Parasites, Hosts and Diseases

"Viraphong Lulitanond"

Brief Communication

A Recombinant Matrix Metalloproteinase Protein from Gnathostoma spinigerum for Serodiagnosis of Neurognathostomiasis: Penchom Janwan, Pewpan M. Intapan, Hiroshi Yamasaki, Porntip Laummaunwai, Kittisak Sawanyawisuth, Chaisiri Wongkham, Chatchai Tayapiwatana, Amnat Kitkhuandee, Viraphong Lulitanond, Yukifumi Nawa, Wanchai Maleewong; Korean J Parasitol 2013;51(6):751-754.
Published online December 31, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.6.751

Abstract
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Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

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Development of Immunochromatographic Test Kit for Rapid Detection of Specific IgG4 Antibody in Whole-Blood Samples for Diagnosis of Human Gnathostomiasis
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Original Articles

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis: Worasak Kaewkong, Pewpan M. Intapan, Oranuch Sanpool, Penchom Janwan, Tongjit Thanchomnang, Porntip Laummaunwai, Viraphong Lulitanond, Pham Ngoc Doanh, Wanchai Maleewong; Korean J Parasitol 2013;51(6):689-694.
Published online December 31, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.6.689

Abstract
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Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

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Picha Suwannahitatorn, Mathirut Mungthin, Ittisak Subrungruang, Lakhanawan Charoensuk, Nithikoon Aksorn, Saiwasan Buathong, Krystyna Cwiklinski
PLOS Neglected Tropical Diseases.2024; 18(8): e0012445. CrossRef
An Approach for Egg Parasite Classification Based on Ensemble Deep Learning
Narut Butploy, Wanida Kanarkard, Pewpan M. Intapan, Oranuch Sanpool
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Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis: Amornmas Kongklieng, Worasak Kaewkong, Pewpan M. Intapan, Oranuch Sanpool, Penchom Janwan, Tongjit Thanchomnang, Viraphong Lulitanond, Pusadee Sri-Aroon, Yanin Limpanont, Wanchai Maleewong; Korean J Parasitol 2013;51(6):651-656.
Published online December 31, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.6.651

Abstract
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Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

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Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time P: Tongjit Thanchomnang, Pewpan M. Intapan, Chairat Tantrawatpan, Viraphong Lulitanond, Sudchit Chungpivat, Piyanan Taweethavonsawat, Worasak Kaewkong, Oranuch Sanpool, Penchom Janwan, Wej Choochote, Wanchai Maleewong; Korean J Parasitol 2013;51(6):645-650.
Published online December 31, 2013; DOI: https://doi.org/10.3347/kjp.2013.51.6.645

Abstract
PDF

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

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