Development of a PCR assay and pyrosequencing for identification of important human fish-borne trematodes and its potential use for detection in fecal specimens

BACKGROUND: Small liver and minute intestinal flukes are highly prevalent in Southeast Asia. Definitive diagnosis of parasite infection is usually achieved parasitologically by finding the fluke eggs in feces. However, their eggs are difficult to differentiate morphologically in fecal samples, even for experienced technicians. The present study developed a PCR assay coupled with DNA pyrosequencing for identification of the fish-borne trematodes (FBT), Opisthorchis viverrini, Clonorchis sinensis, Haplorchis taichui, H. pumilio and Stellantchasmus falcatus, and to evaluate potential detection in fecal specimens, and identification and differentiation of cercarial and metacercarial stages. METHODS: Primers targeting the partial 28S large subunit ribosomal RNA gene were designed and about 46–47 nucleotides were selected as the target region for species identification by a PCR assay coupled with a pyrosequencing technique. RESULTS: The nucleotide variations at 24 positions, which is sufficient for the identification of the five species of FBT were selected. The method could identify O. viverrini and C. sinensis eggs in feces, cercarial and metacercarial stages of O. viverrini, and metacercarial stage of H. pumilio and H. taichui. The detection limit was as little as a single O. viverrini or C. sinensis egg artificially inoculated in 100 mg of non-infected fecal sample (equivalent to 10 eggs per gram), indicating highly sensitivity. The method was found to be superior to the traditional microscopy method and was more rapid than Sanger DNA sequencing. CONCLUSIONS: DNA pyrosequencing-based identification is a valuable tool for differentiating O. viverrini and other Opisthorchis-like eggs, and can be applied to epidemiological studies and for molecular taxonomic investigation of FBT in endemic areas

Effectiveness of Strongyloides Recombinant IgG Immunoreactive Antigen in Detecting IgG and IgG4 Subclass Antibodies for Diagnosis of Human Strongyloidiasis Using Rapid Immunochromatographic Tests

Human strongyloidiasis is an important soil-transmitted helminthiasis that affects millions worldwide and can develop into fatal systemic strongyloidiasis in immunosuppressed patients. We have developed two new rapid and simple-to-use immunochromatographic test (ICT) kits for rapid serodiagnosis that support stool examination for clinical diagnosis. Strongyloides stercoralis recombinant IgG immunoreactive antigen (GenBank: AAB97359.1; rSsIR-based ICT kit) was used for detection of IgG and IgG4 antibodies. The diagnostic efficacy of both kits was evaluated using human serum samples from strongyloidiasis patients, healthy individuals, and those with other parasitosis. At a prevalence of infection of 36.4%, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the rSsIR-based IgG ICT kit were 91.7%, 83.8%, 76.4%, 94.6%, and 86.7%, respectively, and those of the rSsIR-based IgG4 ICT kit were 78.3%, 84.8%, 74.6%, 87.3%, and 82.4% respectively. The concordance between the two kits was 89.7%. The recombinant antigen can be produced to an unlimited extent and the kits can be used as point-of-care diagnostic tools and in large-scale surveys in endemic areas throughout tropical regions without necessitating additional facilities or ancillary supplies

Microbiome Composition and Microbial Community Structure in Mosquito Vectors <i>Aedes aegypti</i> and <i>Aedes albopictus</i> in Northeastern Thailand, a Dengue-Endemic Area

Bacterial content in mosquito larvae and adults is altered by dynamic interactions during life and varies substantially in variety and composition depending on mosquito biology and ecology. This study aimed to identify the microbiota in Aedes aegypti and Aedes albopictus and in water from their breeding sites in northeastern Thailand, a dengue-endemic area. Bacterial diversity in field-collected aquatic larvae and subsequently emerged adults of both species from several locations were examined. The microbiota was characterized based on analysis of DNA sequences from the V3-V4 region of the 16S rRNA gene and exhibited changes during development, from the mosquito larval stage to the adult stage. Aedes aegypti contained a significantly higher number of bacterial genera than did Ae. albopictus, except for the genus Wolbachia, which was present at significantly higher frequencies in male Ae. albopictus (p < 0.05). Our findings also indicate likely transstadial transmission from larva to adult and give better understanding of the microbial diversity in these mosquitoes, informing future control programs against mosquito-borne diseases

Gut microbiota diversity in human strongyloidiasis differs little in two different regions in endemic areas of Thailand.

Human gastrointestinal helminthic infections have a direct and/or indirect effect on the composition of the host gut microbial flora. Here, we investigated the effect of infection with a soil-transmitted intestinal nematode, Strongyloides stercoralis, on the gut microbiota of the human host. We also investigated whether composition of the microbiota in infected persons might vary across endemic regions. Fecal samples were obtained from volunteers from two areas endemic for strongyloidiasis, Khon Kaen Province in northeastern Thailand and Nakhon Si Thammarat Province in southern Thailand. Samples from Khon Kaen were from infected (SsNE) and uninfected (NegNE) individuals. Similarly, samples from the latter province were from infected (SsST) and uninfected (NegST) individuals. DNA sequences of the V3-V4 regions of the bacterial 16S rRNA gene were obtained from the fecal samples. No statistical difference in alpha diversity between groups in terms of richness or diversity were found. Statistical difference in beta diversity was observed only between NegNE and NegST. Some significant differences in species abundance were noted between geographical isolates. The SsNE group had a higher abundance of Tetragenococcus holophilus than did the SsST group, whereas Bradyrhizobium sp. was less abundant in the SsNE than the SsST group. For the uninfected groups, the NegNE had a higher abundance of T. holophilus than the NegST group. Our data showed that S. stercoralis infection leads to only minor alterations in the relative abundance of individual bacterial species in the human gut: no detectable effect was observed on community structure and diversity

Investigating the microbiota of fermented fish products (Pla-ra) from different communities of northeastern Thailand.

DNA-sequencing was performed on the V3-V4 regions of 16S rRNA genes to investigate the microbial diversity of five samples of fermented freshwater fish (pla-ra) from three provinces in northeastern Thailand. The samples had salt concentrations ranging from 7 to 10%, pH values from 4.83 to 7.15, and D-/L-lactic acid concentrations of 90 to 450 mg/l. A total of 598 operational taxonomic units were annotated at various taxonomic ranks based on the SILVA Database. The lactic-acid and halophilic genera Tetragenococcus, Halanaerobium and Lactobacillus were among the dominant taxa of bacteria. The top 20 non-redundant taxa were considered in more detail. In two pla-ra samples, Tetragenococcus muriaticus was commonly identified. Halanaerobium fermentans was the most abundant species in a third sample and co-dominant in another sample. Lactobacillus rennini was dominant in the pla-ra sample from Roi Et Province. Additionally, other beneficial bacteria were detected including Staphylococcus nepalensis, Lactobacillus sakei, Lactobacillus pentosus, Weissella confusa, and Bifidobacterium bifidum. Differences between samples may be due to use of different raw materials, salt concentrations, recipes, processes and fermentation periods. The microbial communities in pla-ra provide a better understanding of the production outcomes of traditional products. Further optimization of the fermentation process, for example by using dominant bacterial taxa in starter cultures, may improve processes of food fermentation, food quality and flavor control, providing useful guidelines for industrial applications

Prevalence of intestinal parasitic infections and genetic differentiation of Strongyloides stercoralis among migrant workers from Myanmar, Lao PDR and Cambodia in northeastern Thailand.

Intestinal parasitic infections (IPIs) remain a public-health problem worldwide, including in countries of the Lower Mekong subregion. Increases in human migration from neighboring countries might cause reemerging parasitic infections, leading to spread of parasites in the landscape. Here, we conducted a cross-sectional study to identify the prevalence of IPIs in migrant workers from Myanmar, Lao PDR, and Cambodia who were dwelling in Nakhon Ratchasima Province, northeastern Thailand. The identification of Strongyloides species and genetic differentiation of worms from migrant workers with different countries of origin was also assessed. Fresh stool samples were collected from 338 migrant workers and examined for evidence of IPIs using agar plate culture (APC) and the formalin-ethyl acetate concentration technique (FECT). Among those nine samples positive for nematodes by APC, the Strongyloides or hookworm species present was confirmed using the polymerase chain reaction (PCR) followed by DNA sequencing. This revealed eight cases of Strongyloides stercoralis infection and one of Necator americanus. Fifty-one out of 338 individuals (15.09%) were positive for IPIs using FECT and APC. Eggs of Opisthorchis-like flukes were the most common parasite (11.83% of samples), followed by S. stercoralis (2.37%), Entamoeba coli (1.50%), hookworm (0.89%), Taenia sp. (0.60%) and Hymenolepis nana (0.30%). The genetic differentiation of S. stercoralis recovered from migrant workers with different countries of origin was analyzed. Specimens of S. stercoralis isolated from workers from Lao PDR, Cambodia and Myanmar were genetically similar to those sequenced from Thailand. However, there were population-genetic differences between S. stercoralis from these Southeast Asian countries and other regions of the world. This study demonstrated that IPIs were prevalent in migrant workers in the northeastern region of Thailand. Our findings provided molecular confirmation of the presence of S. stercoralis and explored the genetic differentiation of S. stercoralis from those infected migrant workers. An effective anti-parasitic drug should be provided for migrant workers and its administration enforced

Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails.

Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies

Comparative assessment of immunochromatographic test kits using somatic antigens from adult Opisthorchis viverrini and IgG and IgG4 conjugates for serodiagnosis of human opisthorchiasis.

Chronic infections of humans with Opisthorchis viverrini and Clonorchis sinensis spanning decades may lead to life-threatening pathology prior to cholangiocarcinoma (CCA), which usually has a poor prognosis. Serological tools can support the parasitological examination in clinical diagnosis and support screening for risk of CCA. We developed novel immunochromatographic test kits using a soluble, somatic tissue extract of adult O. viverrini worms as an antigen and colloidal-gold labeled conjugates of IgG and IgG4 antibodies and evaluated the diagnostic values of both the OvSO-IgG and OvSO-IgG4 kits. For diagnosis of human opisthorchiasis individually, the diagnostic sensitivity, specificity, positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while the 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, for the OvSO-IgG4 kit at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera showed positive reactivity with the OvSO-IgG and OvSO-IgG4 kits, respectively. There was 84.1% (κ-value = 0.649) concordance between the two kits, which was statistically significant (p < 0.001). Both ICT kits can be employed as quick and easy point-of-care diagnostic tools, and hence the OvSO-IgG and OvSO-IgG4 kits can support expanded capacity for clinical diagnosis of human opisthorchiasis and clonorchiasis. These kits may find utility in large-scale surveys in endemic areas where there are limited sophisticated medical facilities or capacity