Partner-Drug Resistance and Population Substructuring of Artemisinin-Resistant Plasmodium falciparum in Cambodia

Plasmodium falciparum in western Cambodia has developed resistance to artemisinin and its partner drugs, causing frequent treatment failure. Understanding this evolution can inform the deployment of new therapies. We investigated the genetic architecture of 78 falciparum isolates using whole-genome sequencing, correlating results to in vivo and ex vivo drug resistance and exploring the relationship between population structure, demographic history, and partner drug resistance. Principle component analysis, network analysis and demographic inference identified a diverse central population with three clusters of clonally expanding parasite populations, each associated with specific K13 artemisinin resistance alleles and partner drug resistance profiles which were consistent with the sequential deployment of artemisinin combination therapies in the region. One cluster displayed ex vivo piperaquine resistance and mefloquine sensitivity with a high rate of in vivo failure of dihydroartemisinin-piperaquine. Another cluster displayed ex vivo mefloquine resistance and piperaquine sensitivity with high in vivo efficacy of dihydroartemisinin-piperaquine. The final cluster was clonal and displayed intermediate sensitivity to both drugs. Variations in recently described piperaquine resistance markers did not explain the difference in mean IC90 or clinical failures between the high and intermediate piperaquine resistance groups, suggesting additional loci may be involved in resistance. The results highlight an important role for partner drug resistance in shaping the P. falciparum genetic landscape in Southeast Asia and suggest that further work is needed to evaluate for other mutations that drive piperaquine resistance

Microscopic Plasmodium falciparum Gametocytemia and Infectivity to Mosquitoes in Cambodia

Although gametocytes are essential for malaria transmission, in Africa many falciparum-infected persons without smear-detectable gametocytes still infect mosquitoes. To see whether the same is true in Southeast Asia, we determined the infectiousness of 119 falciparum-infected Cambodian adults to Anopheles dirus mosquitoes by membrane feeding. Just 5.9% of subjects infected mosquitoes. The 8.4% of patients with smear-detectable gametocytes were >20 times more likely to infect mosquitoes than those without and were the source of 96% of all mosquito infections. In low-transmission settings, targeting transmission-blocking interventions to those with microscopic gametocytemia may have an outsized effect on malaria control and elimination

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

Cluster-randomized trial of monthly malaria prophylaxis versus focused screening and treatment: a study protocol to define malaria elimination strategies in Cambodia

Abstract Background Malaria remains a critical public health problem in Southeast Asia despite intensive containment efforts. The continued spread of multi-drug-resistant Plasmodium falciparum has led to calls for malaria elimination on the Thai-Cambodian border. However, the optimal approach to elimination in difficult-to-reach border populations, such as the Military, remains unclear. Methods/design A two-arm, cluster-randomized controlled, open-label pilot study is being conducted in military personnel and their families at focal endemic areas on the Thai-Cambodian border. The primary objective is to compare the effectiveness of monthly malaria prophylaxis (MMP) with dihydroartemisinin-piperaquine and weekly primaquine for 12 weeks compared with focused screening and treating (FSAT) following current Cambodian national treatment guidelines. Eight separate military encampments, making up approximately 1000 military personnel and their families, undergo randomization to the MMP or FSAT intervention for 3 months, with an additional 3 months’ follow-up. In addition, each treatment cluster of military personnel and civilians is also randomly assigned to receive either permethrin- or sham (water)-treated clothing in single-blind fashion. The primary endpoint is risk reduction for malaria infection in geographically distinct military encampments based on their treatment strategy. Monthly malaria screening in both arms is done via microscopy, PCR, and rapid diagnostic testing to compare both the accuracy and cost-effectiveness of diagnostic modalities to detect asymptomatic infection. Universal glucose-6-phosphate dehydrogenase (G6PD) deficiency screening is done at entry, comparing the results from a commercially available rapid diagnostic test, the fluorescence spot test, and quantitative testing for accuracy and cost-effectiveness. The comparative safety of the interventions chosen is also being evaluated. Discussion Despite the apparent urgency, the key operational elements of proposed malaria elimination strategies in Southeast Asian mobile and migrant populations, including the Military, have yet to be rigorously tested in a well-controlled clinical study. Here, we present a protocol for the primary evaluation of two treatment paradigms – monthly malaria prophylaxis and focused screening and treatment – to achieve malaria elimination in a Cambodian military population. We will also assess the feasibility and incremental benefit of outdoor-biting vector intervention – permethrin-treated clothing. In the process, we aim to define the cost-effectiveness of the inputs required for success including a responsive information system, skilled human resource and laboratory infrastructure requirements, and quality management. Despite being a relatively low transmission area, the complexities of multi-drug-resistant malaria and the movement of vulnerable populations require an approach that is not only technically sound, but simple enough to be achievable. Trial registration ClinicalTrials.gov, ID: NCT02653898 . Registered on 13 January 2016

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

Genetic diversity among Plasmodium vivax isolates along the Thai–Myanmar border of Thailand

International audienceBackground: Knowledge of the population genetics and transmission dynamics of Plasmodium vivax is crucial in predicting the emergence of drug resistance, relapse pattern and novel parasite phenotypes, all of which are relevant to the control of vivax infections. The aim of this study was to analyse changes in the genetic diversity of P. vivax genes from field isolates collected at different times along the Thai–Myanmar border. Methods: Two hundred and fifty-four P. vivax isolates collected during two periods 10 years apart along the Thai– Myanmar border were analysed. The parasites were genotyped by nested-PCR and PCR–RFLP targeting selected polymorphic loci of Pvmsp1, Pvmsp3α and Pvcsp genes. Results: The total number of distinguishable allelic variants observed for Pvcsp, Pvmsp1, and Pvmsp3α was 17, 7 and 3, respectively. High genetic diversity was observed for Pvcsp (H E = 0.846) and Pvmsp1 (H E = 0.709). Of the 254 isolates, 4.3 and 14.6 % harboured mixed Pvmsp1 and Pvcsp genotypes with a mean multiplicity of infection (MOI) of 1.06 and 1.15, respectively. The overall frequency of multiple genotypes was 16.9 %. When the frequencies of allelic variants of each gene during the two distinct periods were analysed, significant differences were noted for Pvmsp1 (P = 0.018) and the Pvcsp (P = 0.033) allelic variants. Conclusion: Despite the low malaria transmission levels in Thailand, P. vivax population exhibit a relatively high degree of genetic diversity along the Thai–Myanmar border of Thailand, in particular for Pvmsp1 and Pvcsp, with indication of geographic and temporal variation in frequencies for some variants. These results are of relevance to monitoring the emergence of drug resistance and to the elaboration of measures to control vivax malaria

Polymorphism patterns in Duffy-binding protein among Thai <it>Plasmodium vivax </it>isolates

<p>Abstract</p> <p>Background</p> <p>The Duffy-binding protein II of <it>Plasmodium vivax </it>(<it>PvDBPII</it>) has been considered as an attractive target for vaccine-mediated immunity despite a possible highly polymorphic nature. Among seven <it>PvDBP </it>domains, domain II has been shown to exhibit a high rate of nonsynonymous polymorphism, which has been suggested to be a potential immune (antibody binding) evasion mechanism. This study aimed to determine the extent of genetic polymorphisms and positive natural selection at domain II of the <it>PvDBP </it>gene among a sampling of Thai <it>P. vivax </it>isolates.</p> <p>Methods</p> <p>The <it>PvDBPII </it>gene was PCR amplified and the patterns of polymorphisms were characterized from 30 Thai <it>P. vivax </it>isolates using DNA cloning and sequencing. Phylogenetic analysis of the sequences and positive selection were done using DnaSP ver 4.0 and MEGA ver 4.0 packages.</p> <p>Results</p> <p>This study demonstrated a high rate of nonsynonymous polymorphism. Using Sal I as the reference strain, a total of 30 point-mutations were observed in the <it>PvDBPII </it>gene among the set of Thai <it>P. vivax </it>isolates, of which 25 nonsynonymous and five synonymous were found. The highest frequency of polymorphism was found in five variant amino acids (residues D384G, R390H, L424I, W437R, I503K) with the variant L424I having the highest frequency. The difference between the rates of nonsynonymous and synonymous mutations estimated by the Nei and Gojobori's method suggested that <it>PvDBPII </it>antigen appears to be under selective pressure. Phylogenetic analysis of <it>PvDBPII </it>Thai <it>P. vivax </it>isolates to others found internationally demonstrated six distinct allele groups. Allele groups 4 and 6 were unique to Thailand.</p> <p>Conclusion</p> <p>Polymorphisms within <it>PvDBPII </it>indicated that Thai vivax malaria parasites are genetically diverse. Phylogenetic analysis of DNA sequences using the Neighbour-Joining method demonstrated that Thai isolates shared distinct alleles with <it>P. vivax </it>isolates from different geographical areas. The study reported here will be valuable for the development of <it>PvDBPII</it>-based malaria vaccine.</p