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

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.

Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia.

BACKGROUND: Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. METHODS: Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. RESULTS: Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. CONCLUSIONS: The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

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.