Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia

Background: Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008–2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. Methods: To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010–2011 from 16 health centres in malaria endemics areas in Cambodia. Results: Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. Conclusions: The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia

Performance of Ultrasensitive Rapid Diagnostic Tests for Detecting Asymptomatic Plasmodium falciparum.

Proposed interventions for eliminating drug-resistant Plasmodium falciparum malaria include the targeting of asymptomatic carriers through screening and treatment. We report on the diagnostic performance of the recently developed ultrasensitive rapid diagnostic test (uRDT) compared with screening with conventional RDTs (cRDT) and polymerase chain reaction (PCR) under field conditions in Cambodia in a total of 2,729 individuals. The P. falciparum positivity by quantitative PCR (qPCR) was 3.8% (26/678) in those screened during active case detection and 0.5% (10/2,051) in the cross-sectional survey. Compared with qPCR, the sensitivity of the uRDTs was 53.8% (95% CI: 33.4-73.4%) when used in active case detection and 60.0% (95% CI: 26.2-87.8%) in the cross-sectional survey. The uRDTs did not show a significant improvement in diagnostic performance over cRDTs when used for active case detection and for a malaria prevalence survey in the context of this low-transmission setting

Reactive case-detection of malaria in Pailin Province, Western Cambodia: lessons from a year-long evaluation in a pre-elimination setting.

BACKGROUND: As momentum towards malaria elimination grows, strategies are being developed for scale-up in elimination settings. One prominent strategy, reactive case detection (RACD), involves screening and treating individuals living in close proximity to passively detected, or "index" cases. This study aims to use RACD to quantify Plasmodium parasitaemia in households of index cases, and identify risk factors for infection; these data could inform reactive screening approaches and identify target risk groups. METHODS: This study was conducted in the Western Cambodian province of Pailin between May 2013 and March 2014 among 440 households. Index participants/index cases (n = 270) and surrounding households (n = 110) were screened for Plasmodium infection with rapid diagnostic tests (RDT), microscopy and real-time polymerase chain reaction (PCR). Participants were interviewed to identify risk factors. A comparison group of 60 randomly-selected households was also screened, to compare infection levels of RACD and non-RACD households. In order to identify potential risk factors that would inform screening approaches and identify risk groups, multivariate logistic regression models were applied. RESULTS: Nine infections were identified in households of index cases (RACD approach) through RDT screening of 1898 individuals (seven Plasmodium vivax, two Plasmodium falciparum); seven were afebrile. Seventeen infections were identified through PCR screening of 1596 individuals (15 P. vivax, and 22 % P. falciparum/P. vivax mixed infections). In the control group, 25 P. falciparum infections were identified through PCR screening of 237 individuals, and no P. vivax was found. Plasmodium falciparum infection was associated with fever (p = 0.013), being a member of a control household (p ≤ 0.001), having a history of malaria infection (p = 0.041), and sleeping without a mosquito net (p = 0.011). Significant predictors of P. vivax infection, as diagnosed by PCR, were fever (p = 0.058, borderline significant) and history of malaria infection (p ≤ 0.001). CONCLUSION: This study found that RACD identified very few secondary infections when targeting index and neighbouring households for screening. The results suggest RACD is not appropriate, where exposure to malaria occurs away from the community, and there is a high level of treatment-seeking from the private sector. Piloting RACD in a range of transmission settings would help to identify the ideal environment for feasible and effective reactive screening methods

Plasmodium knowlesi Infection in Humans, Cambodia, 2007–2010

Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite

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

Spatial clustering and risk factors of malaria infections in Ratanakiri Province, Cambodia

Background: Malaria incidence worldwide has steadily declined over the past decades. Consequently, increasingly more countries will proceed from control to elimination. The malaria distribution in low incidence settings appears patchy, and local transmission hotspots are a continuous source of infection. In this study, species-specific clusters and associated risk factors were identified based on malaria prevalence data collected in the north-east of Cambodia. In addition, Plasmodium falciparum genetic diversity, population structure and gene flows were studied.Method: In 2012, blood samples from 5793 randomly selected individuals living in 117 villages were collected from Ratanakiri province, Cambodia. Malariometric data of each participant were simultaneously accumulated using a standard questionnaire. A two-step PCR allowed for species-specific detection of malaria parasites, and SNPgenotyping of P. falciparum was performed. SaTScan was used to determine species-specific areas of elevated risk to infection, and univariate and multivariate risk analyses were carried out.Result: PCR diagnosis found 368 positive individuals (6.4%) for malaria parasites, of which 22% contained mixed species infections. The occurrence of these co-infections was more frequent than expected. Specific areas with elevated risk of infection were detected for all Plasmodium species. The clusters for Falciparum, Vivax and Ovale malaria appeared in the north of the province along the main river, while the cluster for Malariae malaria was situated elsewhere. The relative risk to be a malaria parasite carrier within clusters along the river was twice that outside the area. The main risk factor associated with three out of four malaria species was overnight stay in the plot hut, a human behaviour associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river.Discussion: Spatial aggregation of malaria parasite carriers, and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings, which can guide targeted supplementary interventions. Consequently, future malaria programmes in the province should implement additional specific policies targeting households staying overnight at their farms outside the village, in addition to migrants and forest workers

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Mosquitoes of the Anopheles gambiae complex display strong preference for human blood-meals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity

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

International audienceBackground 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