Natthawut Kaewpitoon

Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

Background The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Methodology/Principal Findings Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of ∼3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size. Conclusions/Significance A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Prevalence of Opisthorchis viverrini Infection in Nakhon Ratchasima Province, Northeast Thailand

BACKGROUND Opisthorchis viverrini infection is a serious public-health problem in Southeast Asia especially in Lao PDR and Thailand. It is associated with a number of hepatobiliary diseases and the evidence strongly indicates that liver fluke infection is the major etiology of cholangiocarcinoma. OBJECTIVES This study aimed to determine actual levels of Opisthorchis viverrini infection in Nakhon Ratchasima province, Northeast Thailand. METHODS A cross-sectional survey was conducted during a one year period from October 2010 to September 2011. O. viverrini infection was determined using a modified Katos thick smear technique and socio-demographic data were collected using predesigned semi-structured questionnaires. RESULTS A total of 1,168 stool samples were obtained from 516 males and 652 females, aged 5-90 years. Stool examination showed that 2.48% were infected with O. viverrini. Males were slightly more likely to be infected than females, but the different was not statistically significant. O. viverrini infection was most frequent in the 51-60 year age group and was found to be positively associated with education and occupation. Positive results were evident in 16 of 32 districts, the highest prevalence being found in Non Daeng with 16.7%, followed by Pra Thai with 11.1%, Kaeng Sanam Nang with 8.33%, and Lam Ta Men Chai (8.33%) districts. CONCLUSION This study indicates that O viverrini is still a problem in some areas of Nakhon Ratchasima, the patients in this study bing suitable for the purpose of monitoring projects.

Review and Current Status of Opisthorchis viverrini Infection at the Community Level in Thailand

Opisthorchis viverrini is remains a public health problem in Thailand, particularly in the northeast and north regions which have the highest incidences of chonalgiocarcinoma (CCA). O. viverrini causes the disease opithorchiasis, and its has been classified as a group 1 biological carcinogen. Humans, dogs, and cats become infected with O. viverrini by ingesting raw or undercooked fish containing infective metacercariae. The first human cases of O. viverrini infection were reported in Thailand 100 years ago, and its still a problem at the community level. Based on data for the year 2009, more than 6 million people were infected with O. viverrini. Associated medical care and loss of wages in Thailand costs about

Improved Detection of Helicobacter pylori Infection and Premalignant Gastric Mucosa Using "Site Specific Biopsy": a Randomized Control Clinical Trial.

120 million annually. This review highlights the current status of O. viverrini infection in communities of Thailand through active surveillance for the five years period from 2010 and 2015. A total of 17 community-based surveys were conducted, most in the northeast region. Some 7 surveys demonstrated a high prevalence over 20%, and the highest was 45.7%. Most commonly infection was found in age group of 35 years and older, males, and agricultural workers. Although, the national prevalence may be decreasing but the results show that the O. viverrini infection is still high in communities of the northeast region. Therefore, the focus in populations living in northeast Thailand should be screening of infection and changing their eating behavior.

Improved Helicobacter pylori Eradication Rate of Tailored Triple Therapy by Adding Lactobacillus delbrueckii and Streptococcus thermophilus in Northeast Region of Thailand: A Prospective Randomized Controlled Clinical Trial

BACKGROUND Helicobacter pylori infection and premalignant gastric mucosa can be reliably identified using conventional narrow band imaging (C-NBI) gastroscopy. The aim of our study was to compare standard biopsy with site specific biopsy for diagnosis of H. pylori infection and premalignant gastric mucosa in daily clinical practice. MATERIALS AND METHODS Of a total of 500 patients who underwent gastroscopy for investigation of dyspeptic symptoms, 250 patients underwent site specific biopsy using C-NBI (Group 1) and 250 standard biopsy (Group 2). Sensitivity, specificity, and positive and negative predictive values were assessed. The efficacy of detecting H. pylori associated gastritis and premalignant gastric mucosa according to the updated Sydney classification was also compared. RESULTS In group 1 the sensitivity, specificity, positive and negative predictive values for predicting H. pylori positivity were 95.4%, 97.3%, 98.8% and 90.0% respectively, compared to 92.9%, 88.6%, 83.2% and 76.1% in group 2. Site specific biopsy was more effective than standard biopsy in terms of both H. pylori infection status and premalignant gastric mucosa detection (P<0.01). CONCLUSIONS Site specific biopsy using C-NBI can improve detection of H. pylori infection and premalignant gastric mucosa in daily clinical practice.

Correlation between Gastric Mucosal Morphologic Patterns and Histopathological Severity of Helicobacter pylori Associated Gastritis Using Conventional Narrow Band Imaging Gastroscopy

Background and Aim. To evaluate the effect of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to Helicobacter pylori eradication in different periods of therapeutic protocol. Methods. Infected patients were randomized to one-week tailored triple therapy (esomeprazole 20 mg bid, clarithromycin 500 mg bid/metronidazole 400 mg tid if clarithromycin resistant, and amoxicillin 1000 mg bid) with placebo (group 1, n=100); one week of pretreatment with probiotics (group 2, n=100); and one week of pretreatment with probiotic followed by one week of the same probiotics after treatment (group 3, n=100). Result. PP analysis involved 292 patients, 98 in group 1, 97 in group 2, and 97 in group 3. Successful eradication was observed in 229 patients; by PP analysis, the eradication rates were significantly higher (P<0.01, 95% CI; 0.71–0.97) in group 2 and group 3 than group 1. ITT analysis eradication rates were significantly higher in group 2 and group 3 than group 1 (P<0.01 95% CI; 0.72–0.87), and there is no significant difference between the three groups (P=0.32) in terms of adverse events. Conclusion. Adding probiotics before or before and after tailored treatment can improve Helicobacter pylori eradication rates. This trial is registered with Thai Clinical Trials Registry number: TCTR20141209001.

GIS-Based Spatial Statistical Analysis of Risk Areas for Liver Flukes in Surin Province of Thailand

Background and Aim. Identifying specific gastric mucosal morphologic patterns useful for detecting Helicobacter pylori associated gastritis and correlation with histopathological severity. Methods. The endoscopists classified the C-NBI gastroscopic findings into 5 gastric mucosal morphologic patterns as follows: type 1: regular arrangement of collecting venules, type 2: cone-shaped gastric pits, type 3: rod-shaped gastric pits with prominent sulci, type 4: ground glass-like morphology, and type 5: dark brown patches with bluish margin and irregular border. Biopsies of all of the cases were then evaluated by 5 pathologists for definitive Helicobacter pylori diagnosis. Result. Type 1 and type 2 patterns were statistically significant in predicting Helicobacter pylori negative status (58/60, P < 0.01). Type 3, type 4, and type 5 patterns were statistically significant in predicting Helicobacter pylori positive status (132/140, P < 0.01). Furthermore, the sensitivity, specificity, and positive and negative predictive values of type 3, 4, or 5 morphologies for predicting Helicobacter pylori positive were 94.28%, 96.66%, 98.50%, and 87.87%, respectively, correlated well with inflammation grading according to the Sydney classification (P < 0.01). Conclusion. Our study suggests that gastric mucosal morphologic patterns in the Helicobacter pylori infected gastric mucosa can be reliably identified using C-NBI gastroscopy with good correlation with inflammation grading.

The Carcinogenic Liver Fluke Opisthorchis viverrini among Rural Community People in Northeast Thailand: a Cross-Sectional Descriptive Study using Multistage Sampling Technique

It is urgently necessary to be aware of the distribution and risk areas of liver fluke, Opisthorchis viverrini, for proper allocation of prevention and control measures. This study aimed to investigate the human behavior, and environmental factors influencing the distribution in Surin Province of Thailand, and to build a model using stepwise multiple regression analysis with a geographic information system (GIS) on environment and climate data. The relationship between the human behavior, attitudes (<50%; X111), environmental factors like population density (148-169 pop/km2; X73), and land use as wetland (X64), were correlated with the liver fluke disease distribution at 0.000, 0.034, and 0.006 levels, respectively. Multiple regression analysis, by equations OV=-0.599+0.005(population density (148-169 pop/km2); X73)+0.040 (human attitude (<50%); X111)+0.022 (land used (wetland; X64), was used to predict the distribution of liver fluke. OV is the patients of liver fluke infection, R Square=0.878, and, Adjust R Square=0.849. By GIS analysis, we found Si Narong, Sangkha, Phanom Dong Rak, Mueang Surin, Non Narai, Samrong Thap, Chumphon Buri, and Rattanaburi to have the highest distributions in Surin province. In conclusion, the combination of GIS and statistical analysis can help simulate the spatial distribution and risk areas of liver fluke, and thus may be an important tool for future planning of prevention and control measures.

Effect of Pretreatment with Lactobacillus delbrueckii and Streptococcus thermophillus on Tailored Triple Therapy for Helicobacter pylori Eradication: A Prospective Randomized Controlled Clinical Trial

Opisthorchis viverrini infection is a serious public health problem in Southeast Asia especially in the northeast and north of Thailand. Therefore, a cross-sectional survey using multistage sampling was conducted from the rural communities of Surin province, Thailand, during September 2013 to July 2014. O. viverrini infection was determined using Katos thick smear technique. Socio-demographic, information resources, and history data were collected using predesigned semi-structured questionnaires. A total of 510 participants completed interviews and had stools collected. Some 32 (6.47%) participants were infected with O. viverrini. The rate was slightly higher in males (6.61%) than females (6.32%). High frequencies were found in the age groups 61-70 (19.4%) and 71-80 years (19.4%), those involved in agriculture (10.5%), and in primary school (10.3%). The distribution of high infection was found in Tha Tum (16.7%) and Sankha district (16.7%), followed by Samrong Thap (13.3%), Si Narong (13.33%), and Buachet district (13.33%). Chi-square testing indicated that age (61-70 and 71-80 year old), education (primary school) and occupation (agriculture), were significantly associated with O. viverrini infection (p-value<0.05). Of 72.6% participants who had past histories with stool examination, 17.0% of them had been infected with O. viverrini and 43.2% treated with praziquantel. This finding confirmed that O. viverrini is still a problem in Surin province, Thailand, and therefore, interventions are urgently required for mass treatment and health education implementation.

Detection of the Carcinogenic Liver Fluke Opisthorchis viverrini Using a Mini Parasep SF Faecal Parasite Concentrator

BACKGROUND Helicobacter pylori plays an important role in gastric cancer and typical eradication regimens are no longer effective in many countries, including Thailand. The aim of our study was to compare the effect of Lactobacillus delbrueckii and Streptococcus thermophillus on tailored triple therapy for Helicobacter pylori eradication. MATERIALS AND METHODS This prospective single-center study was conducted in Thailand. Helicobacter pylori associated gastritis patients were randomized to 2 groups: group 1 (n=100) was tailored triple therapy with placebo (esomeprazole 20 mg bid, clarithromycin 500 mg bid or metronidazole 400 mg tid if clarithromycin resistance and amoxicillin 1000 mg bid), and group 2 was tailored triple therapy plus pretreatment with probiotic containing yogurt. Successful eradication was defined as both negative histology and negative rapid urease test at four weeks after treatment. RESULTS A total of 200 infected patients were enrolled. PP analysis involved 194 patients: 96 in the tailored triple therapy with placebo group (group 1) and 98 the in tailored triple therapy plus pretreatment with probiotic containing yogurt group (group 2). Successful eradication was observed in 170 (87.6%) patients; by PP analysis, the eradication rate was significantly higher in group 2 (P=0.04, 95%CI; 0.02-0.13) than in group 1. ITT analysis also showed that the value was significantly higher in the tailored triple threapy plus pretreatment with probiotic containing yogurt group (group 2) (89/100; 89%) than in the tailored triple therapy with placebo group (group 1) (P=0.01, 95%CI; 0.04-0.15). In terms of adverse events, there was no significant difference between the two groups. CONCLUSIONS Pretreatment with probiotic containing yogurt can improve Helicobacter pylori eradication rates with tailored triple therapy. Adding probiotics does not reduce adverse effects of the medication.