Dogs are reservoir hosts for possible transmission of human strongyloidiasis in Thailand: molecular identification and genetic diversity of causative parasite species.

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.

Morphological and genetic variation of Wuchereria bancrofti microfilariae in carriers in Thailand, Lao PDR and Myanmar: evaluation using Giemsa-stained thick blood films.

There is geographical variation in the morphology and genetics of Wuchereria bancrofti, the major cause of human lymphatic filariasis. This study aims to compare morphological and genetic variation of W. bancrofti microfilariae recovered from carriers in Lao PDR, Myanmar and Thailand. Six morphological parameters (body length, cephalic space length and width, length of head to nerve ring, body width at nerve ring, Innenkȍrper length and number of column nuclei between the cephalic space and nerve ring) were evaluated from microfilariae in Giemsa-stained thick blood films. A portion of the cytochrome c oxidase subunit 1 gene of mitochondrial DNA was sequenced and analysed. Wuchereria bancrofti microfilariae showed a wide variation in their morphology and morphometry among three countries. Phylogenetic analysis confirmed that all microfilariae belonged to W. bancrofti. Higher mutation frequencies were observed in samples from Myanmar, relative to Thailand and Lao PDR. This study highlights the morphological disparities of microfilariae and genetic variability within W. bancrofti among three geographical locations. We found that reported morphometric differences between localities were less clear-cut than previously thought. Further studies are needed to determine the microfilarial periodicity in Lao PDR.

First molecular identification of Strongyloides fuelleborni in long-tailed macaques in Thailand and Lao People's Democratic Republic reveals considerable genetic diversity.

Strongyloides fuelleborni is a soil-transmitted nematode parasite of non-human primates. The worm is prevalent also in human populations in Africa and South-East Asia. In this study, 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 adult males recovered from faecal samples from long-tailed macaques (Macaca fascicularis) in Thailand and Lao PDR. The prevalence in Thailand was 31.1% (55/177) and in Lao PDR it was 62.1% (41/66), with an overall prevalence of 39.5% (96/243). All 18S rRNA sequences that we obtained (n = 96) showed 100% identity with published S. fuelleborni sequences. The 96 cox1 sequences that we obtained represented 32 new haplotypes. When included with the 17 previously known haplotypes from S. fuelleborni, the cox1 sequences fell into four clusters, which had clear geographical structure. This is the first molecular confirmation of S. fuelleborni in long-tailed macaques in Thailand and Lao PDR. Clearly, awareness needs to be raised of the zoonotic potential of S. fuelleborni. A monitoring programme should be organized, taking into account the role of reservoir hosts (i.e. monkeys) in the natural background of human strongyloidiasis caused by S. fuelleborni.

Genetic variation of Enterobius vermicularis among schoolchildren in Thailand.

Enterobiasis, caused by the nematode Enterobius vermicularis, is a common health problem among schoolchildren in Thailand. We provide the first molecular identification of this nematode from Thai schoolchildren and document genetic variation among E. vermicularis eggs using sequence analyses of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). A cross-sectional parasitological survey was conducted in schoolchildren (n = 491) in five regions of Thailand between May 2015 and December 2016. The diagnosis of Enterobius infection was made using the adhesive tape perianal swab technique. Enterobius eggs were recovered from 43 participants (8.75%). DNA was extracted from these eggs and the cox1 gene and partial ITS2 region amplified using the polymerase chain reaction (PCR). Nineteen amplified PCR products of the cox1 gene (441 bp) and 18 of the ITS2 region (623 bp) were subsequently sequenced. All sequences were identified as belonging to E. vermicularis based on database searches. Phylogenetic analysis and a median-joining network of available E. vermicularis cox1 sequences showed 66 haplotypes. We found haploclusters (types A and B) represented among the Thai sequences. Six haplotypes from Thailand fell into type A (of Nakano et al., 2006) (along with sequences from Japan and Korea) and five haplotypes into type B (with sequences from Japan, Iran, Czech Republic, Greece, Denmark and Sudan). The overall haplotype diversity (Hd) was 0.9888. Transmission of worms with type B haplotypes from primates to humans in Asia or from humans in Europe possibly occurs in Thailand.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces.

Schistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

Abdominal angiostrongyliasis caused by Angiostrongylus cantonensis: a possible cause of eosinophilic infiltration in human digestive tract.

We report the pathological findings of a serologically proven case of Angiostrongylus cantonensis presenting with localized peritonitis followed by eosinophilic meningoencephalitis. The neurological involvement developed 3 days after the occurrence of gastrointestinal symptoms. Similarly to the life cycle in rats, it takes about 2 or 3 days for the larvae to reach the nervous system. The pathological section of the sigmoid colon showed focal eosinophilic infiltration with serosal vessel invasion. In the case reported here, we describe a new possible cause of eosinophilic infiltration in the human digestive tract.

Real-time fluorescence resonance energy transfer PCR with melting curve analysis for the detection of Opisthorchis viverrini in fish intermediate hosts.

A real-time fluorescence resonance energy transfer (FRET) PCR combined with a melting curve analysis was developed for the detection of Opisthorchis viverrini in its fish intermediate host, cyprinoid fishes. Real-time FRET PCR is based on a fluorescence melting curve analysis of a hybrid between an amplicon generated from a family of repeated DNA elements, the pOV-A6 specific probe sequence (Genbank Accession No. S80278), a 162 bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled probes. The real-time FRET PCR could detect as little as a single metacercaria artificially inoculated in 30 fish samples. The O. viverrini infected fishes were distinguished from non-infected fishes and from the genomic DNA of other parasites by their melting temperature. Sensitivity and specificity of this method were both 100% in the laboratory setting and it outperformed the microscopic method on field-collected samples as well. Melting curve analysis is a rapid, accurate, and sensitive alternative for the specific detection of O. viverrini infected fishes. It allows a high throughput and can be performed on small samples. The assay has not only great potential for epidemiological surveys of fish intermediate hosts but it could also be adapted as screening tool for a range of foodborne parasites in freshwater fishes.

Ocular angiostrongyliasis: clinical study of three cases.

PURPOSE: To report three patients with ocular angiostrongyliasis who presented with a variety of clinical findings. METHOD: Retrospective, observational case series. The medical charts, photographs, and electrophysiologic records were reviewed. RESULTS: All patients presented with blurred vision and one had a history of eosinophilic meningitis. In each respective case, only one living larva was found in the anterior chamber, vitreous cavity, and subretinal space. The fundus examination revealed generalized retinal pigment epithelial alteration, subretinal tracks, retinal oedema, macular oedema, and a pale disc. Visually evoked potentials showed delayed latency time in one patient, which represented the secondary optic neuritis. Blood eosinophilia was not detected, and stool examinations did not show Angiostrongylus cantonensis larva or its egg. In both cases of surgical removal, an immature male worm was identified by the parasitologist. After treatment, the visual acuity was slightly improved in all cases. CONCLUSION: This case series illustrated the different ocular manifestations of angiostrongyliasis and that although several treatments were used, the visual outcome was not markedly improved and depended mainly on the initial visual acuity.

Evaluation of immunoglobulin G4 subclass antibody in a peptide-based enzyme-linked immunosorbent assay for the serodiagnosis of human fascioliasis.

SUMMARYTo improve the diagnosis of human fascioliasis caused by Fasciola gigantica, we developed a peptide-based enzyme-linked immunosorbent assay (peptide-based ELISA) based on the detection of specific IgG4 subclass antibody. Two identified B-cell epitopes of F. gigantica cathepsin L1 were synthesized as single synthetic peptides, acetyl-DKIDWRESGYVTELKDQGNC-carboxamide (peptide L) and acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide (peptide V), and their diagnostic potential was evaluated. The sera of 25 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 cholangiocarcinoma patients and 57 healthy controls were analysed. The sensitivity, specificity, accuracy, and positive and negative predictive values of this assay were the same with both peptides at 100%, 99.7%, 99.7%, 96.2% and 100%, respectively. These highly sensitive and specific peptide-based ELISAs for the detection of specific IgG4 antibody could be useful for laboratory diagnosis of human fascioliasis in future large-scale surveys throughout Southeast Asia where this disease is prevalent.

Intraocular angiostrongyliasis: clinical findings, treatments and outcomes.

Ocular angiostrongyliasis, diagnosed by identification of Angiostrongylus cantonensis in any part of the eye, is a very rare manifestation. We report seven cases of intraocular angiostrongyliasis in Srinagarind Hospital, Khon Kaen University, Thailand. From a total of 654 cases of angiostrongyliasis diagnosed between January 1995 and April 2005, 7 cases (1.1%) with ocular manifestations were found. Four men and three women were diagnosed, with a mean age of 32.1 years (range 21-46 years). All of the patients lived in the northeast of Thailand and acquired the infection by eating raw Pila spp. snails, the intermediate host of A. cantonensis. The incubation period lasted from 2 weeks to 2 months. The most common symptom, blurred vision without eosinophilic meningitis, occurred as a presenting symptom in five cases. The other two cases presented with eosinophilic meningitis prior to development of poor visual acuity. Both cases had papilloedema, neck stiffness and eosinophilia without fever. The visual acuity of the patient was mostly lower than 2/60 and, evidently, visual impairment in all patients was caused by retinal pigment epithelial defects. All cases had only one immature A. cantonensis worm in the eye, with the most common site being the intravitreous area. Several treatments, including oral prednisolone, topical prednisolone, argon laser, diode laser, Nd:YAG laser, surgical removal of the parasite and combination therapy, were used. There is no evidence that surgical and laser interventions improve the course of the disease, and both have associated risks. Visual outcome depends on the initial visual defects.

Comparison of the quantitative formalin ethyl acetate concentration technique and agar plate culture for diagnosis of human strongyloidiasis.

The quantitative formalin ethyl acetate concentration technique (QFEC) was compared to agar plate culture (APC) for the detection of Strongyloides stercoralis larvae. QFEC could substitute for APC only when the parasite load was higher than 50 larvae per g of stool. This study serves as a good reminder to those conducting stool exams about the sensitivity and specificity limitations of both techniques.

Genomic characterization of lung flukes, Paragonimus heterotremus, P. siamensis, P. harinasutai, P. westermani and P. bangkokensis by RAPD markers.

Random amplified polymorphic DNA (RAPD) markers were assayed in an attempt to discriminate among five species of Paragonimus. Genomic DNAs of two strains of Paragonimus heterotremus from two provinces in Thailand, Saraburi and Phitsanulok, as well as of P. siamensis, P. harinasutai, P. westermani and P. bangkokensis were extracted and amplified by an arbitrary primer, namely P2 (5-GTTTCGCTCC-3). RAPD patterns showed that those five species were genetically distinct, although they shared genomic DNA to some extent. This primer could also distinguish between two strains of P. heterotremus. The polymorphism observed allowed to construct a relationship dendrogram. The phylogenetic dendrogram showed that the P. heterotremus strains were closest to P. harinasutai, followed by P. siamensis, P. bangkokensis and P. westermani.

Characterisation of Fasciola gigantica adult 27-kDa excretory-secretory antigen in human fascioliasis.

The 27-kDa excretory-secretory antigen partially purified from adult Fasciola gigantica adult worms (FG27) has been used as the sensitive and specific antigen for immunodiagnosis of human fascioliasis. Lectin-blot analyses have shown that the FG27 possesses both N - and O -glycans. In two-dimensional electrophoresis, silver staining, lectin blotting and immunoblotting, FG27 displayed at least four antigenic-glycosylated protein spots at the pI values of 4.8, 4.9, 5.2 and 5.4, respectively. After removing glycan moieties from the antigen by alkaline treatment, the molecular mass of the FG27 was reduced to 26.5 kDa as a prominent deglycosylated band. Immunoblotting analysis has revealed that the 26.5-kDa band reacts with the pooled human fascioliasis sera without any loss of antigenicity. These results suggest that the major antigenicity of the FG27 is due to its protein epitopes.

Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

In vitro excystation of Paragonimus heterotremus metacercariae.

In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39-40 C, and osmolarity of 250-350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5-4 microg/ml), L-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1-8 microM), N-tosyl-L-phenylalanine chloromethyl ketone (0.1-0.4 mM), N alpha-p-tosyl-L-lysine chloromethyl ketone (25-200 microg/ml), iodoacetic acid (0.5-4 mM), and phenylmethylsulfonyl fluoride (1-4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.

Immunoblot evaluation of the specificity of the 29-kDa antigen from young adult female worms Angiostrongylus cantonensis for immunodiagnosis of human angiostrongyliasis.

The antigenic components of Angiostrongylus cantonensis young adult female worm somatic extract (FSE) were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The sera tested were from patients with proven angiostrongyliasis, other parasitic diseases, and healthy adults. Both the sera and cerebrospinal fluid (CSF) were tested from patients with clinical angiostrongyliasis. The CSF from patients with other neurological diseases were also included. Using SDS-PAGE, we found that the FSE comprised more than 30 polypeptides. Immunoblot analysis revealed at least 12 or 13 antigenic bands in patients with proven or clinical angiostrongyliasis, respectively. The patterns of reactivity recognized by the serum and CSF antibodies against FSE were similar. These antigenic components had molecular masses ranging from less than 14.4 to more than 94 kDa. The prominent antigenic band of 29-kDa might serve as a reliable marker for the diagnosis of angiostrongyliasis. The sensitivity, specificity, positive and negative predictive values of immunoblot analysis in this antigenic band were 55.6%, 99.4%, 83.3% and 97.4%, respectively.

Gnathostoma spinigerum: analysis of protein patterns by two dimensional gel electrophoresis.

The protein extracts from male (MS) and female (FS) adults and advanced third-stage larvae (LS) of Gnathostoma spinigerum were separated by high resolution two-dimensional gel electrophoresis (2-DE). The polypeptide spots, as detected by silver staining, were subsequently identified. The spot patterns of LS, MS and FS were highly complex and consisted of more than 75, 44, 52 prominent spots, respectively. In addition, the stage-specific protein patterns were identified. This 2-DE database should provide an important reference for future biological and biochemical studies of G. spinigerum.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis.

Immunodominant antigens of an approximate molecular mass of 27 kD were obtained from an excretory-secretory product of adult Fasciola gigantica by a continuous-elution method. An indirect ELISA using the antigens obtained by this relatively simple procedure was developed for detecting specific antibodies from patients infected with F. gigantica. Sera from patients with other parasitic infections, healthy volunteers, and cholangiocarcinoma were also analyzed. The sensitivity, specificity, and positive and negative predictive values for this ELISA using the fractionated antigens were 100%. The data indicated a possible correlation of antibodies to F. gigantica with cholangiocarcinoma.