Resistance to SP is linked to point mutations that accumulat
Resistance to SP is linked to point mutations that accumulate at several sites in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) (Z)-4-Hydroxytamoxifen of P. falciparum (Roper et al., 2003). These mutations have been identified in codons 16, 51, 59, 108, and 164 in the dhfr gene and codons 436, 437, 540, 581 and 613 in the dhps gene (Wernsdorfer and Noedl, 2003). The combination of a triple dhfr (N51I/C59R/S108N) mutant with a double dhps (A437G/K540E) mutant has been associated with SP treatment failure (Kublin et al., 2002, Mugittu et al., 2004, Omar et al., 2001). Other findings have suggested that the triple dhfr mutant (N51I/C59R/S108N) alone is a useful predictor of treatment failure (Basco et al., 2000, Mombo-Ngoma et al., 2011, Mugittu et al., 2004). High prevalences of double dhfr (C59R/S108N) and triple dhfr/dhps (C59R/S108N+A437G or N51I/S108N+A437G) mutants have been reported in Pakistan (Ghanchi et al., 2011, Khattak et al., 2013, Khatoon et al., 2009). However, these studies lack the in vivo responses of the patients from whom samples were collected. With the support of the WHO, the National Malaria Control of Pakistan has been monitoring the efficacies of recommended ACTs since 2007 to inform national treatment policy. This paper is reporting the efficacies of AS+SP (2007–2012), AL (2012) and DHA+PPQ (2015) from several sites in Pakistan. We are also reporting, for the first time, the molecular markers for SP resistance, together with the clinical outcomes for AS+SP in the same patients.
Discussion Our findings document that the recommended first-line treatment for uncomplicated falciparum malaria in Pakistan remained highly efficacious during the study period, with an ACPR of more than 98%. However, the high prevalence of dhfr and dhps mutations conferring resistance to SP is concerning. Double dhfr C59R/S108N was at or near fixation in the study patients. The substantial number of patients carrying triple dhfr/dhps mutations (C59R/S108N+A437G or N51I/S108N+A437G) in the current and previous surveys (Ghanchi et al., 2011, Khattak et al., 2013, Khatoon et al., 2009) indicates that SP resistance is being established in Pakistan. However, the very low prevalence of triple dhfr (N51I/C59R/S108N) mutations and the lack of the dhfr/dhps quintuple mutation across the sites may explain the high therapeutic efficacy of AS+SP. Studies have shown that the presence of these mutations is linked to increased SP treatment failure (Kublin et al., 2002, Omar et al., 2001, Picot et al., 2009). High AS+SP treatment failure rates associated with a high prevalence of dhfr/dhps quadruple or quintuple mutations were recently reported in India (Mishra et al., 2014) and Somalia (Warsame et al., 2015), leading to treatment policy changes in both countries. In contrast, the sustained high efficacy of AS+SP has been reported in Afghanistan (Awab et al., 2016) and Yemen (Adeel et al., 2015, Atroosh et al., 2016) after its use for more than a decade. The study from Afghanistan indicated a similar prevalence of the S108N mutation and C59R, but other mutations in dhfr and dhps remained rare or absent (Awab et al., 2016), suggesting that the evolution of these mutations is at an early stage. This was true also for Yemen (Al-Hamidhi et al., 2013). Our findings also showed that two other ACTs, AL (second–line treatment) and DHA+PPQ, are highly efficacious for the treatment of uncomplicated falciparum malaria. These combinations have proven to be highly effective in many African and Asian countries (Zani et al., 2014)., but high treatment failure rates with DHA+PPQ were reported in Cambodia (Leang et al., 2015). The emergence and spread of artemisinin resistance in the Greater Mekong sub-region (Ashley et al., 2014, Takala-Harrison et al., 2015) is of global concern. Malaria-endemic countries are, therefore, required to routinely monitor the efficacy of the recommended ACTs at least every two years (WHO, 2010). The mutations in P. falciparum gene encoding kelch (K13) propeller domains should also be monitored as a marker for artemisinin resistance (Ariey et al., 2014).