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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.

Section: Methodsmentioning

confidence: 99%

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

Chaorattanakawee

Saunders

Sea

et al. 2015

“…Samples were considered to have an amplified pfmdr1 gene if the copy number was Ͼ1.5. DNA samples were also genotyped at pfmdr1 codons 86, 184, 1034, and 1042 for single nucleotide polymorphism (SNP) analysis by real-time PCR on an ABI sequence detector 7000 (Applied Biosystems, Warrington, United Kingdom), as previously described (28). Samples were also probed for an additional pfmdr1 SNP (Y1246D) using a PCR-restriction fragment length polymorphism method (29).…”

Section: Methodsmentioning

confidence: 99%

Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

Lanteri

Chaorattanakawee

Lon

et al. 2014

. Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC 50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC 50 s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.

“…All open reading frame sequences of pfcrt were amplified from P. falciparum genomic DNA (63). From clonal lines chosen for further study, the entire open reading frame of pfmdr1, or known polymorphic regions of pfmdr1, were amplified from genomic DNA using primers described previously (44,51). PCR products were directly sequenced using an ABI 3730xl DNA analyzer (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

Griffin

Hoke

Samarakoon

et al. 2012

eThe Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (؊)-quinine and (؉)-quinidine. The potency of (؉)-isomers is greater than the (؊)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC 50 ratio of (؊)/(؉) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (؊) and (؉) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.A lkaloids from the Cinchona tree, exemplified by quinine (QN) and quinidine (QD), have proven to be an important source of antimalarial therapies, especially after resistance to chloroquine (CQ) emerged. QN remains a first-line drug in the treatment of severe malaria in many parts of the world, even with increased use of artemisinin-based combination therapies (70). The Cinchona alkaloids (CA) are aryl amino alcohols where the aryl group is a quinoline (quinoline aminoalcohols). They have four chiral centers, two of which, C-8 and C-9, can have different configurations (66) (Fig. 1). Among the pharmacologically active 8,9-erythro isomers, QN, hydroquinine (HQN), and cinchonidine (CD) all present the S configuration around C-8 and the R configuration at C-9 and are levorotatory (rotate plane polarized light in an anticlockwise [Ϫ] direction). The reverse ordering, R at C-8 and S at C-9, occurs in the respective dextrorotatory (rotate plane polarized light in a clockwise [ϩ] direction) diastereomers, QD, hydroquinidine (HQD), and cinchonine (CN). The 8,9-threo diastereomers 9-epiquinine (EQN) and 9-epiquinidine (EQD) are 8S, 9S, and 8R, 9R, respectively.Against Plasmodium falciparum, the CA diastereomer pairs have a well-established in ...