pfmdr1 GENOTYPING AND IN VIVO MEFLOQUINE RESISTANCE ON THE THAI-MYANMAR BORDER

Molecular markers have been proposed as a method of monitoring malaria drug resistance and could potentially be used to prolong the life span of antimalarial drugs. Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum gene pfmdr1 and increased gene copy number have been associated with in vitro drug resistance but have not been well studied in vivo. In a prospective cohort study of malaria patients receiving mefloquine treatment on the Thai-Myanmar border, there was no significant association between either pfmdr1 SNPs or in vitro drug sensitivity and mefloquine resistance in vivo. Increased pfmdr1 gene copy number was significantly associated with recrudescence (relative risk 2.30, 95% CI 1.27–4.15). pfmdr1 gene copy number may be a useful surveillance tool for mefloquine-resistant falciparum malaria in Thailand

Lon et al. Malaria Journal 2014, 13:96

Blackwater fever in an uncomplicated Plasmodium falciparum patient treated with dihydroartemisinin-piperaquin

Gametocyte Carriage, Antimalarial Use, and Drug Resistance in Cambodia, 2008-2014

Gametocytes are the malaria parasite stages responsible for transmission from humans to mosquitoes. Gametocytemia often follows drug treatment, especially as therapies start to fail. We examined Plasmodium falciparum gametocyte carriage and drug resistance profiles among 824 persons with uncomplicated malaria in Cambodia to determine whether prevalent drug resistance and antimalarial use has led to a concentration of drug-resistant parasites among gametocyte carriers. Although report of prior antimalarial use increased from 2008 to 2014, the prevalence of study participants presenting with microscopic gametocyte carriage declined. Gametocytemia was more common in those reporting antimalarial use within the past year, and prior antimalarial use was correlated with higher IC50s to piperaquine and mefloquine, as well as to increased pfmdr1 copy number. However, there was no association between microscopic gametocyte carriage and parasite drug resistance. Thus, we found no evidence that the infectious reservoir, marked by those carrying gametocytes, is enriched with drug-resistant parasites

Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia

Abstract Background There is currently no standardized approach for assessing in vitro anti-malarial drug susceptibility. Potential alterations in drug susceptibility results between fresh immediate ex vivo (IEV) and cryopreserved culture-adapted (CCA) Plasmodium falciparum isolates, as well as changes in parasite genotype during culture adaptation were investigated. Methods The 50 % inhibitory concentration (IC50) of 12 P. falciparum isolates from Cambodia against a panel of commonly used drugs were compared using both IEV and CCA. Results were compared using both histidine-rich protein-2 ELISA (HRP-2) and SYBR-Green I fluorescence methods. Molecular genotyping and amplicon deep sequencing were also used to compare multiplicity of infection and genetic polymophisms in fresh versus culture-adapted isolates. Results IC50 for culture-adapted specimens were significantly lower compared to the original fresh isolates for both HRP-2 and SYBR-Green I assays, with greater than a 50 % decline for the majority of drug-assay combinations. There were correlations between IC50s from IEV and CCA for most drugs assays. Infections were nearly all monoclonal, with little or no change in merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP) or apical membrane antigen 1 (AMA1) polymorphisms, nor differences in P. falciparum multidrug resistance 1 gene (PfMDR1) copy number or single nucleotide polymorphisms following culture adaptation. Conclusions The overall IC50 reduction combined with the correlation between fresh isolates and culture-adapted drug susceptibility assays suggests the utility of both approaches, as long as there is consistency of method, and remaining mindful of possible attenuation of resistance phenotype occurring in culture. Further study should be done in higher transmission settings where polyclonal infections are prevalent

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 (Pf mdr1 ) 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 in Combination with Artesunate to Treat Multidrug-Resistant P. falciparum Malaria in Cambodia: An Open-Label Randomized Trial

Background: Recent artemisinin-combination therapy failures in Cambodia prompted a search for alternatives. Atovaquone-proguanil (AP), a safe, effective treatment for multidrug-resistant Plasmodium falciparum (P.f.), previously demonstrated additive effects in combination with artesunate (AS). Methods: Patients with P.f. or mixed-species infection (n = 205) in Anlong Veng (AV; n = 157) and Kratie (KT; n = 48), Cambodia, were randomized open-label 1:1 to a fixed-dose 3-day AP regimen +/-3 days of co-administered artesunate (ASAP). Single low-dose primaquine (PQ, 15 mg) was given on day 1 to prevent gametocyte-mediated transmission. Results: Polymerase chain reaction-adjusted adequate clinical and parasitological response at 42 days was 90% for AP (95% confidence interval [CI], 82%-95%) and 92% for ASAP (95% CI, 83%-96%; P =. 73). The median parasite clearance time was 72 hours for ASAP in AV vs 56 hours in KT (P <. 001) and was no different than AP alone. At 1 week postprimaquine, 7% of the ASAP group carried microscopic gametocytes vs 29% for AP alone (P =. 0001). Nearly all P.f. isolates had C580Y K13 propeller artemisinin resistance mutations (AV 99%; KT 88%). Only 1 of 14 treatment failures carried the cytochrome bc1 (Pfcytb) atovaquone resistance mutation, which was not present at baseline. P.f. isolates remained atovaquone sensitive in vitro but cycloguanil resistant, with a triple P.f. dihydrofolate reductase mutation. Conclusions: Atovaquone-proguanil remained marginally effective in Cambodia (≥90%) with minimal Pfcytb mutations observed. Treatment failures in the presence of ex vivo atovaquone sensitivity and adequate plasma levels may be attributable to cycloguanil and/or artemisinin resistance. Artesunate co-administration provided little additional blood-stage efficacy but reduced post-treatment gametocyte carriage in combination with AP beyond single low-dose primaquine