Development of an immunochromatographic point-of-care test for detection of IgG antibody in serodiagnosis of human trichinellosis.

OBJECTIVE: Trichinellosis is a globally distributed food-borne parasitic disease. Initially, diagnosis is usually made based on clinical signs, symptoms, and history of eating raw or undercooked meat. In the present study, an immunochromatographic test (ICT) kit was developed to diagnose trichinellosis by detecting IgG antibodies in sera of infected humans. METHODS: Somatic extract from Trichinella spiralis larvae was used as the antigen for the ICT kit development. Diagnostic efficacy was evaluated using human serum samples from proven trichinellosis patients, healthy persons, and those with other parasitic infections. RESULTS: The diagnostic sensitivity, specificity, positive and negative predictive values and accuracy of the ICT kit were 100.0% (95%; CI 88.8 to 100.0%), 92.5% (95% CI; 86.9 to 96.2%), 73.8% (95% CI; 58.0 to 86.1%), 100.0% (95% CI; 97.3 to 100.0%) and 93.8% (95% CI; 89.2 to 96.9%), respectively. CONCLUSIONS: This ICT diagnostic kit can be used as a testing tool for human trichinellosis. This test should permit rapid detection of infection to enable prompt anthelminthic treatment. It can also be used for retrospective diagnoses and field surveys based at laboratories where sophisticated equipment is lacking.

Possible transmission of Strongyloides fuelleborni between working Southern pig-tailed macaques (Macaca nemestrina) and their owners in Southern Thailand: Molecular identification and diversity.

Human strongyloidiasis is caused by Strongyloides stercoralis, S. fuelleborni fuelleborni and Strongyloides f. kellyi. Strongyloides fuelleborni is a soil-transmitted nematode parasite typically infecting non-human primates. The southern pig-tailed macaque (Macaca nemestrina) is distributed throughout the southern part of Thailand and could be a source of zoonotic transmission of this nematode. Here, we extracted DNA from Strongyloides speciescultured from the feces of southern pig-tailed macaques and their owners. Using PCR and sequencing of the extracted DNA, we compared the nucleotide sequences of these worms using portions of the 18S rDNA hypervariable region IV (HVR-IV) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Sequences from the 18S rRNA gene were obtained from worms from 23 southern pig-tailed macaques and from one owner. These sequences were identical with each other and with all East and Southeast Asian S. fuelleborni sequences (from Japan, Thailand, and Lao PDR) in the GenBank database. A median-joining network of published cox1 sequences (n = 123), in combination with the present 24 new sequences, represented 107 haplotypes distributed among six clusters, which corresponded to geographical localities but did not relate to host species. The S. fuelleborni cox1 sequences from some southern pig-tailed macaques and the one infected owner shared the same cox1 haplotype. This is the first evidence of likely zoonotic transmission of S. fuelleborni from a reservoir host, M. nemestrina.

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.

Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing.

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

Pyrosequencing for rapid molecular identification of Schistosoma japonicum and S. mekongi eggs and cercariae.

Schistosomiasis, which is caused by Schistosoma japonicum and S. mekongi, is a chronic and dangerous widespread disease affecting several countries in Asia. Differentiation between S. japonicum and S. mekongi eggs and/or cercariae via microscopic examination is difficult due to morphological similarities. It is important to identify these etiological agents isolated from animals and humans at the species or genotype level. In this study, a pyrosequencing assay designed to detect S. japonicum and S. mekongi DNA in fecal samples and infected snails was developed and evaluated as an alternative tool to diagnose schistosomiasis. New primers targeting the 18S ribosomal RNA gene were designated for specific amplification. S. japonicum and S. mekongi were identified using a 43-nucleotide pattern of the 18S ribosomal RNA gene and were differentiated using 7 nucleotides within this region. S. japonicum and S. mekongi-infected snails and fecal samples derived from infected mice and rats were differentially detected within a short period of time. The analytical sensitivity of the method enabled the identification of as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and 2 eggs inoculated in 100mg of non-infected fecal sample. To evaluate the comparative efficacy of the assay, identical samples were also analyzed via microscopy and Sanger sequencing. The pyrosequencing technique was found to be superior to the microscopy method and more rapid than the Sanger sequencing method. These results suggest that the pyrosequencing assay is rapid, simple, sensitive and accurate in identifying S. japonicum and S. mekongi in intermediate hosts and fecal samples of the final host.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships.

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.