Optimization of loop mediated isothermal amplification assay (LAMP) for detection of chloroquine resistance in P. vivax malaria.
Chloroquine
/ pharmacology
Malaria, Vivax
/ parasitology
Plasmodium vivax
/ genetics
Nucleic Acid Amplification Techniques
/ methods
Drug Resistance
/ genetics
Humans
Polymorphism, Single Nucleotide
Antimalarials
/ pharmacology
Molecular Diagnostic Techniques
/ methods
Sensitivity and Specificity
Multidrug Resistance-Associated Proteins
/ genetics
Protozoan Proteins
/ genetics
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
27 10 2024
27 10 2024
Historique:
received:
09
02
2024
accepted:
14
10
2024
medline:
28
10
2024
pubmed:
28
10
2024
entrez:
28
10
2024
Statut:
epublish
Résumé
Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.
Identifiants
pubmed: 39465271
doi: 10.1038/s41598-024-76479-7
pii: 10.1038/s41598-024-76479-7
doi:
Substances chimiques
Chloroquine
886U3H6UFF
Antimalarials
0
Mdr1 protein, Plasmodium vivax
0
Multidrug Resistance-Associated Proteins
0
Protozoan Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
25608Informations de copyright
© 2024. The Author(s).
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