An efficient method for building a database of diatom populations for drowning site inference using a deep learning algorithm.

Seasonal or monthly databases of the diatom populations in specific bodies of water are needed to infer the drowning site of a drowned body. However, existing diatom testing methods are laborious, time-consuming, and costly and usually require specific expertise. In this study, we developed an artificial intelligence (AI)-based system as a substitute for manual morphological examination capable of identifying and classifying diatoms at the species level. Within two days, the system collected information on diatom profiles in the Huangpu and Suzhou Rivers of Shanghai, China. In an animal experiment, the similarities of diatom profiles between lung tissues and water samples were evaluated through a modified Jensen-Shannon (JS) divergence measure for drowning site inference, reaching a prediction accuracy of 92.31%. Considering its high efficiency and simplicity, our proposed method is believed to be more applicable than existing methods for seasonal or monthly water monitoring of diatom populations from sections of interconnected rivers, which would help police narrow the investigation scope to confirm the identity of an immersed body.

Diatom DNA barcodes for forensic discrimination of drowning incidents.

The presence of diatoms in victim's internal organs has been regarded as a gold biological evidence of drowning. The idea becomes true at the advent of DNA metabarcoding. Unfortunately, the DNA barcode of diatoms are far from being applicable due to neither consensus on the barcode and nor reliable reference library.In this study we tested 23 pairs of primers, including two new primer pairs, Baci18S (V4 of 18S) and BacirbcL (central region of rbcL), for amplifying fragments of 16S/18S, 23S/28S, COI, ITS and rbcL. A total of five pairs of primers performed satisfactory for diatoms. We used three of them, 18S605 (V2 + V3 of 18S), Baci18S and BacirbcL, to barcode four water samples using next generation sequencing platform. The results showed that these primers worked well for NGS metabarcoding of diatoms. We suggest that 18S605, Baci18S and BacirbcL be barcodes of diatoms and the corresponding primer pairs be used. Considering a quite high proportion of sequences deposited in GenBank were mislabeled, the most urgent task for DNA barcoding of diatoms is to create standard sequences using correctly identified specimens, ideally type specimens.

A novel approach for the forensic diagnosis of drowning by microbiological analysis with next-generation sequencing and unweighted UniFrac-based PCoA.

The diagnosis of drowning is one of the major challenges in forensic practice, especially when the corpse is in a state of decomposition. Novel indicators of drowning are desired in the field of forensic medicine. In the past decade, aquatic bacteria have attracted great attention from forensic experts because they can easily enter the blood circulation with drowning medium, and some of them can proliferate in the corpse. Recently, the advent of next-generation sequencing (NGS) has created new opportunities to efficiently analyze whole microbial communities and has catalyzed the development of forensic microbiology. We presumed that NGS could be a potential method for diagnosing drowning. In the present study, we verified this hypothesis by fundamental experiments in drowned and postmortem-submersed rat models. Our study revealed that detecting the bacterial communities with NGS and processing the data in a transparent way with unweighted UniFrac-based principal coordinates analysis (PCoA) could clearly discriminate the skin, lung, blood, and liver specimens of the drowning group and postmortem submersion group. Furthermore, the acquired information could be used to identify new cases. Taken together, these results suggest that we could build a microbial database of drowned and postmortem-submersed victims by NGS and subsequently use a bioinformatic method to diagnose drowning in future forensic practice.

Diatomological mapping of water bodies in Chongqing section of the Yangtze River and Jialing River.

The diagnosis of drowning is one of the most difficult in forensic medicine. Forensic diatomology has been proposed to be useful in solving the diagnosis of drowning and considered to be a reliable indicator of the site of drowning. The Yangtze River and Jialing River are the main rivers in the Chongqing area (China), and a large number of corpses are found in the rivers every year. However, the distribution of diatoms in the rivers was not fully studied. In the presented study, a Microwave Digestion-Vacuum Filtration-Scanning Electron Microscopy (MD-VF-SEM) method was performed to acquire the qualitative and quantitative data of diatoms of water samples collected from 10 different sites of the Yangtze River and Jialing River in Chongqing section during different seasons. Our study not only created the diatomological maps of water bodies in Chongqing section of the Yangtze River and Jialing River for the first time but also identified some seasonal and site-specific diatoms that can be taken as markers of particular sites or seasons of drowning. The results of our study may provide forensic scientists helpful reference in solving the drowning cases.

Microbial forensics: new breakthroughs and future prospects.

Recent advances in genetic data generation, through massive parallel sequencing (MPS), storage and analysis have fostered significant progresses in microbial forensics (or forensic microbiology). Initial applications in circumstances of biocrime, bioterrorism and epidemiology are now accompanied by the prospect of using microorganisms (i) as ancillary evidence in criminal cases; (ii) to clarify causes of death (e.g., drownings, toxicology, hospital-acquired infections, sudden infant death and shaken baby syndromes); (iii) to assist human identification (skin, hair and body fluid microbiomes); (iv) for geolocation (soil microbiome); and (v) to estimate postmortem interval (thanatomicrobiome and epinecrotic microbial community). When compared with classical microbiological methods, MPS offers a diverse range of advantages and alternative possibilities. However, prior to its implementation in the forensic context, critical efforts concerning the elaboration of standards and guidelines consolidated by the creation of robust and comprehensive reference databases must be undertaken.

[Diagnosis of drowning by detecting gyrB and 16S rRNA genes of Aeromonas hydrophila using PCR-capillary electrophoresis].

OBJECTIVE: To establish a method for diagnosis of freshwater drowning by amplifying gyrB and 16S rRNA genes of Aeromonas hydrophila using PCR coupled with capillary electrophoresis (CE). METHODS: DNA samples were extracted from human, 18 planktons (including Candida albicans, Aeromonas hydrophila, and 16 species of algae), and 30 cases of tissue samples (including the lung, liver, and kidney, all examined with microwave digestion-vacuum filtration-automated scanning electron microscopy) from human cadavers, including 28 freshwater drowning victims and 2 with natural death. The DNA samples were amplified with the primer AH (for gyrB gene) and primer Ah (for 16S rRNA gene), and the products were analyzed with CE. RESULTS: PCR amplification followed by CE yielded negative results for DNA of human, Candida albicans and 16 species of algae, whereas a positive result was found for Aeromonas hydrophila DNA with PCR products of 195 bp (with primer AH) and 350 bp (with primer Ah). In the 28 drowning cases, the detection rates of Aeromonas hydrophila using primer AH were 96.4% in the lung tissue, 71.4% in the liver tissue, and 60.7% in the kidney, as compared with the rates of 75.0%, 42.9%, and 32.1% using primer Ah, respectively. The positive rates for Aeromonas hydrophila in the organs of the drowning victims were 82.1% and 53.6% with primer AH and primer Ah, respectively. The detection showed negative results in the 2 cases of natural deaths. The two primers produced significantly different detection rates of Aeromonas hydrophila (P<0.05). CONCLUSION: PCR coupled with CE for detecting gyrB gene of Aeromonas hydrophila has a high sensitivity in assisting a diagnosis of freshwater drowning. Detection of both the gyrB gene and 16S rRNA gene of Aeromonas hydrophila can yield more convincing evidence of the diagnosis of freshwater drowning.

[The updated techniques for the detection of diatomic plankters in the corpse after death by drowning].

The authors report investigations carried out with a view to improving the method for the detection of diatomic plankters for the purpose of forensic medical expertise of death by drowning. The methods for studying diatomic plankton present in blood, pericardial fluid, and contents of the sphenoid bone sinus are proposed to be employed for the purpose of differential diagnostics of death by drowning. Specific approaches to sampling cadaveric materials, their preparation and examination by dark field and phase contrast microscopy are described. It was shown that the application of the proposed methods for the investigation of diatomic plankton in cadaveric biological fluids ensures preservation of all planktonic species and thereby significantly improves the quality of analysis and decreases the time needed to perform forensic medical expertise.

The presence of diatom algae in a tracheal wash from a German Wirehaired Pointer with aspiration pneumonia.

A 7-year-old spayed female German Wirehaired Pointer was presented with difficulty breathing after being found seizing in a water-filled drainage ditch while out hunting. Aspirates from a tracheal wash contained numerous degenerate neutrophils, fewer macrophages, some of which contained basophilic debris, low numbers of extracellular diatoms, and a single intracellular short bacterial rod. As the dog continued to clinically decline and could not be weaned from oxygen support, the owners chose euthanasia. The major necropsy finding was a severe granulomatous bronchopneumonia that was likely due to aspiration of foreign material based on the microscopic presence of plant-like material, bi-refringent crystalline material, non-cellular debris, and occasional fungal structures. Diatoms are a class of algae that live primarily in water. Diatom analysis has been used, with some controversy, in human forensics to assist in documenting drowning as the cause of death. In this case, given the clinical history, the presence of diatoms and inflammation in the tracheal wash were interpreted as a likely result of the aspiration of surface water. To our knowledge, this is the first reported case of diatoms observed in a cytologic specimen in a nonhuman mammal with aspiration pneumonia.

False-positive diatom test: a real challenge? A post-mortem study using standardized protocols.

The main criticism of the validity of the diatom test for the diagnosis of drowning is based on the potential ante- and post-mortem penetration of diatoms and the finding of diatoms in bodies of non-drowned human beings. However, qualitative and quantitative studies on diatoms in organs of the non-drowned have yielded both conflicting and contradictory results. In the present study, we have analysed under standardised methods the diatom content in several organs of 14 non-drowned human bodies. Overall, only 9 diatoms (6 entire, 3 fragmented) were disclosed in 6 of the 14 non-drowned bodies. Each of these 6 cadavers had only a single "positive" organ. Six diatoms were found in the bone marrow, 2 in the lung, and one in the pleural liquid. No diatoms were recovered from the brain, liver, kidney, or blood samples of any of these 14 bodies. Moreover, in five additional cadavers, whose lungs were injected, prior autopsy, with a 3.5L solution containing a bi-cellulate diatom culture (Thalassiosira baltica, Thalassiosira levanderi) via tracheostomy, a few diatoms appeared in the pleural cavity and in the blood from the left heart chamber, but none in any other internal organs investigated. The results of the presented study demonstrate that the issue of the false-positive diatom test should not be a logical impediment to the performance of the diatom method. However, strict and standardized protocols aimed at avoiding contamination during sample preparation must be used, appropriate separation values set and taxonomic analysis of all diatoms performed.

Successful liver transplantation from donor with Plesiomonas shigelloides sepsis after freshwater drowning: case report and review of literature on gram-negative bacterial aspiration during drowning and utilization of organs from bacteremic donors.

BACKGROUND: Plesiomonas shigelloides is a freshwater, non-fermentative gram-negative bacillus associated with diarrheal disease. Rare cases of invasive infection in human beings usually involve immunosuppressed individuals. METHODS: We report a patient who underwent successful liver transplantation (LT) using a graft from a 14-year-old boy who had drowned in a freshwater lake. PUBMED was searched for both reported drowning victims with sepsis and outcomes of LT using organs from infected donors. RESULTS: Our patient received prophylactic piperacillin-tazobactam, which was switched to cefepime one day after transplantation when gram-negative bacteria grew in blood cultures of the donor. The next day, the organism was identified as P. shigelloides resistant to third- and fourth-generation cephalosporins; ciprofloxacin was given for seven days, and surveillance cultures remained negative. After an uneventful course the patient was discharged on day 10 after LT without signs of infection and is alive with a well-functioning graft. Literature review revealed one case of P. shigelloides in a potential allograft, in which the organism was isolated from heart valves of a drowning victim; the organs were discarded. Reports of freshwater drowning show that bacteremia is universally found post-mortem. Isolated pathogens correspond to specimens from the drowning site, with Aeromonas spp. being the most common and many other microorganisms described anecdotally. Livers from infected donors have been used, in most cases with good results if the recipient and, when possible, donor were treated appropriately; however, cases of fatal pathogen transmission have been reported. CONCLUSION: This is the first reported case of a LT using a graft from a donor with P. shigelloides sepsis. Drowning victims should be considered potentially infected with rare pathogens and therefore represent extended-criteria donors.

[Prospects for the use of bacteriological method to establish the fact of death by drowning].

Bacteriological studies of cadaveric blood for the presence of Pseudomonas putida and Pseudomonas fluorescence have demonstrated that identification of these microorganisms may be considered as an evidence of death by drowning. These bacteria are typical "aqueous" organisms non-pathogenic for man. They do not grow at a temperature of 41 degrees C and therefore are usually absent in normal human microflora. It is concluded that the establishment of the fact and location of death by drowning based on the analysis of plankton composition should be supplemented by identification of various forms of Pseudomonas in cadaveric blood.

A novel method for the diagnosis of drowning by detection of Aeromonas sobria with PCR method.

The acid digestion method has been widely used for the diagnosis of death by drowning, but it is not always sensitive. However, there has been no definitive method to replace acid digestion until now. We speculate that bacteria are more useful markers than plankton for the diagnosis of death by drowning. In this study, from the preserved blood samples of 32 freshwater drowning cases, specific DNA fragments of Aeromonas sobria, one of the most common aquatic bacteria, were examined using PCR. The DNA fragments of the bacterium were detected from 27 of 32 cases with first round PCR or nested-PCR. The remaining 5 cases in which bacterial DNA was not detected had longer storage periods for the blood samples and shorter time intervals from drowning to death. These results indicate that the present method can be applied to the diagnosis of death by drowning.

Evaluation of four digestive methods for extracting diatoms.

OBJECTIVE: To compare the merit of four digestive methods (nitric acid plus hydrogen peroxide, proteinase K, nitric acid in Disorganization Can and Soluene-350) for extracting diatoms in order to choose the best digestive method for the diagnosis of drowning. METHODS: Liver, kidney and bone marrow of rabbits were minced and then digested by four digestive methods separately with the following indices compared: (1) time demanded for complete digestion; (2) degree of digestion for different tissues; (3) the reclaiming ratio of diatoms; (4) the degree of digestive destruction to diatoms. RESULTS: For sufficiently digesting the same tissue, the demanded times for the different methods ranked from the longest to the shortest were as follows: Soluene-350, proteinase K, nitric acid plus hydrogen peroxide, nitric acid in Disorganization Can. Nitric acid in Disorganization Can method and nitric acid plus hydrogen peroxide method digested the tissues more thoroughly than proteinase K, than Soluene-350 methods. For Cyclotella and Cybella, proteinase K method reclaimed most diatoms and nitric acid plus hydrogen peroxide method reclaimed less, while nitric acid in Disorganization Can and Soluene-350 methods reclaimed the least. For Navicula, the majority of diatoms could be extracted using proteinase K method, but only a few diatoms with other three methods. Under scanning electron microscopy (SEM), the structure of diatoms remained almost perfect after digestion with proteinase K, but destroyed to some extent with other three methods. CONCLUSION: This study demonstrated that different diatoms (in fresh or sea water) have different resistance to different digestive reagents. As far as the reliability and applicability of the diatom test is concerned, proteinase K method is of the best choice, nitric acid plus hydrogen peroxide can be its substitute. Soluene-350 cannot be used for extracting sea water diatoms.

Diatoms in forensic expertise of drowning--a Macedonian experience.

Results obtained by examination of 22 human cases suspected for drowning, one human case of death other than drowning and several tests on laboratory rats were used as a basis for evaluation of diatom method as supportive in forensic expertise of drowning. The recovery of diatoms from various examined organs, their qualitative and quantitative composition, if properly treated without the possibility of contamination, can be a reliable proof of the time and place of drowning. The priority of organ examination (external microflora determination, lungs, brain, heart (and/or blood), stomach, liver and kidney, and finally bone marrow) is discussed and established as well as the basic future research on cases suspected of drowning, but also on non-drowned victims and laboratory animals.

Diatom detection in the diagnosis of death by drowning.

A medicolegal/algological collaboration lasting several years aimed at developing methods for dealing with dead bodies found in water where the circumstances are not clear, has led to an interdisciplinary procedure. To enable algological analysis, sample preservation and preparation must be free from contamination or carry-over at the beginning of the autopsy, although it should be noted that the demands on the digestion method are very high. One or more water samples from the site of drowning (from surface and bed) should be taken. Microscopic-algological analysis should record quantitative (diatom density), qualitative (species) and morphological (description of diatom valves) details for every sample. Furthermore, the species index and dominance identity similarity indices are calculated during the analysis procedure. The algological conclusions are based primarily on the separation values of Kater as well as on pair-matching. The final report is the result of interdisciplinary collaboration.

Diatoms and homicide.

Six illustrative cases of homicidal drowning are reported in which the diatom test for drowning was a useful adjunct to the medicolegal investigation of death. In all six cases, diatom frustules were recovered from the femoral bone marrow. In five cases, diatoms extracted from the bone marrow were compared with diatoms obtained from samples of putative drowning medium. In all of these case, the same types of diatoms were observed in both the water samples and the marrow. In an additional case, watery fluid from the maxillary sinus contained the same diatom types as were present in the femoral bone marrow. Four of the six cases were found submerged in water and had autopsy findings consistent with drowning. In the remaining two cases, the bodies were found on land; one case was grossly decomposed and one body was extensively burned since the body was set afire on dry land after death. In some of the case drowning was associated with blunt force head injury (one case), sharp force injuries of the chest (one case), or strangulation (three cases). These results indicate that the diatom test for drowning is an important adjunct to the medicolegal investigation of homicidal drowning, particularly in those cases were autopsy and scene findings do not imply drowning as a cause of death.

The diagnostic value of the diatom test for drowning, I. Utility: a retrospective analysis of 771 cases of drowning in Ontario, Canada.

The utility and validity of the diatom test for drowning was studied using a retrospective analysis of 771 cases of drowning mostly from Ontario, Canada, over the period 1977 to 1993. In this article (part one), the utility of the test was assessed using an analysis of test outcomes. In the companion article (part two), the validity of the test was assessed by analyzing the relationship between test outcome and characteristics of diatoms in the bone marrow and samples of putative drowning medium. In the present study, freshwater drownings accounted for 738 of the cases and 33 cases were drownings in bathtubs, pools, or toilets. Diatoms were recovered from the femoral bone marrow of 205 cases (28%) of freshwater drowning and four cases (12%) of domestic water drowning. There was a monthly variation in the frequency of positive test outcomes that could not be explained by seasonal differences in the total number of drownings. However, the monthly variation was strongly correlated with the periodic cycle of diatom blooms that occurs in freshwater. Positive diatom tests were characterized by a limited number of distinctive diatom species per case, and a restricted quantity and size range of diatom frustules. These results indicate that the diatom test for drowning will identify approximately one in three victims of freshwater drowning and may be useful in the assessment of deaths occurring in bathtubs. The correlation of the outcome of the diatom test for drowning with diatom blooms provides further evidence for the reliability of the test.

The diagnostic value of the diatom test for drowning, II. Validity: analysis of diatoms in bone marrow and drowning medium.

In part two of this series, we investigated the characteristics of diatom frustules recovered from bone marrow and samples of putative drowning medium in case of freshwater drownings. A total of 52 cases of freshwater drowning with diatoms in the femoral bone marrow in which a sample of the putative drowning medium was also collected were available for analysis. The same types of diatoms were found in the bone marrow and putative drowning medium in 47 cases (90%) indicating that the water samples were representative of the site of drowning in at least 90% of freshwater drownings. In the remaining 5 cases (10%), the diatoms in the water samples did not match those in the bone marrow indicating that the site of body and water sample recovery were likely geographically remote from the site of drowning. In cases with matching diatoms in the bone marrow and drowning medium, the diatoms were consistently the smallest and most abundant types represented in the water samples. In addition, there were highly stereotyped physical characteristics of typical "drowning-associated" diatoms with small pennate diatoms representing the most common type of frustule found in the bone marrow. In an additional 34 cases of putative drowning in water that lacked detectable diatoms, 29 cases (85%) lacked diatoms in the bone marrow. Analysis of the diatom content of samples of putative drowning medium by month revealed that winter months had the highest frequency of samples devoid of diatoms. These data indicate that the true positive rate of the diatom test for drowning is at least 90% and that small pennate frustules are most commonly associated with drowning, particular in non winter months.

Ocular surface flora in drowning victims.

Contaminated allograft donor tissue represents a potential source of infection unique to keratoplasty. We prospectively studied perilimbal cultures of drowning victims over 30 months to determine if the ocular surface flora of drowning victims was unique. Twenty-eight donor eyes were cultured from 14 drowning victims. Ninety-three percent of limbal cultures were positive for one or more organisms, compared to results of a previous study of surface contamination of donor globes in which 65% were found to be culture positive. Fifty-seven percent (16 of 28) of donor eyes grew at least one streptococcal species, while 46% (13 of 28) grew two or more streptococcal species. A previous study identified streptococcal species in 7.9% of perilimbal cultures from nondrowning donor eyes, which demonstrates the unique effect of drowning on the ocular flora. The incidence of gram-negative isolates was also markedly higher in drowning victims, compared to previous studies of donor globes. This prospective study has shown that the ocular surface flora of drowning victims harbors markedly increased numbers of streptococcal species as well as gram-negative organisms. These findings demand careful globe decontamination, and emphasize the need for appropriate antibiotic coverage in corneal storage media.