquots and DNA-RNA extracts obtained from the original samples. Records should be securely stored. Although currently the only European official recommendation regarding the chain of custody for legal purposes is aimed at seized drugs (Council Recommendation of 30 March 2004 regarding guidelines for taking samples of seized drugs. Official Journal C086, 06/04/2004 P.0010-0011), it is expected that this rule will soon be extended to other forensic issues.

  2. B)

    Bioterrorism

    Intentional misuse of biological agents can lead to bioterrorism, i.e. transfer of this agent to a third party for harmful purposes [14]. Biological agents have been classified into categories (class A–C) according to their risk for public health, their ease of dissemination and social disruption http://www.bt.cdc.gov/agent/agentlist-category.asp#a [14]. Table 5 provides recommendations on sampling of main class A, B and C microorganisms, according to their clinical presentation [3, 14–16].

    Table 5 Bioterrorism agents: clinical presentation and sampling of main microorganismsa
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  3. C)

    Cell and tissue transplantation

    Reducing the risk of transmission of infectious diseases when implanting/transplanting cell/tissue allografts can be achieved following existing guidelines [9]. To maximize microbiological safety, the medical history and physical examination of the potential donor should be investigated. Serological/molecular tests are performed to exclude infection of the donor with Treponema pallidum, human immunodeficiency virus, hepatitis B and C virus and, when relevant, human T-cell lymphotropic virus. In addition, the absence of aerobic and anaerobic bacteria, yeast and filamentous fungi is evaluated on the final product of cell/tissue allografts [17, 18]. Table 6 presents an example of a Belgian sampling protocol tailored to each type of cell/tissue graft [19].

    Table 6 Sampling protocol for different cell/tissue allografts (based on [19])
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  4. D)

    Paleomicrobiology

    Anthropologists and archaeologists dealing with the study of mummified bodies/partially degraded corpses should take special preventive measures aimed to: minimize the risk of cross-contamination, DNA degradation and ancient DNA contamination with contemporary DNA; and avoid the risk of acquiring infection during handling. It is reasonable to undertake a risk assessment and an exposure control plan based on the specific features of the archaeological site under study. Any agreed measure should be in place at the beginning of specimen collection. Specific criteria to confirm authenticity and to avoid contamination are required [20].

    Manipulation of ancient human tissues should take place in facilities specifically dedicated and physically distant from the extraction area. Instrumentation tools should always be cleaned with bleach (10 % sodium hypochlorite) in between samples. The samples should be immediately placed in sterile containers or tubes at 4 °C, protected from light and humidity, and transported to the laboratory as soon as possible.

    More specific recommendations for the collection of the different types of suitable specimens include:

    • Mummified bodies: The tissue type selection for microbiological sampling is based on integrity. Specimens are acquired by aseptic dissection and stored in desiccated chambers. Samples are pulverized under liquid nitrogen. Whenever possible, 400 mg of pulverized tissue is left available for DNA extraction [10]. DNA is more stable in bones than in soft tissues and this seems to be independent of the specific anatomical origin of the samples [21].

    • Skeletal remains: Selected sections of skeletal elements can be removed using a rotational cutting tool (Dremel tool) on the lowest setting to reduce heat, in order to avoid DNA denaturing [22]. The bone external and internal surfaces should be first cleaned using a radial saw or a Dremel tool to file about 2–4 cm of bone surfaces. If 0.5 % sodium hypochlorite solution is also used to decontaminate, samples should then be rinsed thoroughly afterwards. The radial saw is not appropriate in small bones, the surface can be cleaned with swabs soaked in sterile water. After decontamination, bones are cut into small fragments and UV (ultra violet light) irradiated on their surface. Before extraction, the fragments are pulverized with a freezer mill or a mixer mill [22].

    • Tooth powder: Intact teeth should be thoroughly cleaned with sterile water and then UV irradiated (see above). If their surfaces are heavily contaminated, they can be cleaned with a scalpel or decontaminated with 0.5 % sodium hypochlorite and washed with sterile water. The teeth are then longitudinally fractured with a cutting wheel, sectioning the cementoenamel junction to expose the pulp chamber. This is opened and the remnants of the dental pulp (which are powdery in ancient teeth), are scraped off and transferred into sterile tubes in order to be pulverized with a freezer mill [23].

    • Dental calculus: can be extracted directly from dental pieces using a curette and placed in a 1.5-ml tube. The samples are treated with 500 mL of 4–6 % sodium hypochlorite for 1 minute to eliminate surface contaminants, and then washed three times in double-distilled water to eliminate the sodium hypochlorite [24].

    • Coprolites: Remove the surface first, then UV irradiate the sample. The core of the coprolites needs to be grounded and rehydrated by immersion in a 0.5 % aqueous solution of trisodium phosphate for 72 h. The ancient DNA is extracted by physical-chemical treatment [25].

Biosafety considerations

In any of the above described scenarios, biosafety rules should always be a priority. Microorganisms can be transmitted through inhalation, ingestion or inoculation (intravenous, subcutaneous, direct contact with skin and mucous membranes). Whether or not an infection will develop depends on the exposure route, the bio-burden, the virulence and the immune status of the host. Since all incoming material is potentially infectious, standard safety precautions should be followed both in the autopsy room and in the laboratory [26]. In case of occupational exposure to infectious agents a written protocol should be in place and a training of laboratory personnel is required. Vaccination of personnel should be considered. Disposal of waste should follow regulatory requirements [15]. Table 7 shows the most common microorganisms, hazardous material and specific protection measures that should be taken in the post-mortem environment [27].

Table 7 Common microorganisms with their hazards in postmortem environment [16, 27]
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Discussion

The success of PMM depends on the adequacy of the post-mortem sampling protocol and strategy [28]. The forensic pathologist needs to contemplate whether PMM should be carried out or not. There are specific indications where PMM is required: to confirm the presence of an unproven infection; when the cause of death is unknown (sudden or unexpected death); to evaluate the efficacy of antimicrobial therapy in eradicating an infection [28]; during the investigation of malpractice related to an infectious disease; and in the archaeological scenario, where it is helpful to identify infectious diseases in human skeletal and ancient remains, especially in mummified bodies [20]. It is crucial to confirm the absence of microorganisms in cells/tissues retrieved in order to confirm that these are safe for transplantation/implantation.

We envisioned four practical sub-groups of case scenarios of SD which could benefit from PMM. The utility of PMM in SUDI (sudden unexpected death in infancy) and in SD in young adults has been demonstrated in recent reports. In one of them, a potential pathogen was found in 57 out of 116 (49 %) infant cases. The use of a complete protocol of multisite microbiological investigations was important for the identification of cases where these pathogens were the cause of death [7]. Recent developments in PMM and histopathology have increased the identification of an infectious disease as the cause of death in infants, therefore reducing the number of cases that are regarded as “sudden infant death syndrome—SIDS”. In patients aged 0–34 years, PMM detected the organism responsible for the death in 17 out of 23 (73.9 %) cases [29]. PMM is relevant for public health in those fulminating fatal infections requiring notification and/or when there is a need to treat the contacts of the deceased [4] or in the recognition of emerging infections.

Bacterial, fungal and viral infections have been transmitted by different types of cell/tissue allografts [30]. Currently, strict control steps occur at various levels of the cell/tissue banking processes with the aim to identify potentially contaminated/infected donors [30], which include donor selection, physical examination, serology, environmental control during tissue procurement, processing, packaging and final microbiological cultures. To the best of our knowledge, no national or international guidelines have ever addressed post-mortem microbiological sampling methods. So far, they only stated that cell and tissue material should be free from bacteria and fungi. Therefore, the Belgian Superior Health Council aimed at establishing such specific recommendations regarding the type of cell/tissue material, the minimum quantity of material, the culture methods and the specific characteristics of detection and reporting of microbiological culture results for cell and tissue allografts.

In addition, the use of hybridisation, real-time PCR, and sequencing informs novel anthropological and paleopathological research. These techniques are an aid in the identification of, for instance, leprosy, malaria, Chagas disease, plague, syphilis, diphtheria or typhoid fever [20]. Moreover, microbiology laboratories performing forensic/post-mortem analyses should perform their own appropriate validation studies on post-mortem samples to evaluate the application of both “in-house” and commercial serological or molecular assays, due to the fact that many commercial assays don’t include these specimens in their developmental validation.

PMM contributes to the search for the cause of death in forensic pathology [3], and its strength is enforced when using standardized sampling protocols at autopsy. These sampling protocols should be adapted to the specific post-mortem circumstances. The closer to the moment of death the samples are obtained, the higher the quality and the success rate. To obtain useful microbiological results, sampling should be adequate and thorough. The tests performed need to be selected from a broad portfolio, according to the infection suspected and using cost-benefit criteria. The preferential sites for peripheral blood sampling in PMM differ from those used in routine clinical practice. As an example, blood sampling in routine clinical practice is preferably not performed via the femoral vein because of the high yield of contaminants. At post-mortem, femoral or external iliac blood vessels are sampled in situ (during the abdominal time) and after clamping the common iliac vein. Once the microbiological analyses are concluded, the forensic microbiologist and forensic pathologist need to assess the microbiological results obtained in relation to the circumstances of death and to clinical, macroscopic and histopathological findings [5, 7]. In the near future, new technologies such as the analysis of non-nucleotidic biomolecules [31] are promising for their contribution to the success of forensic microbiology.

In conclusion, the sampling protocols proposed here aim to standardize the microbiological sampling for the most frequent situations seen in the daily forensic and post-mortem practice. Wider implementation of post-mortem sampling recommendations will allow a better interpretation of the microbiological results related to forensic/post-mortem cases and a better understanding of the mechanisms and manner of death related to unsuspected infectious diseases in forensic practice.