Geographic diversity of Helicobacter pylori in cadavers: Forensic estimation of geographical origin

Forensic Science International 229 (2013) 7–12

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Geographic diversity of Helicobacter pylori in cadavers: Forensic estimation of geographical origin Sayaka Nagasawa *, Hisako Motani-Saitoh, Hiroyuki Inoue, Hirotaro Iwase Department of Legal Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 December 2011 Received in revised form 11 January 2013 Accepted 18 February 2013 Available online 11 April 2013

A method for determining the geographical origin of unidentified cadavers by determining the genotype of Helicobacter pylori, which is latent in one-half of the world’s population, was developed. In the first stage, DNA was extracted from samplings at 5 points in the gastric mucosa of 177 individuals randomly selected from cadavers undergoing medico-legal autopsy. 16S-rDNA of H. pylori DNA was detected by polymerase chain reaction (PCR) in 101 cadavers (57.0%); by sex, 74 of 123 (60.1%) males and 28 of 54 (46.4%) females were positive. There were no significant differences in H. pylori detection rate among the 5 sampling points of the gastric mucosa, cause of death, or age. In the second stage, amplified fragments of H. pylori vacA regions s and m from 17 individuals with the following ethnic backgrounds were sequenced: Japanese, 10; Chinese, 2; South Korean, 1; Taiwanese, 1; Thai, 1; Afghan, 1; and Filipino, 1. A phylogenetic tree constructed with these and 28 previously reported H. pylori strain sequences revealed 3 major gene clusters consisting of East Asian type I (Japanese, South Korean and Chinese), Western type II, and Southeast Asia type III. The Taiwanese and Filipino samples deviated from the clusters type III to which they typically belong. The ultimate aim of the present study was to develop a more accurate method of determining of geographic origin of unidentified cadavers through the combination of the present method with other, virus-based methods H. pylori DNA was detected from over half of the cadavers tested and vacA genotypes showed specificity to geographical origin. Therefore, these results suggest that the H. pylori genome provides valuable additional information for tracing the geographical origin of unidentified cadavers. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: H. pylori DNA polymorphism vacA

1. Introduction In many areas worldwide, increasing internationalization and the occurrence of large-scale disasters has resulted in growing numbers of unidentified cadavers. This is an important forensic problem. In Japan, 1000 or more deceased individuals are not identified upon death every year, giving a total of more than 16,000 unidentified deceased individuals [1]. Following the Great East Japan Earthquake and tsunami in 2011, about 3000 people remain missing and there are about 300 unidentified bodies as of June 2012. In 8 European nations—Denmark, Finland, Greece, Ireland, Portugal, Luxembourg, Spain, and Germany, there were 3035 unidentified deceased individuals during the 4-year period from

* Corresponding author at: Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel.: +81 43 226 2078; fax: +81 43 226 2079. E-mail addresses: [email protected], [email protected], [email protected] (S. Nagasawa). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.02.028

1994 to 1998, of which 2228 were subsequently identified and 807 remain unidentified [2]. As methods of personal identification, fingerprints, dental records and human DNA typing are reliable methods. Other methods targeting personal characteristics, such as facial appearance and personal belonging, are also used. However, when such information is lacking, personal identification becomes difficult and results in the case remaining unsolved. Therefore, methods that can quickly and easily narrow down the possible geographic origins of unidentified cadavers would be extremely valuable. However, there are no effective ways to estimate the geographical origins of humans based on human DNA or morphological character analysis. Therefore, tests that utilize the distinct geographical distributions of DNA polymorphisms of latent human viruses have been developed in recent years. Various methods targeting different viruses have been developed by Ikegaya and colleagues: John Cunningham virus (JCV) in the kidneys [3,4], BK virus (BKV) in the tubular epithelium [5,6], and Epstein-Barr virus (EBV) in B-lymphocytes [4]. Furthermore, we reported the use of this method for the varicella-zoster virus (VZV) in trigeminal ganglia [7]. A DNA chip has already been

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developed for detection of JC virus, and it is used in forming professional opinions regarding geographical origin [8]. Since the state of cadavers at autopsy varies widely and is dependent on the cause of death, time elapsed since death, areas damaged, the degree of decomposition, it is more effective to use samples from multiple organs than from only one type of organ. Here, we selected Helicobacter pylori based on the following characteristics: (1) high infection rate, (2) infection primarily occurring early in life and usually persisting for a lifetime, (3) DNA polymorphisms that are geographically unique, as noted for the previously reported viruses, and (4) tendency to infect the gastric mucosa, which remains intact in many cases despite general decomposition and which is easy to collect during forensic autopsy. H. pylori is a Gram-negative microaerobic bacteria that colonizes the human stomach [9]. Today, one-half of the world’s population is infected with H. pylori with prevalence ranging from 15% in developed countries [10] to more than 90% in developing areas [11,12]. Epidemiological studies have shown that acquisition of H. pylori infection primarily occurs early in life; infection occurs within families and usually lasts for a lifetime unless eradicated [13]. Genetic exchange among distinct strains is rare, even during superinfections [14]. H. pylori could, therefore, provide a window into human origins and migration [15]. H. pylori strains from different geographical areas exhibit clear phylogeographic features. In addition, population-wide sequence diversity is greater for H. pylori than for most other bacteria [16] and is approximately 50-fold more diverse than that of humans [17]. Analysis of geographical genetic diversity and genotyping of H. pylori isolates is often accomplished by the examination of two genes encoding proteins associated with virulence [18,19]: cytotoxin-associated (CagA) protein, encoded by cagA, and vacuolating cytotoxin (VacA), which is encoded by vacA. cagA is a marker for the presence of a pathogenicity island (cag PAI) and is divided into two types, East Asian CagA and Western CagA. The East Asian type CagA is only observed in H. pylori isolates from East Asian populations, whereas the Western type CagA is widely distributed among isolates from European, South and Central Asian, North and South American and African populations [20]. However, approximately 20% to 40% of isolates from Europe and Africa are CagA-negative. Another virulence factor, VacA, is associated with epithelial cell damage. This cytotoxin is present in all H. pylori strains [21] and comprises two variable parts in the region of the vacA gene: the sregion (s1 and s2) and the m-region (ma and m2). Essentially, all East Asian H. pylori strains are of the vacA s1 type. Within East Asian counties, the m1 type predominates in Japan and Korea, whereas the prevalence of the m2 types gradually increases in the southern parts of East Asia [22,23]. In this study, we used 177 cadavers submitted for forensic autopsy to examine H. pylori DNA vacA region polymorphism and the stability of H. pylori DNA after death. The cadavers were selected at random without regard to age, clinical history, sex, cause of death and time since death. Further, molecular phylogenetic analysis of 17 samples of known geographic origin (10 Japanese, 2 Chinese, 1 South Korean, 1 Taiwanese, 1 Filipino, 1 Thai and 1 Afghan) was performed using the s- and m-regions of the vacA region. A novel method was developed to determine the geographical origin of unidentified deceased individuals by examination of H. pylori vacA genotype polymorphisms that are associated with the geographical distribution of the bacterium.

Afghan, and 3 Filipino) randomly selected from those having undergone forensic autopsy at the Department of Legal Medicine, Graduate School of Medicine, Chiba University between 2006 and 2011. The samples were collected using sterile scissors and tweezers and kept frozen at 80 8C prior to DNA extraction. The gender and age distributions of the cadavers are shown in Fig. 1. The average age at the time of death was 55.6 years old (55.8 for males and 55.0 for females). The subjects had no known active H. pylori infection at the time of autopsy. 2.2. DNA extraction Genomic DNA was extracted from about 25 mg of tissue using a QIAamp1 DNA Mini Kit (Qiagen, Hilden, Germany) and eluted in 100 ml of sterile water at the final step. 2.3. Polymerase chain reaction (PCR) amplification Extracted DNA was amplified using the primers of 16S-rDNA which listed in Table 1. DNA samples which had been purified from cultivated H.plyori extracted from the patient’s gastric mucous membrane (gifted from Prof. Azuma) were used as positive controls and sterilized water was used as a negative control. Reactions (20 ml) contained 50 pmol of each of the primers, 0.2 mM of dNTP, 1 PCR buffer (Applied Biosystems, Foster City, CA, USA) and 1.25 U of AmpliTaq Gold1 DNA polymerase (Applied Biosystems). Amplification for the 16S-rDNA reactions was carried out as follows: 95 8C for 11 min, then 45 cycles of 95 8C for 30 s, 56 8C for 30 s, and 72 8C for 30 s, followed by a final extension step at 72 8C for 10 min. Two PCR reactions were conducted for each tissue extraction, and PCR products were separated by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining. To confirm the presence of H. pylori, samples that were positive for H. pylori on PCR were subjected to sequencing and confirmed H. pylori using an ABI PRISM1 310 Genetic Analyzer (Applied Biosystems). For the vacA s- and m-regions, amplifications were carried out using primers listed in Table 1 that were designed using DNASIS1 pro (Hitachi, Software, Yokohama, Japan). Amplification was carried out as follows: denaturation at 95 8C for 11 min, then 45 cycles of 95 8C for 30 s, 58 8C for 30 s, and 72 8C for 30 s, followed by a final extension at 72 8C for 10 min. 2.4. Sequencing VacA regions were sequenced for 17 samples from 10 randomly selected from the Japanese with clear birth and growing being Japan at random and all non-Japanese cadavers (2 Chinese, 1 South Korean, 1 Taiwanese, 1 Thai, 1 Afghan, and 1 Filipino) that tested positive for H. pylori DNA. PCR products were subjected to cycle sequencing using a BigDye1 Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). Cycle sequencing was performed in 10-ml reaction volumes containing vacA s- and m-region primers at a final concentration of 0.25 pmol/ml. The amplification conditions were 25 cycles of 30 s at 96 8C, 15 s at 50 8C, and 2 min at 60 8C. Cycle sequencing products were purified using a Quick step 2 PCR Purification Kit (Edge Biosystems, Gaithersburg). DNA sequencing was performed on an ABI 310 Avant DNA sequencer (Applied Biosystems). 2.5. Phylogenetic tree analysis Molecular phylogenetic trees were constructed based on the sequence analysis of combining both vacA regions. An alignment of the vacA s- and mregion sequences determined in this study, as well as the reference sequences of 28 strains was made using DNASIS1 Pro software (Hitachi, Software, Yokohama, Japan). The aligned sequences were analyzed using the neighbor-joining method and a phylogenetic tree was constructed using DNASIS1 pro with 1000 bootstrap iterations. The phylogenetic tree was visualized using the NJ plot program.

35 30 25 20

Male

15

Female

10 5 0

2. Materials and methods 2.1. Collection of samples Tissues from 5 points of the gastric mucosa (cardia, upper and lower greater curvatures, pylorus, and lesser curvature) were obtained from 177 cadavers (male 123 and female 54; 162 Japanese, 4 Chinese, 3 South Korean, 1 Taiwanese, 3 Thai, 1

Fig. 1. Gender and age distributions of 177 cadavers. There were 123 males and 54 females ranging in age from 2 to 89 years old. There were 9 male and 4 female cadavers classified as ‘‘unknown’’, but these were estimated to be 30–90 years old.

S. Nagasawa et al. / Forensic Science International 229 (2013) 7–12

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Table 1 Target amplification regions and primers for PCR and sequencing. Gene

PCR/sequencing

Primers

Primer sequence

Reference

0

0

16S-rDNA

PCR

16S rRNA-F 16S rRNA-R

5 -TAAGAGATCAGCCTATGTCC-3 50 -TCCCACGCTTTAAGCGCAAT-30

Kumar et al. (2008) Kumar et al. (2008)

vacA s-region

Both

LMVAS-F LMVAS-R

50 -AAAATCAATCGCCCTCTGGTTTCTC-30 50 -TTCCCCCAATCCCAACCTCCATCAAT-30

This study This study

vacA m-region

Both

LMVAM-F LMVAM-R

50 -GCCCCTTGGAATTATTTTGACGCTA-30 50 -ATCCCGCATGCTTTAATGTC-30

This study This study

2.6. Statistical methods The relationship between the H. pylori-positive rate and age, the 5 gastric sampling positions, and cause of death were tested by the rank test using Spearman’s correlation coefficients.

3. Results The cadavers included in this study were selected at random, regardless of age, sex, cause of death, postmortem interval and condition. The distribution of age and sex of the cadavers is shown in Fig. 1. H. pylori 16S-rDNA was detected in 57%. The H. pylori positive rate for males (60.1%, 74/123) was higher than that for females (46.4%, 28/56) as shown in Fig. 2. H. pylori DNA became detectable for individuals in their teens, and the positive rate increased with increasing age; the youngest and oldest individuals to test positive for H. pylori were 18 (male) and 89 (female) years old, respectively. Infection rates were greater than 50% in individuals over 40 years old and older, which is consistent with a previous finding of 50% rates reported in previous epidemiological studies. The influence of cause of death and postmortem interval on the detection of H. pylori DNA is shown in Table 2. H. pylori DNA was detected in 57.7% (71/123) of individuals who died of external causes. Individuals who died of internal causes had similar H. pylori DNA-positive rates (54.8%, 17/31). H. pylori DNA was also detected in subjects who died of chemical poisoning by overdose of herbicides and anti-psychotic drugs. Spearman’s correlation coefficients showed no statistically significant differences in positive rate among the causes of death (p < 0.05). Regarding postmortem interval, 153 cadavers were estimated to have within 1 week of death, and H. pylori DNA was detected in 52.9% (81/153) of these individuals. Furthermore, for postmortem interval of 1 week to <1 month, H. pylori DNA was detected in 15 (less than 40 years old, 2 and older than 40 years old, 13) of 19 cadavers (less than 40 years old, 4 and older than 40 years old, 15). For postmortem interval of 1 month, all 5 cadavers (unknown age, 2 and >60 years old, 3) were positive, and 1 mummified cadaver with an estimated postmortem interval of several months

Fig. 2. Human gastric mucosa samples positive for H. pylori by age and sex. The youngest and oldest positive individuals were an 18-year-old male and an 89-yearold female, respectively. The PCR-positive rate increased with advanced age and exceeded 50% at the 40 s.

to 2 years, the longest in this study, was H. pylori DNA-positive. These results suggest that neither the cause of death nor time after death affects the detection of H. pylori DNA. Next, we examined the relationship between samples that were H. pylori DNA-positive and the presence or absence of gastric findings on autopsy. Gastric findings comprised macroscopic findings during autopsy and overall findings obtained from biochemical, histopathological and other laboratory tests performed at autopsy. As shown in Table 3, samples were collected from 28 individuals showing gastric findings, and H. pylori DNA was detected in 75.0% of these samples. Regarding gastric submucosal hemorrhage and discoloration, some were due to post mortem changes and could not be distinguished from diseases of the stomach. Additionally, 1 of 2 samples that were H. pylorinegative were from cases with gastric perforation, the cause of perforation was based on an injury that was due to an external cause. For the remaining 146 individuals with no gastric findings on autopsy, the H. pylori DNA-positive rate was 54.7%. In 3 individuals, a post-operative scar from the resection of stomach cancer was observed and but H. pylori DNA was not detected. Although the relevance of the presence of gastric findings and H. pylori was not elucidated, it is obvious that H. pylori DNA was detectable in over 50% of samples that had no gastric findings in cadavers. The H. pylori DNA-positive rate in each of 5 points of the gastric mucosa taken to determine the most suitable source of tissue samples was similar at 52.5% (93/177) in the cardiac part and lesser greater curvature, 53.1% (94/177) in the upper greater curvature, Table 2 Detection rate by cause of death and postmortem time. Number of cases

Number of H. pylori-positive cases

(%)

123 14 26 3 2 61 7 10

71 11 17 2 2 29 6 4

57.7 78.5 65.3 66.6 100 47.5 85.7 56.5

2. Internal cause Cardiac disease Cerebral disorder Other

31 14 5 12

17 8 4 5

54.8 50 80 41.6

3. Unknown

23

13

56.5

153 19 5

81 15 5

52.9 78.9 100

Cause of death 1. External cause Burns Drowning Freezing Starvation Injury Asphyxia Poisoning

Postmortem interval 1 week About 1 month 1 year

16S-rDNAregions of DNA extracted from 5 gastric mucosal sites were amplified by PCR. Samples were designated as positive for H. pylori if the 16S-rDNA region was detected from at least 1 site.

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Table 3 Comparison of H. pylori-positive status for cases with and without gastric findings.a Number of cases Finding Submucosal hemorrhage or change in color Ulcer Perforation Tumor No findings Post-operation

Number of H. pyloripositive cases

(%)

28 15

21 10

75 66.6

8 3 2

8 1 2

100 33.3 100

149 3

80 0

53.6 0

a Gastric findings comprised macroscopic findings during autopsy and overall findings obtained from biochemical, histopathological and other laboratory tests performed at autopsy.

50.8% (90/177) in the lesser curvature, and 51% (92/177) in the pylorus. Spearman’s correlation coefficient showed no statistically significant differences in the detection rates of H. pylori DNA at each of the 5 points of the gastric mucous membrane (p < 0.05). In 101 H. pylori DNA-positive samples, 75, 13, 5, 3 and 5 were positive at all 5 points, 4, 3, 2 and 1 points, respectively. Finally, vacA s- and m-region DNA segments were amplified by PCR and sequenced in samples from the randomly collected 10 Japanese and other foreigner cadavers of known geographic origin and previously reported reference sequences from 28 samples. As shown in Fig. 3, The sequenced vacA genes were classified into 3 major clusters: (1) East Asian type I, consisting of Japan, China, and South Korea; (2) Western type II, consisting of Russia, the Americas, and Europe; and (3) Southeast Asia type III, consisting of Thailand, Hong Kong, Taiwan, and Vietnam. Phylogenetic tree analysis of the H. pylori vacA s- and m-regions from the cadavers examined in this study and those described in previous studies revealed that all strains could be classified into one of these genotypes with high bootstrap values. 4. Discussion Gastric H. pylori is common among middle-aged and elderly Japanese. A seroepidemiological study of H. pylori infection among apparently healthy residents of Sapporo found a prevalence of 70– 80% in individuals born before 1950 [24]. For residents born after 1950, the frequency of H. pylori infection increased by approximately 1% with the birth year [24]. Overall, the prevalence of H.

Fig. 3. Phylogenetic tree of H. pylori strain types based on vacA s- and m-regions from 17 cadavers (10 Japanese, 2 Chinese, 1 South Korean, 1 Taiwanese, 1 Thai, 1 Afghan, and 1 Filipino) submitted for medico-legal autopsy which showed F and 32 reference strains which showed R.vacA genes were classified into 3 major clasters: (1) East Asian type I, Western type II, and Southeast Asia type III.

pylori infection has decreased over the past few decades; based on serum samples from 1015 healthy people living in several prefectures in central Japan [25], overall H. pylori seropositivity was 72.7% in 1974, 54.5% in 1984, and 39.3% in 1994. In a 2007 study of 777 university students with a mean age of 19.6 years, H. pylori prevalence was only 14.7% [26]. Previous studies used serum samples to detect H. pylori, but in forensic autopsies, it is difficult to extract blood due to decomposition of the cadavers and hemolysis. In 1994, Judice et al. found no difference in H. pylori detection rates based on PCR amplification and the anti-H. pylori IgG antigen detection methods. In this study, therefore, we used PCR amplification to detect H. pylori DNA from 5 points of the human gastric mucosa, which remains relatively intact even when decomposition has progressed. Consequently, H. pylori DNA was detected in over 50% of forensic autopsy samples, despite the purported decline in the prevalence of H. pylori infection. External causes of death such as injury due to traffic accidents, burns, or drowning are often associated with severe damage and decomposition, and various body parts are often damaged or missing. In this study, except for one case in which the stomach was carbonized by a burn, gastric mucosal samples could be collected from all cadavers with external causes as the cause of death. Among the present cases, the trigeminal nerve was lost as a result of head injury in 2 cases and blood could not be extracted in 26 cases due to coagulation and decomposition due to burns or drowning, and H. pylori DNA was detected in 57.7% of cases. Additionally, H. pylori was detectable from the stomach, which can also contain many medicines and agricultural chemicals. Regarding postmortem interval, Castillo-Rojas et al. [27] reported detecting H. pylori DNA in Mexican pre-Columbian mummies dating to circa 1350 BC [27]. In this study, we also detected H. pylori DNA from a mummified body, which was estimated to have died several months to 2 years prior. There was no information regarding the age, sex, or cause of death of this individual. The detection rate of H. pylori DNA increased with postmortem interval, but since there were many elderly people among the samples with long postmortem interval, it was considered that the influence of age was greater than the influence of postmortem interval. These findings confirm that the condition of the cadaver has only a minimal influence on the ability to detect H. pylori DNA in the gastric mucosa. A number of studies reporting the relationship between stomach disease and H. pylori infection based on the detection of specimens according to presence or absence of symptoms and using various study protocols indicate a prevalence of 90% among the gastric disease cohort [28–31]. However, the present study is the first to review actual gastric findings and H. pylori DNA detection. Gastric findings on autopsy were observed in 28 individuals, and H. pylori DNA was detected in 21 (75%) of these. Gastric submucosal hemorrhage and discoloration among these individuals were likely due to post-mortem changes and, therefore, should not be considered to be linked to gastric diseases. Accordingly, individuals with gastric diseases, i.e., ulcers, perforations, and tumors, showed a H. pylori DNA detection rate of approximately 75%. The present findings corroborate previous reports that suggest that H. pylori colonization is related to gastric disease. On the other hand, of the remaining 149 individuals with no gastric findings, 80 (54.7%) were found to carry H. pylori DNA, while no gastric findings were observed in 53.6% of individuals carrying H. pylori. These findings show that H. pylori DNA is detected at high rates, even in the absence of gastric findings, which in turn suggests the importance of gastric mucosal sampling in the analysis of H. pylori DNA, irrespective of gastric findings. In 1990, a new system of classification, known as the ‘Sydney System’, was announced at the World Congress of Gastroenterology in Sydney, Australia [32]. As endoscopy has been gaining in

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popularity in both clinical and research settings, the Sydney System was established in an attempt to avoid diagnostic confusion. This system recommends biopsy from 4 points: 2 each from the antrum and corpus [32]. Moreover, the updated Sydney System released in 1994 recommends that additional biopsy specimens be taken from the incisura angularis [33]. Applying the biopsy sampling protocol of the updated Sydney System was used to the detect H. pylori DNA in cadavers at 5 detection points in the stomach, consisting of the cardia, upper and lower greater curvatures, pylorus, and lesser curvature. There were no statistically significant differences in the H. pylori detection rates among the 5 points, but the number of sites detected varied from 1 to 5. As mentioned previously, H. pylori DNA colonizes in the gastric mucosa irrespective of gastric findings. These findings suggest that in order to analyze H. pylori DNA, samples should be collected from at least 2 gastric mucosal sites but better from 5 points, regardless of gastric findings. The 16S-rDNA region was used to ascertain the geographic origin of the cadavers. In order to effectively estimate the geographic origin of unidentified cadavers, it is necessary to use a gene that has a high detection rate, narrow detection region in bps, and has high region specificity. Previously, Falush et al. reported that H. pylori can be broadly classified into 6 groups (Africa 1, Africa 2, NE Africa, Asia 2, East Asia, and Europe) based on multi-locus sequence typing of several housekeeping genes and a virulence factor gene [15]. However, this method requires a large number of gene segments, and applying it to cadavers could produce diminished detection rates. The 16S rDNA polymorphisms for H. pylori were reported by Sushil et al. in 2011, but regionspecificity has not been described. We, therefore, applied the DNA polymorphisms of the vacA gene for which region-specificity has already been reported and which is universal to H. pylori. The vacA gene contains at least 2 polymorphic sites in the s- and m-regions. Numerous studies have reported that the differences in these 2 polymorphisms lead to obvious differences in clinical symptoms. We already know that the s-region has alleles s1 and s2 and that the m-region has alleles m1 and m2. A study by Yamaoka et al. in 2009 reported that the s1 type was detected mainly in East Asia, the m1 type is predominant in Japan and South Korea, and the m2 type is detected in southern parts of East Asia, such as Vietnam and Hong Kong [20]. Yamaoka et al. further indicated that the vacA s1 type is subdivided into s1a, s1b, and s1c, and that the m1 type is subdivided into m1a, m1b, and m1c. The vacA s1c and m1b types are typical of H. pylori from East Asia and the s1a and m1c types are common in South Asia [23]. The vacA m1c genotype is also found in strains from Central Asia (ethnic Kazakhs). The m1a type is typical of Africans and ethnic Europeans. Both the s1a and s1b types are common in ethnic European strains, and s1b types are especially common in strains from the Iberian Peninsula and Latin America [21,23]. The s1b type is also predominant in Africa. Thus, we analyzed the genome sequence of the s- and m-regions, and the sequence analysis results of both domains were combined for generating the phylogenetic tree analysis was conducted. Consequently, vacA was classified according to 3 major clusters consisting of the East Asian type I, including Japan, China and South Korea, the Western type II, including Russia, the Americas, and Europe, and the Southeast Asia type III, including Thailand, Hong Kong, Taiwan, and Vietnam. All of the Japanese (n = 10), South Korean (n = 1), and Chinese (n = 2) cadavers examined in the present study were classified as type I, the single Thai cadaver was classified as type III, and the single Afghan and Filipino-Western cadavers were classified as type II. Filipinos are typically classified in the type III cluster, but the background details of the sample examined in the present study are unknown. The Western-type classification in the present case may be due to previous generations of Filipinos being infected with Western-type strains

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based on the Philippines’ status as a former Spanish colony and the primarily interfamily horizontal transmission of H. pylori infection. Similarly, Taiwanese usually belong to type III, but the cadaver in the present study was classified as type I. This may be due to the fact that the individual was recorded as being an ethnic Taiwanese but had lived in Japan from childhood. These findings demonstrate that the methods of the present study adequately reflect both the geographic and latent origin of the cadavers analyzed. Among the viruses used to determine the geographic origin of the unidentified cadavers, JCV has been detected in 45% of kidney samples, BKV in 30.5% of blood samples, and VZV in almost 100% of trigeminal ganglia samples. The H. pylori DNA detection rate of the present study was 57.06%, which was lower than that of VZV but higher than that of JCV and EBV. However, H. pylori prevalence will tend to decrease by improvement of public health and eradication in the future. H. pylori DNA can be detected consistently, regardless of the postmortem interval, and compared with other organs, the gastric mucosa can be collected with a high degree of certainty, irrespective of the cause of death. These attributes suggest that H. pylori DNA resident in the stomach represents a new source of information, in addition to previously reported viruses. The present study is the first report on the medico-legal application of H. pylori genotype classification. It should be noted that the origin of an unidentified cadavers determined by the H. pylori strains detected with the present method cannot be assured, as there are limits to the degree of certainty with which geographical origin that can be ascertained. The method provides information that is valuable only for tracking the geographic origin. We recommend combining the present method with previously reported virus-based methods. However, this method meets the demands of an investigative team aiming to obtain a greater amount of information regarding the unidentifiedcadavers. Information on geographic origin is valuable since it differs from information on race that is provided by polymorphism analysis of the human genome. Genotyping of JCV is being used by the Japanese police in order to determine the geographic origin of unidentified cadavers. If combined with information regarding the genotypes of JCV, BKV, EBV, and VZV, information on the H. pylori genotype will provide additional valuable information for determining the geographic origin of unidentified cadavers. References [1] M. Grandi, H.J. Kaatsch, Unidentified cadavers and human remains in the EU: an unknown issue, Int. J. Legal Med. 113 (2000) N2–N3. [2] National Police Agency Homepage. 2011. http://www.npa.go.jp/ (accessed December 2011) [3] H. Ikegaya, H. Iwase, C. Sugimoto, Y. Yogo, JC virus genotyping offers a new means of tracing the origins of unidentified cadavers, Int. J. Legal Med. 116 (2002) 242– 245. [4] H. Ikegaya, H. Iwase, Trial for the geographical identification using JC viral genotyping in Japan, Forensic Sci. Int. 139 (2004) 169–172. [5] H. Ikegaya, P.J. Saukko, R. Tertti, K.P. Metsarinne, M.J. Carr, B. Crowley, K. Sakurada, H.Y. Zzheng, T. Kitamura, Y. Yogo, Identification of a genomic subgroup of BK polymavirus spread in European populations, J. Gen. Virol. 87 (2006) 3201–3208. [6] H. Ikegaya, H. Motani, P. Saukko, K. Sato, T. Alutsu, K. Sakurada, BK virus genotype distribution offers information of tracing the geographical origins of unidentified cadaver, Forensic Sci. Int. 173 (2007) 41–46. [7] H. Inoue, H. Motani-Saitoh, K. Sakurada, H. Ikegaya, D. Yajima, M. Hayakawa, Y. Sato, K. Otsuka, K. Kobayashi, H. Iwase, Determination of the geographical origin of unidentified cadavers based on geographical differences in genotype of varicella-zoster virus, J. Med. Virol. 82 (2010) 903–908. [8] H. Ikegaya, H. Iwase, P.J. Saukko, T. Akutsu, K. Sakurada, M. Yoshino, JC viral DNA chip allows geographical localization of unidentified cadavers for rapid identification, J. Med. Virol. 2 (2010) 54–60. [9] B.J. Marshall, J. Warren, Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration, Lanset 16 (1984) 1311–1315. [10] P.B. Ernst, B.D. Gold, Helicobacter pylori in childhood: new insights for managing infection in children, J. Pediatr. Gastroenterol. Nutr. 28 (1999) 462–473. [11] F. Megraud, M.P. Brassens-Rabbe, F. Denis, A. Belbouri, D.Q. Hoa, Seroepidemiology of Campylobacter pylori infection in various populations, J. Clin. Microbiol. 27 (1989) 1870–1873.

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