Molecular discrimination of Opisthorchis-like eggs from residents in a rural community of central Thailand.

BACKGROUND: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination. METHODOLOGY/PRINCIPAL FINDINGS: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR. CONCLUSIONS/SIGNIFICANCE: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite.

Development and evaluation of PCR methods based on cytochrome c oxidase subunit one (cox1) and NADH dehydrogenase subunit one gene (nad1) to detect Opisthorchis viverrini in human fecal samples.

Opisthorchis viverrini is highly prevalent throughout Southeast Asia. Chronic infection of this parasite leads to cholangiocarcinoma (CCA), a fatal bile duct cancer. The early and accurate detection of this parasite is very important; therefore, new PCR methods targeting cytochrome c oxidase subunit one and NADH dehydrogenase subunit one gene to detect O. viverrini in fecal specimens have been developed. Ninety O. viverrini-positive human fecal samples were used in this study. The PCR sensitivity of both genes was compared with internal transcribe spacer 2 (ITS2)-PCR. The sensitivity of cox1-PCR and nad1-PCR was 66.7 and 50 %, respectively. The sensitivity of cox1-PCR and nad1-PCR achieved 89.1 and 71.7 % in specimens containing O. viverrini eggs of >100 eggs per gram (EPG). Additionally, these primers can be used to provide the information on genetic diversity from mitochondrial genes of O. viverrini.

Incidence and risk factors of blastocystis infection in orphans at the Babies' Home, Nonthaburi Province, Thailand.

BACKGROUND: Blastocystis infection is one of the most common intestinal protozoan infections reported in Thai population of all age groups for which epidemiological information is important to understand patterns of transmission for developing methods ofprevention and control for each specific group. The authors aimed to study prevalence, incidence and riskfactors associated with Blastocystis infection in orphans and childcare workers. Additionally, subtypes ofBlastocystis were identified MATERIAL AND METHOD: A retrospective cohort study of Blastocystis sp. was conducted in orphans aged less than 5 years and their childcare workers at Babies' Home, Nonthaburi Province, Thailand. A base line survey was conducted in December 2009 and afollow-up survey was conducted in April 2010. A total of 336 and 331 stool samples were collected. Blastocystis infection was examined using short-term in vitro cultivation in Jones's medium supplemented with 10% fetal calf serum. To analyze subtypes ofBlastocystis sp., PCR-RFLP of the small subunit ribosomal RNA gene was performed. RESULTS: Theprevalence ofBlastocystis infection in December 2009 and April 2010 were 8.1% and 13.3%, respectively The inlcidence rate ofBlastocystis infection was 1.6/100 person-months. Subtype analysis ofBlastocystis sp. in December 2009 and in April 2010 showed that subtype 3 was the most predominant (76% and 76%), followed by subtype 1 (16% and 20%), and unidentified subtype (8% and 4%), respectively. Subtype 3 is of human origin, thus person-to-person transmission is considered a major route ofBlastocystis infection in this population. CONCLUSION: Person-to-person transmission of Blastocystis infection in orphans living in the same house had been proposed, thus the prevalence and incidence of Blastocystis infection could be used to reflect the hygienic condition in the orphanage. Infection prevention and control practice can be effectively implemented.

A follow-up study of Opisthorchis viverrini infection after the implementation of control program in a rural community, central Thailand.

BACKGROUND: Opisthorchis viverrini infection is still one of the public health problems in Thailand. Our recent cohort study conducted in a rural community in central Thailand showed that the incidence rate of O. viverrini infection in 2002-2004 was 21.6/100 person-years. Conventional control activities including case diagnosis and treatment, hygienic defecation promotion and health education focusing on avoiding raw fish consumption was implemented. This study aimed to re-assess the status of infection after implementation of intervention programs, using both quantitative and qualitative methods in 2007-2009. METHODS: A prospective cohort study was conducted to evaluate the incidence and risk factors of O. viverrini infection. Stool examination methods including wet preparation, Kato and formalin-ethyl acetate concentration technique were performed for the detection of O. viverrini eggs. A standardized questionnaire was used to assess risk behavior. In addition, qualitative information was collected from both O. viverrini negative and positive villagers using focus group discussions. RESULTS: The incidence of O. viverrini infection was 21.4/100 person-years. Consumption of chopped raw fish salad, Koi pla and age 60 years and older were independently associated with O. viverrini infection, similar to our previous study. Findings from the qualitative study, indicated that inadequate knowledge, misbeliefs, and social and cultural mores were important factors leading to the maintenance of risk behaviors. Moreover, unhygienic defecation and insufficient diagnosis and treatment were found to facilitate O. viverrini transmission. CONCLUSION: Although the conventional control program had been used in the study population, the incidence of O. viverrini infection remained the same. Precise and regular health education and promotion targeting the main risk factor, Koi pla consumption, improving diagnosis and treatment, and promoting hygienic defecation should be used in the prevention and control program.

Incidence and risk factors of hookworm infection in a rural community of central Thailand.

A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2-14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9-11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.

Assessment of the association between Blastocystis infection and irritable bowel syndrome.

OBJECTIVE: To assess the association between Blastocystis infection and irritable bowel syndrome (IBS) in Thai patients, Phramongkutklao Hospital. MATERIAL AND METHOD: A case-control study was conducted at Phramongkutklao Hospital, Bangkok, Thailand during 2007-2008. A total of 126 subjects were enrolled into the study. Sixty-six persons were enrolled in the IBS group and 60 persons were enrolled in the control group. Intestinal parasitic infections were determined using wet preparation and formalin-ethyl acetate concentration. Short-term in vitro culture was performed to detect Blastocystis sp. Additionally, stool specimens were cultured for pathogenic bacteria. Patients' history and physical examination were also recorded. RESULTS: Of 66 IBS patients, 61 (92.4%) had abdominal pain, 50 (78.1%) had diarrhea, and 14 (21.9%) had constipation. In vitro cultivation revealed that 10.0% and 16.7% were positive for Blastocystis sp. in the control and IBS group, respectively. There was no significant difference of the prevalence of Blastocystis infection between these 2 groups (p = 0.203). CONCLUSION: Blastocystis infection was a common parasitic infection which was predominant in both IBS and control group. The presence of Blastocystis sp. in stool did not imply gastrointestinal symptoms in the IBS patients. Further study of subtype characterization of Blastocystis sp. in a larger sample size of the case-control study might reveal a possible relationship between Blastocystis sp. and IBS patients.

PBS incubation, a simple technique to release miracidia of Opisthorchis-like eggs for DNA extraction.

This study describes a simple technique for releasing miracidia from Opisthorchis-like eggs before DNA extraction by keeping PBS washed specimens at 4 degrees C for two days. Using PCR amplification, the sensitivity of the PBS incubation technique was as good as that obtained from freeze-thaw technique. Moreover, this low-cost technique is less laborious.

Incidence and risk factors of Opisthorchis viverrini infections in a rural community in Thailand.

Opisthorchis viverrini infection is one of the major public health problems in Thailand. Although information about transmission of this parasite to humans is well established, infections are still prevalent in the northern and northeastern Thailand. Thus, prospective epidemiologic information is needed for developing more effective public health interventions. A cohort study to identify incidence and risk factors of O. viverrini infection was conducted in a rural community in Thailand during December 2002-February 2004. The incidence rate of opisthorchiasis was 21.6/100 person-years. The independent factors associated with opisthorchiasis were an age > 60 years and consuming chopped raw fish salad (Koi pla). Thus, avoiding this raw fish salad should be emphasized in the national control program.

Genotypic characterization of Enterocytozoon bieneusi in specimens from pigs and humans in a pig farm community in Central Thailand.

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.

Prevalence of intestinal parasitic infections in military personnel and military dogs, Thailand.

OBJECTIVE: To determine the prevalence of intestinal parasitic infections and risk factors among military personnel and military dogs at the Military Dog Center, Veterinary and Remount Department, Royal Thai Army, Thailand. MATERIAL AND METHOD: A cross-sectional study was conducted in January 2006 to examine intestinal parasitic infections using wet preparation and, formalin-ethyl acetate concentration. Modified acid fast and gram-chromotrope stains were used to identify Cryptosporidium spp. and microsporidia, respectively. Culture for Blastocystis was performed using Jone's medium. Genotypic characterization of Blastocystis and Giardia duodenalis were also determined using PCR-RFLP. To determine the risk factors and outcomes of intestinal parasitic infections, standardized questionnaires were used in the present study. RESULTS: Of 317 military personnel, the prevalence of intestinal parasitic infections was 22.4%. Blastocystis was the most predominant intestinal protozoa infection of 14.5% while G. duodenalis was only 1.3%. The prevalence of other helminthic infections were 4.8% which were Strongyloides stercoralis (2.5%), Hookworm (1.0%), Opisthorchis viverrini (1.0%), and Taenia spp. (0.3%), respectively. Blastocystis subtype 1 was identified in 25 positive culture specimens while all 4 positive of G. duodenalis were analyzed as Assemblage B, subgenotype IV. The presented data could not indicate that intestinal parasitic infections and blastocystosis in this army population were significantly linked to risk association among groups with regard to rank, age group, working unit, area of residence, animal contact, source and treatment of drinking water. Of 189 military dogs, the prevalence of intestinal parasitic infections was only 3.7% which was Blastocystis sp. (2.6%), S. stercoralis (0.5%), and Entamoeba coli (0.5%), respectively. CONCLUSION: The predominant intestinal parasites found in this population, such as Blastocystis sp. and G duodenalis transmit to humans via fecal-oral route so that improvement of sanitation and personal hygiene should be emphasized.

Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand.

In January 2005, a survey of intestinal parasitic infections was performed in a primary school, central Thailand. Of 675 stool samples, Blastocystis was identified with a prevalence of 18.9%. Genetic characterization of Blastocystis showed subtype 1 (77.9%) and subtype 2 (22.1%). Study of the water supply in this school was performed to find the possible sources of Blastocystis. Blastocystis from one water sample was identified as subtype 1, which had a nucleotide sequence of small subunit (SSU) ribosomal RNA (rRNA) gene that was 100% identical to that of Blastocystis infected in schoolchildren. Our information supports the evidence of water-borne transmission in this population.

Multiple modes of transmission of giardiasis in primary schoolchildren of a rural community, Thailand.

In February 2005, we conducted a cross-sectional study to determine the prevalence and the risk factors of giardiasis in 531 primary schoolchildren of a rural community, Chacheongsao province, Thailand. Using both sedimentation and flotation techniques to detect Giardia duodenalis, the prevalence of giardiasis was 6.2%. Assemblage A, subgenotype II and assemblage B, subgenotype IV were identified by PCR-RFLP of glutamate dehydrogenase gene. Our data might indicate that, in this population, only assemblage A, subgenotype II of G. duodenalis was transmitted via water. Using multivariate analysis, significant risk factors for giardiasis were children of age 5-9 years, households with > or = 3 children under the age of 12 years, low parental educational level, drinking bottled water, and living in close contact with dogs. Washing hands before meals had a protective effect. From these significant risk factors, multiple modes of transmission of G. duodenalis were suggested in this population.

Teaching medical ethics: Phramongkutklao College of Medicine's model.

BACKGROUND: Third-year (first preclinical year) Royal Thai Army (RTA) medical cadets were exposed to a rural community in a community medicine field activity. The poor, kind rural people gave good conditions for student's professional development and empathy. OBJECTIVES: To report a community medicine field work activity for professional development of the RTA medical school. MATERIAL AND METHOD: Descriptive analysis on two occasions to evaluate the third-year RTA medical cadets' impressions immediately after the community medicine field activity in a rural area in central Thailand and one year later using baseline scores of cadets' impressions against our institute's objectives. RESULTS: At the end of the field work, 90.9% of the cadets reported they achieved several institute objectives. The four most frequent mentioned were ethical development (70.6%), professionalism (84.3%), human relationship & communication skills (92.2%), and capability to work in the cmmunity (64.7%). Furthermore, RTA medical cadets rated the staff as good role models (51%). CONCLUSION: The community field work course provided a good environment for the third year medical cadets in building morality and professional development to be good doctors.

The usefulness of short-term in vitro cultivation for the detection and molecular study of Blastocystis hominis in stool specimens.

When in vitro cultivation was used as the 'gold standard' for the detection of Blastocystis hominis in stool specimens, simple smear and trichrome staining showed sensitivities of 16.7% and 40.2% and specificities of 94% and 80.4%, respectively. In vitro cultivation also enhanced PCR amplification for the detection of B. hominis in stool specimens. Our data show the usefulness of in vitro cultivation for the detection and molecular study of B. hominis in stool specimens.

Evidence of waterborne transmission of Blastocystis hominis.

A cross-sectional study was performed in February 2001 to evaluate the prevalence and risk factors of Blastocystis hominis infection in army personnel who resided in an army base in Chonburi, Thailand. A total of 904 army personnel were enrolled in this study. Short-term in vitro cultivation was used to detect B. hominis in stool samples. In this population, B. hominis was the parasite most frequently found, and was identified in 334 of 904 stool specimens (36.9%). A significant association between B. hominis infection and symptoms was identified that might emphasize the role of B. hominis as a human pathogen. After adjustment for potential confounders, significantly increased risk of being infection with B. hominis was associated with being a private, working in a specific unit, and consuming unboiled drinking water. Thus, waterborne transmission of B. hominis infection was indicated at this army base. However, other modes of transmission cannot be ruled out.

Prevalence of Blastocystis hominis carriage in Thai army personnel based in Chonburi, Thailand.

Blastocystis hominis is one of the most common intestinal parasites found in humans. The prevalence of B. hominis carriage in personnel who worked in the First Army Support Command, Chonburi, Thailand, was determined. Forty-four percent of 616 stool samples were positive for B. hominis using both simple smear and concentration (Formalin/ethyl acetate) techniques. The usefulness of the concentration technique in the diagnosis of B. hominis has been confirmed. Of 272 positive specimens, 75% were detected by simple smear and 25% were additionally identified by the concentration technique. The prevalence of B. hominis carriage was highest among the privates who had lived longest in this command. This finding indicates that they might have acquired B. hominis during their stay at this base. The significantly high prevalence of B. hominis carriage was found in the Ordnance Ammunition Battalion. In this battalion, privates who had education lower than the secondary school level had significantly greater risk of B. hominis carriage.