Study of the population genetic structure of Opisthorchis-like eggs in northern Thailand using mitochondrial genes.

BACKGROUND: Opisthorchis-like eggs are a public health problem in northern and northeastern Thailand. However, the genetic epidemiology and structure of these parasites in northern Thailand are unknown. Thus, this study investigated their population genetic structure using cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) nucleotide sequences. METHODOLOGY/PRINCIPAL FINDINGS: A study was conducted in the hill tribe regions of Chiang Mai Province, northern Thailand. Internal transcribed spacer 2 polymerase chain reaction and restriction fragment length polymorphism were used to distinguish 205 positive feces samples for Opisthorchis-like eggs. The results showed that the prevalence of O. viverrini and Haplorchis taichui was 10.5% and 38.2%, respectively, and the co-infection rate was 37.2%. To determine the genetic structure of O. viverrini and H. taichui using cox1 and nad1 genes, genetic analysis was performed using 30 randomly chosen fecal samples for Opisthorchis-like eggs. Pairwise FST analysis indicated that O. viverrini and H. taichui displayed nonsignificant genetic differentiation within Chiang Mai Province and between interpopulations from different geographic areas. Moreover, within the intrapopulation in Chiang Mai Province, cox1 presented higher gene flow than nad1 in O. viverrini, while nad1 demonstrated higher gene flow than cox1 in H. taichui. The neutrality tests based on Fu's Fs indicated population expansion and selective sweep from bottleneck or hitchhiking in O. viverrini and H. taichui populations, supported by haplotype network patterns. Phylogenetic tree analysis based on cox1 and nad1 revealed the monophyly of O. viverrini and H. taichui and genetic relationships with other isolates collected from Thailand, Lao People's Democratic Republic (PDR), and Vietnam. CONCLUSIONS/SIGNIFICANCE: This study investigated the molecular discrimination and genetic structure of Opisthorchis-like eggs in northern Thailand. The genetic information derived from this study could be associated with the background, molecular epidemiology, and disease severity of these parasites.

Comparison of stool examination techniques to detect Opisthorchis viverrini in low intensity infection.

Opisthorchiasis, caused by Opisthorchis viverrini, remains the public health significance in Thailand, particularly in the northeastern region. Number of parasitological techniques is available for diagnosis. However, the detection the parasite's eggs in stool still referred as gold standard. Today, most people living in the endemic areas harbored the light infection. In this study, we compared the performance of formalin-ethyl acetate concentration technique (FECT), Kato-Katz technique, fecal parasite concentrator kit (FPCK) and direct simple smear technique for O. viverrini egg examination in stool. The results revealed that the FECT gave the best sensitivity (91.0%) followed by Kato-Katz technique, FPCK and direct simple smear techniques. Interestingly, the FECT showed the highest sensitivity in both groups of egg per gram (EPG) <50 (94.3%) and EPG ≥ 50 (100%). The FPCK and direct simple smear technique exhibited the higher sensitivity in EPG ≥ 50 group compared with EPG < 50 group (p < 0.05). However, the sensitivity of FPCK was not statistically differed from that direct simple smear in EPG <50 and EPG ≥50 group. In conclusion, the FECT showed the highest efficiency to detect O. viverrini egg in stool, followed by Kato-Katz technique, FPCK and direct simple smear technique. Substituting FPCK (Mini Parasep® Kit) for FECT and Kato-Katz method is not recommended for low intensity infection.

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.

Identification of genotypes of Enterocytozoon bieneusi from stool samples from human immunodeficiency virus-infected patients in Thailand.

We identified genotypes of Enterocytozoon bieneusi from 33 stool samples of Thai human immunodeficiency virus (HIV)-infected adult patients. Genotype D was identified at the highest frequency (36.4%), while genotype E was the second most common (15.1%). Genotypes O and PigEBITS 7, previously found only in pigs, were observed in Thai HIV-infected patients. Phylogenetic analysis supported a zoonotic nature for E. bieneusi.

Transmission of Enterocytozoon bieneusi genotype a in a Thai orphanage.

A cross-sectional study of Enterocytozoon bieneusi infection in children who lived in an orphanage in Bangkok, Thailand was conducted in April 2003. Two hundred ninety stool specimens were collected and examined under light microscopy after staining with gram-chromotrope. Confirmation of E. bieneusi was done using transmission electron microscopy. Of 290 samples, 12 (4.1%) were positive for E. bieneusi. Genotypic characterization of 10 E. bieneusi showed that all were genotype A, which might indicate the same source of infection. Multivariate analysis showed that orphans who were 12-23 months old, girls, and living in one particular house were independently associated with E. bieneusi infection. Our study suggests that E. bieneusi infection in this orphanage might be transmitted person to person.

Spore shedding pattern of Enterocytozoon bieneusi in asymptomatic children.

Stool samples from seven human immunodeficiency virus (HIV)-negative and two HIV-positive children with asymptomatic Enterocytozoon bieneusi infections were daily examined to quantify spore shedding using Gram-chromotrope staining under light microscopy. The spore shedding pattern and intensity in these children was variable. Mean spore concentrations in the stool samples from these children ranged from 2.4 x 10(2) to 1.2 x 10(5) spores per gram. Light microscopy could detect spores in stool specimens for 9-33 days, while PCR was able to detect E. bieneusi in stool specimens for 3-40 days longer. This suggests that light microscopy may not detect low levels of spore shedding. Considering that the asymptomatic group are a potential source of infection, detection methods with a higher sensitivity should be used.

Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens.

An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.