Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia.

BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Prevalence of CYP2D6 Genotypes and Predicted Phenotypes in a Cohort of Cambodians at High Risk for Infections with Plasmodium vivax.

Clinical failure of primaquine (PQ) has been demonstrated in people with CYP450 2D6 genetic polymorphisms that result in reduced or no enzyme activity. The distribution of CYP2D6 genotypes and predicted phenotypes in the Cambodian population is not well described. Surveys in other Asian countries have shown an approximate 50% prevalence of the reduced activity CYP2D6 allele *10, which could translate into increased risk of PQ radical cure failure and repeated relapses, making interruption of transmission and malaria elimination difficult to achieve. We determined CYP2D6 genotypes from 96 volunteers from Oddor Meanchey Province, Cambodia, an area endemic for Plasmodium vivax. We found a 54.2% frequency of the *10 allele, but in approximately half of our subjects, it was paired with a normal activity allele, either *1 or *2. The prevalence of *5, a null allele, was 9.4%. Overall predicted phenotype percentages were normal metabolizers, 46%; intermediate metabolizers, 52%; and poor metabolizers, 1%.

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

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.

Using Amplicon Deep Sequencing to Detect Genetic Signatures of Plasmodium vivax Relapse.

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.

Fighting the good fight: the role of militaries in malaria elimination in Southeast Asia.

Despite significant progress in malaria control in the Greater Mekong Subregion (GMS), malaria is still endemic, with more than 30 million people infected annually. Important gaps remain in case management, service delivery, prevention, and vector control, particularly in hard-to-reach mobile populations. Rapidly evolving drug resistance has created a new urgency to move aggressively toward elimination. However, no clear and cost-effective strategy has been identified. Although GMS militaries are under-recognized as a malaria transmission reservoir, they are an important focal point for elimination activities, given their high mobility, frequent malaria exposure, and potential for asymptomatic carriage. At the same time, military organizational capacity and proximity to other mobile populations could facilitate elimination efforts if relevant political barriers could be overcome. Here, we review considerations for military involvement in regional malaria elimination efforts.

Efficacy of two versus three-day regimens of dihydroartemisinin-piperaquine for uncomplicated malaria in military personnel in northern Cambodia: an open-label randomized trial.

INTRODUCTION: Emerging antimalarial drug resistance in mobile populations remains a significant public health concern. We compared two regimens of dihydroartemisinin-piperaquine in military and civilians on the Thai-Cambodian border to evaluate national treatment policy. METHODS: Efficacy and safety of two and three-day regimens of dihydroartemisinin-piperaquine were compared as a nested open-label evaluation within a malaria cohort study in 222 otherwise healthy volunteers (18% malaria-infected at baseline). The first 80 volunteers with slide-confirmed Plasmodium falciparum or vivax malaria were randomized 1:1 to receive either regimen (total dose 360 mg dihydroartemisinin and 2880 mg piperaquine) and followed weekly for up to 6 months. The primary endpoint was malaria recurrence by day 42. Volunteers with vivax infection received primaquine at study discharge with six months follow-up. RESULTS: Eighty patients (60 vivax, 15 falciparum, and 5 mixed) were randomized to dihydroartemisinin-piperaquine. Intention-to-treat all-species efficacy at Day 42 was 85% for the two-day regimen (95% CI 69-94) and 90% for the three-day regimen (95% CI 75-97). PCR-adjusted falciparum efficacy was 75% in both groups with nearly half (45%) still parasitemic at Day 3. Plasma piperaquine levels were comparable to prior published reports, but on the day of recrudescence were below measurable in vitro piperaquine IC50 levels in all falciparum treatment failures. CONCLUSIONS: In the brief period since introduction of dihydroartemisinin-piperaquine, there is early evidence suggesting declining efficacy relative to previous reports. Parasite IC50 levels in excess of plasma piperaquine levels seen only in treatment failures raises concern for clinically significant piperaquine resistance in Cambodia. These findings warrant improved monitoring of clinical outcomes and follow-up, given few available alternative drugs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01280162.