TY - JOUR
T1 - A method for amplicon deep sequencing of drug resistance genes in plasmodium falciparum clinical isolates from India
AU - Rao, Pavitra N.
AU - Uplekar, Swapna
AU - Kayal, Sriti
AU - Mallick, Prashant K.
AU - Bandyopadhyay, Nabamita
AU - Kale, Sonal
AU - Singh, Om P.
AU - Mohanty, Akshaya
AU - Mohanty, Sanjib
AU - Wassmer, Samuel C.
AU - Carlton, Jane M.
N1 - Funding Information:
This work, including the efforts of Pavitra Nagesh Rao, Swapna Uplekar, Sriti Kayal, Prashant K Mallick, Nabamita Bandyopadhyay, Sonal Kale, Om P Singh, Sanjib Mohanty, Samuel Crocodile Wassmer, and Jane M. Carlton, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (U19AI089676).
Publisher Copyright:
© 2016, American Society for Microbiology. All Rights Reserved.
PY - 2016/6
Y1 - 2016/6
N2 - 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.
AB - 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.
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U2 - 10.1128/JCM.00235-16
DO - 10.1128/JCM.00235-16
M3 - Article
C2 - 27008882
AN - SCOPUS:84970028964
SN - 0095-1137
VL - 54
SP - 1500
EP - 1511
JO - Journal of Clinical Microbiology
JF - Journal of Clinical Microbiology
IS - 6
ER -