{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T15:42:33Z","timestamp":1783525353900,"version":"3.55.0"},"reference-count":35,"publisher":"American Society of Tropical Medicine and Hygiene","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2017,1,11]]},"abstract":"<jats:p>We developed a combined conventional polymerase chain reaction (PCR) and real-time PCR (qPCR)-based assay for detecting and discriminating between <jats:italic>Opisthorchis viverrini<\/jats:italic> and <jats:italic>Haplorchis taichui<\/jats:italic> parasite infections. The first PCR amplifies the mitochondrial cytochrome c oxidase subunit I (<jats:italic>COI<\/jats:italic>) genes of parasites, and differential diagnosis is achieved by performing qPCR with specific primers and SYBR Green I. The detection limit of the assay was found to be 2.0 \u00d7 10<jats:sup>2<\/jats:sup> plasmid copies in a test in which a stool sample was spiked with a single egg, which is equivalent to 5 eggs per gram (EPG). The testing of 34 clinical stool samples that had been demonstrated to contain \u201c<jats:italic>Opisthorchis<\/jats:italic>-like\u201d eggs by microscopy showed that the novel assay exhibited a sensitivity of 100% for \u201c<jats:italic>Opisthorchis<\/jats:italic>-like\u201d parasitic infections, and 71% and 91% of these samples were found to be infected with <jats:italic>O. viverrini<\/jats:italic> and <jats:italic>H. taichui<\/jats:italic>, respectively. A further four parasitic infections were diagnosed in the 16 negative samples, and the microscopic findings of these samples were confirmed to be false negatives by sequencing analysis. The assay also displayed high specificity during the testing of 10 samples containing other common parasites. The fact that our qPCR SYBR Green I\u2013based assay detected submicroscopic traces of parasitic DNA and was able to differentiate between parasites that produce eggs with similar morphologies indicates that it has a good potential for development of diagnostic application to use in areas where multiple parasites coexist.<\/jats:p>","DOI":"10.4269\/ajtmh.16-0165","type":"journal-article","created":{"date-parts":[[2016,11,8]],"date-time":"2016-11-08T03:44:29Z","timestamp":1478576669000},"page":"221-228","source":"Crossref","is-referenced-by-count":16,"title":["Development of a PCR Assay for Diagnosing Trematode (Opisthorchis and Haplorchis) Infections in Human Stools"],"prefix":"10.4269","volume":"96","author":[{"given":"Pheophet","family":"Lamaningao","sequence":"first","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."},{"name":"Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seiji","family":"Kanda","sequence":"additional","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."},{"name":"Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sakhone","family":"Laimanivong","sequence":"additional","affiliation":[{"name":"Center of Malariology, Parasitology, and Entomology, Ministry of Health, Vientiane, Lao People's Democratic Republic."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takaki","family":"Shimono","sequence":"additional","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."},{"name":"Regenerative Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew Waleluma","family":"Darcy","sequence":"additional","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amphay","family":"Phyaluanglath","sequence":"additional","affiliation":[{"name":"Clinical Laboratory, Mahosot Hospital, Ministry of Health, Vientiane, Lao People's Democratic Republic."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nobuyuki","family":"Mishima","sequence":"additional","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshimasa","family":"Nishiyama","sequence":"additional","affiliation":[{"name":"Department of Public Health, Kansai Medical University, Osaka, Japan."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2795","reference":[{"key":"ref1","series-title":"PLoS Med","first-page":"e201","article-title":"Liver fluke induces cholangiocarcinoma","volume":"4","author":"Sripa","year":"2007"},{"key":"ref2","series-title":"J Parasitol","first-page":"1165","article-title":"A focus of human infection by Haplorchis taichui (Trematoda: Heterophyidae) in the southern Philippines","volume":"90","author":"Belizario","year":"2004"},{"key":"ref3","series-title":"Korean J Parasitol","first-page":"439","article-title":"Intestinal helminths recovered from humans in Xieng Khouang Province, Lao PDR with a particular note on Haplorchis pumilio infection","volume":"53","author":"Chai","year":"2015"},{"key":"ref4","series-title":"Trans R Soc Trop Med Hyg","first-page":"247","article-title":"Diversity of human intestinal helminthiasis in Lao PDR","volume":"103","author":"Sayasone","year":"2009"},{"key":"ref5","series-title":"Korean J Parasitol","first-page":"213","article-title":"High prevalence of liver and intestinal fluke infections among residents of Savannakhet Province in Laos","volume":"45","author":"Chai","year":"2007"},{"key":"ref6","series-title":"Korean J Parasitol","first-page":"243","article-title":"High prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and other helminth infections among people in Khammouane province, Lao PDR","volume":"47","author":"Chai","year":"2009"},{"key":"ref7","series-title":"Korean J Parasitol","first-page":"339","article-title":"Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR","volume":"48","author":"Chai","year":"2010"},{"key":"ref8","series-title":"Acta Trop","first-page":"81","article-title":"Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions","volume":"109","author":"Sato","year":"2009"},{"key":"ref9","series-title":"J Clin Microbiol","first-page":"366","article-title":"Improvement of PCR for detection of Opisthorchis viverrini DNA in human stool samples","volume":"46","author":"Duenngai","year":"2008"},{"key":"ref10","series-title":"Acta Trop","first-page":"26","article-title":"Evaluation of PCR based coprodiagnosis of human opisthorchiasis","volume":"97","author":"Stensvold","year":"2006"},{"key":"ref11","series-title":"J Clin Microbiol","first-page":"3879","article-title":"Detection of Opisthorchis viverrini in human stool specimens by PCR","volume":"40","author":"Wongratanacheewin","year":"2002"},{"key":"ref12","series-title":"Parasitol Res","first-page":"905","article-title":"PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers","volume":"100","author":"Muller","year":"2007"},{"key":"ref13","series-title":"Parasitol Int","first-page":"90","article-title":"Families Opisthorchiidae and Heterophyidae: are they distinct?","volume":"61","author":"Thaenkham","year":"2012"},{"key":"ref14","series-title":"Acta Trop","first-page":"26","article-title":"Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker","volume":"103","author":"Thaenkham","year":"2007"},{"key":"ref15","series-title":"J Clin Microbiol","first-page":"1517","article-title":"PCR Diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in a Lao Community in an area of endemicity and comparison of diagnostic methods for parasitological field surveys","volume":"47","author":"Lovis","year":"2009"},{"key":"ref16","series-title":"Parasitol Int","first-page":"178","article-title":"Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP)","volume":"61","author":"Arimatsu","year":"2012"},{"key":"ref17","series-title":"Korean J Parasitol","first-page":"689","article-title":"Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis","volume":"51","author":"Kaewkong","year":"2013"},{"key":"ref18","series-title":"Parasitol Res","first-page":"89","article-title":"Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis","volume":"111","author":"Sanpool","year":"2012"},{"key":"ref19","series-title":"Exp Parasitol","first-page":"109","article-title":"Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination","volume":"112","author":"Le","year":"2006"},{"key":"ref20","series-title":"Biotechniques","first-page":"130","article-title":"Continuous fluorescence monitoring of rapid cycle DNA amplification","volume":"22","author":"Wittwer","year":"1997"},{"key":"ref21","series-title":"Anal Biochem","first-page":"154","article-title":"Product differentiation by analysis of DNA melting curves during the polymerase chain reaction","volume":"245","author":"Ririe","year":"1997"},{"key":"ref22","series-title":"J Microbiol Methods","first-page":"295","article-title":"Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis","volume":"51","author":"Nicolas","year":"2002"},{"key":"ref23","series-title":"J Clin Microbiol","first-page":"2756","article-title":"Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene","volume":"38","author":"Pietila","year":"2000"},{"key":"ref24","series-title":"J Clin Microbiol","first-page":"1511","article-title":"SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability","volume":"42","author":"Papin","year":"2004"},{"key":"ref25","series-title":"Korean J Parasitol","first-page":"51","article-title":"Prevalence of helminthic infections among inhabitants of Lao PDR","volume":"52","author":"Eom","year":"2014"},{"key":"ref26","series-title":"Parasitology","first-page":"1479","article-title":"Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes","volume":"135","author":"Saijuntha","year":"2008"},{"issue":"(Suppl 2)","key":"ref27","series-title":"Southeast Asian J Trop Med Public Health","first-page":"17","article-title":"Nucleotide sequence of mitochondrial CO I and ribosomal ITS II genes of Opisthorchis viverrini in northeast Thailand","volume":"32","author":"Ando","year":"2001"},{"key":"ref28","series-title":"J Clin Microbiol","first-page":"1300","article-title":"Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions","volume":"28","author":"Wilde","year":"1990"},{"key":"ref29","series-title":"J Clin Microbiol","first-page":"4463","article-title":"Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat","volume":"38","author":"Abu Al-Soud","year":"2000"},{"key":"ref30","series-title":"Int J Lab Hematol","first-page":"267","article-title":"BsaXI\/RFLP analysis of initial or selectively reamplified PCR product is unreliable in detecting the V617F mutation in JAK2","volume":"33","author":"Shepard","year":"2011"},{"key":"ref31","series-title":"ScientificWorldJournal","first-page":"893981","article-title":"Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis","volume":"2014","author":"Cai","year":"2014"},{"key":"ref32","series-title":"Exp Parasitol","first-page":"353","article-title":"Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples","volume":"120","author":"Umesha","year":"2008"},{"key":"ref33","series-title":"Trans R Soc Trop Med Hyg","first-page":"538","article-title":"Human Opisthorchis and Haplorchis infections in Laos","volume":"85","author":"Giboda","year":"1991"},{"key":"ref34","series-title":"Korean J Parasitol","first-page":"305","article-title":"Hyperendemicity of Haplorchis taichui infection among Riparian people in Saravane and Champasak province, Lao PDR","volume":"51","author":"Chai","year":"2013"},{"issue":"(Suppl 1)","key":"ref35","series-title":"Acta Trop","first-page":"S158","article-title":"Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos","volume":"120","author":"Sripa","year":"2011"}],"container-title":["The American Society of Tropical Medicine and Hygiene"],"original-title":[],"link":[{"URL":"https:\/\/www.ajtmh.org\/view\/journals\/tpmd\/96\/1\/article-p221.xml","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.ajtmh.org\/downloadpdf\/journals\/tpmd\/96\/1\/article-p221.xml","content-type":"text\/html","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/www.ajtmh.org\/downloadpdf\/journals\/tpmd\/96\/1\/article-p221.xml","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,7]],"date-time":"2024-08-07T22:44:08Z","timestamp":1723070648000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.ajtmh.org\/view\/journals\/tpmd\/96\/1\/article-p221.xml"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,1,11]]},"references-count":35,"journal-issue":{"issue":"1"},"URL":"https:\/\/doi.org\/10.4269\/ajtmh.16-0165","relation":{},"ISSN":["0002-9637","1476-1645"],"issn-type":[{"value":"0002-9637","type":"print"},{"value":"1476-1645","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,1,11]]}}}