Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

Abstract

Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC 50 ] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.

Atovaquone-Proguanil Remains a Potential Stopgap Therapy for Multidrug-Resistant Plasmodium falciparum in Areas along the Thai-Cambodian Border

“…Susceptibility was measured by histidine-rich protein-2 (HRP-2) enzyme-linked immunosorbent assay (ELISA) testing on fresh isolates within 4 h of phlebotomy after being incubated for 72 h in 0.5% AlbuMAX with RPMI medium on drug-coated plates, according to previously published methods (13,14). Figure 1 shows the ex vivo susceptibility results for all isolates, and the values were comparable to our other recent ex vivo observations for all drugs except piperaquine, which was more resistant (15). The atovaquone-susceptible P. falciparum W2 clone was used as an established reference (16); the highly ATQ-resistant C2B clone was also used (17).…”

“…2). Following this reanalysis, piperaquine resistance in some clones was higher than what we had previously reported for this study, with 22 of 92 (24%) evaluable isolate IC 50 s substantially Ͼ50 nM, the highest value seen from previous years (2009 to 2012 [15]). Further, isolates from subjects with P. falciparum recrudescence had a significantly increased median IC 50 of 40.3 nM, compared to 28.6 nM for those without P. falciparum recrudescence (P Ͻ 0.05, Mann-Whitney U test).…”

Understanding artemisinin-resistant malaria

“…Since high ACT failure rates in SEA have only been observed where resistance to the partner drug exists, it is likely that piperaquine resistance has indeed emerged. 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].…”

Dihydroartemisinin–piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study

“…In view of these findings and the lack of a genetic marker, piperaquine resistance in western Cambodia has not been confirmed, although increasing piperaquine IC 50 values in northern Cambodia suggest that it may be emerging. 18 …”