Helicobacter pylori Infection in Thailand: A Nationwide Study of the CagA Phenotype.

BACKGROUND: The risk to develop gastric cancer in Thailand is relatively low among Asian countries. In addition, the age-standardized incidence rate (ASR) of gastric cancer in Thailand varies with geographical distribution; the ASR in the North region is 3.5 times higher than that in the South region. We hypothesized that the prevalence of H. pylori infection and diversity of CagA phenotype contributes to the variety of gastric cancer risk in various regions of Thailand. METHODS: We conducted a nationwide survey within Thailand. We determined H. pylori infection prevalence by detecting H. pylori, using histochemical and immunohistochemical methods. The anti-CagA antibody and anti-East-Asian type CagA antibody (α-EAS Ab), which showed high accuracy in several East Asian countries, were used to determine CagA phenotype. RESULTS: Among 1,546 patients from four regions, including 17 provinces, the overall prevalence of H. pylori infection was 45.9% (710/1,546). Mirroring the prevalence of H. pylori infection, histological scores were the lowest in the South region. Of the 710 H. pylori-positive patients, 93.2% (662) were immunoreactive with the anti-CagA antibody. CagA-negative strain prevalence in the South region was significantly higher than that in other regions (17.9%; 5/28; p < 0.05). Overall, only 77 patients (11.6%) were immunoreactive with the α-EAS Ab. There were no differences in the α-EAS Ab immunoreactive rate across geographical regions. CONCLUSIONS: This is the first study using immunohistochemistry to confirm H. pylori infections across different regions in Thailand. The prevalence of East-Asian type CagA H. pylori in Thailand was low. The low incidence of gastric cancer in Thailand may be attributed to the low prevalence of precancerous lesions. The low incidence of gastric cancer in the South region might be associated with the lower prevalence of H. pylori infection, precancerous lesions, and CagA-positive H. pylori strains, compared with that in the other regions.

An improved method for the diatom test utilizing DNA binding ability of silica.

In order to devise a better forensic test for diatoms, the DNA binding ability of the diatom frustule constructing by silica, in the presence of chaotropic ions were utilized. It was proved that the diatoms were able to be captured via λDNA using silica-coated magnetic beads (Mag beads), followed by isolation and purification from the Mag beads as a solid phase by substituting the chaotropic agent with ultrapure water. Five cases of drowning, three in freshwater and two in seawater, were applied to the present method and similar results as the usual diatom test were obtained. Specimens of lung and other organs were rendered clearly visible, with elimination of foreign impurities. The present method appears applicable for detection of diatoms indirectly using PCR amplification of bound DNA or directly staining of the DNA.

A simple DNA coprecipitation method for the detection of diatoms in heart blood.

We developed a method for detecting and enumerating diatoms in the heart blood of drowning victims and evaluate its utility for diagnosing death by drowning. For purification of diatoms from blood, the DNA binding ability of the diatom frustule in the presence of a chaotropic agent was utilized. The procedure is basically the same as the commonly used method for DNA purification from blood using Proteinase K treatment and denaturation by a chaotropic agent. DNA adsorbed to the diatom (DNA/diatom complex) is recovered by ethanol precipitation, and the DNA is subsequently digested using DNase. Purified diatoms could be clearly observed under a microscope. Diatoms spiked in the blood of non-drowned cadavers (n=15) were well recovered, and were detected in heart blood from all drowning victims (n=22). The mean number of diatoms found in 5 ml of blood from drowning victims was 7.8±5.8 (mean±SD), and the number of diatoms detected in the blood of the left ventricle (6.1±5.8) was approximately two times higher than that of the right ventricle (3.0±2.7, p<0.05). These results suggest that this simple and safe method can become an effective tool for diagnosing the cause of death as drowning.

Whole-exome sequencing of a unique brain malformation with periventricular heterotopia, cingulate polymicrogyria and midbrain tectal hyperplasia.

We report a case of an infant with unique and unreported combinations of brain anomalies. The patient showed distinctive facial findings, severe delay in psychomotor development, cranial nerve palsy and seizures. Brain magnetic resonance imaging performed at 5 days of age revealed complex brain malformations, including heterotopia around the mesial wall of lateral ventricles, dysmorphic cingulate gyrus, and enlarged midbrain tectum. The patient unexpectedly died at 13 months of age. Postmortem pathological findings included a polymicrogyric cingulate cortex, periventricular nodular heterotopia, basal ganglia and thalamic anomalies, and dysmorphic midbrain tectum. Potential candidate genes showed no abnormalities by traditional PCR-based sequencing. Whole-exome sequencing confirmed the presence of novel gene variants for filamin B (FLNB), guanylate binding protein family member 6, and chromosome X open reading frame 59, which adapt to the autosomal recessive mode or X-linked recessive mode. Although immunohistochemical analysis confirmed the expression of FLNB protein in the vessel walls and white matter in autopsied specimens, there may be functional relevance of the compound heterozygous FLNB variants during brain development.

STR and mitochondrial DNA SNP typing of a bone marrow transplant recipient after death in a fire.

Personal identification of a house fire victim is described. About 5 years prior to death, the victim had been underwent bone marrow transplantation (BMT) with a graft from an unrelated donor as treatment for acute myelogenous leukemia. Clinically, the victim had been in remission at the time of death. Typing of STRs and sequencing of mitochondrial DNA (mtDNA) were performed using blood from the heart as well as several soft (psoas major muscle, uterine muscle and mucous membrane of the urinary bladder) and hard (costal cartilage and nail) tissues. STR genotypes and amelogenin from each of the tissue samples were successfully typed, and the parentage was identified. The blood STR types demonstrated no relationship with those from other tissues. None of the blood STR loci showed extra peaks arising from those of the recipient. Therefore, the blood stem cells were assumed to have been altered to those of the donor. The genotypes of mtDNA control regions were also examined. The electropherogram of hypervariable region II (nucleotide positions 29-408) obtained from the blood revealed a similar length heteroplasmy, suggesting microchimerism of the blood. Sequence analysis of mtDNA might be applicable as a more sensitive method for determination of chimerisms after BMT.

Analysis of virulence factors of Helicobacter pylori isolated from a Vietnamese population.

BACKGROUND: The incidence of gastric cancer differs among countries in Asia, and it has been suggested that virulence factors associated with Helicobacter pylori are partly responsible. The aim of this study was to investigate several genetic factors regarded as virulence or molecular epidemiologic markers in H. pylori isolates from Vietnamese subjects. RESULTS: The cagA, vacA and cag right-end junction genotypes of 103 H. pylori strains from Vietnam (54 from Hanoi and 49 from Ho Chi Minh) were determined by PCR and sequencing. Three types of deletion in the region located upstream of the cagA Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region were identified: the 39-bp deletion type, the 18-bp deletion type, and the no-deletion type. The majority of strains studied (77%; 80/103) had the 18-bp deletion irrespective of geographical location in the country or clinical outcome. All of the 39-bp and 18-bp deletion-type strains possessed the East Asian type cagA repeat region. The type II cag right-end junction genotype was predominant (84%). The vacA m1 genotype was significantly more common in strains isolated in Hanoi, where the incidence of gastric cancer is higher, than in strains from Ho Chi Minh. CONCLUSION: Pre-EPIYA-region typing of the cagA gene could provide a new genetic marker of H. pylori genomic diversity. Our data support the hypothesis that vacA m1 is closely associated with gastric carcinogenesis.

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type.

Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA-positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori-infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up-regulated by adhesion to epithelial cells.

Immunohistochemical diagnosis of the cagA-gene genotype of Helicobacter pylori with anti-East Asian CagA-specific antibody.

Cytotoxin-associated antigen A (CagA) protein produced by Helicobacter pylori is proposed to be associated with the pathogenesis of gastric cancer as well as gastritis and gastroduodenal ulcer. It has been reported that the CagA of H. pylori widespread in East Asian countries, where the mortality rate due to gastric cancer is high, is structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. In this study, we generated an antibody, East Asian CagA-specific antibody (alpha-EAS Ab), which is specifically immunoreactive with East Asian CagA but not with Western CagA. The CagA was immunohistochemically detected at the surface of the gastric mucosa. Interestingly, positive immunoreactivity was also detected in the nucleus and cytoplasm of the infected gastric epithelium, suggesting that CagA may play some pathogenic role in both the nucleus and cytoplasm. Immunohistochemistry of 47 gastric biopsy specimens detected East Asian CagA-positive H. pylori in 43 cases. In 46 of the 47 cases examined, the data obtained by immunohistochemistry were completely consistent with those obtained by sequencing of the cagA gene of the isolated strain, suggesting that our immunohistochemical method is reliable and useful for diagnosis of the cagA genotype.

Population data of six STR loci using hexaplex PCR amplification and typing system, "Midi-6" in four regional populations in Japan.

The genetic differences of the allele frequency distributions for six STR loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290) among regions in Japan were examined using our recently designed hexaplex amplification and typing system, "Midi-6" newly named, to construct a database in the Japanese population. Genotypes at six loci were analyzed in 198, 200, 175, and 196 individuals from the area of Akita, Nagoya, Oita, and Okinawa, respectively, in Japan. The allele frequency distributions were significantly different (p<0.05) at from one to five loci among the four populations when compared pairwise. Significant differences were also observed at two or three loci between Oita- or Okinawa-Japanese and the "pooled" population (n=769), respectively. However, since F(ST) (theta) values were extremely low (<0.05), ranging from 0.0020 to 0.0118 for six loci, genetic differentiation within the pooled Japanese population was negligible. Therefore, it suggested that the data of the allele frequencies at six loci in the pooled population would be employed as the base of calculation for statistical probabilities.