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.     

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.     

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 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.     

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.     

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.     

Marine bacteria comprise a possible indicator of drowning in seawater

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.     

Blood strontium concentration related to the length of the agonal period in seawater drowning cases

The levels of ventricular blood strontium (Sr) from 70 seawater drowning victims were compared with their diagnosis of drowning based mainly on certain criteria selected from their autopsy report. From this comparison, intervals of either the difference of Sr concentration between the left and the right ventricle blood (LVSr-RVSr) or the Sr concentration in the left ventricle blood (LVSr), appear to be related to different time-lapses of the agonal period of drowning. In the aim to diagnose drownings, intervals of both LVSr-RVSr and LVSr were proposed to characterize three different agonal periods in seawater drowning cases: instantaneous death (ID), fast vital-submersion drowning (FVSD) and common vital-submersion drowning (CVSD).     

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.     

Study of the diagnostic value of iron in fresh water drowning

The aim of our study was to test the diagnostic value of iron (Fe) in fresh water drowning by investigating the postmortem levels of hemodilution in drowning cases compared to control cases. Twenty-six typical fresh water drowning cases were selected from 128 immersion cases autopsied in our Department of Forensic Pathology between 1998 and 2004. The exclusion criteria were a long postmortem interval and other causes of death than drowning. For all selected cases, the diagnosis of drowning was based on the presence of autopsy findings and positive diatom test. A control population of 12 cases was also selected. For each drowning and control case, iron blood levels were measured in the left ventricle (LV) and right ventricle (RV) of the heart. The mean difference of iron concentration (RVFe-LVFe) between the drowning group and the control group was statistically compared. Furthermore, iron measurements were performed in 19 drowning cases showing advanced putrefaction. The mean difference of iron concentration was significantly higher in the drowning cases compared with controls (P<0.001). All drowning cases showed hemodilution. No overlap was found in the RVFe-LVFe levels between the two groups. Resuscitation attempts seemed to have no effect on the results. In cases of drowning showing advanced putrefaction, the iron test was not reliable because biochemical iron measurement was often prevented by no sufficient blood in the heart or postmortem clots. In conclusion, according to our results, iron seems to be a good biochemical marker in fresh water drowning with a short postmortem interval.     

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.     

Plasmatic levels of atrial natriuretic peptide (ANP) in drowning. A pilot study

Bodies found in water may cause problems for forensic pathologists who have to differentiate drowning from postmortem immersion or fresh from salt water drowning. The exact physiopathology of drowning is still controversial and complementary tests can not exactly establish the exact cause of death if macroscopic findings at autopsy are not conclusive. We have employed atrial natriuretic peptide (ANP) as a marker in an experimental series of fresh and salt water drowning, comparing their results with a non-drowned control series. There are differences between the plasma basal levels of the control series (79 pg/ml) and the levels in animals drowned in fresh water (358 pg/ml, P less than 0.001) and between control and rabbits drowned in salt water (190 pg/ml, P less than 0.001). According to these values, there are also differences between fresh and salt water drowned animals (P less than 0.001). We propose this peptide as a new marker in cases of drowning, with the ability to differentiate drowning from postmortem immersion and fresh from salt water drowning.     

测定血锶含量鉴定溺死的法医学意义

为评价血锶含量测定对诊断鉴定溺死的实用价值,作者用家兔进行实验,分别测定溺死与死后入水家兔左心血中锶含量。结果证实:1.死后较短时间内血锶含量测定确可鉴定溺死;2、死后24小时内仅可帮助鉴定溺死。实验表明水中浸泡时间因素对血锶含量确有显著影响。两例实际检案的应用观察,较实验性研究为佳,值得进一步研究。     

Serum biochemical markers in drowning: diagnostic efficacy of Strontium and other trace elements

The aim of our study was to analyse several serum biochemical markers in order to evaluate the discriminant capacity of trace elements individually and jointly in drowning (seawater drowning and freshwater drowning) and their correlation with age, sex, postmortem interval, time in water and concentrations of the trace elements in the drowning medium. Sixty-seven cases of drowning (53 seawater drownings (SWD); 14 freshwater drownings (FWD) and 73 control cases (other asphyxias, n=44, and other causes of death, n=29) were selected according to the scene, cause and circumstances of death, together with autopsy findings. Serum strontium (Sr), magnesium (Mg), sodium (Na), chloride (Cl), calcium (Ca), iron (Fe), urea, creatinine (Cr) and cardiac Troponine T (cTn-T) were measured in the left ventricle (Lv), right ventricle (Rv) and peripheral blood. Lv-Rv differences for each marker and Sr, Mg, Na, Cl, Ca and Fe concentrations in the drowning medium were determinated. Mean concentrations of Sr, Cl and Mg in both ventricles and peripheral serum and Lv-Rv differences and Ca Lv and Na Rv were significantly higher in cases of drowning than for other causes of death. In SWD, Sr, Mg, Ca, Na and Cl were significantly higher in Lv than in Rv as a result of aspirating water. In contrast, haemodilution is evident from the significantly higher levels of Fe and urea in Rv than in Lv in cases of SWD, and from the higher Mg and Cr levels in Rv in FWD. In the case of SWD, serum levels of Sr are confirmed as the best parameter for diagnosis, although other trace elements may also be useful, such as the serum concentrations of Mg and Cl. In the case of FWD, the joint determination of Sr and other biochemical markers, especially Fe, may increase correct diagnosis.     

Pediatric drowning: a 20-year review of autopsied cases: I. Demographic features

Drowning is a significant cause of childhood mortality in the developed world. Males and preschool-aged children are overrepresented in most series of pediatric drowning. To obtain a better understanding of age- and gender-related factors, a retrospective review of autopsy records over a 20-year period (1984-2003) for cases of drowning (exclusive of bathtub drownings) was performed. Sixty-three consecutive cases of drowning were identified in 45 males and 18 females (ratio 2.5; P = 0.0007). The ages ranged from 9 months to 17 years, and most cases occurred in the 0- to 4-year age group (57%). Boys tended to be older than girls (5 years 9 months versus 4 years 4 months; P = 0.2133). Sites of drowning were open water (48%) and pools (44%), with children drowning in open water being significantly older than those drowning in pools (6 years 6 months versus 4 years 6 months; P = 0.0462). Most boys drowned in open water (53%), whereas most girls drowned in pools (50%). Resuscitation was attempted after the drowning episode in 84% of cases, with children aged 4 years or less having significantly higher rates than older children (P = 0.0346). The present study highlights differences in the pattern of drowning according to age and sex and may aid in developing targeted educational and preventative campaigns.     

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.     

微波消解-扫描电镜联用法在溺死诊断中的应用

目的探讨微波消解-扫描电镜联用法在溺死诊断中的应用价值。方法收集已知死因的尸体标本105例,其中水中尸体85例(生前溺死70例,死后抛尸入水15例),陆地自然死亡尸体20例。水中死亡案例同时收集落水处水样。分别用微波消解-扫描电镜联用法(方法 A)和硝酸破机-光镜联用法(方法 B)对上述尸体的离体肺、肝、肾、骨髓组织及水样进行硅藻定性、定量检测。结果①溺死尸体的肺、肝、肾、骨髓中及落水处水样硅藻检出率:A法分别为100%、94.3%、92.9%、82.9%、100%,硅藻检验阳性率为100%;B法分别为90%、62.9%、51.4%、28.6%、92.9%,硅藻检验阳性率为65.7%。②两种方法检出的硅藻种类与落水处水样中硅藻的种类均一致。③两种方法在死后入水尸体离体的肺中也检出少量硅藻(<3个/2g肺组织),但在死后入水尸体的其它脏器及陆地自然死亡尸体脏器中均未检出硅藻。结论微波消解-扫描电镜联用法较硝酸破机-光镜联用法对尸体离体组织脏器中的硅藻检出率高,方法灵敏,定性准确。     

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.     

微波消解-扫描电镜联用检测脏器内硅藻

目的介绍一种微波消解-扫描电镜联用检测脏器内硅藻的新方法。方法大白兔30只,随机分为生前溺死组（n=15）和死后溺尸组（n=15）。提取兔肺、肝、肾、股骨骨髓和现场珠江水样,采用建立的微波消解-扫描电镜联用法检测脏器组织和水样中的硅藻,并与硝酸乙醇消解-扫描电镜联用的方法进行比较。结果生前溺死组的兔肺、肝、肾、股骨骨髓组织中大多数观测到与现场珠江水样一致的硅藻,微波消解-扫描电镜联用法检测脏器内硅藻的平均含量分别为：2505.2个/2g肺,18.7个/10g肝,6.5个/10g肾,6.3个/0.5g骨髓;肺与肝、肾、骨髓检出硅藻的阳性比例为86.7%,脏器硅藻检出含量和硅藻检验阳性比例均明显高于硝酸乙醇消解-扫描电镜联用法所测值。死后溺尸组的兔脏器组织均未观测到硅藻。结论微波消解-扫描电镜联用法检测脏器内硅藻,高效、安全、环保,硅藻检验灵敏度高,降低了劳动强度,提高了定性定量分析准确度,且能有效避免污染,在法医学溺死鉴定中具有良好的应用价值。