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Background: Dual hrp2/hrp3 genes deletions in P. falciparum isolates are increasingly reported in malaria-endemic countries and can produce false negative RDT results leading to inadequate case management. Data on the frequency of hrp2/hrp3 deleted parasites are rarely available and it has become necessary to investigate the issue in Burkina Faso., Methods: Plasmodium falciparum-positive dried blood spots were collected during a cross-sectional household survey of the malaria asymptomatic children from Orodara, Gaoua, and Banfora. Amplicons from the target regions (exon 2 of hrp2 and hrp3 genes) were generated using multiplexed nested PCR and sequenced according to Illumina's MiSeq protocol., Results: A total of 251 microscopically positive parasite isolates were sequenced to detect hrp2 and hrp3 gene deletions. The proportion of RDTs negative cases among microscopy positive slides was 12.7% (32/251). The highest prevalence of negative RDTs was found in Orodara 14.3% (5/35), followed by Gaoua 13.1%(24/183), and Banfora 9.1% (3/33). The study found that 95.6% of the parasite isolates were wild type hrp2/ hrp3 while 4.4% (11/251) had a single hrp2 deletion. Of the 11 hrp2 deletion samples, 2 samples were RDT negative (mean parasitaemia was 83 parasites/ μL) while 9 samples were RDT positive with a mean parasitaemia of 520 parasites /μL (CI95%: 192-1239). The highest frequency of hrp2 deletion 4/35 (11.4%) was found in Orodara, while it was similar in the other two sites (< 3.5%). No single deletion of the hrp3 or dual deletion hrp2/3 gene was detected in this study., Conclusion: These results demonstrate that P. falciparum isolates lacking hrp2 genes are present in 4.4% of samples obtained from the asymptomatic children population in three sites in Burkina Faso. These parasites are circulating and causing malaria, but they are also still detectable by HRP2-based RTDs due to the presence of the intact pfhrp3 gene., (© 2023. The Author(s).)

Background: Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments., Methods: In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria., Results: We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests., Conclusions: The emergence and spread of P. falciparum lineages with both Pfkelch13 -mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.)., (Copyright © 2023 Massachusetts Medical Society.)

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13- propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2 , earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance., Competing Interests: BS, SD, KR, SM, JS, NG, ID, KS, JB, KW, JS, TY, LR, EL, FA, CC, IS, AG, RN, CN, AK, AU, SS, OK, MN, MW, RL, FN, TA, PR, DM, DF No competing interests declared, (© 2021, Stokes et al.)

Background: Mass drug administration (MDA) can rapidly reduce the burden of Plasmodium falciparum (Pf). However, concerns remain about its contribution to select for antimalarial drug resistance., Methods: We used Sanger sequencing and real-time PCR to determine the proportion of molecular markers associated with antimalarial resistance (k13, pfpm2, pfmdr1 and pfcrt) in Pf isolates collected before (n = 99) and after (n = 112) the implementation of two monthly MDA rounds with dihydroartemisinin-piperaquine (DHAp) for two consecutive years in Magude district of Southern Mozambique., Results: None of the k13 polymorphisms associated with artemisinin resistance were observed in the Pf isolates analyzed. The proportion of Pf isolates with multiple copies of pfpm2, an amplification associated with piperaquine resistance, was similar in pre- (4.9%) and post-MDA groups (3.4%; p = 1.000). No statistically significant differences were observed between pre- and post-MDA groups in the proportion of Pf isolates neither with mutations in pfcrt and pfmdr1 genes, nor with the carriage of pfmdr1 multiple copies (p>0.05)., Conclusions: This study does not show any evidence of increased frequency of molecular makers of antimalarial resistance after MDA with DHAp in southern Mozambique where markers of antimalarial resistance were absent or low at the beginning of the intervention., Competing Interests: The authors have declared that no competing interests exist.

Background: Over the last decade, artemisinin-based combination therapy (ACT) has contributed substantially to the decrease in malaria-related morbidity and mortality. The emergence of Plasmodium falciparum parasites resistant to artemisinin derivatives in Southeast Asia and the risk of their spread or of local emergence in sub-Saharan Africa are a major threat to public health. This study thus set out to estimate the proportion of P. falciparum isolates, with Pfkelch13 gene mutations associated with artemisinin resistance previously detected in Southeast Asia., Methods: Blood samples were collected in two sites of Bangui, the capital of the Central African Republic (CAR) from 2017 to 2019. DNA was extracted and nested PCR were carried out to detect Plasmodium species and mutations in the propeller domain of the Pfkelch13 gene for P. falciparum samples., Results: A total of 255 P. falciparum samples were analysed. Plasmodium ovale DNA was found in four samples (1.57%, 4/255). Among the 187 samples with interpretable Pfkelch13 sequences, four samples presented a mutation (2.1%, 4/187), including one non-synonymous mutation (Y653N) (0.5%, 1/187). This mutation has never been described as associated with artemisinin resistance in Southeast Asia and its in vitro phenotype is unknown., Conclusion: This preliminary study indicates the absence of Pfkelch13 mutant associated with artemisinin resistance in Bangui. However, this limited study needs to be extended by collecting samples across the whole country along with the evaluation of in vitro and in vivo phenotype profiles of Pfkelch13 mutant parasites to estimate the risk of artemisinin resistance in the CAR.

Background: Today, the development of new and well-tolerated anti-malarial drugs is strongly justified by the emergence of Plasmodium falciparum resistance. In 2014-2015, a phase 2b clinical study was conducted to evaluate the efficacy of a single oral dose of Artefenomel (OZ439)-piperaquine (PPQ) in Asian and African patients presenting with uncomplicated falciparum malaria., Methods: Blood samples collected before treatment offered the opportunity to investigate the proportion of multidrug resistant parasite genotypes, including P. falciparum kelch13 mutations and copy number variation of both P. falciparum plasmepsin 2 (Pfpm2) and P. falciparum multidrug resistance 1 (Pfmdr1) genes., Results: Validated kelch13 resistance mutations including C580Y, I543T, P553L and V568G were only detected in parasites from Vietnamese patients. In Africa, isolates with multiple copies of the Pfmdr1 gene were shown to be more frequent than previously reported (21.1%, range from 12.4% in Burkina Faso to 27.4% in Uganda). More strikingly, high proportions of isolates with multiple copies of the Pfpm2 gene, associated with piperaquine (PPQ) resistance, were frequently observed in the African sites, especially in Burkina Faso and Uganda (> 30%)., Conclusions: These findings were considered to sharply contrast with the recent description of increased sensitivity to PPQ of Ugandan parasite isolates. This emphasizes the necessity to investigate in vitro susceptibility profiles to PPQ of African isolates with multiple copies of the Pfpm2 gene and estimate the risk of development of PPQ resistance in Africa. Trial registration Clinicaltrials.gov reference: NCT02083380. Study title: Phase II efficacy study of artefenomel and piperaquine in adults and children with P. falciparum malaria. https://clinicaltrials.gov/ct2/results?cond=&term=NCT02083380&cntry=&state=&city=&dist= . FSFV: 23-Jul-2014; LSLV: 09-Oct-2015.

Plasmodium falciparum artemisinin-resistant parasites can be evaluated by examining polymorphisms in the kelch ( PfK13 ) domain. A total of 69 samples from patients with falciparum malaria were analyzed. All samples were from areas in states in Brazil where the parasite was endemic: Acre ( n = 14), Amapá ( n = 15), Amazonas ( n = 30), and Pará ( n = 10). After DNA alignment with the 3D7 reference sequence, all samples were found to be wild type. These data provide a baseline for PfK13 and reinforce the pertinence of artemisinin combination therapy in Brazilian areas., (Copyright © 2018 Gomes et al.)

Background: Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas., Methods: A semi-automated workflow based on the easyMAG ® platform and the Argene Solution ® (bioMérieux, Marcy l'Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing)., Results: By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients., Conclusion: The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.

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.

To limit the spread and impact of anti-malarial drug resistance and react accordingly, surveillance systems able to detect and track in real-time its emergence and spread need to be strengthened or in some places established. Currently, surveillance of anti-malarial drug resistance is done by any of three approaches: (1) in vivo studies to assess the efficacy of drugs in patients; (2) in vitro/ex vivo studies to evaluate parasite susceptibility to the drugs; and/or (3) molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance. These methods are complementary, as they evaluate different aspects of resistance; however, standardization of methods, especially for in vitro/ex vivo and molecular techniques, is lacking. The World Health Organization has developed a standard protocol for evaluating the efficacy of anti-malarial drugs, which is used by National Malaria Control Programmes to conduct their therapeutic efficacy studies. Regional networks, such as the East African Network for Monitoring Antimalarial Treatment and the Amazon Network for the Surveillance of Antimalarial Drug Resistance, have been set up to strengthen regional capacities for monitoring anti-malarial drug resistance. The Worldwide Antimalarial Resistance Network has been established to collate and provide global spatial and temporal trends information on the efficacy of anti-malarial drugs and resistance. While exchange of information across endemic countries is essential for monitoring anti-malarial resistance, sustainable funding for the surveillance and networking activities remains challenging. The technology landscape for molecular assays is progressing quite rapidly, and easy-to-use and affordable new techniques are becoming available. They also offer the advantage of high throughput analysis from a simple blood spots obtained from a finger prick. New technologies combined with the strengthening of national reference laboratories in malaria-endemic countries through standardized protocols and training plus the availability of a proficiency testing programme, would contribute to the improvement and sustainability of anti-malarial resistance surveillance networks worldwide.

Background: Owing to the emergence of multiresistant Plasmodium falciparum parasites in Southeast Asia, along with the impressive decrease in the efficacy of the endoperoxide compound artemisinin and of artemisinin-based combination therapies, the development of novel antimalarial drugs or combinations is required. Although several antiplasmodial molecules, such as endoperoxide-based compounds, are in advanced research or development, we do not know whether resistance to artemisinin derivatives might impact the efficacy of these new compounds., Objectives: To address this issue, the antiplasmodial efficacy of trioxaquines, hybrid endoperoxide-based molecules, was explored, along with their ability to select in vitro resistant parasites under discontinuous and dose-escalating drug pressure., Methods: The in vitro susceptibilities of artemisinin- and trioxaquine-resistant laboratory strains and recent Cambodian field isolates were evaluated by different phenotypic and genotypic assays., Results: Trioxaquines tested presented strong cross-resistance with artemisinin both in the artemisinin-resistant laboratory F32-ART5 line and in Cambodian field isolates. Trioxaquine drug pressure over 4 years led to the in vitro selection of the F32-DU line, which is resistant to trioxaquine and artemisinin, similar to the F32-ART lineage. F32-DU whole genome sequencing (WGS) revealed that resistance to trioxaquine was associated with the same non-synonymous mutation in the propeller domain of the K13 protein (M476I) that was found in the F32-ART lineage., Conclusions: These worrisome results indicate the risk of cross-resistance between artemisinins and endoperoxide-based antiplasmodial drugs in the development of the K13 mutant parasites and question the usefulness of these molecules in the future therapeutic arsenal., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

One of the fundamental steps toward malaria control is the use of antimalarial drugs. The success of antimalarial treatment can be affected by the presence of drug-resistant populations of Plasmodium falciparum. To assess resistance, we used molecular methods to examine 351 P. falciparum isolates collected from 4 sentinel sites in Mozambique for K13, pfmdr1, pfcrt, and pfdhps polymorphisms and for plasmepsin2 (pfpm2) and pfmdr1 copy numbers. We found multiple copies of pfpm2 in 1.1% of isolates. All isolates carried K13 wild-type alleles (3D7-like), except 4 novel polymorphisms (Leu619Leu, Phe656Ile, Val666Val, Gly690Gly). Prevalence of isolates with pfcrt mutant (K76T) allele was low (2.3%). Prevalence of isolates with pfdhps mutant alleles (A437G and K540E) was >80%, indicating persistence of sulfadoxine/pyrimethamine resistance; however, markers of artemisinin were absent, and markers of piperaquine resistance were low. Piperaquine resistance isolates may spread in Mozambique as dihydroartemisinin/piperaquine drug pressure increases.

Artemisinin-based combination therapies (ACTs) are the cornerstone of current strategies for fighting malaria. Over the last decade, ACTs have played a major role in decreasing malaria burden. However, this progress is being jeopardized by the emergence of artemisinin-resistant Plasmodium falciparum parasites. Artemisinin resistance was first detected in western Cambodia in 2008 and has since been observed in neighboring countries in southeast Asia. The problem of antimalarial drug resistance has recently worsened in Cambodia, with reports of parasites resistant to piperaquine, the latest generation of partner drug used in combination with dihydroartemisinin, leading to worrying rates of clinical treatment failure. The monitoring and the comprehension of both types of resistance are crucial to prevent the spread of multidrug-resistant parasites outside southeast Asia, and particularly to Africa, where the public health consequences would be catastrophic. To this end, new tools are required for studying the biological and molecular mechanisms underlying resistance to antimalarial drugs and for monitoring the geographic distribution of the resistant parasites. In this review, we detail the major advances in our understanding of resistance to artemisinin and piperaquine and define the challenges that the malaria community will have to face in the coming years., (© The American Society of Tropical Medicine and Hygiene.)

Background: Serological markers for exposure to different Plasmodium species have recently been used in multiplex immunoassays based on the Luminex technology. However, interpretation of the assay results requires consideration of the half-life of specific antibodies against these markers. Therefore, the aim of the present study was to document the half-life of malaria specific serological makers, as well as assessing the sensitivity of these markers to pick up recent changes in malaria exposure., Methods: A recently developed multiplex immunoassay was used to measure the intensity of antibody (Ab) responses against 19 different Plasmodium specific antigens, covering different human malaria parasites and two vector saliva antigens. Therefore, 8439 blood samples from five cross-sectional surveys in Ratanakiri, Cambodia, were analysed. These involve a random selection from two selected surveys, and an additional set of blood samples of individuals that were randomly re-sampled three, four or five times. A generalized estimating equation model and linear regression models were fitted on log transformed antibody intensity data., Results: Results showed that most (17/21) Ab-responses are higher in PCR positive than PCR negative individuals. Furthermore, these antibody-responses follow the same upward trend within each age group. Estimation of the half-lives showed differences between serological markers that reflect short- (seasonal) and long-term (year round) transmission trends. Ab levels declined significantly together with a decrease of PCR prevalence in a group of malaria endemic villages., Conclusion: For Plasmodium falciparum, antibodies against LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be a reflexion of recent (range from 6 to 8 months) exposure in the Mekong Subregion. PvEBP is the only Plasmodium vivax Ag responding reasonably well, in spite of an estimated Ab half-life of more than 1 year. The use of Ab intensity data rather dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance.

Background: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale., Methods: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci., Results: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay., Conclusions: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

Background: Malaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine-pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparum dhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively., Methods: Blood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing., Results: In the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr-pfdhps mutant was noted., Conclusion: pfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 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 Grande Comore showed polymorphisms (N490H, N554K, and E596G)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: 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., Methods: 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., Results: 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., Conclusion: 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.

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 after 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 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of 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., (Copyright © 2015, American Association for the Advancement of Science.)

Background: 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., Methods: 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., Results: 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., Discussion: 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.

Background: 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., Methods: 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&#95;1343700)., Result: 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., Conclusion: 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.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine., (© The American Society of Tropical Medicine and Hygiene.)

Reduced Plasmodium falciparum sensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivity in vitro at the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins., (© 2014 John Wiley & Sons Ltd.)

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&#95;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., Competing Interests: The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Malaria research units within the Institut Pasteur international network (RIIP-Palu) located in Africa, in South-East Asia and in South America, work for many years in close collaboration with the National malaria control programmes. Relying on technical platforms with well-equipped laboratories and scientific expertise, they are at the forefront of research on the antimalarial drug resistance by working together for training young scientists and developping similar protocols allowing comprehensive comparisons. Including fundamental and operational researches, they conduct regional and international projects which aim (1) to detect the emergence of antimalarial drugs resistant parasites and to evaluate their spatio-temporal distribution, (2) to develop in vitro and molecular tools, (3) to identify epidemiological factors involved in the emergence and the spread of antimalarial drugs resistant parasites and (4) to understand the molecular and cellular mechanisms implicated in resistance. In this review, will be presented methodological approaches and data obtained since 2000., (© 2013 médecine/sciences – Inserm.)

To date, 11 studies conducted in different countries to test the association between Plasmodium falciparum Na(+)/H(+) exchanger gene (pfnhe-1; PF13&#95;0019) polymorphisms and in vitro susceptibility to quinine have generated conflicting data. In this context and to extend our knowledge of the genetic polymorphism of Pfnhe gene, we have sequenced the ms4760 locus from 595 isolates collected in the Comoros (N = 250; an area with a high prevalence of chloroquine and sulfadoxine-pyrimethamine resistance) and Madagascar (N = 345; a low drug-resistance area). Among them, 29 different alleles were observed, including 8 (27%) alleles not previously described. Isolates from the Comoros showed more repeats in block II (DNNND), which some studies have found to be positively associated with in vitro resistance to quinine, compared with isolates from Madagascar. Additional studies are required to better define the mechanisms underlying quinine resistance, which involve multiple gene interactions.

Background: Early diagnosis, as well as prompt and effective treatment of uncomplicated malaria, are essential components of the anti-malaria strategy in Madagascar to prevent severe malaria, reduce mortality and limit malaria transmission. The purpose of this study was to assess the performance of the malaria rapid diagnostic tests (RDTs) used by community health workers (CHWs) by comparing RDT results with two reference methods (microscopy and Polymerase Chain Reaction, PCR)., Methods: Eight CHWs in two districts, each with a different level of endemic malaria transmission, were trained to use RDTs in the management of febrile children under five years of age. RDTs were performed by CHWs in all febrile children who consulted for fever. In parallel, retrospective parasitological diagnoses were made by microscopy and PCR. The results of these different diagnostic methods were analysed to evaluate the diagnostic performance of the RDTs administered by the CHWs. The stability of the RDTs stored by CHWs was also evaluated., Results: Among 190 febrile children with suspected malaria who visited CHWs between February 2009 and February 2010, 89.5% were found to be positive for malaria parasites by PCR, 51.6% were positive by microscopy and 55.8% were positive by RDT. The performance accuracy of the RDTs used by CHWs in terms of sensitivity, specificity, positive and negative predictive values was greater than 85%. Concordance between microscopy and RDT, estimated by the Kappa value was 0.83 (95% CI: 0.75-0.91). RDTs stored by CHWs for 24 months were capable of detecting Plasmodium falciparum in blood at a level of 200 parasites/μl., Conclusion: Introduction of easy-to-use diagnostic tools, such as RDTs, at the community level appears to be an effective strategy for improving febrile patient management and for reducing excessive use of anti-malarial drugs.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Background: Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves., Methods: Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the south-east of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC(50s) to pyrimethamine was performed on adapted isolates., Results: Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC(50)) < 10 nM. The geometric mean of IC(50) of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC(50) of the triple mutant parasite (13,804 nM)., Conclusion: The IC(50)s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC(50)s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the Pfdhfr I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.

The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr (pfdhfr) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.

We sought to test the association of polymorphisms in Plasmodium falciparum nhe-1 (Pfnhe-1, gene PF13&#95;0019) with in vitro susceptibility to quinine, which was previously reported in a limited number of reference strains or culture-adapted isolates. Determination of in vitro susceptibility to quinine, genotyping of Pfnhe-1 ms4760 microsatellite and polymorphism in codon 76 of Pfcrt were performed for 83 isolates obtained from symptomatic malaria-infected travelers returning from various African countries to France or from subjects living in Madagascar. Nineteen different ms4760 microsatellite profiles of Pfnhe-1 were found including 14 not previously described. Multivariate analysis showed no significant association between the in vitro susceptibility to quinine with particular ms4760 profiles. Contrary to previous reports, we only observed that the number of NHNDNHNNDDD repeats was positively associated with the increased IC50 of QN (P = 0.01). We concluded that the studied polymorphisms in Pfnhe-1 did not appear as valid molecular markers of in vitro susceptibility to quinine in P. falciparum isolates from Africa. Because we did not include any isolate of Asian origin in our series, these results did not exclude the possibility of regional associations, for example in South-East Asia.

Malaria remains a major health problem in Madagascar. Over past decades, the burden of malarial disease has fluctuated over time, partly in line with the successes and failures of antimalarial policy. In the 1950s and 1960s, a sharp decline in malaria transmission was observed in the central highlands due to indoor spraying with DDT and to the massive use of chloroquine by the population. Following this, the discontinuation of the 'nivaquinization' policy was followed by devastating outbreaks in the central highlands in the 1980s. Currently, the rate of in vitro chloroquine-resistant Plasmodium falciparum isolates does not exceed 5%. This figure appears disconnected from the high level of clinical treatment failure (near 40%). pfcrt mutant isolates are found in less than 1% of isolates on the Island. Conversely, pfmdr1 mutant isolates are found in more than 60% of isolates and may be responsible for the bulk of resistance to chloroquine in Madagascar. Other antimalarials remain generally effective in Madagascar. Recent clinical and in vitro data support the complete efficacy of the combination artesunate-amodiaquine in Madagascar. As such, this artemisinin combination therapy should play a central role in the control and possible elimination of P. falciparum malaria in Madagascar

The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.

We have developed a High-Resolution DNA Melting method to detect mutations related to Plasmodium falciparum resistance. This method is based on real-time PCR followed by High Resolution Melting ramping from 67 degrees C to 80 degrees C with fluorescence data acquisition set at 0.1 degrees C increments. The accuracy of the technique was assessed using 177 P. falciparum clinical isolates and two reference strains. Results perfectly matched those obtained by DNA sequencing for some important genetic markers of P. falciparum resistance. This technique could be of great value for epidemiological studies, especially in developing countries.

Background: Rapid diagnostic tests (RDTs) are becoming increasingly indispensable in malaria management, as a means of increasing the accuracy of diagnosis. The WHO has issued recommendations, but the selection of the most suitable RDT remains difficult for users in endemic countries. The genetic variability of the antigens detected with RDTs has been little studied, but may affect the sensitivity of RDTs. This factor has been studied by comparisons between countries at continental level, but little information is available concerning antigen variability within a given country., Methods: A country-wide assessment of polymorphism of the PfHRP2, PfHRP3, pLDH and aldolase antigens was carried out in 260 Plasmodium falciparum and 127 Plasmodium vivax isolates, by sequencing the genes encoding these antigens in parasites originating from the various epidemiological strata for malaria in Madagascar., Results: Higher levels of polymorphism were observed for the pfhrp2 and pfhrp3 genes than for the P. falciparum and P. vivax aldolase and pldh genes. Pfhrp2 sequence analysis predicted that 9% of Malagasy isolates would not be detected at parasite densities < or = 250 parasites/mul (ranging from 6% in the north to 14% in the south), although RDTs based on PfHRP2 detection are now recommended in Madagascar., Conclusion: These findings highlight the importance of training of health workers and the end users of RDTs in the provision of information about the possibility of false-negative results for patients with clinical symptoms of malaria, particularly in the south of Madagascar.

To determine risk for drug-resistant malaria parasites entering Madagascar from Comoros Islands, we screened travelers. For the 141 Plasmodium falciparum isolates detected by real-time PCR, frequency of mutant alleles of genes associated with resistance to chloroquine and pyrimethamine was high. International-level antimalarial policy and a regional antimalarial forum are needed.

BackgroundArtesunate-amodiaquine (AS-AQ) is one of the most widely used artemisinin-based combination therapies (ACTs) to treat uncomplicated Plasmodium falciparum malaria in Africa. We investigated the impact of different dosing strategies on the efficacy of this combination for the treatment of falciparum malaria.MethodsIndividual patient data from AS-AQ clinical trials were pooled using the WorldWide Antimalarial Resistance Network (WWARN) standardised methodology. Risk factors for treatment failure were identified using a Cox regression model with shared frailty across study sites.ResultsForty-three studies representing 9,106 treatments from 1999-2012 were included in the analysis; 4,138 (45.4%) treatments were with a fixed dose combination with an AQ target dose of 30 mg/kg (FDC), 1,293 (14.2%) with a non-fixed dose combination with an AQ target dose of 25 mg/kg (loose NFDC-25), 2,418 (26.6%) with a non-fixed dose combination with an AQ target dose of 30 mg/kg (loose NFDC-30), and the remaining 1,257 (13.8%) with a co-blistered non-fixed dose combination with an AQ target dose of 30 mg/kg (co-blistered NFDC). The median dose of AQ administered was 32.1 mg/kg [IQR: 25.9-38.2], the highest dose being administered to patients treated with co-blistered NFDC (median = 35.3 mg/kg [IQR: 30.6-43.7]) and the lowest to those treated with loose NFDC-25 (median = 25.0 mg/kg [IQR: 22.7-25.0]). Patients treated with FDC received a median dose of 32.4 mg/kg [IQR: 27-39.0]. After adjusting for reinfections, the corrected antimalarial efficacy on day 28 after treatment was similar for co-blistered NFDC (97.9% [95% confidence interval (CI): 97.0-98.8%]) and FDC (98.1% [95% CI: 97.6%-98.5%]; P = 0.799), but significantly lower for the loose NFDC-25 (93.4% [95% CI: 91.9%-94.9%]), and loose NFDC-30 (95.0% [95% CI: 94.1%-95.9%]) (P

Plasmodium falciparum has evolved resistance to almost all antimalarial drugs through genomic changes –SNPs and copy number variants (CNVs) – and a nonspecific stress-response-based survival such as the ring-stage temporary growth arrest (TGA). The ring-stage can develop into asexual or sexual stages or stop growing when conditions are unfavorable. The ring-stage shows extreme plasticity under adverse conditions, such as artemisinin exposure. The ring-stage TGA stage exhibits an innate and passive resistance to many stressors, which ensures that some individuals in the population can survive in the host. P. falciparum ring-stage TGA may rely on environmental sensing and extracellular communication between the parasites. Ring-stage TGA may be an underestimated parasite reservoir, especially in asymptomatic individuals. No treatment that specifically targets growth-arrested ring stages is currently available. Malaria is a life-threatening tropical disease caused by parasites of the genus Plasmodium, of which Plasmodium falciparum is the most lethal. Malaria parasites have a complex life cycle, with stages occurring in both the Anopheles mosquito vector and human host. Ring stages are the youngest form of the parasite in the intraerythrocytic developmental cycle and are associated with evasion of spleen clearance, temporary growth arrest (TGA), and drug resistance. This formidable ability to survive and develop into mature, sexual, or growth-arrested forms demonstrates the inherent population heterogeneity. Here we highlight the role of the ring stage as a crossroads in parasite development and as a reservoir of surviving cells in the human host via TGA survival mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plasmodium vivax is the most widespread human malaria parasite, but has received much less attention than Plasmodium falciparum during the past 50 years of research. Plasmodium vivax was historically seen as causing only benign disease, but this view has recently changed, with increased recognition of the burden of vivax malaria, as well as numerous case reports of severe malaria or death caused by this parasite. The complexity of P. vivax biology is characteristic of specific features of the parasite, and recent years have seen major progress in our understanding of this complexity. In this review, we analyze the latest advances in the field, describing the constraints that the unique features of P. vivax place on drug treatments aimed at controlling or eliminating it. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]