Pakistan has set a target to reduce the malaria burden by 75% in high endemic districts and to eliminate malaria in low endemic districts by 2020 [2]. However, if artemisinin resistance continues to spread westward from neighbouring regions in South East Asia, then efforts in further controlling and eliminating malaria will be largely hampered [38]. Early surveillance of antimalarial drug resistance through molecular epidemiology studies, is in such, essential for achieving these targets. This study provides an update on the current prevalence of molecular markers associated with drug resistance in Pakistan after the introduction of AL as first-line treatment in 2017, including a first baseline assessment of polymorphisms in the pfcoronin gene which has been associated with decreased in vitro sensitivity to artemisinin.
No SNPs associated with artemisinin resistance were observed in the pfk13 propeller domain, nor in pfcoronin. One other study has to date, assessed the prevalence of polymorphisms in pfk13 in Pakistan [39]. This study reported nine non-synonymous and four synonymous mutations among 209 samples collected in 2016–2017. None of these SNPs correspond to mutations that have been associated with artemisinin resistance. These findings are in line with in vivo assessments of ACT efficacy in Pakistan. There has been no evidence of reduced ACT efficacy in Pakistan as of yet, with adequate clinical and parasitological responses of greater than 98% after treatment with AS + SP, AL, and dihydroartemisinin-piperaquine [40]. However, the recent increase in AS + SP treatment failures in the North Eastern states of India, bordering with Myanmar, is a matter of concern for the continued westward spread of ACT resistance across from Southeast Asia [17]. Especially since molecular data have indicated that resistance to the SP partner drug is being established in Pakistan, although mutations that confer a high risk of SP treatment failure are still rare or non-existent [39, 40].
AL, on the other hand, remains efficacious in the North Eastern region of the India [41, 42], and is currently the recommended first line treatment for P. falciparum in the whole of the WHO Eastern Mediterranean Region, covering Afghanistan, Djibouti, Pakistan, Somalia and Sudan [1]. The efficacy of AL has been monitored in each of these countries, except in Djibouti. All therapeutic efficacy studies in these countries have showed low rates (< 5%) of AL treatment failure, supporting its continued use as first-line treatment. However, whilst no SNPs associated with artemisinin resistance were observed in pfk13 and pfcoronin, the pfmdr1 N86 and D1246 alleles which have been associated with reduced sensitivity to lumefantrine [25, 28–36] were present in 83.8% and 100% of the isolates, respectively (Table 2). The pfmdr1 N86 allele in particular, as well as increased pfmdr1 copy number, have shown to be significant independent risk factors for recrudescence in patients treated with AL [36]. The prevalence of pfmdr1 N86 has increased since the introduction of ACTs in Pakistan in 2007, with prevalence’s ranging from 55–67% in 2005–2007, and 96% in 2011, (Table 3) [43–45]. The pfmdr1 D1246 has been consistently reported in 100% of isolates since 2007. Pfmdr1 copy number was not analysed in this study due to its previously reported very low or non-existent presence in Pakistan.
Table 3
Prevalence of polymorphisms in pfmdr1 and pfcrt before introduction of AL in Pakistan.
| 2005–2007 (N = 240) [43] | 2007 (N = 28) [44] | 2011 (N = 171) [45] | 2018–2019 (N = 179) [current study] |
Pfmdr1 N86Y | | | | |
N86 | 43% | 67% | 80% | 77% |
86Y | 45% | 33%1 | 4% | 16% |
N86Y (mixed) | 12% | Not stated1 | 16% | 7% |
Pfmdr1 Y184F | | | | |
Y184 | ND | ND | 75% | 83% |
184F | ND | ND | 0% | 10% |
Y184F (mixed) | ND | ND | 25% | 6% |
Pfmdr1 D1246Y | | | | |
D1246 | ND | 100% | 100% | 100% |
1246Y | ND | 0% | 0% | 0% |
D1246Y (mixed) | ND | 0% | 0% | 0% |
Pfmdr1 CNV | | | | |
Single copy | ND | 99.6% (232/233)2 | 100% | ND |
Multiple copy | ND | 0.4% (1/233)2 | 0% | ND |
Pfcrt K76T | | | | |
K76 | 7% | 0% | 0% | 2% |
76T | 93% | 100% | 100% | 91% |
K76T (mixed) | 0% | 0% | 0% | 07% |
1 Proportion of infections containing pfmdr1 86Y, proportion of which were mixed infections not stated. |
2 (n/number of samples successfully analysed) |
ND = not determined; CNV = copy number variation |
Over a decade of wide-scale use of AL, mainly in African counties, has selected for pfcrt K76 and the pfmdr1 haplotype N86/184F/D1246, with a parallel decline in pfcrt 76T and the pfmdr1 86Y/Y184/1246Y haplotype [31, 33, 35, 46–49]. Parasites harbouring the pfmdr1 NFD haplotype have shown to able to re-infect patients whose lumefantrine blood concentrations were 15-fold higher than for parasites carrying the pfmdr1 YYY haplotype [32]. Despite this wide scale selection of SNPs associated with reduced sensitivity of lumefantrine, AL remains highly effective across sub-Saharan Africa [50]. However, tracking changes in prevalences of molecular markers of drug resistance can be a sensitive indicator of the selection of parasite populations, and may signal early decreases in drug susceptibility. In addition, decreasing efficacy of the partner drug may expose the artemisinin component of the ACT to selective pressure, and could facilitate emergence of new foci of resistance to artemisinin, as observed in the Greater Mekong Subregion [36]. Hence, in addition to therapeutic efficacy studies, further monitoring of the selection of molecular markers associated with both artemether and lumefantrine resistance in Pakistan, as in this study, is considered to be a critical tool to detect and prevent the spread of artemisinin resistance, and for preserving the efficacy of AL in the area [36].
Finally, the observed high prevalence (98.3%) of the chloroquine resistance associated pfcrt 76T allele is in line with previous assessments [43–45, 51, 52], and provides molecular evidence of chloroquine resistant P. falciparum in Pakistan. Although chloroquine was removed from the national guidelines for treatment of P. falciparum malaria more than ten years ago, chloroquine is still the recommended in the treatment of P. vivax and unconfirmed malaria infections [1]. Improper diagnosis of malaria, due to presumptive diagnosis based on clinical grounds and/or lack of diagnostic facilities (which are common practices in resource-limited countries such as Pakistan), is likely to sustain a high exposure of P. falciparum to chloroquine, maintaining the near fixation of the chloroquine resistant pfcrt 76T allele.