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.

The parasitic nematodes, Strongyloides stercoralis and Strongyloides fuelleborni, can infect humans and non-human primates. We amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides from humans in the study area in Thailand, where people have frequent contact with long-tailed macaques. Fresh stool samples were obtained from 213 people and were examined using the agar plate culture method. The overall prevalence of Strongyloides infection was 8.92% (19/213). From a total of 19 worms (one per infected person), 18 adult males had 18S rRNA sequences identical with that of S. stercoralis and one adult female had a sequence almost identical with that of S. fuelleborni. A median-joining network of cox1 sequences revealed nine new haplotypes from S. stercoralis, and an overall haplotype diversity (Hd) of 0.9309. The single haplotype of S. fuelleborni was also new and contributed to an overall haplotype diversity for that species of 0.9842. This is the first molecular identification of S. stercoralis and S. fuelleborni in a human community having contact with long-tailed macaques in Thailand. It is also the first report of S. fuelleborni infecting a human in Thailand.

Opisthorchis viverrini is endemic in the South East Asian region, especially in Cambodia, Lao People's Democratic Republic, Vietnam and Thailand, but there have been no previous records from Myanmar. During stool surveys of rural populations in three regions of Lower Myanmar, Opisthorchis-like eggs were found in 34 out of 364 (9.3%) participants by stool microscopy after using the modified formalin-ether concentration technique. DNA was extracted from these positive stool samples and a portion of the mitochondrial cytochrome c oxidase subunit I (cox1) gene was amplified using the polymerase chain reaction and then sequenced. DNA sequences, successfully obtained from 18 of 34 positive samples (Bago Region, n = 13; Mon State, n = 3; Yangon Region, n = 2), confirmed that the eggs were of O. viverrini. Sequences showed 99.7% identity with O. viverrini mitochondrial cox1 (GenBank accession no. JF739555) but 95%, 88.7%, 82.6% and 81.4% identities with those of Opisthorchis lobatus from Lao People's Democratic Republic (GenBank accession nos. HQ328539-HQ328541), Metorchis orientalis from China (KT239342), Clonorchis sinensis from China (JF729303) and Opisthorchis felineus from Russia (EU921260), respectively. When alignement with other Opisthorchiidae trematodes, 81% similarity with Metorchis bilis from Czech Republic (GenBank accession nos. KT740966, KT740969, KT740970) and Slovakia (GenBank accession nos. KT740971-KT740973), 84.6% similarity with Metorchis xanthosomus from Czech Republic (GenBank accession no. KT740974), 78.6% similarity with M. xanthosomus from Poland (GenBank accession no. KT740968) and 82.2% similarity with Euamphimerus pancreaticus from Czech Republic (GenBank accession no. KT740975) were revealed. This study demonstrated, for the first time, O. viverrini from rural people in Myanmar using molecular methods and is an urgent call for surveillance and control activities against opisthorchiasis in Myanmar.

Strongyloidiasis is a major soil-transmitted helminth (STH) disease that affects people worldwide. We present updated data on prevalence in the Lao People's Democratic Republic (Lao PDR) in 2015, arising from a community cross-sectional helminthiasis survey. Fecal samples were collected from 327 individuals across three provinces in Lao PDR (Luang Prabang in the north, Khammouane in the center, and Champasack in the south). Agar plate culture and Kato-Katz methods were used to examine duplicate stool samples from each participant to detect Strongyloides stercoralis and co-infecting helminths. Overall prevalences of S. strercoralis human hookworm, Taenia spp., Trichuris trichiura, Ascaris lumbricoides, and Enterobius vermicularis were 41.0, 28.1, 4.9, 4.0, 1.5, and 0.9 %, respectively. The prevalence of miscellaneous trematodiases (including opisthorchiasis) was 37.9 % and of Schistosoma mekongi infection was 0.3 %. Strongyloidiasis is a current major STH disease in Lao PDR. We also report the molecular-phylogenetic identification of S. stercoralis adult males collected from 40 representative human strongyliodiasis fecal samples. DNA was extracted, amplified, and sequenced from a portion of the mitochondrial cox1 gene and the nuclear 18S ribosomal DNA. Phylogenetic analyses indicated that all specimens sequenced belonged to S. stercoralis (Bavay, 1876) Stiles and Hassall, 1902. The cox1 sequences exhibited great diversity (24 haplotypes) in Lao PDR. This is the first molecular identification and report of genetic diversity of S. stercoralis in humans from Lao PDR. An effective parasite control program is needed to reduce the serious health impacts.

We identified genomic safe harbor sites (GSH) in the human blood fluke, Schistosoma mansoni and developed a CRISPR-focused protocol for insertion of a reporter transgene into a representative GSH. The protocol employed ribonuclear protein complexes of Cas9 nuclease, three overlapping guide RNAs, and phosphorothioate-modified, double stranded donor DNAs encoding green fluorescent protein driven by a strong endogenous promoter. Gene-editing efficiencies of >20% and reporter transgene fluorescence of >50% of gene-edited eggs were obtained by five days after CRISPR transfection. These methods and results advance functional genomics for multicellular parasites, and represent a tractable path towards transgenic schistosomes using homology directed repair catalyzed transgene insertion. Identification and characterization of GSH is expected to facilitate consistent transgene activity with neutral influence on the host cell genome and, concurrently, provide a privileged locus for transgene activity. This approach should be adaptable to platyhelminths generally.

BackgroundTwo important helminths, Strongyloides stercoralis (an intestinal roundworm) and Opisthorchis viverrini (a liver fluke), are endemic in northeast Thailand. There have been national campaigns in place aimed at the control and eradication of soil-transmitted helminthiasis and opisthorchiasis in Thailand for several decades. However, these helminths still exist and raise concerns regarding public health. This study aimed to evaluate the current prevalence of S. stercoralis and O. viverrini infections in rural communities in northeast Thailand. The data from this study will be useful to improve strategies for future helminth prevention and control.MethodsA cross-sectional study was conducted from December 2016 to June 2017 in Mueang Khon Kaen district in Khon Kaen, Thailand. The participants were selected using a simple random sampling method. Demographic data were collected using a questionnaire. Stool samples were collected and processed using agar plate culture to determine the presence of S. stercoralis infection and an in-house formalin-ethyl acetate concentration technique to determine the presence of O. viverrini and other intestinal parasite infections (IPIs).ResultsIn total, 602 persons were enrolled. However, only 526 were analyzed for S. stercoralis and 387 for O. viverrini risk factors. The overall prevalence of S. stercoralis infection was 23.0% (95% confidence interval [95%CI]: 19.4 to 26.6). The prevalence of O. viverrini infection and IPIs other than S. stercoralis was 20.4% (95%CI: 16.5 to 24.8). The prevalence of O. viverrini infection was 19.4% (95%CI: 15.6 to 23.7). Male sex was significantly associated with S. stercoralis infection [Adjusted Odds Ratio (aOR) 4.0; 95%CI: 2.5 to 6.2; P-value < 0.001]. Males were significantly more likely to be infected with O. viverrini and other IPIs (aOR 4.1; 95%CI: 2.3 to 7.2, P-value < 0.001).ConclusionsThis study demonstrated that the updated prevalence of intestinal parasite infections is still high in rural communities in northeast Thailand, especially that of strongyloidiasis and opisthorchiasis.

Background: Human gnathostomiasis is a serious tropical disease, which is often overlooked. There is an urgent need to improve tools to aid the potential diagnosis of the disease in endemic regions. To overcome this, we produced the immunochromatographic test (ICT) kit for a rapid and simple diagnosis of human gnathostomiasis. Findings: The recombinant protein (named rGslic18) was applied to ICT kit as the antigen. The diagnostic value of ICT kit was evaluated using serum samples from parasitologically proven and clinically suspected gnathostomiasis patients, healthy volunteers and patients with other parasitic diseases. The ICT kit exhibited quite high sensitivity (93.75 %) and specificity (97.01 %). Conclusions: The ICT kit is simple, convenient and easy to implement and expected to provide reliable diagnostic results for human gnathostomiasis. It also will be a promising diagnostic tool not only for large-scale epidemiological surveys in endemic or remote areas where diagnostic facilities are poor but also for a rapid clinical diagnosis in the bedside laboratory.

Human strongyloidiasis is a deleterious gastrointestinal disease mainly caused by Strongyloides stercoralis infection. We aimed to study the possible transmission of S. stercoralis between humans and pet animals. We isolated Strongyloides from humans and domestic dogs in the same rural community in north-east Thailand and compared the nucleotide sequences of derived worms using portions of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and 18S ribosomal RNA (18S rRNA) genes. Twenty-eight sequences from the 18S rRNA gene were obtained from worms derived from humans (n = 23) and dogs (n = 5), and were identical with S. stercoralis sequences (from Thailand, Cambodia, Lao PDR and Myanmar) published in the GenBank database. The 28 cox1 sequences from humans and dogs showed high similarity to each other. The available published cox1 sequences (n = 150), in combination with our 28 sequences, represented 68 haplotypes distributed among four clusters. The 28 samples from the present study represented eight haplotypes including four new haplotypes. Dogs and humans shared the same haplotypes, suggesting the possibility of zoonotic transmission from pet dogs to humans. This is of concern since dogs and humans live in close association with each other.