Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People’s Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.     

Molecular identification of Ascaris lumbricoides and Ascaris suum recovered from humans and pigs in Thailand,Lao PDR,and Myanmar

Parasitology Research - Ascaris lumbricoides is the largest roundworm known from the human intestine while Ascaris suum is an internal parasite of pigs. Ascariasis, caused by Ascaris lumbricoides,...     

Intestinal parasitic infections and risk factors among Myanmar migrant workers in northeast Thailand

Objective: To determine the prevalence and associated factors of intestinal parasitic infections in migrant workers in Nakhon Ratchasima Province, Northeast Thailand. Methods: A cross-sectional study was conducted from August 2017 to July 2018 in 600 Myanmar migrant workers. Questionnaires were employed for collecting the demographic data of participants. Stool samples were collected and examined using the formalinether concentration technique. Risk factors for intestinal parasitic infections were determined using multiple logistic regressions analyses. Results: The overall infection rate of intestinal parasitic infections was 27.67%(166/600). Among the intestinal helminthes observed, hookworm was most abundant(8.67%) followed by Trichuris trichiura(8.50%), Opisthorchis viverrini(4.17%), Ascaris lumbricoides(1.50%), Strogyloides stercoralis(1.17%) and Hymenolepis nana(0.5%). Meanwhile, Entamoeba coli was the most prevalent intestinal protozoa(4.33%, 26/600) followed by Endolimax nana(1.33%), Entamoeba histolytica complex(1.17%), Blastocystis sp.(1.0%) and Giardia duodenalis(0.17%). The study found significant associations between gender and Strogyloides stercoralis infection(ORadj=5.61, 95% CI=1.18–26.70, P=0.03), workers aged 30 years old were likely to have a lower risk of the T. trichiura infection(ORadj=0.45, 95% CI= 0.23–0.89). Moreover, the history of consuming raw or undercooked cyprinoid fish was a risk factor of Opisthorchis viverrini infection(ORadj=2.82, 95% CI=1.22–6.49, P=0.015). Conclusions: There remains a high prevalence of intestinal parasitic infections among Myanmar migrant workers in the study area and therefore health screenings for all migrant workers in Thailand are required.     

Rapid detection of Brugia malayi in mosquito vectors using a real-time fluorescence resonance energy transfer PCR and melting curve analysis

We developed real-time fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) combined with melting curve analysis for detection of Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET PCR is based on a fluorescence melting curve analysis of hybrid formed between amplicons generated from a family of repeated DNA element, 153-bp HhaI repeated sequence, specific to genus Brugia and specific fluorophore-labeled probes. The B. malayi-infected mosquitoes were differentiated from Wuchereria bancrofti-infected and uninfected mosquitoes and from genomic DNA of Dirofilaria immitis--and Plasmodium falciparum--infected human red blood cells and human leukocytes by their melting temperature. Sensitivity and specificity were both 100%. Melting curve analysis produces a rapid, accurate, and sensitive alternative for specific detection of B. malayi in mosquitoes, allows high throughput, and can be performed on small samples. This method has the potential for endemic area mapping or monitoring effect of brugian filariasis mass treatment programs.     

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.     

Identification of antigenic proteins in Strongyloides stercoralis by proteomic analysis

Parasitology Research - Strongyloides stercoralis is an intestinal helminth that infects people worldwide. Hyperinfection or disseminated human strongyloidiasis can involve vital organs, leading to...     

Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People's Democratic Republic by specific hybridization probe-based real-time fluorescence resonance energy transfer PCR method

Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini, was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific hybridization probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini-infected snails in Southeast Asian countries.     

Three Human Gnathostomiasis Cases in Thailand with Molecular Identification of Causative Parasite Species

Human gnathostomiasis is one of the important food-borne parasitic zoonoses. The disease is caused by a spirurid roundworm of the genus Gnathostoma. Here, we describe three parasitological confirmed cases of human gnathostomiasis, caused by Gnathostoma spinigerum, in a hospital in Thailand during 2004–2012. Clinical characteristics, treatment, and outcome of cases were revealed. Parasites were accidentally recovered from patients and morphologically identified as Gnathostoma species. Confirmed diagnosis and identification of causative parasite species was made by DNA extraction of the recovered worms, followed by a polymerase chain reaction (PCR) of the second internal transcribed spacer region (ITS2) of DNA and the partial cytochrome c oxidase subunit 1 (cox-1) gene. Sequences corresponding to ITS2 and cox-1 were similar to G. spinigerum. To our knowledge, this study represents the first molecular confirmation that recovered G. spinigerum is a causative agent of human infection in Thailand.