{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T06:05:20Z","timestamp":1782453920341,"version":"3.54.5"},"reference-count":41,"publisher":"American Society of Tropical Medicine and Hygiene","issue":"4","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2023,10,4]]},"abstract":"ABSTRACT.<\/jats:title>\nThere are many techniques for malaria diagnosis. Currently, the nested polymerase chain reaction (PCR) method based on a small subunit ribosomal RNA gene (18S rRNA) has been used as a confirmatory method. However, this method is time-consuming, laborious, and costly. Therefore, the objective of this study was to develop nested multiplex PCR for Plasmodium<\/jats:italic> species identification using the dihydropterin pyrophosphokinase-dihydropteroate synthase (hppk-dhps<\/jats:italic>) gene. Genus- and species-specific primers for the hppk-dhps<\/jats:italic> gene were designed. The performance of the novel nested multiplex PCR was compared with 18S rRNA nested PCR. A total of 115 blood samples were used in this study, including 84 infected samples and 31 uninfected samples. Analysis of the blood samples by nested multiplex PCR targeting the hppk-dhps<\/jats:italic> gene identified 81 infected cases. The level of agreement between this novel method and 18S rRNA nested PCR was 97.4%. Further, the novel method successfully detected all human malaria parasites except Plasmodium ovale<\/jats:italic> and detected mixed Plasmodium falciparum<\/jats:italic>\/Plasmodium vivax<\/jats:italic> infections. The sensitivity and specificity obtained from this novel method were 96.4% and 100%, respectively. The limit of detection of the hppk-dhps<\/jats:italic> nested multiplex PCR for P. falciparum<\/jats:italic> and P. vivax<\/jats:italic> was 500 parasites\/\u00b5L and 4 parasites\/\u00b5L, respectively. The lowest parasite gDNA detected by this method was 0.5\u2009ng\/\u00b5L for P. falciparum<\/jats:italic> and 0.1\u2009ng\/\u00b5L for P. vivax<\/jats:italic>. These results corroborate that the hppk-dhps<\/jats:italic> gene is a novel amplification target for the detection of human malaria. This novel target PCR-based method is a beneficial approach for malaria diagnosis, as well as species identification and differentiation.<\/jats:p>","DOI":"10.4269\/ajtmh.23-0130","type":"journal-article","created":{"date-parts":[[2023,9,11]],"date-time":"2023-09-11T15:22:58Z","timestamp":1694445778000},"page":"770-777","source":"Crossref","is-referenced-by-count":1,"title":["A Novel Nested Multiplex Polymerase Chain Reaction Assay for Malaria Diagnosis Using the Hydroxymethyl Dihydropterin Pyrophosphokinase-Dihydropteroate Synthase (hppk-dhps) Gene"],"prefix":"10.4269","volume":"109","author":[{"given":"Natpasit","family":"Chaianantakul","sequence":"first","affiliation":[{"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tippawan","family":"Sungkapong","sequence":"additional","affiliation":[{"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kanokwan","family":"Nikhomkham","sequence":"additional","affiliation":[{"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kansuda","family":"Sanseewong","sequence":"additional","affiliation":[{"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sawanya","family":"Kornsang","sequence":"additional","affiliation":[{"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2795","reference":[{"key":"b1","series-title":"World Malaria Report 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