{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T23:58:09Z","timestamp":1790035089527,"version":"4.0.1"},"reference-count":30,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2019,5,10]],"date-time":"2019-05-10T00:00:00Z","timestamp":1557446400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"publisher","award":["3\/1\/3\/PDF (11)\/2016-HRD"],"award-info":[{"award-number":["3\/1\/3\/PDF (11)\/2016-HRD"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJERPH"],"abstract":"<jats:p>Sulphadoxine and pyrimethamine (SP) have been used as long-acting partner antimalarial drugs in artemisinin combination therapy (ACT) for falciparum malaria. The emergence and increasing spread of SP resistance in malaria-endemic areas have become a challenge for the control of malaria. Therefore, regular monitoring of the mutation status of partner drugs is important for the better management of drug policy. There are limitations with traditional molecular methods and there is an urgent need for an easy method for diagnosis of drug resistance. In this study we have introduced and developed a novel single nucleotide polymorphism loop-mediated isothermal amplification (SNP\u2013LAMP) approach based on a hydroxynaphthol blue (HNB) indicator for the easier and quicker detection of pyrimethamine resistance in Plasmodium falciparum malaria. To implement this novel approach, many sets of LAMP primers were designed and tested. Finally, one set of forward inner primer M1 (FIPM1) of LAMP primer was selected that specifically distinguishes pyrimethamine-resistant P. falciparum malaria. The LAMP reactions were optimized at 60\u201366 \u00b0C for 45 min. High sensitivity (7 parasites\/\u00b5L) was observed with 10\u22124 fold dilutions (&lt;2 ng DNA) of genomic DNA. Moreover, this approach has the potential to be applied even in laboratories unfamiliar with PCR or other molecular methods, and in future, this can be helpful for the better management of anti-malarial policy.<\/jats:p>","DOI":"10.3390\/ijerph16091635","type":"journal-article","created":{"date-parts":[[2019,5,13]],"date-time":"2019-05-13T03:57:07Z","timestamp":1557719827000},"page":"1635","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Development and Evaluation of a Novel HNB Based Isothermal Amplification Assay for Fast Detection of Pyrimethamine Resistance (S108N) in Plasmodium falciparum"],"prefix":"10.3390","volume":"16","author":[{"given":"Madhvi","family":"Chahar","sequence":"first","affiliation":[{"name":"Division of Epidemiology and Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anup","family":"Anvikar","sequence":"additional","affiliation":[{"name":"Division of Epidemiology and Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neena","family":"Valecha","sequence":"additional","affiliation":[{"name":"Division of Epidemiology and Clinical Research, National Institute of Malaria Research, Sector-8 Dwarka, New Delhi 110077, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,5,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.actatropica.2012.02.004","article-title":"Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: Would it come to clinical reality as a point-of-care test?","volume":"122","author":"Karanis","year":"2012","journal-title":"Acta Trop."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/S1473-3099(10)70214-0","article-title":"Antimalarial drug resistance of Plasmodium falciparum in India: Changes over time and space","volume":"11","author":"Shah","year":"2011","journal-title":"Lancet Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/s11908-010-0099-y","article-title":"Drug-Resistant Malaria: The Era of ACT","volume":"12","author":"Lin","year":"2010","journal-title":"Curr. Infect. Dis. Rep."},{"key":"ref_4","unstructured":"WHO (2010). Guidelines for the Treatment of Malaria, WHO."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1128\/AAC.48.3.879-889.2004","article-title":"Plasmodium falciparum isolates in India exhibit a progressive increase in mutations associated with sulfadoxine-pyrimethamine resistance","volume":"48","author":"Ahmed","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mohapatra, P.K., Sarma, D.K., Prakash, A., Bora, K., Ahmed, M.A., Sarma, B., Goswami, B.K., Bhattacharyya, D.R., and Mahanta, J. (2014). Molecular evidence of increased resistance to anti-folate drugs in Plasmodium falciparum in North-East India: A signal for potential failure of artemisinin plus sulphadoxine-pyrimethamine combination therapy. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0105562"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1186\/1475-2875-12-247","article-title":"A clinical and molecular study of artesunate + sulphadoxine-pyrimethamine in three districts of central and eastern India","volume":"12","author":"Srivastava","year":"2013","journal-title":"Malar. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1546","DOI":"10.1128\/AAC.50.4.1546-1549.2006","article-title":"Quadruple mutations in dihydrofolate reductase of Plasmodium falciparum isolates from Car Nicobar Island, India","volume":"50","author":"Ahmed","year":"2006","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1046\/j.1365-3156.2000.00632.x","article-title":"Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from India and Thailand: A molecular epidemiologic study","volume":"5","author":"Biswas","year":"2000","journal-title":"Trop. Med. Int. Health"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/0163-7258(90)90017-V","article-title":"The dihydrofolate reductase-thymidylate synthetase gene in the drug resistance of malaria parasites","volume":"48","author":"Hyde","year":"1990","journal-title":"Pharmacol. Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"E63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1006\/mcpr.2002.0415","article-title":"Accelerated reaction by loop-mediated isothermal amplification using loop primers","volume":"16","author":"Nagamine","year":"2002","journal-title":"Mol. Cell. Probes"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1006\/bbrc.2001.5921","article-title":"Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation","volume":"289","author":"Mori","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"41119","DOI":"10.1038\/srep41119","article-title":"Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum","volume":"7","author":"Chahar","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.exppara.2018.05.001","article-title":"Evaluation of four novel isothermal amplification assays towards simple and rapid genotyping of chloroquine resistant Plasmodium falciparum","volume":"190","author":"Chahar","year":"2018","journal-title":"Exp. Parasitol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1016\/j.parint.2016.10.024","article-title":"Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick","volume":"66","author":"Yongkiettrakul","year":"2017","journal-title":"Parasitol. Int."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1038\/nature12876","article-title":"A molecular marker of artemisinin-resistant Plasmodium falciparum malaria","volume":"505","author":"Ariey","year":"2014","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1007\/s12010-010-9088-8","article-title":"Applications of loop-mediated isothermal DNA amplification","volume":"163","author":"Fu","year":"2011","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Lee, D., Kim, E.J., Kilgore, P.E., Kim, S.A., Takahashi, H., Ohnishi, M., Anh, D.D., Dong, B.Q., Kim, J.S., and Tomono, J. (2015). Clinical evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Neisseria meningitidis in cerebrospinal fluid. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0122922"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"167","DOI":"10.2144\/000113072","article-title":"Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue","volume":"46","author":"Goto","year":"2009","journal-title":"Biotechniques"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.theriogenology.2011.01.020","article-title":"A rapid improved method for sexing embryo of water buffalo","volume":"76","author":"Zoheir","year":"2011","journal-title":"Theriogenology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2895","DOI":"10.1128\/JCM.43.6.2895-2903.2005","article-title":"Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay","volume":"43","author":"Parida","year":"2005","journal-title":"J. Clin. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.jviromet.2008.04.011","article-title":"Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP)","volume":"151","author":"Curtis","year":"2008","journal-title":"J. Virol. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1038\/nprot.2008.57","article-title":"Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products","volume":"3","author":"Tomita","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_25","first-page":"297","article-title":"An innovative test for rapid diagnosis of malaria","volume":"27","author":"Mohan","year":"2014","journal-title":"Natl. Med. J. India"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2866","DOI":"10.1128\/JCM.00355-10","article-title":"Mitochondrial DNA targets increase sensitivity of malaria detection using loop-mediated isothermal amplification","volume":"48","author":"Polley","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1180","DOI":"10.1093\/infdis\/jiu252","article-title":"Field evaluation of a real-time fluorescence loop-mediated isothermal amplification assay, RealAmp, for the diagnosis of malaria in Thailand and India","volume":"210","author":"Patel","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.exppara.2013.03.031","article-title":"Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP)","volume":"134","author":"Surabattula","year":"2013","journal-title":"Exp. Parasitol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/j.mimet.2010.12.004","article-title":"Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus","volume":"84","author":"Hanaki","year":"2011","journal-title":"J. Microbiol. Meth."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.gene.2012.04.049","article-title":"A loop-mediated isothermal amplification method for rapid detection of the multidrug-resistance gene cfr","volume":"504","author":"Qi","year":"2012","journal-title":"Gene"}],"container-title":["International Journal of Environmental Research and Public Health"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-4601\/16\/9\/1635\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:50:47Z","timestamp":1760187047000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-4601\/16\/9\/1635"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,5,10]]},"references-count":30,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2019,5]]}},"alternative-id":["ijerph16091635"],"URL":"https:\/\/doi.org\/10.3390\/ijerph16091635","relation":{},"ISSN":["1660-4601"],"issn-type":[{"value":"1660-4601","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,5,10]]}}}