Comparing malaria risk exposure in rural Cambodia population using GPS tracking and questionnaires.

BACKGROUND: The Great Mekong Subregion has attained a major decline in malaria cases and fatalities over the last years, but residual transmission hotspots remain, supposedly fueled by forest workers and migrant populations. This study aimed to: (i) characterize the fine-scale mobility of forest-goers and understand links between their daily movement patterns and malaria transmission, using parasites detection via real time polymerase chain reaction (RT PCR) and the individual exposure to Anopheles bites by quantification of anti-Anopheles saliva antibodies via enzyme-linked immunosorbent assay; (ii) assess the concordance of questionnaires and Global Positioning System (GPS) data loggers for measuring mobility. METHODS: Two 28 day follow-ups during dry and rainy seasons, including a GPS tracking, questionnaires and health examinations, were performed on male forest goers representing the population at highest risk of infection. Their time spent in different land use categories and demographic data were analyzed in order to understand the risk factors driving malaria in the study area. RESULTS: Malaria risk varied with village forest cover and at a resolution of only a few kilometers: participants from villages outside the forest had the highest malaria prevalence compared to participants from forest fringe's villages. The time spent in a specific environment did not modulate the risk of malaria, in particular the time spent in forest was not associated with a higher probability to detect malaria among forest-goers. The levels of antibody response to Anopheles salivary peptide among participants were significantly higher during the rainy season, in accordance with Anopheles mosquito density variation, but was not affected by sociodemographic and mobility factors. The agreement between GPS and self-reported data was only 61.9% in reporting each kind of visited environment. CONCLUSIONS: In a context of residual malaria transmission which was mainly depicted by P. vivax asymptomatic infections, the implementation of questionnaires, GPS data-loggers and quantification of anti-saliva Anopheles antibodies on the high-risk group were not powerful enough to detect malaria risk factors associated with different mobility behaviours or time spent in various environments. The joint implementation of GPS trackers and questionnaires allowed to highlight the limitations of both methodologies and the benefits of using them together. New detection and follow-up strategies are still called for.

Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant Plasmodium falciparum in Cambodia.

BACKGROUND: In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. OBJECTIVES: To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. METHODS: For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. RESULTS: Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. CONCLUSIONS: The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs.

Clinical and In Vitro Resistance of Plasmodium falciparum to Artesunate-Amodiaquine in Cambodia.

BACKGROUND: Artesunate-amodiaquine is a potential therapy for uncomplicated malaria in Cambodia. METHODS: Between September 2016 and January 2017, artesunate-amodiaquine efficacy and safety were evaluated in a prospective, open-label, single-arm observational study at health centers in Mondulkiri, Pursat, and Siem Reap Provinces, Cambodia. Adults and children with microscopically confirmed Plasmodium falciparum malaria received oral artesunate-amodiaquine once daily for 3 days plus single-dose primaquine, with follow-up on days 7, 14, 21, and 28. The primary outcome was day-28 polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR). An amodiaquine parasite survival assay (AQSA) was developed and applied to whole genome sequencing results to evaluate potential amodiaquine resistance molecular markers. RESULTS: In 63 patients, day-28 PCR-adjusted ACPR was 81.0% (95% confidence interval [CI], 68.9-88.7). Day 3 parasite positivity rate was 44.4% (28/63; 95% CI, 31.9-57.5). All 63 isolates had the K13(C580Y) marker for artemisinin resistance; 79.4% (50/63) had Pfpm2 amplification. The AQSA resistance phenotype (≥45% parasite survival) was expressed in 36.5% (23/63) of isolates and was significantly associated with treatment failure (P = .0020). Pfmdr1 mutant haplotypes were N86/184F/D1246, and Pfcrt was CVIET or CVIDT at positions 72-76. Additional Pfcrt mutations were not associated with amodiaquine resistance, but the G353V mutant allele was associated with ACPR compared to Pfmdr1 haplotypes harboring F1068L or S784L/R945P mutations (P = .030 and P = .0004, respectively). CONCLUSIONS: For uncomplicated falciparum malaria in Cambodia, artesunate-amodiaquine had inadequate efficacy owing to amodiaquine-resistant P. falciparum. Amodiaquine resistance was not associated with previously identified molecular markers.

Choosing interventions to eliminate forest malaria: preliminary results of two operational research studies inside Cambodian forests.

BACKGROUND: Rapid elimination of Plasmodium falciparum malaria in Cambodia is a goal with both national and international significance. Transmission of malaria in Cambodia is limited to forest environments, and the main population at risk consists of forest-goers who rely on forest products for income or sustenance. The ideal interventions to eliminate malaria from this population are unknown. METHODS: In two forested regions of Cambodia, forest-goers were trained to become forest malaria workers (FMWs). In one region, FMWs performed mass screening and treatment, focal screening and treatment, and passive case detection inside the forest. In the other region, FMWs played an observational role for the first year, to inform the choice of intervention for the second year. In both forests, FMWs collected blood samples and questionnaire data from all forest-goers they encountered. Mosquito collections were performed in each forest. RESULTS: Malaria prevalence by PCR was high in the forest, with 2.3-5.0% positive for P. falciparum and 14.6-25.0% positive for Plasmodium vivax among forest-goers in each study site. In vectors, malaria prevalence ranged from 2.1% to 9.6%, but no P. falciparum was observed. Results showed poor performance of mass screening and treatment, with sensitivity of rapid diagnostic tests equal to 9.1% (95% CI 1.1%, 29.2%) for P. falciparum and 4.4% (95% CI 1.6%, 9.2%) for P. vivax. Malaria infections were observed in all demographics and throughout the studied forests, with no clear risk factors emerging. CONCLUSIONS: Malaria prevalence remains high among Cambodian forest-goers, but performance of rapid diagnostic tests is poor. More adapted strategies to this population, such as intermittent preventive treatment of forest goers, should be considered.

Correction: Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis.

[This corrects the article DOI: 10.1371/journal.pmed.1003084.].

Quantification of glucose-6-phosphate dehydrogenase activity by spectrophotometry: A systematic review and meta-analysis.

BACKGROUND: The radical cure of Plasmodium vivax and P. ovale requires treatment with primaquine or tafenoquine to clear dormant liver stages. Either drug can induce haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, necessitating screening. The reference diagnostic method for G6PD activity is ultraviolet (UV) spectrophotometry; however, a universal G6PD activity threshold above which these drugs can be safely administered is not yet defined. Our study aimed to quantify assay-based variation in G6PD spectrophotometry and to explore the diagnostic implications of applying a universal threshold. METHODS AND FINDINGS: Individual-level data were pooled from studies that used G6PD spectrophotometry. Studies were identified via PubMed search (25 April 2018) and unpublished contributions from contacted authors (PROSPERO: CRD42019121414). Studies were excluded if they assessed only individuals with known haematological conditions, were family studies, or had insufficient details. Studies of malaria patients were included but analysed separately. Included studies were assessed for risk of bias using an adapted form of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Repeatability and intra- and interlaboratory variability in G6PD activity measurements were compared between studies and pooled across the dataset. A universal threshold for G6PD deficiency was derived, and its diagnostic performance was compared to site-specific thresholds. Study participants (n = 15,811) were aged between 0 and 86 years, and 44.4% (7,083) were women. Median (range) activity of G6PD normal (G6PDn) control samples was 10.0 U/g Hb (6.3-14.0) for the Trinity assay and 8.3 U/g Hb (6.8-15.6) for the Randox assay. G6PD activity distributions varied significantly between studies. For the 13 studies that used the Trinity assay, the adjusted male median (AMM; a standardised metric of 100% G6PD activity) varied from 5.7 to 12.6 U/g Hb (p < 0.001). Assay precision varied between laboratories, as assessed by variance in control measurements (from 0.1 to 1.5 U/g Hb; p < 0.001) and study-wise mean coefficient of variation (CV) of replicate measures (from 1.6% to 14.9%; p < 0.001). A universal threshold of 100% G6PD activity was defined as 9.4 U/g Hb, yielding diagnostic thresholds of 6.6 U/g Hb (70% activity) and 2.8 U/g Hb (30% activity). These thresholds diagnosed individuals with less than 30% G6PD activity with study-wise sensitivity from 89% (95% CI: 81%-94%) to 100% (95% CI: 96%-100%) and specificity from 96% (95% CI: 89%-99%) to 100% (100%-100%). However, when considering intermediate deficiency (<70% G6PD activity), sensitivity fell to a minimum of 64% (95% CI: 52%-75%) and specificity to 35% (95% CI: 24%-46%). Our ability to identify underlying factors associated with study-level heterogeneity was limited by the lack of availability of covariate data and diverse study contexts and methodologies. CONCLUSIONS: Our findings indicate that there is substantial variation in G6PD measurements by spectrophotometry between sites. This is likely due to variability in laboratory methods, with possible contribution of unmeasured population factors. While an assay-specific, universal quantitative threshold offers robust diagnosis at the 30% level, inter-study variability impedes performance of universal thresholds at the 70% level. Caution is advised in comparing findings based on absolute G6PD activity measurements across studies. Novel handheld quantitative G6PD diagnostics may allow greater standardisation in the future.

Significant Efficacy of a Single Low Dose of Primaquine Compared to Stand-Alone Artemisinin Combination Therapy in Reducing Gametocyte Carriage in Cambodian Patients with Uncomplicated Multidrug-Resistant Plasmodium falciparum Malaria.

Since 2012, a single low dose of primaquine (SLDPQ; 0.25 mg/kg of body weight) with artemisinin-based combination therapies has been recommended as the first-line treatment of acute uncomplicated Plasmodium falciparum malaria to interrupt its transmission, especially in low-transmission settings of multidrug resistance, including artemisinin resistance. Policy makers in Cambodia have been reluctant to implement this recommendation due to primaquine safety concerns and a lack of data on its efficacy. In this randomized controlled trial, 109 Cambodians with acute uncomplicated P. falciparum malaria received dihydroartemisinin-piperaquine (DP) alone or combined with SLDPQ on the first treatment day. The transmission-blocking efficacy of SLDPQ was evaluated on days 0, 1, 2, 3, 7, 14, 21, and 28, and recrudescence by reverse transcriptase PCR (RT-PCR) (gametocyte prevalence) and membrane feeding assays with Anopheles minimus mosquitoes (gametocyte infectivity). Without the influence of recrudescent infections, DP-SLDPQ reduced gametocyte carriage 3-fold compared to that achieved with DP. Of 48 patients tested on day 0, only 3 patients were infectious to mosquitoes (∼6%). Posttreatment, three patients were infectious on day 14 (3.5%, 1/29) and on the 1st and 7th days of recrudescence (8.3%, 1/12 for each); this overall low infectivity precluded our ability to assess its transmission-blocking efficacy. Our study confirms the effective gametocyte clearance of SLDPQ when combined with DP in multidrug-resistant P. falciparum infections and the negative impact of recrudescent infections due to poor DP efficacy. Artesunate-mefloquine (ASMQ) has replaced DP, and ASMQ-SLDPQ has been deployed to treat all patients with symptomatic P. falciparum infections to further support the elimination of multidrug-resistant P. falciparum in Cambodia. (This study has been registered at ClinicalTrials.gov under identifier NCT02434952.).

Impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on Plasmodium vivax drug susceptibility in Cambodia.

BACKGROUND: Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite's intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. OBJECTIVES: To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. METHODS: In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. RESULTS: Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4-22.3) chloroquine, 3.4 nM (IQR 2.1-5.0) mefloquine and 4.6 nM (IQR 2.7-7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. CONCLUSIONS: P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017-18.

Forest malaria in Cambodia: the occupational and spatial clustering of Plasmodium vivax and Plasmodium falciparum infection risk in a cross-sectional survey in Mondulkiri province, Cambodia.

BACKGROUND: After a marked reduction in malaria burden in Cambodia over the last decades, case numbers increased again in 2017-2018. In light of the national goal of malaria elimination by 2025, remaining pockets of high risk need to be well defined and strategies well-tailored to identify and target the persisting burden cost-effectively. This study presents species-specific prevalence estimates and risk stratification for a remote area in Cambodia. METHODS: A cross-sectional survey was conducted in 17 villages in the high-incidence province Mondulkiri in the dry season (December 2017 to April 2018). 4200 randomly selected participants (2-80 years old) were tested for Plasmodium infection by PCR. Risk of infection was associated with questionnaire-derived covariates and spatially stratified based on household GPS coordinates. RESULTS: The prevalence of PCR-detectable Plasmodium infection was 8.3% (349/4200) and was more than twice as high for Plasmodium vivax (6.4%, 268) than for Plasmodium falciparum (3.0%, 125, p < 0.001). 97.8% (262/268) of P. vivax and 92.8% (116/125, p < 0.05) of P. falciparum infections were neither accompanied by symptoms at the time of the interview nor detected by microscopy or RDT. Recent travels to forest sites (aOR 2.17, p < 0.01) and forest work (aOR 2.88, p < 0.001) were particularly strong risk factors and risk profiles for both species were similar. Large village-level differences in prevalence of Plasmodium infection were observed, ranging from 0.6% outside the forest to 40.4% inside. Residing in villages at the forest fringe or inside the forest compared to outside was associated with risk of infection (aOR 2.14 and 12.47, p < 0.001). Villages inside the forest formed spatial hotspots of infection despite adjustment for the other risk factors. CONCLUSIONS: Persisting pockets of high malaria risk were detected in forested areas and in sub-populations engaging in forest-related activities. High levels of asymptomatic infections suggest the need of better case detection plans and the predominance of P. vivax the implementation of radical cure. In villages inside the forest, within-village exposure was indicated in addition to risk due to forest activities. Village-level stratification of targeted interventions based on forest proximity could render the elimination efforts more cost-effective and successful.

A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

Recrudescence, Reinfection, or Relapse? A More Rigorous Framework to Assess Chloroquine Efficacy for Plasmodium vivax Malaria.

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes. Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area. Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones. Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Hemolytic Dynamics of Weekly Primaquine Antirelapse Therapy Among Cambodians With Acute Plasmodium vivax Malaria With or Without Glucose-6-Phosphate Dehydrogenase Deficiency.

BACKGROUND: Hemoglobin (Hb) data are limited in Southeast Asian glucose-6-phosphate dehydrogenase (G6PD) deficient (G6PD-) patients treated weekly with the World Health Organization-recommended primaquine regimen (ie, 0.75 mg/kg/week for 8 weeks [PQ 0.75]). METHODS: We treated Cambodians who had acute Plasmodium vivax infection with PQ0.75 and a 3-day course of dihydroartemisinin/piperaquine and determined the Hb level, reticulocyte count, G6PD genotype, and Hb type. RESULTS: Seventy-five patients (male sex, 63) aged 5-63 years (median, 24 years) were enrolled. Eighteen were G6PD deficient (including 17 with G6PD Viangchan) and 57 were not G6PD deficient; 26 had HbE (of whom 25 were heterozygous), and 6 had α-/ß-thalassemia. Mean Hb concentrations at baseline (ie, day 0) were similar between G6PD deficient and G6PD normal patients (12.9 g/dL [range, 9‒16.3 g/dL] and 13.26 g/dL [range, 9.6‒16 g/dL], respectively; P = .46). G6PD deficiency (P = <.001), higher Hb concentration at baseline (P = <.001), higher parasitemia level at baseline (P = .02), and thalassemia (P = .027) influenced the initial decrease in Hb level, calculated as the nadir level minus the baseline level (range, -5.8-0 g/dL; mean, -1.88 g/dL). By day 14, the mean difference from the day 7 level (calculated as the day 14 level minus the day 7 level) was 0.03 g/dL (range, -0.25‒0.32 g/dL). Reticulocyte counts decreased from days 1 to 3, peaking on day 7 (in the G6PD normal group) and day 14 (in the G6PD deficient group); reticulocytemia at baseline (P = .001), G6PD deficiency (P = <.001), and female sex (P = .034) correlated with higher counts. One symptomatic, G6PD-deficient, anemic male patient was transfused on day 4. CONCLUSIONS: The first PQ0.75 exposure was associated with the greatest decrease in Hb level and 1 blood transfusion, followed by clinically insignificant decreases in Hb levels. PQ0.75 requires monitoring during the week after treatment. Safer antirelapse regimens are needed in Southeast Asia. CLINICAL TRIALS REGISTRATION: ACTRN12613000003774.

Households or Hotspots? Defining Intervention Targets for Malaria Elimination in Ratanakiri Province, Eastern Cambodia.

BACKGROUND: Malaria "hotspots" have been proposed as potential intervention units for targeted malaria elimination. Little is known about hotspot formation and stability in settings outside sub-Saharan Africa. METHODS: Clustering of Plasmodium infections at the household and hotspot level was assessed over 2 years in 3 villages in eastern Cambodia. Social and spatial autocorrelation statistics were calculated to assess clustering of malaria risk, and logistic regression was used to assess the effect of living in a malaria hotspot compared to living in a malaria-positive household in the first year of the study on risk of malaria infection in the second year. RESULTS: The crude prevalence of Plasmodium infection was 8.4% in 2016 and 3.6% in 2017. Living in a hotspot in 2016 did not predict Plasmodium risk at the individual or household level in 2017 overall, but living in a Plasmodium-positive household in 2016 strongly predicted living in a Plasmodium-positive household in 2017 (Risk Ratio, 5.00 [95% confidence interval, 2.09-11.96], P < .0001). There was no consistent evidence that malaria risk clustered in groups of socially connected individuals from different households. CONCLUSIONS: Malaria risk clustered more clearly in households than in hotspots over 2 years. Household-based strategies should be prioritized in malaria elimination programs in this region.

De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

BACKGROUND: Mosquitoes are colonized by a large but mostly uncharacterized natural virome of RNA viruses, and the composition and distribution of the natural RNA virome may influence the biology and immunity of Anopheles malaria vector populations. RESULTS: Anopheles mosquitoes were sampled in malaria endemic forest village sites in Senegal and Cambodia, including Anopheles funestus, Anopheles gambiae group sp., and Anopheles coustani in Senegal, and Anopheles hyrcanus group sp., Anopheles maculatus group sp., and Anopheles dirus in Cambodia. The most frequent mosquito species sampled at both study sites are human malaria vectors. Small and long RNA sequences were depleted of mosquito host sequences, de novo assembled and clustered to yield non-redundant contigs longer than 500 nucleotides. Analysis of the assemblies by sequence similarity to known virus families yielded 115 novel virus sequences, and evidence supports a functional status for at least 86 of the novel viral contigs. Important monophyletic virus clades in the Bunyavirales and Mononegavirales orders were found in these Anopheles from Africa and Asia. The remaining non-host RNA assemblies that were unclassified by sequence similarity to known viruses were clustered by small RNA profiles, and 39 high-quality independent contigs strongly matched a pattern of classic RNAi processing of viral replication intermediates, suggesting they are entirely undescribed viruses. One thousand five hundred sixty-six additional high-quality unclassified contigs matched a pattern consistent with Piwi-interacting RNAs (piRNAs), suggesting that strand-biased piRNAs are generated from the natural virome in Anopheles. To functionally query piRNA effect, we analyzed piRNA expression in Anopheles coluzzii after infection with O'nyong nyong virus (family Togaviridae), and identified two piRNAs that appear to display specifically altered abundance upon arbovirus infection. CONCLUSIONS: Anopheles vectors of human malaria in Africa and Asia are ubiquitously colonized by RNA viruses, some of which are monophyletic but clearly diverged from other arthropod viruses. The interplay between small RNA pathways, immunity, and the virome may represent part of the homeostatic mechanism maintaining virome members in a commensal or nonpathogenic state, and could potentially influence vector competence.

Correction to: De novo profiling of RNA viruses in Anopheles malaria vector mosquitoes from forest ecological zones in Senegal and Cambodia.

Following the publication of this article [1], the authors reported that the original shading in columns 3 and 4 of Table 3, which indicated the presence or absence of viruses in each library, had been removed during typesetting.

In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia.

BACKGROUND: Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments. OBJECTIVES: To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity. METHODS: In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine. RESULTS: Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7-18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1-11.8) for Pfk13 WT parasites and was 12.9 nM (9.5-20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5-20.5) versus 9.1 nM (7.9-10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene. CONCLUSIONS: Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria.

Molecular characterization and mapping of glucose-6-phosphate dehydrogenase (G6PD) mutations in the Greater Mekong Subregion.

BACKGROUND: Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. METHODS: Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. RESULTS: G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. CONCLUSIONS: Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

The tolerability of single low dose primaquine in glucose-6-phosphate deficient and normal falciparum-infected Cambodians.

BACKGROUND: The WHO recommends single low-dose primaquine (SLDPQ, 0.25 mg/kg body weight) in falciparum-infected patients to block malaria transmission and contribute to eliminating multidrug resistant Plasmodium falciparum from the Greater Mekong Sub region (GMS). However, the anxiety regarding PQ-induced acute haemolytic anaemia in glucose-6-phosphate dehydrogenase deficiency (G6PDd) has hindered its use. Therefore, we assessed the tolerability of SLDPQ in Cambodia to inform national policy. METHODS: This open randomised trial of dihydroartemisinin-piperaquine (DHAPP) + SLDPQ vs. DHAPP alone recruited Cambodians aged ≥1 year with acute uncomplicated P. falciparum. Randomisation was 4:1 DHAPP+SLDPQ: DHAPP for G6PDd patients and 1:1 for G6PDn patients, according to the results of the qualitative fluorescent spot test. Definitive G6PD status was determined by genotyping. Day (D) 7 haemoglobin (Hb) concentration was the primary outcome measure. RESULTS: One hundred nine patients (88 males, 21 females), aged 4-76 years (median 23) were enrolled; 12 were G6PDd Viangchan (9 hemizygous males, 3 heterozygous females). Mean nadir Hb occurred on D7 [11.6 (range 6.4 ─ 15.6) g/dL] and was significantly lower (p = 0.040) in G6PDd (n = 9) vs. G6PDn (n = 46) DHAPP+SLDPQ recipients: 10.9 vs. 12.05 g/dL, Δ = -1.15 (95% CI: -2.24 ─ -0.05) g/dL. Three G6PDn patients had D7 Hb concentrations < 8 g/dL; D7-D0 Hbs were 6.4 ─ 6.9, 7.4 ─ 7.4, and 7.5 ─ 8.2 g/dL. For all patients, mean (range) D7-D0 Hb decline was -1.45 (-4.8 ─ 2.4) g/dL, associated significantly with higher D0 Hb, higher D0 parasitaemia, and receiving DHAPP; G6PDd was not a factor. No patient required a blood transfusion. CONCLUSIONS: DHAPP+SLDPQ was associated with modest Hb declines in G6PD Viangchan, a moderately severe variant. Our data augment growing evidence that SLDPQ in SE Asia is well tolerated and appears safe in G6PDd patients. Cambodia is now deploying SLDPQ and this should encourage other GMS countries to follow suit. TRIAL REGISTRATION: The clinicaltrials.gov reference number is NCT02434952 .

Contribution to Malaria Transmission of Symptomatic and Asymptomatic Parasite Carriers in Cambodia.

Background: Eliminating falciparum malaria in Cambodia is a top priority, requiring the implementation of novel tools and strategies to interrupt its transmission. To date, few data are available regarding the contributions to malaria transmission of symptomatic and asymptomatic carriers. Methods: Direct-membrane and skin feeding assays (DMFAs, SFAs) were performed, using Anopheles minimus and Anopheles dirus, to determine infectivity of symptomatic falciparum-infected patients and malaria asymptomatic carriers; a subset of the latter were followed up for 2 months to assess their transmission potential. Results: By microscopy and real-time polymerase chain reaction, Plasmodium falciparum gametocyte prevalence rates were, respectively, 19.3% (n = 21/109) and 44% (n = 47/109) on day (D) 0 and 17.9% (n = 5/28) and 89.3% (n = 25/28) in recrudescent patients (Drec) (RT-PCR Drec vs D0 P = .002). Falciparum malaria patient infectivity was low on D0 (6.2%; n = 3/48) and in Drec (8.3%; n = 1/12). Direct-membrane feeding assays and SFAs gave similar results. None of the falciparum (n = 0/19) and 3 of 28 Plasmodium vivax asymptomatic carriers were infectious to mosquitoes, including those that were followed up for 2 months. Overall, P. falciparum gametocytemias were low except in a few symptomatic carriers. Conclusions: Only symptomatic falciparum malaria patients were infectious to mosquito vectors at baseline and recrudescence, highlighting the need to detect promptly and treat effectively P. falciparum patients.

Adapting Reactive Case Detection Strategies for falciparum Malaria in a Low-Transmission Area in Cambodia.

Reactive case detection around falciparum malaria cases in Cambodia presents a low output. We improved it by including individuals occupationally coexposed with index case patients and using polymerase chain reaction-based diagnosis. The positivity rate increased from 0.16% to 3.9%.