Decreasing pfmdr1 Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is Regaining In Vitro Susceptibility to Mefloquine

Abstract

b Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS؉MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies-a molecular marker for MQ resistance-in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falciparum is regaining in vitro susceptibility to MQ.A rtemisinin-based combination therapy (ACT) is used worldwide to treat uncomplicated Plasmodium falciparum malaria. Artesunate plus mefloquine (ASϩMQ) was adopted as Cambodia's first-line treatment in 2000. ASϩMQ treatment failures were first observed in 10 to 20% of patients in Pailin and Battambang Provinces, Western Cambodia, in 2003 to 2004 (1, 2). The presence of multiple (i.e., Ն2) pfmdr1 copies-a genetic marker of MQ resistance-was associated with ASϩMQ failures and reduced parasite in vitro susceptibility to MQ not only in Pailin and Battambang but in neighboring Pursat and Kampot Provinces as well (3)(4)(5). This development, along with emerging evidence of reduced in vitro susceptibility to MQ in other provinces (6), prompted Cambodia's National Malaria Control Program to adopt dihydroartemisinin-piperaquine (DHA-PPQ) as the firstline ACT in Western Cambodia in 2008 and in the entire country in 2010. Unfortunately, recent clinical studies suggest that the efficacy of DHA-PPQ is rapidly declining in five Western Cambodian provinces: Pursat, Battambang, Pailin, Oddar Meancheay, and Preah Vihear. For example, DHA-PPQ treatment failures were observed in 25% and 11% of patients in Pailin and Pursat in 2010 (7) and in 36% of patients in Oddar Meancheay in 2013 (8). The rapidly increasing prevalence of DHA-PPQ failures in these provinces, likely due to entrenched artemisinin resistance (9, 10) and suspected PPQ resistance (7, 8), demands additional evaluations of newer antimalarial drugs (11, 12), as well as reevaluation of previously used ACTs.Only a few ACTs, including ASϩMQ and artemether-lumefantrine (AL), are presently available for widespread use in Cambodia to replace DHA-PPQ as it fails. Although ASϩMQ and AL treatment failures have previously been reported from Battambang and Pursat (2, 13), the clinical efficacy of these regimens in the setting of DHA-PPQ resistance is not known. However, studies have suggested a lack of cross-resistance between PPQ and MQ in vitro; for example, pfmdr1 amplification is associated with decreased susceptibility to MQ but increased susceptibility to PPQ (14). Given these and related findings (15), and the fact that earlier use of DHA-PPQ was associated with decreasing prevalence of multiple pfmdr1 copies in Pailin (from 33% in 2005 to 5% in 2007) (16), we hypothesized that the recent substantially reduced use of ASϩMQ in Pursat and Preah Vihear would select for parasites that have regained sensitivity to MQ. To explore this hyp...

Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt

“…In fact, miners on the Guiana Shield readily cross borders between Brazil, French Guiana, and Suriname and regularly automedicate with Artecom (33)(34)(35). This clue to the putative mechanism of resistance to PPQ is potentially of considerable importance, because PPQ treatment failure is a major and increasing concern in southeast Asia (36)(37)(38). Therefore, the association between PfCRT haplotypes and PPQ treatment failure should be further explored in southeast Asia, where polymorphisms in the pfcrt gene, notably in exon 10, were found to accompany mutations in the pfK13 gene associated with artemisinin resistance (39).…”

Antimalarial Drug Resistance: A Threat to Malaria Elimination

“…. Similarly, increased copy number of Pfmdr-1 increases resistance to mefloquine, but conversely increases the sensitivity to chloroquine and to piperaquine (Leang et al 2013Duru et al 2015;Lim et al 2015;Amaratunga et al 2016) P. falciparum bifunctional dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) Located on chromosome 4 (Pfdhfr-ts gene, PF3D7_0417200, previous ID: MAL4P1.161, PFD0830w), Pfdhfr-ts encodes an enzyme involved in the pathway of the folate synthesis (Foote and Cowman 1994;Gregson and Plowe 2005). DHFR-TS is the target of the antifolate drugs such as pyrimethamine and proguanil (metabolized in vivo to the active form cycloguanil).…”

“…Possible strategies include drug rotation between different ACTs, in particular DHA-piperaquine and artesunate-mefloquine. It has been shown that withdrawal of mefloquine as antimalarial treatment is followed by the recovery of mefloquine sensitivity in P. falciparum, resulting from the quick loss of mdr-1 gene amplification, which exerts an important fitness cost in the absence of drug pressure (Preechapornkul et al 2009;Leang et al 2013Leang et al , 2015Duru et al 2015;Lim et al 2015;Amaratunga et al 2016). This strategy is currently implemented in large parts of Cambodia suffering from high failure rates with DHA-piperaquine.…”

Understanding artemisinin-resistant malaria

“…While this possibility is further suggested by increasing piperaquine IC 50 values within multiple study sites over time (unpublished data; [52]), more robust evidence of piperaquine resistance is needed to identify its genetic markers, elucidate its molecular mechanism, and discover new drugs that circumvent it. Meanwhile, artesunate-mefloquine may be an effective treatment for DHA-piperaquine failures, as suggested by a contemporaneous reduction in mefloquine IC 50 values and disappearance of the multi-copy pfmdr1 genotype – a molecular marker of mefloquine resistance [35, 52, 53]. …”