Rutchanee Rodpai

First report and molecular identification of Opisthorchis viverrini infection in human communities from Lower Myanmar

Opisthorchis viverrini is endemic in the South East Asian region, especially in Cambodia, Lao Peoples 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 Peoples 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.

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.

First molecular identification and genetic diversity of Strongyloides stercoralis and Strongyloides fuelleborni in human communities having contact with long-tailed macaques in Thailand

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.

Genetic diversity of Taenia saginata (Cestoda: Cyclophyllidea) from Lao People’s Democratic Republic and northeastern Thailand based on mitochondrial DNA

BackgroundTaenia saginata is a tapeworm found in cattle worldwide. Analysis of genetic diversity in different geographical populations of T. saginata not only helps to understand the origin, transmission and spread of this organism, but also to evaluate the selection pressures acting on T. saginata and how it is responding to them. However, there are few reports of the genetic variability of T. saginata populations in different regions of the world, including Lao PDR and Thailand. We report the genetic diversity of T. saginata populations in Lao PDR and northeastern Thailand together with sequences of T. saginata from other countries deposited in GenBank.ResultsMitochondrial cox1 sequence analysis revealed that 15 and 8 haplotypes were identified in 30 and 21 T. saginata isolates from Lao PDR and northeastern Thailand, respectively. Fifty-three haplotypes were identified from 98 sequences. Phylogenetic tree and haplotype network analyses revealed that global isolates of T. saginata were genetically divided into five groups (A, B, C1, C2 and D). Taenia saginata isolates from Lao PDR and northeastern Thailand belonged to either Group A or B. Taenia saginata from western Thailand clustered in groups C1, C2 and D, and populations from the northeast and western Thailand were found to be genetically distinct. Taenia saginata isolates in Lao PDR and Thailand were also found to be genetically diverse but the degree of genetic differentiation was low.ConclusionsTaenia saginata populations from Lao PDR and northeastern Thailand are genetically distinct from the population in western Thailand and it is proposed that T. saginata has been dispersed by different transmission routes in Southeast Asia.

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’- and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ωl-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines followed the exposure to those of ωl-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas surrounding ωl-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ωl and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.Soluble egg antigen (SEA) and excretory-secretory (ES) products of the egg of Schistosoma mansoni contain a glycosylated T2 family ribonuclease termed omega-1 (ω1). Following release from the egg ω1 instructs antigen presenting cells to induce naive CD4+ T cells to mature into T helper 2 effectors that, in turn, establish the Th2 immunological phenotype characteristic of schistosomiasis. Here schistosome eggs were either transiently exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) of 20 nt complementary to exon 1 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and this sgRNA. Subsequently some eggs also were transduced with a single stranded oligodeoxynucleotide donor transgene that coded for six stop codons, flanked by 50 nt-long 5′- and 3′-homology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the predicted DSB revealed ~6% of the reads (total reads 2×61620×106) were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.18% insertion of the donor transgene. ω1-encoding mRNAs were reduced > 80%, and soluble egg antigen (SEA) from ω1-mutated eggs exhibited markedly reduced ribonuclease activity, indicative that programmed Cas9 cleavage had mutated the ω1 gene. Following tail vein injection of schistosome eggs into BALB/c mice, the volume of pulmonary granulomas surrounding wild type eggs was 18-fold greater than ω1-mutated eggs, 6.21×10 −2 ± 1.61×10 −3 vs. 0.34×10 −2 ± 0.12×10 -4 mm 3 , respectively. In parallel, whereas wild-type SEA polarized Th2 cytokine responses including IL-4 and IL-5 in human monocyte/T cell co-cultures, significantly reduced levels of these cytokines followed the exposure to ω1-mutated SEA. In overview, programmed genome editing was active and facile in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by HDR and/or NHEJ, and mutation of ω1 impeded the capacity of schistosome eggs to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. These findings demonstrated for the first time the utility of CRISPR/Cas9-based genome editing for functional genomics for schistosomes.

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

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, has a worldwide distribution. Here, we have provided the first molecular identification of Ascaris eggs and adults recovered from humans and pigs in Thailand, Lao PDR, and Myanmar. We amplified and sequenced nuclear ribosomal DNA (ITS1 and ITS2 regions) and mitochondrial DNA (cox1 gene). Sequence chromatograms of PCR-amplified ITS1 region revealed a probable hybrid genotype from two human ascariasis cases from Chiang Mai Province, northern Thailand. All complete ITS2 sequences were identical and did not differ between the species. Phylogenetic trees and haplotype analysis of cox1 sequences showed three clusters with 99 haplotypes. Forty-seven samples from the present study represented 14 haplotypes, including 7 new haplotypes. To our knowledge, this is the first molecular confirmation of Ascaris species in Thailand, Lao PDR, and Myanmar. Zoonotic cross-transmission of Ascaris roundworm between pigs and humans probably occurs in these countries.

Current high prevalences of Strongyloides stercoralis and Opisthorchis viverrini infections in rural communities in northeast Thailand and associated risk factors

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.

Molecular Identification of Trichuris suis and Trichuris trichiura Eggs in Human Populations from Thailand, Lao PDR, and Myanmar

Trichuris trichiura is a soil-transmitted helminth infecting human populations globally. Human cases caused by Trichuris suis and Trichuris vulpis have also been reported. Molecular identifications of Trichuris species infecting human populations in Lao PDR and Myanmar are lacking. Here, we explored molecular data obtained from Trichuris eggs recovered from human fecal samples from these countries and compared these with new and existing data from Thailand. Nuclear ribosomal DNA (18S and ITS2) sequences were amplified from Trichuris eggs and sequenced. Forty-one samples showed 99-100% similarity in their 18S sequences to published sequences of T. trichiura and one sample showed 99% similarity to a sequence of T. suis. Similarly, 41 samples showed 92-100% similarity in their ITS2 sequences to published sequences of T. trichiura and one sample showed 94-97% similarity to sequences of T. suis. This study is the first molecular confirmation of human infection with T. suis in northeast Thailand and the first molecular confirmation of the species of Trichuris infecting humans in Lao PDR and Myanmar.

Human liver fluke Opisthorchis viverrini (Trematoda, Opisthorchiidae) in Central Myanmar: New records of adults and metacercariae identified by morphology and molecular analysis

Opisthorchis-like metacercariae were found in cyprinoid fish, Puntius brevis, bought from markets in the Bago region, Central Myanmar. Adult worms recovered from experimentally-infected hamsters resembled Opisthorchis viverrini. DNA was extracted from adults and metacercariae. A portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the internal transcribed spacer 2 (ITS2) regions were amplified using the polymerase chain reaction and then sequenced. The sequences confirmed that the flukes were O. viverrini. In phylogenetic analyses, sequences of O. viverrini, including our new sequences, clustered in a group with high bootstrap support for ITS2 (80%) and the cox1 gene (99%). Interestingly, ITS2 and cox1 sequences of O. viverrini and O. lobatus were very similar, raising a question about the identity of the latter. This is the first report of O. viverrini in cyprinoid fish in Central Myanmar, and only the second report of the species in Myanmar. It is an urgent warning against consuming raw or semi-cooked freshwater fish dishes. Development of an effective food-safety strategy should be provided for the prevention and control of opisthorchiasis and other foodborne diseases.

Identification of antigenic proteins in Strongyloides stercoralis by proteomic analysis

Strongyloides stercoralis is an intestinal helminth that infects people worldwide. Hyperinfection or disseminated human strongyloidiasis can involve vital organs, leading to lethal outcomes. We analyzed immunoproteomics of antigenic spots, derived from S. stercoralis third-stage larvae and reacted with human strongyloidiasis sera, by two-dimensional gel electrophoresis and immunoblotting. Of 26 excised antigenic spots analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry, 20 proteins were identified. Most proteins were associated with enzymes involved in the metabolic process, energy generation, and oxidation–reduction. The proteins relate to promotion of worm development, cell division, cell signaling and transportation, and regulation of muscular contraction. Identification of antigenic proteins shows promise in helping to discover potential diagnostic protein markers or vaccine candidates for S. stercoralis infection.