Tippayarat Yoonuan

Copro-molecular identification of infections with hookworm eggs in rural Lao PDR.

The prevalence of hookworm eggs in fecal samples, by modified cellophane thick-smear technique, was found to be 30.0% (61/203 samples) in Lahanam Village, Savannakhet Province, Lao PDR. The hookworm eggs were morphologically heterogeneous, so that identification was confirmed by copro-PCR with specific primers for hookworms and Trichostrongylus spp.; 12 samples were positive for Necator americanus, 19 for Ancylostoma spp., with one mixed infection of both. Sequencing of the Ancylostoma spp. copro-PCR products found A. duodenale, and also the animal hookworms, A. caninum and A. ceylanicum. Moreover, Trichostrongylus spp.-specific copro-PCR revealed a significant rate of infection (43/203; 21.2%). Sequencing confirmed the zoonotic species, T. colubriformis. PCR/sequencing is useful for differentiating parasite species. The positives by the Kato-Katz method, and by the combined hookworm/Trichostrongylus specific-PCR, were comparable, although the agreement between the two methodologies was only 50%.

Short Report : Human Trichostrongylus colubriformis Infection in a Rural Village in Laos

In Lahanam Village, Savannakhet Province, Laos, 125 of 253 villagers (49.4%) were found by fecal examination to harbor hookworm eggs. The eggs were heterogeneous in morphology and size, suggesting infections of mixed nematode species. To confirm the hookworm egg species, on a voluntary basis, 46 hookworm egg-positive participants were treated with albendazole, and post-treatment adult worms were collected from purged fecal samples. The common human hookworm was found in only 3 participants; 1 case of Necator americanus, and 2 cases of Ancylostoma duodenale. In contrast, adult Trichostrongylus worms were expelled from most participants (43 of 46, 93.5%). The Trichostrongylus species were confirmed by morphology and internal transcribed spacer 2 sequences; all worms were of the same species (T. colubriformis). In addition, some Trichostrongylus worms were obtained from a goat in the same village and identified as T. colubriformis. The results suggested that T. colubriformis was the main zoonotic species causing hookworm infections in the village.

Molecular and serological survey on taeniasis and cysticercosis in Kanchanaburi Province, Thailand.

A community-based field survey on taeniasis and cysticercosis was performed in two villages in Thong Pha Phum District, Kanchanaburi Province, central Thailand, where 3 Taenia species, T. solium, T. saginata and T. asiatica, are sympatrically occurring. Four (0.6%) out of 667 stool samples were egg-positive for Taenia sp. by Kato-Katz technique. Three out of those four persons and other three persons who were Taenia egg-negative but having a recent (<1 year) history of discharging worms in stool were treated with niclosamide. One Taenia egg-positive woman was not treated because of severe ascites. After treatment, three persons expelled long strobilae with scolices and two persons expelled strobilae without scolex. One Taenia egg-positive person did not expel any worms post-treatment. Among 5 persons, four expelled a single worm, whereas one expelled multiple worms, may be 6 worms but not confirmed by detection of scolices. One scolex was armed with hooklets, whereas 2 others did not. Multiplex PCR of 10 expelled proglottids (including 6 estimated worms from one patient) revealed that one sample was T. solium, one T. saginata, and 8 T. asiatica. A total of 159 residents agreed to receive a serological test for cysticercosis. By ELISA using partially purified glycoprotein antigen, 9 cases, 5 and 4 from villages A and B respectively, were found to be sero-positive. The five and an additional sample on the border line from village A were evaluated using confirmative immunoblot using recombinant chimeric antigen. Among the six samples, four including the border line sample were confirmed to be cysticercosis by immunoblotting. One of the 4 persons had neurological symptoms with nodular lesions in the brain by computed tomography. These 4 confirmed or suspected cysticercosis cases were free of T. solium worms, but two of them including confirmed NCC case had a past (>1 year) history of expelling proglottids in the stool.

Monophyly of Opisthorchis viverrini populations in the lower Mekong Basin, using mitochondrial DNA nad1 gene as the marker.

The liver fluke, Opisthorchis viverrini, causes serious public-health problems in the Lower Mekong Basin. This study aimed to clarify whether O. viverrini populations may be genetically divided into sub-specific taxa. We collected 6 populations of O. viverrini from different places in Cambodia, Lao PDR, and Thailand, along both sides of the Mekong River, and analyzed the population structure of these using the mitochondrial nad1 gene as a marker. The results of the DNA polymorphism measurements, by theta-w (thetaw) and -pi (thetapi) values, neutrality tests, and mismatch distribution, suggested that the population of O. viverrini has expanded under the influence of purifying selection and selective sweep. The analysis of molecular variance (AMOVA) test revealed no significant genetic differences among the O. viverrini populations on opposite sides of the Mekong River. O. viverrini haplotypes occurred in multiple populations, and no distinct geographical clade. The star-like haplotype network confirmed a demographic expansion of the O. viverrini population. Overall, the genetic data from these populations suggested that the postulated existence of an O. viverrini species complex should be rejected. The bio-geographical diversity of O. viverrini populations should be explored further, using other appropriate markers and a wider range of samples from geographically different areas.

Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): A New Record of Small Liver Flukes In the Greater Mekong Sub-Region

Abstract Metacercariae, morphologically similar to those of small liver flukes, were found to parasitize red-tailed snakehead fish, Channa limbata, collected from the city of Vientiane, Lao Peoples Democratic Republic. Adult worms that were recovered from experimentally-infected hamsters showed characteristics distinctly different from Opisthorchis viverrini, but closely similar to Opisthorchis lobatus, which was first reported in poultry (Anas sp.) from Pakistan. The present study aimed to redescribe O. lobatus based on the adult worms recovered from experimentally-infected hamsters. Additionally, it aimed to document the genetic relationships among O. lobatus and other opisthorchiid liver flukes using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the internal transcribed spacer 2 (ITS2) region. DNA alignment of the O. lobatus and O. viverrini COI partial sequences (330 bp) showed 3.03% fixed differences (2.72% of amino acids changed) while the ITS2 region (350 bp) indicated a 0.86% difference for nucleotides. Species boundaries between the 2 parasites were determined by neighbor-joining analysis using the molecular sequence data. The phenogram confirmed that O. lobatus was distinctly different from O. viverrini, representing the first reported instance of O. lobatus in the Greater Mekong Sub-region (GMS) and the first record of C. limbata as the second intermediate host of a small liver fluke. Questions regarding human infection and the extent of the geographic distribution of these species should be investigated further.

Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples

Opisthorchiasis, which can lead to cholangiocarcinoma in cases of chronic infection, is a major public health problem in Southeast Asian countries. The trematode, Opisthorchis viverrini, is the causative agent of the disease. Accurate and rapid monitoring of O. viverrini is crucial for disease prevention and containment. Therefore, in this study we sought to develop a novel species-specific real-time PCR assay for detecting O. viverrini using environmental DNA (eDNA). The diagnostic sensitivity of the newly developed real-time PCR assay was similar to that of the traditional PCR assay for 50 fecal samples collected in Lao PDR (21 and 19 samples were positive by real-time PCR and traditional PCR, respectively). The efficacy of eDNA analysis and its applicability in the field were tested using a total of 94 environmental water samples collected from 44 sites in Savannakhet, Lao PDR during May and October 2015 and February 2016. O. viverrini eDNA was detected in five samples by real-time PCR, indicating the presence of the fluke in the area and the risk of infection for individuals consuming fish from these water sources. The application of eDNA analysis would facilitate the identification of O. viverrini endemic hotspots and contribute to the ecological control of opisthorchiasis, and this strategy can be applied to other eukaryotic water pathogens.

Prevalence and clinical aspects of human Trichostrongylus colubriformis infection in Lao PDR

There have been few studies on human trichostrongyliasis in Southeast Asia, information on its clinical manifestations is also sparse. Trichostrongyliasis occurs predominantly in areas where poor hygiene is common especially where human/animal feces are used as a fertilizer, thereby contaminating vegetables and stream water. The intimate coexistence of domestic animals and humans explains the prevalence of Trichostrongylus infection in such areas. The goal of the current study was to determine the prevalence of trichostrongyliasis among villagers in Thakamrien village, Sonkon district, Savannakhet province, Laos, and to investigate potential relationships between clinical features, laboratory data, and severity of infection. Of 272 villagers examined, 160 (58.8%) were determined positive for helminthic infections by fecal examination, and 59 (36.9%) of these were infected with Trichostrongylus. Only 58 cases were in the inclusion criteria of the study and then underwent further assessment, including a questionnaire on personal behaviors, physical examination, and laboratory tests. Villagers in the trichostrongyliasis group were more likely than the control group to have consumed fresh vegetables, not washed their hands before meals or after using the toilet, and to have had close contact with herbivorous animals (goats and cows). Similarly, villagers in the trichostrongyliasis group were more likely than the control group to have a history of loose feces, rash, or abdominal pain; however, no obvious clinical symptoms were observed during physical examination of the trichostrongyliasis patients. The degree of infection was determined by both fecal egg counts and quantification of adult worms after deworming. Laboratory data were evaluated for any relationship with severity of infection. No significant differences were found in laboratory values between the trichostrongyliasis and control groups, with most values being within normal limits; however, both groups had high eosinophil counts. This study demonstrated that the useful clinical characteristics of trichostrongyliasis patients include history of loose feces, rashes, and abdominal pain, as well as in personal behaviors, such as the regular consumption of fresh vegetables, lack of hand washing, and close contact with cattle.

Sero-epidemiological survey of gnathostomiasis in Lao PDR

While human gnathostomiasis cases have been reported sporadically in Lao PDR since 1975, little is known about the disease in this country. We aimed to investigate sero-prevalence of gnathostomiasis and Gnathostoma species in Lao PDR. One village each in the north, central and south regions of Lao PDR was selected as the study sites. Overall, 125 (29.8%) of 420 sera from the randomly selected participants were sero-positive by immunoblot technique, with anti-Gnathostoma IgG antibody against the 24 kDa fraction. The sero-prevalence was high in the central (47.1%) and south (38.6%), but low (3.6%) in the north. Risk factor analyses revealed that the consumption of raw/undercooked fish was significantly associated with Gnathostoma sero-positivity (95% CI 1.05-17.05, P=0.042). The sero-positivity significantly increased with the age of the participants. Several fish, swamp eels, and frogs collected from central and southern Lao were infected with G. spinigerum advanced 3rd-stage larvae. Channa limbata (red-tailed snakehead fish) was identified as a natural second intermediate host of G. spinigerum. Eggs of G. spinigerum were found in dog feces collected in the south. Gnathostomiasis is endemic in central and southern Laos, so that preventive measures should be introduced for people living in these regions.

Patterns of trematode infections of Opisthorchis viverrini (Opisthorchiidae) and Haplorchis taichui (Heterophyidae) in human populations from two villages in Savannakhet Province, Lao PDR

The liver fluke, Opisthorchis viverrini, and the minute intestinal fluke, Haplorchis taichui, are prevalent in many Asian countries. This study analysed the patterns of infections of O. viverrini and H. taichui in Lahanam and Thakhamlien villages (Savannakhet Province, Lao PDR), in two cross-sectional investigations. Out of a total of 207 human participants, post-anthelmintic treatment positivity rates for expelled worms were 170 (82.1%) for H. taichui and 65 (31.4%) for O. viverrini. Both these species co-exist in the study villages. When each parasite was analysed separately, H. taichui infections reached a plateau among people aged >20 years. Opisthorchis viverrini infection rates were highest in the age group 21-30 years, with decreasing infection rates after the age of 30. Our findings indicated that fish-borne trematode infections were more prevalent among adults. Fish, common intermediate hosts, were acquired in the study area for analysis. The examination of 35 species of fish as intermediate hosts found O. viverrini metacercariae in only six species, and these were found mostly during the month of November. Many farmers who live on the rice fields obtain their food from their immediate environment, including these intermediate-host fish, potentially putting them at greater risk of O. viverrini infection. By contrast, H. taichui metacercariae were found in three species of fish obtained from the market, meaning that anyone could consume them and become infected. If people who work in rice fields limit the species of fish they consume, or avoid consuming raw fish during the month of November, they may reduce their risk of O. viverrini infection.

Nematode infection among ruminants in monsoon climate (Ban-Lahanam, Lao PDR) and its role as food-borne zoonosis

Trichostrongylids infection has gained significant public health importance since Trichostrongylus spp. infections have been reported in humans in Lao PDR. In this study, gastrointestinal nematodes were identified and the intensity of infections was determined in goats and cattle, which are animals greatly used for meat production in Lahanam Village, Lao PDR. The total number of goats and bovines was 23 and 29, respectively, pertaining to 32 households surveyed in the area. Feacal samples were randomly collected from 14 goats and 11 bovines. Ninety three percent (13/14) of goats and 36% (3/11) of cattle were infected, with an average of 1,728 and 86 eggs per gram of faeces (EPG), respectively. Coproculture showed Trichostrongylus spp. (goats 16%; bovines 48%), Haemonchus spp. (goats 69%; bovines 37%), Cooperia spp. (bovines 8%) and Oesophagostomum spp. (goats 15%; bovines 6%). After performing the necropsy on an adult goat, Trichuris spp. was also found. We confirmed the presence of Oesophagostomum spp., H. contortus and T. colubriformis by morphology and DNA sequencing analysis of the ITS region of rDNA. Due to interactions between humans and goats in Lahanam Village and high EPG results, the diagnosis of species and the intensity of gastrointestinal nematode infection in these animals are important public-health issues. Other ruminant parasites, such as Oesophagostomum and Haemonchus, found in caprines and bovines, are reported to be causes of zoonosis and their presence in humans should be investigated in future field surveys in this area.