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RESEARCH

Malaria Molecular Epidemiology

In Cambodia, malaria continues to be a major cause for public health and economic burden. Its elimination is given high priority by the government and partners. Forest villagers in the eastern and northern provinces are at high risk of malaria, with all age groups suffering infection; children under the age of five years are at highest risk of severe disease due to their lack of immunity. 

RESEARCH

Malaria Molecular Epidemiology

In Cambodia, malaria continues to be a major cause for public health and economic burden. Its elimination is given high priority by the government and partners. Forest villagers in the eastern and northern provinces are at high risk of malaria, with all age groups suffering infection; children under the age of five years are at highest risk of severe disease due to their lack of immunity. 

Elsewhere, malaria is an occupational disease with specific high-risk groups, including forestry workers, new settlers and mobile/migrant populations who have come into forested areas, and soldiers, and their families, serving in the forests.

The five Plasmodium species known to cause malaria in humans (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi) are present in Cambodia. Currently, the number of malaria cases recorded in the public sector is estimated to ~40,000. P. falciparum was the predominant species among confirmed malaria cases until 2010 (CNM data).

Although there has been a steady reduction in the total number of clinically diagnosed and treated malaria cases as well as in the severe case fatality rate over the last thirteen years, morbidity due to malaria remain high compared to other countries in the region. Malaria in Cambodia is also a key contributor to anaemia, complications during pregnancy, low-birth weight and poor child growth.

In addition, multidrug resistant strains of Plasmodium falciparum are common, particularly in the west of the country. As with earlier antimalarials, we are now facing to the emergence of artemisinin and piperaquine resistance while no suitable alternative currently exists for first-line treatments of falciparum malaria. As previously observed with chloroquine resistance in the last century, artemisinin-resistant parasites represent a major threat to worldwide goals of malaria eradication and the potential to devastate sub-Saharan Africa by increasing childhood mortality.

Major Areas of Research at IPC Scientific projects are built around the malaria elimination concept in Southeast Asia. They are mainly focused on four major areas of research:

      • Supporting and evaluating the impact of strategies against malaria implemented by National Malaria Control Programmes.
      • Conduct researches focused on P. falciparum artemisinin resistant parasites.
      • Conduct researches on vivax malaria & other emerging Plasmodium sp.
      • Transmission of Plasmodium by Anopheline mosquitoes.

Projects

Novel cross-border approaches to optimise identification of asymptomatic and artemisinin-resistant plasmodium infection in mobile populations crossing Cambodian borders

Human population movement across country borders presents a real challenge for malaria control and elimination efforts in Cambodia and its neighbouring countries. To quantify Plasmodium infection among the border-crossing population, including asymptomatic and artemisinin resistant (AR) parasites, three official border crossing points, one from each of Cambodia’s borders with Thailand, Laos and Vietnam, were selected for sampling. A total of 3206 participants (of 4110 approached) were recruited as they crossed the border, tested for malaria and interviewed. By real-time polymerase chain reaction (RT-PCR), 5.4% of all screened individuals were found to harbor Plasmodium parasites. The proportion was highest at the Laos border (11.5%). Overall there were 97 P. vivax (55.7%), 55 P. falciparum (31.6%), two P. malariae (1.1%) and 20 mixed infections (11.5%). Of identified infections, only 20% were febrile at the time of screening. Of the 24 P. falciparum samples where a further PCR was possible to assess AR, 15 (62.5%) had mutations in the K13 propeller domain gene, all from participants at the Laos border point. Malaria rapid diagnostic test (RDT) pLDH/HRP-2 identified a positivity rate of 3.2% overall and sensitivity compared to RT-PCR was very low (43.1%). Main individual risk factors for infection included sex, fever, being a forest-goer, poor knowledge of malaria prevention methods and previous malaria infection. Occupation, day of the week and time of crossing (morning vs. afternoon) also appeared to play an important role in predicting positive cases. This study offers a novel approach to identify asymptomatic infections and monitor AR parasite flow among mobile and migrant populations crossing the borders. Similar screening activities are recommended to identify other hot borders and characterise potential hot spots of AR. Targeted “customised” interventions and surveillance activities should be implemented in these sites to accelerate elimination efforts in the region.


Malaria PCR detection in Cambodian low-transmission settings: dried blood spots versus venous blood samples

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 mL) and different volumes of venous blood (50 µL, 200 µL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 µL, 200 µL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ~100-fold lower than 5 µL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 µL DBS method missed 27%of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.


Plasmodium prevalence and artemisinin-resistant falciparum malaria in Preah Vihear Province, Cambodia: a cross-sectional population-based study

Intensified efforts are urgently needed to contain and eliminate artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion. Médecins Sans Frontières plans to support the Ministry of Health in eliminating P. falciparum in an area with artemisinin resistance in the north-east of Cambodia. As a first step, the prevalence of Plasmodium spp. and the presence of mutations associated with artemisinin resistance were evaluated in two districts of Preah Vihear Province. A cross-sectional population-based study using a two-stage cluster sampling was conducted in the rural districts of Chhaeb and Chey Saen, from September to October 2013. In each district, 30 clusters of 10 households were randomly selected. In total, blood samples were collected for 1,275 participants in Chhaeb and 1,224 in Chey Saen. Prevalence of Plasmodium spp. was assessed by PCR on dried blood spots. Plasmodium falciparum positive samples were screened for mutations in the K13-propeller domain gene (PF3D7_1343700). The prevalence of Plasmodium spp. was estimated at 1.49% (95% CI 0.71–3.11%) in Chhaeb and 2.61% (95% CI 1.45–4.66%) in Chey Saen. Twenty-seven samples were positive for P. falciparum, giving a prevalence of 0.16% (95% CI 0.04–0.65) in Chhaeb and 2.04% (95% CI 1.04–3.99%) in Chey Saen. Only 4.0% of the participants testing positive presented with fever or history of fever. K13-propeller domain mutant type alleles (C580Y and Y493H) were found, only in Chey Saen district, in seven out of 11 P. falciparum positive samples with enough genetic material to allow testing. The overall prevalence of P. falciparum was low in both districts but parasites presenting mutations in the K13-propeller domain gene, strongly associated with artemisinin-resistance, are circulating in Chey Saen. The prevalence might be underestimated because of the absentees – mainly forest workers – and the workers of private companies who were not included in the study. These results confirm the need to urgently develop and implement targeted interventions to contain and eliminate P. falciparum malaria in this district before it spreads to other areas.


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

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. 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 SNP genotyping 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. 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 behavior associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river. 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.


Implementation and application of a multiplex assay to detect malaria-specific antibodies: a promising tool for assessing malaria transmission in Southeast Asian pre-elimination areas

Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life. An assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia. A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults.The multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection.


Tolerability and safety of weekly primaquine against relapse of Plasmodium vivax in Cambodians with glucose-6-phosphate dehydrogenase deficiency

Primaquine is used to prevent Plasmodium vivax relapse; however, it is not implemented in many malaria-endemic countries, including Cambodia, for fear of precipitating primaquine-induced acute haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficiency (G6PDd). Reluctance to use primaquine is reinforced by a lack of quality safety data. This study was conducted to assess the tolerability of a primaquine regimen in Cambodian severely deficient G6PD variants to ascertain whether a weekly primaquine could be given without testing for G6PDd. From January 2013 to January 2014, Cambodians with acute vivax malaria were treated with dihydroartemisinin/piperaquine on days (D) 0, 1 and 2 with weekly doses of primaquine 0.75 mg/kg for 8 weeks (starting on D0, last dose on D49), and followed until D56. Participants’ G6PD status was confirmed by G6PD genotype and measured G6PD activity. The primary outcome was treatment completion without primaquine toxicity defined as any one of: (1) severe anaemia (haemoglobin [Hb] <7 g/dL), (2) a >25 % fractional fall in Hb from D0, (3) the need for a blood transfusion, (4) haemoglobinuria, (5) acute kidney injury (an increase in baseline serum creatinine >50 %) or (6) methaemoglobinaemia >20 %. We enrolled 75 patients with a median age of 24 years (range 5–63); 63 patients (84 %) were male. Eighteen patients were G6PDd (17/18 had the Viangchan variant) and had D0 G6PD activity ranging from 0.1 to 1.5 U/g Hb (median 0.85 U/g Hb). In the 57 patients with normal G6PD (G6PDn), D0 G6PD activity ranged from 6.9 to 18.5 U/g Hb (median 12 U/g Hb). Median D0 Hb concentrations were similar (P = 0.46) between G6PDd (13 g/dL, range 9.6–16) and G6PDn (13.5 g/dL, range 9–16.3) and reached a nadir on D2 in both groups: 10.8 g/dL (8.2–15.3) versus 12.4 g/dL (8.8–15.2) (P = 0.006), respectively. By D7, five G6PDd patients (27.7 %) had a >25 % fall in Hb, compared to 0 G6PDn patients (P = 0.00049). One of these G6PDd patients required a blood transfusion (D0–D5 Hb, 10.0–7.2 g/dL). No patients developed severe anaemia, haemoglobinuria, a methaemoglobin concentration >4.9 %, or acute kidney injury. Vivax-infected G6PDd Cambodian patients demonstrated significant, mostly transient, falls in Hb and one received a blood transfusion. Weekly primaquine in G6PDd patients mandates medical supervision and pre-treatment screening for G6PD status. The feasibility of implementing a package of G6PDd testing and supervised primaquine should be explored.


Field trial evaluation of the performances of point-of-care tests for screening G6PD  deficiency in Cambodia

User-friendly, accurate, point-of-care rapid tests to detect glucose-6-phosphate dehydrogenase deficiency (G6PDd) are urgently needed at peripheral level to safely recommend primaquine for malaria elimination. The CareStart G6PD RDT (AccessBio, New Jersey, USA), a novel rapid diagnostic test and the most commonly used test, the fluorescent spot test (FST) were assessed against the quantitatively measured G6PD enzyme activity for detecting G6PDd. Subjects were healthy males and non-pregnant females aged 18 years or older residing in six villages in Pailin Province, western Cambodia. Of the 938 subjects recruited, 74 (7.9%) were severe and moderately severe G6PD deficient (enzyme activity, 30%), mostly in male population; population median G6PD activity was 12.0 UI/g Hb. The performances of the CareStart G6PD RDT and the FST, according to different cut-off values used to define G6PDd were very similar. For the detection of severe and moderately severe G6PDd (enzyme activity<30%, 3.6 UI/g Hb) in males and females, sensitivity and negative (normal status) predictive value were 100% for both point-of-care tools. When the G6PDd cut-off value increased (from 40% to 60%), the sensitivity for both PoCs decreased: 93.3% to 71.7% (CareStart G6PD RDT, p51026) and 95.5% to 73.2% (FST, p51026) while the specificity for both PoCs remained similar: 97.4% to 98.3% (CareStart G6PD RDT, p=0.23) and 98.7% to 99.6% (FST, p=0.06). The cut-off values for classifying individuals as normal were 4.0 UI/g Hb and 4.3 UI/g Hb for the CareStart G6PD RDTand the FST, respectively. The CareStart G6PD RDT reliably detected moderate and severe G6PD deficient individuals (enzyme activity <30%), suggesting that this novel point-of-care is a promising tool for tailoring appropriate primaquine treatment for malaria elimination by excluding individuals with severe G6PDd for primaquine treatment.


Use of Plasmodium falciparum culture-adapted field isolates for in vitro exflagellation-blocking assay

A major requirement for malaria elimination is the development of transmission-blocking interventions. In vitro transmission-blocking bioassays currently mostly rely on the use of very few Plasmodium falciparum reference laboratory strains isolated decades ago. To fill a piece of the gap between laboratory experimental models and natural systems, the purpose of this work was to determine if culture-adapted field isolates of P. falciparum are suitable for in vitro transmission-blocking bioassays targeting functional maturity of male gametocytes: exflagellation. Plasmodium falciparum isolates were adapted to in vitro culture before being used for in vitro gametocyte production. Maturation was assessed by microscopic observation of gametocyte

morphology over time of culture and the functional viability of male gametocytes was assessed by microscopic counting of exflagellating gametocytes. Suitability for in vitro exflagellation-blocking bioassays was determined using dihydroartemisinin and methylene blue. In vitro gametocyte production was achieved using two isolates from French Guiana and two isolates from Cambodia. Functional maturity of male gametocytes was assessed by exflagellation observations and all four isolates could be used in exflagellation-blocking bioassays with adequate response to methylene blue and dihydroartemisinin. This work shows that in vitro culture-adapted P. falciparum field isolates of different genetic background, from South America and Southeast Asia, can successfully be used for bioassays targeting the male gametocyte to gamete transition, exflagellation.


Malaria rapid diagnostic test as point-of-care test: study protocol for evaluating the VIKIA Malaria Ag Pf/Pan

Malaria rapid diagnostic tests (RDTs) are generally considered as point-of-care tests. However, most of the studies assessing the performance of malaria RDTs are conducted by research teams that are not representative of the classical end-users, who are typically unskilled in traditional laboratory techniques for diagnosing malaria. To evaluate the performance of a malaria RDT by end-users in a malaria-endemic area, a study protocol was designed and the VIKIA® Malaria Ag Pf/Pan test, previously evaluated in 2013, was re-evaluated by representative end-users. Twenty end-users with four different profiles in seven communes in Kampot Province (Cambodia) were selected. A set of 20 calibrated aliquots, including negative samples, low positive samples (200 parasites/μL of Plasmodium falciparum and Plasmodium vivax) and high positive samples (2,000 parasites/μL of P. falciparum and P. vivax) was used. Testing was performed directly by the end-users without any practical training on the VIKIA® Malaria Ag Pf/Pan kit. All results obtained by the end-users were consistent with the expected results, except for the low positive (200 parasites/μL) P. vivax aliquot (35% of concordant results). No significant difference was observed between the different end-users. End-user interviews evaluating ease-of-use and ease-of-reading of the VIKIA® Malaria Ag Pf/Pan kit recorded 159 positive answers and only one negative answer. Out of 20 end-users, only one considered the test was not easy to perform with the support of the quick guide. The data presented in this study clearly demonstrate that the performance of the VIKIA® Malaria Ag Pf/Pan test when performed by traditional end-users in field conditions is similar to that obtained by a research team and that this RDT can be considered as a point-of-care tool/assay. Furthermore, the protocol designed for this study could be used systematically in parallel to conventional evaluation studies to determine the performance of malaria RDTs in field conditions.

K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases, and measured ring-stage survival rates following drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13–49% to 0.3–2.4% following the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2–29% following the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared to reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia

The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. The presence of non-reference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10−12). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of resistance to artemisinin mutations and independent emergence of the same mutation in different geographic areas. K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.


Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012-2013

The emergence of Plasmodium falciparum resistance to artemisinin and its derivatives, manifested as delayed parasite clearance following the treatment, has developed in Southeast Asia. The spread of resistance to artemisinin from Asia to Africa may be catastrophic for malaria control and elimination worldwide. Recently, mutations in the propeller domain of the Kelch 13 (k13) gene (PF3D71343700) were associated with in vitro resistance to artemisinin and with delayed clearance after artemisinin treatment in southern Asia. The aim of the study was to characterize the genetic variability of k13 and to evaluate the molecular resistance to artemisinin for the first time in Senegal. Plasmodium falciparum isolates were collected from 138 malaria patients in Dakar and its districts during the rainy season of October 2012 to January 2013 at the Hôpital Principal de Dakar. The k13 gene was amplified using nested PCR and sequenced. A very limited variability within the k13 gene in Senegalese P. falciparum isolates was identified. No polymorphism was detected in the six k13-propeller blades. Only two mutations, T149S (6.3%) and K189T (42.2%), and one (N) or two (NN) asparagine insertion at the codon 142 (4.7 and 6.3%, respectively) were detected in the Plasmodium/Apicomplexa-specific domain. None of the polymorphisms associated with artemisinin resistance in Southeast Asia was detected in the 138 P. falciparum from Dakar. The present data do not suggest widespread artemisinin resistance in Dakar in 2012–2013. Notably, the C580Y, R539T or Y493H substitutions that were associated with in vitro resistance or delayed parasite clearance in Southeast Asia were not observed in Dakar, nor were any of the polymorphisms observed in parasites from Southeast Asia, nor the M476I mutation that was selected in vitro with artemisinin pressure in a African parasite line.


Plasmodium falciparum K13-Propeller Polymorphisms Isolated from Patients in Mayotte in 2013-2014

Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013-2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from the Grande Comore showed polymorphism (N490H, N554K and E596G).


Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P<10_3). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC50) of PP were independent of treatment outcomes, in contrast to median mefloquine IC50, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P_0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P<0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested.


Histone methyltransferase inhibitors are orally bioavailable, fast-acting molecules with activity against different species causing malaria in humans

Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC50s) of<50 nM against drug-sensitive laboratory strains and multidrug-resistant field isolates, including artemisinin-refractory Plasmodium falciparum isolates. Activities against ex vivo clinical isolates of both P. falciparum and Plasmodium vivax were similar, with potencies of 300 to 400 nM. Sexual-stage gametocyte inhibition occurs at micromolar levels; however, mature gametocyte progression to gamete formation is inhibited at submicromolar concentrations. Parasite reduction ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria.

Plasmodium vivax: Diversity and Population Structure across Four Continents

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11microsatellitemarkers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (FST>0.2). Outside Central Asia FST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.


Reduced polymorphism in the Kelch propeller domain in Plasmodium vivax isolates from Cambodia

Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanakiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism.


Support to National Authorities

The following summarizes key support to Cambodian National Authorities during 2019 as part of our ongoing programs and projects.

MMEU works under the umbrella of the Cambodian Ministry of Health, as do all IPC research units. Specifically, MMEU is a main collaborator and a main technical support of the Cambodia National Malaria Control Program at CNM. In particular, MMEU provides its support to drug efficacy studies that are conducted yearly in Cambodia. Additionally, MMEU is involved in quality assurance and quality control (QA/QC) for malaria screening among Cambodian UN peacekeepers sent on international missions abroad.

Teaching & Training

The Malaria Molecular Epidemiology Unit staff are involved in the international master’s degree in infectious disease at the University ofHealth Science Phnom Penh and the University of Paris Saclay. Benoit Witkowski teaches a 2.5h course on malaria parasites evolution; Amélie Vantaux teaches a 2.5h course on host manipulation by parasites, while Nimol Khim teaches a 4h bioinformatics course.

PhD students:

      • Melissa Mairet-Khedim: P. falciparum resistance (since January 2017)
      • Camille Roesch: biology of P. vivax invasion (since January 2017)
      • Kutub Ashraf: P. vivax liver stage (since April 2018)
      • Anais Pepey: Ecology of malaria transmission (since November 2018)

Interns

      • Beatrice Tappy (February-July 2019) Synergy of antimalarial drugs
      • Daniel Calixto (March-July 2019) Novel antimalarial drugs
      • IPIN researcher Mobility: Sandrine Nsango (March-May 2019) DHA-PPQ resistance in Cameroon

Team

Benoit Witkowski

Head of unit

Nimol Khim

Deputy Head of unit

Amelie Vantaux

Contractual Researcher

Jean Popovici

Contractual Researcher

Main National Partners

Main International Partners

OPEN HOURS