A method for amplicon deep sequencing of drug resistance genes in plasmodium falciparum clinical isolates from India

Pavitra N. Rao, Swapna Uplekar, Sriti Kayal, Prashant K. Mallick, Nabamita Bandyopadhyay, Sonal Kale, Om P. Singh, Akshaya Mohanty, Sanjib Mohanty, Samuel C. Wassmer, Jane M. Carlton

Research output: Contribution to journalArticle

Abstract

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

Original languageEnglish (US)
Pages (from-to)1500-1511
Number of pages12
JournalJournal of Clinical Microbiology
Volume54
Issue number6
DOIs
StatePublished - Jun 1 2016

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High-Throughput Nucleotide Sequencing
Plasmodium falciparum
Drug Resistance
India
Genes
Mutation
Malaria
Single Nucleotide Polymorphism
Ions
Endemic Diseases
Chloroquine
Antimalarials
Computational Biology
Genotype
Therapeutics
Infection

ASJC Scopus subject areas

  • Microbiology (medical)

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A method for amplicon deep sequencing of drug resistance genes in plasmodium falciparum clinical isolates from India. / Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.; Carlton, Jane M.

In: Journal of Clinical Microbiology, Vol. 54, No. 6, 01.06.2016, p. 1500-1511.

Research output: Contribution to journalArticle

Rao, PN, Uplekar, S, Kayal, S, Mallick, PK, Bandyopadhyay, N, Kale, S, Singh, OP, Mohanty, A, Mohanty, S, Wassmer, SC & Carlton, JM 2016, 'A method for amplicon deep sequencing of drug resistance genes in plasmodium falciparum clinical isolates from India', Journal of Clinical Microbiology, vol. 54, no. 6, pp. 1500-1511. https://doi.org/10.1128/JCM.00235-16
Rao, Pavitra N. ; Uplekar, Swapna ; Kayal, Sriti ; Mallick, Prashant K. ; Bandyopadhyay, Nabamita ; Kale, Sonal ; Singh, Om P. ; Mohanty, Akshaya ; Mohanty, Sanjib ; Wassmer, Samuel C. ; Carlton, Jane M. / A method for amplicon deep sequencing of drug resistance genes in plasmodium falciparum clinical isolates from India. In: Journal of Clinical Microbiology. 2016 ; Vol. 54, No. 6. pp. 1500-1511.
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