Oranuch Sanpool

Molecular identification of Paragonimus species by DNA pyrosequencing technology

DNA pyrosequencing for PCR amplicons is an attractive strategy for the identification of microorganisms because of its short time performance for large number of samples. In this study, the primers targeting the fragment of ITS2 region of nuclear ribosomal RNA gene were newly developed for pyrosequencing-based identification of 6 Paragonimus species, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus macrorchis, Paragonimus siamensis and Paragonimus westermani. Pyrosequencing determination of 39 nucleotides of partial ITS2 region could discriminate 6 Paragonimus species, and could also detect intra-species genetic variation of P. macrorchis. This DNA pyrosequencing-based identification can be a valuable tool to improve species-level identification of Paragonimus in the endemic areas.

Molecular Markers for Detection and Differentiation of Plasmodium falciparum and Plasmodium vivax in Human Blood Samples by Pyrosequencing

ABSTRACT PCR amplification coupled with pyrosequencing was used to measure molecular markers that could be used to detect and differentiate Plasmodium falciparum and Plasmodium vivax in human blood samples. The detection rates were in agreement with the results of Giemsa-stained film microscopy, which is the current gold standard for detection. This method provides an exciting alternative for malaria diagnosis.

Molecular Identification of a Case of Paragonimus pseudoheterotremus Infection in Thailand

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Southeast Asia, Paragonimus heterotremus is the only proven causative pathogen. Recently, a new Paragonimus species, P. pseudoheterotremus, was found in Thailand. This species is genetically similar to P. heterotremus and is considered as a sister species. However, infectivity or pathogenicity of P. pseudoheterotremus to humans remains unclear. We report the first confirmed human pulmonary paragonimiasis case caused by P. pseudoheterotremus infection. After polymerase chain reaction/sequencing of the DNA extracted from Paragonimus eggs in the sputum of the patient, partial internal transcribed spacer 2 and cytochrome c oxidase subunit 1 sequences were approximately identical (98-100%) with those of P. pseudoheterotremus. For P. heterotremus, the partial internal transcribed spacer 2 sequence was approximately identical (99-100%), but the partial mitochondrial cytochrome c oxidase subunit 1 sequence showed a similarity of 90-95%.

Current Status of Human Hookworm Infections, Ascariasis, Trichuriasis, Schistosomiasis Mekongi and Other Trematodiases in Lao People's Democratic Republic

Soil-transmitted helminth (STH) infections, and schistosomiasis and other trematodiases often have a high prevalence in developing countries. Here, we present updated information on the prevalence of these parasites in the Lao Peoples Democratic Republic (Lao PDR) in 2012, arising from the annual national helminthiasis survey. Fecal specimens were collected from 8,610 inhabitants of 12 provinces and one municipality (Bokeo, Houaphan, Luang Namtha, Luang Prabang, Oudomxay, and Phongsaly Provinces from northern Lao PDR; Bolikhamxay and Xieng Khouang Provinces and Vientiane Municipality from the central part of the country; and Attapeu, Champasak, Saravan, and Sekong Provinces from southern Lao PDR). The overall prevalence of three major STHs, Ascaris lumbricoides, Trichuris trichiura, and hookworms (Necator americanus and Ancylostoma duodenale) were 11.6%, 8.5%, and 25.0%, respectively. Prevalence of Schistosoma mekongi infection was 0.1%, and of miscellaneous trematodiases (including opisthorchiasis) was 14.0%. Clearly, the nationwide parasite control project is still necessary to reduce morbidity caused by helminthic diseases.

Molecular Detection of Ancylostoma duodenale, Ancylostoma ceylanicum, and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.

Molecular Identification of Trichinella papuae from a Thai Patient with Imported Trichinellosis

Previously, we reported the presence of imported trichinellosis in a Thai worker returning from Malaysia, who presented with progressive generalized muscle hypertrophy and weakness after eating wild boar meat. This work analyzed a partial small subunit of a mitochondrial ribosomal RNA gene of Trichinella larvae isolated from the patient. The results showed complete identity with a mitochondrial RNA gene of Trichinella papuae (GenBank accession no. EF517130). This is the first report of imported trichinellosis in Thailand caused by T. papuae. It is possible that T. papuae is widely distributed in the wildlife of Southeast Asia.

Nine human sparganosis cases in Thailand with molecular identification of causative parasite species.

Human sparganosis is one of the neglected diseases but important food-borne parasitic zoonoses. The disease is caused by larvae (spargana) of diphyllobothriidean tapeworm. Here, we describe nine cases of human sparganosis, caused by Spirometra erinaceieuropaei in a hospital in Thailand during 2001-2012. Clinical characteristics, treatment, and outcome of cases were revealed. Diagnosis and identification of causative parasite species was made by histopathological investigations followed by a polymerase chain reaction-based molecular method using formalin-fixed paraffin embedded tissues. The DNA samples were extracted from tissues and a partial fragment of cytochrome c oxidase subunit 1 (cox1) gene was amplified for the detection of parasitic DNA. Infection could be prevented by increasing activities on health communication by responsible public health agencies.

Molecular Evidence of Trichostrongylus colubriformis and Trichostrongylus axei Infections in Humans from Thailand and Lao PDR

Human trichostrongylosis has been reported in Thailand. Recent reports in Lao Peoples Democratic Republic concerning species identification urged us to investigate species distribution in Thailand. We report eight human cases in Thailand and Lao Peoples Democratic Republic that were found to be infected by Trichostrongylus colubriformis and T. axei identified and confirmed by molecular techniques. This evidence is the first molecular evidence of human T. colubriformis and T. axei infection in Thailand. Infection by these two species was apparently epidemic in these areas. It is necessary to proceed with more comprehensive veterinary and epidemiologic studies to enable the practical prevention and control of this parasitic zoonosis.

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.