Detection of marine and freshwater bacterioplankton in immersed victims: Post-mortem bacterial invasion does not readily occur

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.     

A new molecular approach to help conclude drowning as a cause of death: Simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes

We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.     

A promising microbiological test for the diagnosis of drowning

A number of biological and chemical tests have been developed over the years to determine whether a person was drowned. This study focuses on the potential of a microbiological test for detecting common bacterial markers of water faecal pollution such as faecal coliforms (FC) and faecal streptococci (FS) as possible indicators of drowning. A promising previous study was carried out on central and peripheral blood samples of 42 drowned victims (20 cases in saltwater and 22 cases in freshwater) and 30 not-drowned bodies. To improve the accuracy of our previous results and also in order to investigate a possible cause of a false positive due to pulmonary passive diffusion and subsequently endogenous or exogenous bacterial invasion of the blood in the post-mortem interval (PMI), the FC and FS test was applied to bodies submerged in water but died from causes other than drowning. In the present study, blood samples collected from the left ventricle (LV), right ventricle (RV), femoral artery (FA) and, femoral vein (FV) of 10 drowned victims (5 cases in freshwater and 5 cases in seawater) and 3 not-drowned individuals with bodies submerged in water for a while after death have been analysed. Preliminary results are in agreement with other reports dealing with diatoms and marine bacteria that suggest to exclude the hypothesis of a passive penetration of sufficient quantities of drowning medium into circulation after death or during the agonal period. Based on our results there is also no evidence of a relevant dissemination of endogenous micro-flora from the gastrointestinal tract affecting the FS and FC test. There are still several other factors that could influence the applicability of post-mortem FS and FC cultures for the diagnosis of drowning and they need further investigations. The present article provides only a glimpse of the potential of the FS and FC test as bacteriological method for the diagnosis of drowning.     

A microbiological test for the diagnosis of death by drowning

The present study was aimed at demonstrating the diffusion of sea water or freshwater into the bloodstream as a consequence of water aspiration due to drowning. The study was carried out on 42 study group subjects who died by drowning in salt water (20 cases) and freshwater (22 cases) and 30 control group subjects who died from causes other than drowning. For 25 cases we obtained water samples from the aquatic locations where the victims were found. The blood samples of study and control groups were analyzed to search for faecal coliforms (FC) and faecal streptococci (FS) bacteria. The presence of FC and FS was showed by the development of blue and red colonies, respectively. From left ventricular (LV) and right ventricular (RV) blood cultures of the 20 sea drowning victims we always isolated FS and FC, whereas 19 (95%) femoral arterial (FA) and 18 (90%) femoral venous (FV) blood cultures were positive for both faecal bacteria. Related to freshwater victims, LV blood cultures showed FS presence for all the 22 cases studied (100%) and FC presence for 20 cases (90.91%). Blood cultures from RV, FV, and FA showed various patterns of faecal bacteria presence. The analysis of 25 water samples from the aquatic locations where the victims were found showed the presence of FC and FS bacteria. Blood cultures from the 30 control subjects uniformly proved the absence of faecal bacteria.     

Factors affecting strontium absorption in drownings

This study examines the effects of age, gender, a cold water medium versus warm water medium, and salinity on strontium levels determined in left ventricular blood in drownings. Significant differences in the amount of strontium absorbed into the bloodstream (p<0.001) were detected between individuals who drowned in fresh water versus those drowning in seawater, and between those drowning in cold water versus warm water (p=0.030). However, no significant differences were noted in the strontium concentrations of left ventricular blood according to gender or age.     

Production of carbon monoxide in cadavers

Carbon monoxide (CO), total hemoglobin (Hb) and carboxyhemoglobin (HbCO) in the blood and reddish discolored body cavity fluids of cadavers which had not been exposed to fire and CO were analyzed. In 13 cadavers found on land, the maximum saturation of HbCO in the blood was 3.6%, and was 10.1% in the body cavity fluids. There was only one case in which the HbCO saturations in the body cavity fluids were more than 10%. In seven drowned bodies found in fresh water, the highest HbCO saturation in the blood was 6.1%, and was 44.1% in the body cavity fluids. There were three cases in which the HbCO saturations in the body cavity fluids were more than 10%. In 12 drowned bodies found in sea water, the HbCO saturations in the blood were not more than 6.2%, and the maximum saturation of HbCO in the body cavity fluids was 83.7%. There were eight cases in which the HbCO saturations in the body cavity fluids were more than 10%. The results seem to indicate that the interpretation of HbCO saturation in the blood would not be affected significantly by the postmortem formation of CO, and that body cavity fluids should not be used for CO determination.     

Identification of decomposition volatile organic compounds from surface-deposited and submerged porcine remains

Cadaver dogs are routinely used internationally by police and civilian search organisations to locate human remains on land and in water, yet little is currently known about the volatile organic compounds (VOCs) that are released by a cadaver underwater; how this compares to those given off by a cadaver deposited on land; and ultimately, how this affects the detection of drowned victims by dogs. The aim of this study was to identify the VOCs released by whole porcine (Sus scrofa domesticus) cadavers deposited on the surface and submerged in water using solid phase microextraction gas chromatography mass spectrometry (SPME GC–MS) to ascertain if there are notable differences in decomposition odour depending on the deposition location.For the first time in the UK, the volatile organic compounds (VOCs) from the headspace of decomposing porcine cadavers deposited in both terrestrial and water environments have been detected and identified using SPME-GCMS, including thirteen new VOCs not previously detected from porcine cadavers. Distinct differences were found between the VOCs emitted by porcine cadavers in terrestrial and water environments. In total, seventy-four VOCs were identified from a variety of different chemical classes; carboxylic acids, alcohols, aromatics, aldehydes, ketones, hydrocarbons, esters, ethers, nitrogen compounds and sulphur compounds. Only forty-one VOCs were detected in the headspace of the submerged pigs with seventy detected in the headspace of the surface-deposited pigs. These deposition-dependent differences have important implications for the training of cadaver dogs in the UK. If dog training does not account for these depositional differences, there is potential for human remains to be missed.Whilst the specific odours that elicit a trained response from cadaver dogs remain unknown, this research means that recommendations can be made for the training of cadaver dogs to incorporate different depositions, to account for odour differences and mitigate the possibility of missed human remains operationally.     

The use of bacteriological analysis of the blood in the diagnosis of death by drowning

Investigation of blood samples from the femoral vein of subjects drowned in water and died of other causes (control) was carried out. Penetration of microflora contained in water into the bloodflow of the drowned people was shown. Possibility of using bacteriologic analysis of the postmortal blood to diagnose death from drowning was stated. Ps. putida and Ps. fluorescens. that were not present in the blood of persons who had died of causes other than drowning were recommended as test bacteria. Selective media and methods to isolate the bacteria from the cadaveric blood were chosen.     

Comparison between Temperature Gradient Gel Electrophoresis of Bacterial 16S rDNA and Diatom Test for Diagnosis of Drowning

When a body is discovered in water, it is difficult to conclude whether the cause of death was drowning, even today. Although diatom testing by the digestive method is classical, we hypothesized that aquatic bacteria, as well as diatoms, might be detected in drowned bodies, and conducted temperature gradient gel electrophoresis (TGGE)‐targeting 16S rDNA. DNA was extracted from the site water, and from heart blood and liver samples from 27 bodies concluded as drowning deaths by autopsy and subjected to TGGE after amplification of 16S rDNA by polymerase chain reaction. We observed whether the feature point of each 16S rDNA from the site water and blood or liver samples matched. Considerably higher correspondence was observed in drowned bodies, and the rate was higher than that achieved with the digestive method. Moreover, TGGE is safer than the digestive method. Our study suggests that this method can aid diagnosis of drowning.     

What is the incidence and significance of "dry-lungs" in bodies found in water?

BACKGROUND: Drowning without aspiration of liquid, generally attributed to death from asphyxia while submerged and in laryngospasm, has been reported to occur in approximately 10% to 15% of drowning victims. OBJECTIVES: The occurrence of "dry-drowning" recently has been questioned and the hypothesis developed that "dry-lungs" in bodies found dead in the water could conceal more natural deaths than previously recognized. METHODS: Based on 578 selected adult victims who presumably drowned, we analyzed the correlation between the cases with a low combined lung/pleura liquid weight (< 1000 g and < 750 g) and a wide set of individual, circumstantial, and postmortem (PM) variables, using multivariate logistic regression analysis. Victims with lung weight < 1000 g were screened for long-QT syndrome (LQTS) founder mutations in KCNQ1 and KCNH2 genes. RESULTS: Of the 578 victims, 120 (20.7%) had a lung weight of < 1000 g, and 22 of these (3.8%) of < 750 g. Multivariate analysis showed a significant correlation for women (P < 0.001), for women aged 65 years or older (P < 0.001), and for men with prolonged PM submersion time (P < 0.001). "Normal" lungs were found in only 8 (1.4%) victims. Low-weight (< 1000 g), overdistended lungs with no sign of liquid penetration were seen in 11 (1.9%). No LQTS founder mutations were detected. CONCLUSIONS: The actual incidence of death of persons found in water who have normal lungs or do not have penetration of liquid into their airways, based on our study, is much lower (below 2%) than currently assumed.     

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.     

The forensic medical evaluation of the results of determining diatomaceous plankton in the diagnosis of drowning

Evaluation of demonstrative value of method based on diatom plankton determination as a diagnostical sign of death due to drowning was performed. Plankton was detected in the viscera in 37% of cases (as a whole 376 bodies of people who drowned in fresh water were examined). Plankton was detected in aspiration type of drowning and its mixed variant. In spastic and reflector types of drowning plankton wasn't detected. According to author's opinion this method is an objective diagnostical test to determine the cause of death of people when their bodies are found in water.     

Marine taphonomy: adipocere formation in a series of bodies recovered from a single shipwreck.

Taphonomy of marine environments has been studied mostly from individual cases. The formation of adipocere, or "grave-wax," is an important indicator of the postmortem interval. In the present paper, the conditions and the timing of adipoceric formation are observed in a series of 15 cadavers recovered at different times, over a period of 433 days, from the same contained environment. Initial foci of adipocere on the subcutaneous tissue of the cadavers were detected as early as 38 days from the time of immersion in cold (10-12 degrees C) sea water. The discrepancies between our findings and previous reports on the correlation between time since death and decomposition stages in marine environments are discussed.     

Experimental study on postmortem formation of carbon monoxide

Rats were drowned and kept immersed for 1 month in either boiled city water, or boiled or unboiled fresh water collected from a river. A small amount of carbon monoxide (CO) formed after death and a low carboxyhemoglobin (HbCO) saturation was found in blood and thoracic cavity fluid of the animals immersed in boiled city water and in boiled fresh water. A considerable amount of CO and a high HbCO saturation was observed in blood and thoracic cavity fluid in two out of three rats immersed in unboiled fresh water at 4-6 degrees C, and in one out of three at 6-16 degrees C. It is suggested that microorganisms in the water, in which the rats were drowned and kept immersed, and low temperatures of around 5 degrees C during storage, played an important role in the postmortem formation of carbon monoxide.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

溺死尸体器官硅藻检出率季节性差异

目的 研究溺水死亡者肺、肝、肾、骨髓等器官淡水硅藻检出率可能存在的季节性差异,为法医学解决水中尸体死因鉴定提供技术帮助与科学证据.方法 收集2009～2011年1、4、7、10月沈阳市浑河中溺死尸体每月(组)各5例;45只家兔分相应月份生前、死后入水组及对照组,分别于当月发现水中尸体后第2天入水,取人及兔肺、肝、肾、股骨骨髓及现场水样备检,应用酶消化法检测各组器官中硅藻数量、种属并与现场水样比对.结果 各组溺死尸体肺中均检出多量硅藻且种属与现场水样一致,春、秋及冬季溺死尸体肝、肾中仅可检出少量体积较小的小环藻等中心纲硅藻,夏季肝、肾及骨髓中均未检出硅藻;各组溺死家兔肺中均检出少量小环藻等中心纲硅藻,肝、肾及骨髓中未检出硅藻.结论 硅藻种群的季节性变化及不同种群硅藻体积的差异可能造成肝、肾等大循环器官中硅藻检出率的季节性差异;仅肺中检出硅藻,肝、肾中硅藻检验结果阴性并不能排除溺死可能.     

Pink teeth in a series of bodies recovered from a single shipwreck

Pink teeth have most often been observed in victims of drowning but have also been reported in subjects who died suddenly and unnaturally. There is general agreement that there is no obvious connection between the occurrence of pink teeth and the cause of death, but the condition of the surroundings (especially humidity) must certainly play an important role in the development of the pink-tooth phenomenon. The frequency and distribution of postmortem pink coloration of the teeth have been studied among a representative sample of 52 cadavers. All the bodies were victims of a single shipwreck that occurred on March 13, 1997, in the middle of the Otranto Canal (Mediterranean Sea). The bodies were recovered from the seawater after approximately 7 months. A distinct pink coloration of the teeth was found in only 18 cadavers (13 females and 5 males) of ages ranging between 13 and 60 years. The phenomenon was more pronounced in younger individuals due to age-related changes of the root canal, less penetrable by the pigment responsible for the postmortem pink staining. By histochemical methods and autofluorescence, hemoglobin and its derivatives have been identified as the most likely pigments responsible for this postmortem process that can be considered analogous to postmortem lividity. These data are consistent with previous reports on pink teeth, indicating that the diffusion of the blood in the pulp into the dentinal tubules causes the red discoloration of the teeth. Based on the results, the pigmentation is more prominent on the teeth with single roots rather than in the posterior teeth with multiple roots.     

顶空GC／MS法检验血液中的硫化氢

目的建立血液中硫化氢的气相色谱质谱联用分析方法。方法取心血3mL-5mL,置于20mL顶空瓶中,加入1g氯化钠,加3mL-5mL蒸馏水,加入2mol／L盐酸1mL,加盖密封,混匀后于80℃水浴中加热20min。取液上气体0．5mL进样分析。结果在中毒死亡者的心血中检出硫化氢,保留时间参考值为3．6min。结论该方法可用于刑事案件中硫化氢的快速分析。     

Detection of diverse aquatic microbes in blood and organs of drowning victims: first metagenomic approach using high-throughput 454-pyrosequencing

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.     

Postmortem formation of carbon monoxide

Since carbon monoxide (CO) production after death was suggested in a drowned body, CO and carboxyhemoglobin (HbCO) levels in blood and body cavity fluids of cadavers which were not exposed to fire and CO have been analyzed. CO released from the tissues was determined by gas chromatography and gas chromatography-mass spectrometry, and the total concentration of hemoglobin (Hb) was measured as cyanmethemoglobin (CNmHb). The HbCO level was calculated by the ratio of CO content and CO-binding capacity. CO levels (ml/100 g at STP) of the seven cases in which blood and body cavity fluids could be collected ranged from 0.13 to 0.87 in blood and 0.02 to 0.80 in body cavity fluids. HbCO levels in blood and body cavity fluids were from 0.3 to 6.0% and from 2.3 to 44.1%, respectively. In a typical case showing postmortem formation of CO, the CO levels in body cavity fluids were higher than that in blood. It is suggested that CO in a putrefied body is due to CO in blood prior to death and the CO formed by the decomposition of Hb, myoglobin and other substances during putrefaction. The significance of HbCO levels in body cavity fluids of cases with marked postmortem decomposition seems difficult to interpret without the value of HbCO in blood.