Forensic Science International 229 (2013) 712

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Forensic Science International

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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 unidentied cadavers by determining the genotype
of Helicobacter pylori, which is latent in one-half of the worlds population, was developed. In the rst
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 signicant differences in H. pylori detection rate among the
5 sampling points of the gastric mucosa, cause of death, or age. In the second stage, amplied 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 unidentied 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 specicity to geographical origin. Therefore, these results suggest that the H. pylori
genome provides valuable additional information for tracing the geographical origin of unidentied
cadavers.
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 unidentied cadavers. This is an important forensic
problem. In Japan, 1000 or more deceased individuals are not
identied upon death every year, giving a total of more than 16,000
unidentied deceased individuals [1]. Following the Great East
Japan Earthquake and tsunami in 2011, about 3000 people remain
missing and there are about 300 unidentied bodies as of June
2012. In 8 European nationsDenmark, Finland, Greece, Ireland,
Portugal, Luxembourg, Spain, and Germany, there were 3035
unidentied 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: nagasawa.s@graduate.chiba-u.jp, nagasawa.s@chiba-u.jp,
sayaka.30oct.19honey@gmail.com (S. Nagasawa).
0379-0738/$ see front matter 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 identied and 807

remain unidentied [2].
As methods of personal identication, ngerprints, 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 identication becomes difcult
and results in the case remaining unsolved. Therefore, methods
that can quickly and easily narrow down the possible geographic
origins of unidentied 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

S. Nagasawa et al. / Forensic Science International 229 (2013) 712

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 worlds
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 unidentied 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 nal
step.
2.3. Polymerase chain reaction (PCR) amplication
Extracted DNA was amplied using the primers of 16S-rDNA which listed in
Table 1. DNA samples which had been puried from cultivated H.plyori extracted
from the patients 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). Amplication 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 nal 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 conrm the presence of H. pylori, samples that were positive
for H. pylori on PCR were subjected to sequencing and conrmed H. pylori using an
ABI PRISM1 310 Genetic Analyzer (Applied Biosystems).
For the vacA s- and m-regions, amplications were carried out using primers
listed in Table 1 that were designed using DNASIS1 pro (Hitachi, Software,
Yokohama, Japan). Amplication 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 nal 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 nal concentration of 0.25 pmol/ml. The
amplication 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 puried using a Quick step 2 PCR Purication
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 classied as unknown, but these were estimated to be 3090 years old.

S. Nagasawa et al. / Forensic Science International 229 (2013) 712

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

PCR/sequencing

Primers

Primer sequence

Reference

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
Spearmans correlation coefcients.

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 nding of 50% rates reported in previous epidemiological studies. The inuence 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. Spearmans
correlation coefcients showed no statistically signicant 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 mummied
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
ndings on autopsy. Gastric ndings comprised macroscopic
ndings during autopsy and overall ndings obtained from
biochemical, histopathological and other laboratory tests performed at autopsy. As shown in Table 3, samples were collected
from 28 individuals showing gastric ndings, 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 ndings
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 ndings and H.
pylori was not elucidated, it is obvious that H. pylori DNA was
detectable in over 50% of samples that had no gastric ndings 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 amplied by

PCR. Samples were designated as positive for H. pylori if the 16S-rDNA region was
detected from at least 1 site.

S. Nagasawa et al. / Forensic Science International 229 (2013) 712

10

Table 3
Comparison of H. pylori-positive status for cases with and without gastric ndings.a
Number
of cases
Finding
Submucosal
hemorrhage or
change in color
Ulcer
Perforation
Tumor
No ndings
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 ndings comprised macroscopic ndings during autopsy and overall
ndings 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. Spearmans correlation coefcient showed no statistically
signicant 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 amplied 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 classied 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 classied 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 classied 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 difcult 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 amplication and the anti-H. pylori IgG antigen
detection methods. In this study, therefore, we used PCR
amplication 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 trafc 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 mummied 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
inuence of age was greater than the inuence of postmortem
interval. These ndings conrm that the condition of the cadaver
has only a minimal inuence 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 [2831]. However, the present study is
the rst to review actual gastric ndings and H. pylori DNA
detection. Gastric ndings 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 ndings 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 ndings, 80 (54.7%) were found to carry H. pylori DNA,
while no gastric ndings were observed in 53.6% of individuals
carrying H. pylori. These ndings show that H. pylori DNA is
detected at high rates, even in the absence of gastric ndings,
which in turn suggests the importance of gastric mucosal sampling
in the analysis of H. pylori DNA, irrespective of gastric ndings.
In 1990, a new system of classication, known as the Sydney
System, was announced at the World Congress of Gastroenterology in Sydney, Australia [32]. As endoscopy has been gaining in

S. Nagasawa et al. / Forensic Science International 229 (2013) 712

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 signicant 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 ndings. These ndings 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 ndings.
The 16S-rDNA region was used to ascertain the geographic
origin of the cadavers. In order to effectively estimate the
geographic origin of unidentied cadavers, it is necessary to use
a gene that has a high detection rate, narrow detection region in
bps, and has high region specicity. Previously, Falush et al.
reported that H. pylori can be broadly classied 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 regionspecicity has not been described. We, therefore, applied the DNA
polymorphisms of the vacA gene for which region-specicity 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 classied 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 classied as type I, the single Thai cadaver was
classied as type III, and the single Afghan and Filipino-Western
cadavers were classied as type II. Filipinos are typically classied
in the type III cluster, but the background details of the sample
examined in the present study are unknown. The Western-type
classication in the present case may be due to previous
generations of Filipinos being infected with Western-type strains

11

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 classied 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 ndings demonstrate
that the methods of the present study adequately reect both the
geographic and latent origin of the cadavers analyzed.
Among the viruses used to determine the geographic origin of
the unidentied 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 rst report on the medico-legal application
of H. pylori genotype classication. It should be noted that the
origin of an unidentied 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 unidentiedcadavers.
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 unidentied 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 unidentied cadavers.
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