Application of a simple enzymatic digestion method for diatom detection in the diagnosis of drowning in putrified corpses by diatom analysis

The reliability and applicability of quantitative and qualitative diatom analysis by an enzymatic digestion method in the diagnosis of drowning of putrified bodies has been evaluated. The authors report the analysis of water and organ samples of 12 immersion cases using light microscopy. This study included control organ samples from the bodies of persons who died from causes other than drowning. Organ samples were treated by both chemical and enzymatic methods, the first one using concentrated nitric acid and the second proteinase K. Diatoms were present in most organ samples of the immersed corpses; no diatoms could be found in the control samples. Our experience was that the enzymatic method seemed to be more convenient in terms of rapidity, safety and environmental protection than chemical digestion. The number of diatoms recovered with both methods was similar. Qualitative and quantitative analysis of both water and organ samples of immersion cases supported the diagnosis of death by drowning in 41.6% of the putrified cases studied. The authors suggest that diatom analysis using enzymatic digestion of organs can be used as a criterion for positive diagnosis of drowning in cases involving putrified bodies.     

Real-time PCR assay for the detection of picoplankton DNA distribution in the tissues of drowned rabbits

The detection of plankton DNA is one of the important methods for the diagnosis of drowning from postmortem tissues. This study investigated the quantities of picoplankton (Cyanobacteria) DNA in the lung, liver, kidney tissues and blood of drowned and non-drowned rabbits, and the sensitivity of detection of picoplankton DNA by polymerase chain reaction (PCR) detect for the diagnosis of death from drowning. For this purpose, the DNA of the 16S ribosomal RNA gene of picoplankton was quantitatively assayed from the tissues of drowned and non-drowned rabbits immersed in water after death. Each of the liver, kidney and lung tissues and blood were obtained from drowned and non-drowned rabbits. Picoplankton DNA in the tissues was extracted using the DNeasy® Blood & Tissue kit to determine the yield of picoplankton DNA from each tissue. TaqMan real-time PCR was performed for quantitative analysis of picoplankton DNA. Target DNA was detected in the liver, kidney and lung samples obtained from the drowned rabbits, while no picoplankton DNA was detected in the non-drowned rabbit tissues (except in lung samples). The results verified that direct PCR for the detection of picoplankton DNA is useful for the diagnosis of drowning. Although we observed seasonal changes in the quantity of picoplankton in river water, we were able to detect DNA from various organs of drowned bodies during the season when picoplankton were not the most abundant.     

Phytoplankton gene detection in drowned rabbits

We have developed a sensitive and specific polymerase chain reaction (PCR) method for identifying phytoplankton in cases of death by drowning, and we have designed four primer pairs, EG1, EG2, SK1 and SK2, for chlorophyll-related genes of Euglena gracilis and Skeletonema costatum, which are commonly distributed in all types of water. In order to evaluate the usefulness of this method for diagnosis of drowning, we have used this method for detection of plankton genes in non-drowned rabbits submerged after death and in decomposed drowned rabbits. Plankton DNA was identified in lung samples obtained from the non-drowned rabbits because of postmortem plankton penetration into the respiratory system, and plankton DNA was identified in liver and kidney samples obtained from the decomposed drown rabbits. The results show that our new PCR method is a useful method for diagnosing drowning.     

A novel PCR method for identifying plankton in cases of death by drowning

We present a new PCR method for identifying plankton in cases of death by drowning. We designed four primer pairs for chlorophyll-related genes of Euglena gracilis (EG) and Skeletonema costatum (SK), which are commonly distributed in water. The primers were selected from sequences coding chloroplast/chlorophyll apoprotein of EG (EG1 and EG2) and fucoxanthin-chlorophyll a/c harvesting protein of SK (SK1 and SK2). With EG1 or EG2, up to 2 fg of EG-DNA was identified, and 0.2 pg of SK-DNA was detectable with SK1 or SK2. No PCR products were amplified from green vegetables (komatsuna, spinach, parsley) or human DNA with the four primer pairs. Regardless of the origin, seawater or fresh water, most diatoms were detectable with primer pairs of EG1 and EG2. With SK1, only Centrales diatoms were identified, and five diatom strains originating from seawater were detectable with SK2. EG1 and EG2 gave rise to PCR products from most water samples. By using Percoll, plankton was easily isolated from human tissue or blood samples and good results of PCR analysis were obtained in cases of death by drowning.     

Sternal aspirate sampling of Bacillariophyceae (diatoms) and Cyanobacteria in suspected drowning cases

A diagnosis of drowning is not always possible based on the traditional autopsy findings. The most widely used ancillary methods are based on the detection of diatoms and other waterborne organisms in the organs of the systemic circulation by light microscope or polymerase chain reaction (PCR). One of the greatest concerns is sample contamination. Bone marrow is a favourable source because the compact bone protects the sample from water ingress in the case of advanced decay.In our pilot study, we aimed to adopt sternal bone marrow aspiration - which is a widely used technique in haematology - for postmortem sampling.Control experiments of non-drowning victims showed that cleaning the skin over the sternum can prevent external contamination.Sternal aspirate samples were taken from seven suspected drowning victims along with lung, spleen, and femoral bone marrow samples. All specimens were examined for the presence of diatoms by light microscope and Cyanobacteria-specific DNA by PCR.We were able to obtain bone marrow aspirates from all cases without complications. In four of the sternal samples both diatoms and cyanobacterial DNA were detected, while one additional sternum sample was tested positive with PCR, but no diatom shells were detectable.Sternal bone marrow aspiration is simple and quick, which can be performed at the beginning of an autopsy, minimizing the chance of contamination. We have shown that this sampling method can be adopted for postmortem diatom testing. This minimally invasive technique might be used in virtual autopsy (postmortem computed tomography, PMCT) settings without opening body cavities.     

Haematic silicon in drowning

The aim of this paper was to evaluate silicon (Si) concentration in human whole ventricular blood as a further potential chemical marker in the diagnosis of drowning. We employed an acidic digestion for the extraction of soluble Si, and an alkaline digestion for the determination of total Si, including particulate matter, both arising from drowning medium. 29 suspected drowning situations, 24 in fresh water (Fw) and 5 in seawater (Sw), were examined. The difference in Si concentration between the left and right ventricular blood (Si Δ_L–R) was measured and alkaline Si Δ_L–R seems, indeed, a potentially significant complementary tool in the diagnosis of Fw drowning, because insoluble silicon fraction does not undergo hemo-dilution or hemo-concentration, and the Δ_L–R is not affected by exogenous factors. In spite of the limited number of cases investigated, a good correlation was observed between the analytical results and the macro-microscopic autoptic findings.     

The detection of picoplankton 16S rDNA in cases of drowning

Picoplankton belonging to theSynechococcus genus in cyanobacteria (approximately 1 μm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria by microscopy. In this study we attempted to use picoplankton for the diagnosis of drowning by PCR analysis of the 16S ribosomal DNA (rDNA). We designed primers complementary to the variable regions of 16S rDNA of the picoplankton we had sequenced. A comparison was made of the PCR products from the three picoplanktons, five other cyanobacteria,Melosira (diatom),Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human lung tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The isolation of the picoplankton from human lung tissue increased the sensitivity of PCR more than a hundred-fold. The specific PCR products of the picoplankton were obtained from formalin-fixed drowning tissue. Molecular biological diagnosis of drowning was successful using picoplankton 16S rDNA.     

Contribution to the determination of the place of death by drowning – A study of diatoms' biodiversity in Douro river estuary

The role of the investigation of diatoms' presence in organs and body fluids of an individual found dead in a liquid medium and the relevant contribution to the forensic diagnosis of drowning remain controversial. Furthermore, the absence of an exact and well-defined method for diatoms' analysis makes its study a challenging task.Considering this medico-legal problem and the absence of forensic studies on this subject in Portugal, this work aimed to determine the drowning place of dead individuals based on the analysis of diatom species found in different tissues (lung, liver, kidney, bone marrow) and stomach content. Diatom species found in biological samples were compared with those present in the liquid medium where the corpses were found. A total of 37 cases of death by drowning in Oporto metropolitan area were studied. A seasonal database of the diatom species found in Douro river estuary was built based on water samples collected at nine selected places. Diatoms' extractions were performed by a chemical method using 37% (w/w) hydrochloridric acid for the biological samples and 96% (w/w) sulfuric acid for water samples.Diatoms were found in 63% of total cases but only in lung and gastric content samples. The absence of diatoms in other organs is probably related with a quick death, which may have stopped blood circulation almost immediately, preventing diatom contamination of the other organs.A strong relationship between the diatom species found in the biological samples and those found in water samples of the respective drowning place was observed. Due to the high anthropogenic influence on the Douro estuary no significant differences were observed between the five sampling places, making it extremely difficult to determine the exact estuary location of the drowning.The importance of the creation of a diatom database of the potential drowning places (e.g., rivers, seas, lakes) becomes clear in this study. It also shows that, in cases of drowning, the collection of a water sample from the drowning place is crucial. This is the only way to allow a rigorous comparison of the diatom species in water and biological samples.     

Microwave Digestion—Vacuum Filtration-Automated Scanning Electron Microscopy as a sensitive method for forensic diatom test

Diagnosis of drowning is one of the most difficult issues in forensic practice. A number of methods have been developed over the years to determine whether a person was drowned. Microwave Digestion—Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) method we developed is a new qualitative and quantitative method of diatom test for diagnosis of drowning. The new method is based on microwave digestion technique, vacuum filtration, and automated SEM, which would achieve a maximal recovery of diatoms and identify diatoms easily by SEM with high resolution. This study was designed to evaluate the sensitivity of this method, the recovery of diatom, and loss ratio of centrifugation, which were compared using the MD-VF-Auto SEM method and the conventional acid digestion method. Two groups of samples were designed in the study. Groups A (n?=?20) and B (n?=?20) were performed by MD-VF-Auto SEM method and the conventional acid digestion method, respectively. In addition, another eight water samples were centrifuged, and the diatoms in the supernatant and precipitate were counted and measured, respectively, in order to find out how many diatoms were lost after centrifugation. The difference between the two groups was statistically highly significant, and about 34 % of diatoms were lost after centrifugation at 4,000 rpm for 15 min. The results showed that the MD-VF-Auto SEM method was more sensitive and specific.     

Diatoms and drowning

Summary An examination is made of the applicability of quantitative and qualitative diatom analysis to the diagnosis of death by drowning, definition of the environment in which drowning occurred, and delimitation of the area where it occurred. The material comprises 107 bodies of subjects known or suspected to have died by drowning together with a control series of 15 bodies of subjects over 30 years of age who had died of various diseases on land.Whenever diatoms were found in the greater circulatory organs they were also found in the lungs, and when none were present in the lungs none were found in the other organs either. No diatoms or fragments of diatoms were found in the samples from the control subjects. All the fresh, well-preserved bodies for which death by drowning could be regarded as certain from the macroscopic autopsy findings and police reports, the cases used to test the method, gave quantitative diatom results that supported a diagnosis of water aspiration.The diatoms identified in the qualitative analyses served well to describe the ecological properties of the environments in which death had taken place, and the site of drowning could be defined by means of comparative water samples provided that sufficient diatoms were present, the local environment was not too homogeneous or the diatoms were not of quite different species due to a completely unknown location of death.     

An experimental study of postmortem decomposition of methomyl in blood

Methomyl (S-methyl-1-N-[(methylcarbamoyl)oxy]thioacetimidate) is a carbamate pesticide. It has been noted that in some cases of methomyl poisoning, methomyl is either not detected or detected only in low concentrations in the blood of the victims. However, in such cases, methomyl is detected at higher concentrations in the vitreous humor than in the blood. This indicates that methomyl in the blood is possibly decomposed after death. However, the reasons for this phenomenon have been unclear. We have previously reported that methomyl is decomposed to dimethyl disulfide (DMDS) in the livers and kidneys of pigs but not in their blood. In addition, in the field of forensic toxicology, it is known that some compounds are decomposed or produced by internal bacteria in biological samples after death. This indicates that there is a possibility that methomyl in blood may be decomposed by bacteria after death. The aim of this study was therefore to investigate whether methomyl in blood is decomposed by bacteria isolated from human stool. Our findings demonstrated that methomyl was decomposed in human stool homogenates, resulting in the generation of DMDS. In addition, it was observed that three bacterial species isolated from the stool homogenates, Bacillus cereus, Pseudomonas aeruginosa, and Bacillus sp., showed methomyl-decomposing activity. The results therefore indicated that one reason for the difficulty in detecting methomyl in postmortem blood from methomyl-poisoning victims is the decomposition of methomyl by internal bacteria such as B. cereus, P. aeruginosa, and Bacillus sp.     

Using oral microbial DNA analysis to identify expirated bloodspatter

Distinguishing expirated bloodstains (blood forced by airflow out of the nose, mouth or a chest wound) from impact spatter (blood from gunshots, explosives, blunt force trauma and/or machinery accidents) is an important challenge in forensic science. Streptococcal bacteria are only found in the human mouth and saliva. This study developed a polymerase chain reaction (PCR) method that detects DNA from these bacteria as a sensitive tool to detect the presence of saliva. The PCR method was very specific to human oral streptococci, with no PCR product being made from human DNA or DNA from other microbes that were tested. It was also very sensitive, detecting as little as 60 fg of target DNA. The PCR amplification gave product with 99 out of 100 saliva samples tested. PCR was not inhibited by the presence of blood and could detect target DNA in expirated bloodstains in a range of materials and for up to 92 days after deposit on cardboard or cotton fabric. In a blind trial, the PCR method was able to distinguish three mock forensic samples that contained expirated blood from four that did not. Our data show that bacteria present in the oral cavity can be detected in bloodstains that contain saliva and therefore can potentially be used as a marker in forensic work to distinguish mouth-expirated bloodstains from other types of bloodstains.     

CMV-DNA detection in parenchymatous organs in cases of SIDS

A nested PCR approach has been developed especially for the detection of small amounts of cytomegalovirus (CMV) DNA in autopsy samples. Lung tissue and submandibular glands in 118 cases of infant death (92 SIDS cases, 13 natural deaths due to other defined causes and 13 unnatural deaths) were investigated by this technique and compared to the results obtained by other CMV detection methods (histology, immunohistochemistry, in situ hybridization and PCR). CMV-DNA could be detected in the lung tissue in 7 cases of SIDS using nested PCR. Compared to conventional PCR (3 positive cases in lung tissue) the nested approach always gave glear results and showed less additional bands. In all cases where CMV could be detected in the lungs, positive results were also obtained in the submandibular glands. The nested PCR method proved to be a more sensitive technique than the other detection methods including PCR and hot start, and. even minimal amounts of target DNA could be detected in the presence of human and bacterial background DNA.     

Species identification based on the point mutations of histo-blood group ABO genes by PCR-RFLP and direct sequencing

Using the PCR-restriction fragment length polymorphism (RFLP) and direct sequencing methods, 181 bp DNA fragments at the ABO blood group locus of human and eight different primates were examined. Through PCR amplification and digestion of the product with Hha-1 restriction enzyme, each of the amplified fragments of human, chimpanzee and green monkey was cut into two fragments of 147 and 34 bp, and the corresponding fragment of the other primates was digested into three fragments of 115, 34 and 32 bp. The 181 bp fragments of chimpanzee and green monkey were digested with Mva-1 into three fragments of 82, 58 and 41 bp, and 69, 58 and 54 bp, while the fragments of human and the other primates were separated into two fragments of 123 and 58 bp. Thus, using Hha-1 and Mva-1, human PCR-amplified product of a part of the ABO gene could be discriminated from those of the other primates examined. In addition, by direct sequencing of the 181 bp DNA fragment, two Mva-1 recognition sites and one Hha-1 recognition site were found in the fragments of chimpanzee and green monkey, one Mva-1 site and one Hha-1 site were detected in humans, and one Mva-1 site and two Hha-1 sites in the other primates. These results corresponded well with the data of PCR-RFLP. This method has a good potential for species identification at the DNA level. Received: 27 January 1997 / Received in revised form: 17 March 1997     

The number of diatoms recovered from the lungs and other organs in drowning deaths in bathwater

We assessed the plankton levels using the diatom test in the lungs and organs of the circulatory system of nine postmortem cases of bathwater drowning and one case of death due to ischemic heart disease (IHD) while bathing, as well as in the associated bathwater. The number of planktons detected in the lungs was not related to the postmortem period or the process of drowning, but was related to the plankton levels detected in the bathwater taken from the scene. In one case of bathwater drowning, the diatom distribution of the four pulmonary lobes (165.0-280.0planktons/100g sample) and other organs (3.6-13.6/100g) highly corresponded to the pattern of 'usual' drowning in water which contains a lot of diatoms. In this case, the bathwater taken from the scene contained a considerable amount of planktons (16.3/100ml water), which suggests that, when the bathwater is relatively 'dirty', the diatom test may be helpful in reaching a diagnosis of drowning. In another case of drowning, a considerable number of diatoms was detected relatively evenly in all four pulmonary lobes (58.3-141.7/100g), although no planktons were detected in distant organs. In this case, the bathwater also contained a substantial number of planktons (18.8/100ml), which suggests that a relatively even distribution of planktons in each pulmonary lobe may also support a diagnosis of drowning based on the autopsy findings when the bathwater is relatively 'dirty'. In the other seven cases of drowning, few planktons were detected in the lungs (0-18.2/100g) and other organs (0-9.1/100g) as well as in the associated bathwater (0-3.1/100ml). These cases suggest that if autopsy findings indicate drowning, the low levels of diatoms detected in both the pulmonary lobes and the bathwater may indicate drowning in clean water. In contrast, in one case of death due to IHD while bathing, no planktons were detected in the organs except for the lower lobe of the right lung (11.8/100g) and the left kidney (9.1/100g), although the bathwater contained a sufficient number of planktons by the diatom test (21.3/100ml). This case suggests that the diatom test may be helpful in distinguishing drowning and other causes of death in bathwater by comparison of the diatom levels in the organs and the bathwater. All of these results indicate that the diatom test may be helpful to differentiate ambiguous cases of drowning in bathwater. However, in this study, although special care and precautions were undertaken, contamination with some planktons could not be prevented. For accurate interpretation and diagnosis, examination and comparison of test results from all four pulmonary lobes, at least four distant organs, and the associated bathwater from the scene of death are necessary together with careful consideration of autopsy findings.     

A quantitative comparison analysis of diatoms in the lung tissues and the drowning medium as an indicator of drowning

The presence of diatoms in the lung tissues, internal organs and bone marrow is considered as the supportive evidence in the diagnosis of death by drowning. Generally, the diatoms detected in the lung tissues are regarded as insignificant since these diatoms can be detected in the lung tissues of the postmortem immersion bodies. In this study, we analyzed the relationships between the numbers of the diatoms in the lung tissues and the drowning medium. We made a comparison analysis between the diatoms in the lung tissues and the drowning medium using the ratio of diatom numbers in both samples (L/D ratio), utilizing Microwave Digestion - Vacuum Filtration - Automated Scanning Electron Microscopy method. Our data indicate that the L/D ratios in victims of the drowning group were higher than the postmortem immersion group. A higher L/D ratio provides valuable information about the cause of death in drowning victims. Quantitative diatom analysis in the lung tissues, especially combined with the diatom analysis of the drowning medium, provides supportive evidence in determining if a body recovered in water was due to drowning or not.     

Bioluminescent bacteria have potential as a marker of drowning in seawater: Two immersed cadavers retrieved near estuaries

We detected numerous bioluminescent bacteria in blood samples from two cadavers that had been immersed in estuarine environments. Autopsy, diatomaceous and toxicological findings indicated death by drowning, which agreed with environmental aspects and the findings of police investigations. Bioluminescent bacteria appeared in blood samples cultured on selective agar containing 2%, 3% and 4% NaCl after about 18 h. Blood from the left side of the heart, the right side of the heart and the femoral vein generated 7.0 × 10², 2.0 × 10⁴ and 8.0 × 10² cfu/ml of blood (case 1), and 1.8 × 10⁴, 1.1 × 10³ and 2.5 × 10¹ cfu/ml (case 2) of bioluminescent colonies, respectively, in agar containing 4% NaCl. Homologous analysis based on the 16S rRNA gene also identified the bioluminescent colonies as Vibrio fischeri and V. harveyi, which normally inhabit seawater. This simple assay might serve as an additional indicator to support a conclusion of death by drowning together with the diatom test.     

Feasibility of analysis of the <Emphasis Type="Italic">SCN5A</Emphasis> gene in paraffin embedded samples in sudden infant death cases at the Pretoria Medico-Legal Laboratory,South Africa

To determine variations in the SCN5A gene linked to inherited cardiac arrhythmogenic disorders in sudden, unexplained infant death (SUID) cases examined at the Pretoria Medico-Legal Laboratory, South Africa. A retrospective study was conducted on SUID cases and controls, analyzing DNA extracted from archived formalin-fixed, paraffin-embedded (FFPE) myocardial tissue samples as well as blood samples. A total of 48 FFPE tissue samples (cases), 10 control FFPE tissue samples and nine control blood samples were included. DNA extracted from all samples was used to test for variations in the SCN5A gene by using high resolution melt (HRM) real-time PCR and sequencing. Genetic analysis showed 31 different single nucleotide variants in the entire study population (n = 67). Five previously reported variants of known pathogenic significance, and 14 variants of benign clinical significance, were identified. The study found 12 different variants in the cases that were not published in any database or literature and were considered novel. Of these novel variants, two were predicted as “probably damaging” with a high level of certainty (found in four case samples), one (identified in another case sample) was predicted to be “possibly damaging” with a 50% chance of being disease-causing, and nine were predicted to be benign. This study shows the significant added value of using genetic testing in determining the cause of death in South African SUID cases. Considering the high heritability of these arrhythmic disorders, post mortem genetic testing could play an important role in the understanding of the pathogenesis thereof and could also aid in the diagnosis and treatment of family members at risk, ultimately preventing similar future cases.