Surveillance of genetic markers associated with Plasmodium falciparum resistance to artemisinin-based combination therapy in Pakistan, 2018–2019

BACKGROUND: The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether–lumefantrine as first-line treatment in 2017. METHODS: Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. Plasmodium falciparum infections were confirmed by rapid diagnostic test or microscopy. Plasmodium falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism. RESULTS: No artemisinin resistance associated SNPs were identified in the pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections. CONCLUSION: This study provides an update on the current prevalence of molecular markers associated with reduced P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.