Therapeutic efficacy and safety of artesunate + amodiaquine and artemether + lumefantrine in treating uncomplicated Plasmodium falciparum malaria in children on the rainy south-east coast of Madagascar.

Malaria is a major public health problem in Madagascar, particularly in coastal areas. We conducted a randomized, controlled, parallel-group study of artemisinin-based combination therapy (ACT) in Mananjary and Farafangana, two localities on the rainy south-east coast of Madagascar, from March to September 2018. The efficacy and safety of artesunate + amodiaquine (ASAQ) and artemether + lumefantrine (AL) were assessed according to the WHO protocol with a 28-day follow-up. Children aged 6 months to 14 years with uncomplicated Plasmodium falciparum malaria were randomized to receive ASAQ or AL for three days (1:1). 347/352 (98.5%) randomized patients reached the study endpoint on day 28. Crude adequate clinical and parasitological response (ACPR) rates were 100% (95% CI: 98.8-100%) in the ASAQ group and 96% (95% CI: 93.1-98.9%) in the AL group (per protocol population). However, the PCR-corrected ACPR rate was 97.7% (95% CI: 95.4-100%) in the AL group. Two cases of recrudescence and three of re-infection were observed. Mild and moderate adverse events, including gastrointestinal and/or nervous disorders, were reported in 11.9% (42/352) of patients. We found that ASAQ and AL were safe and efficacious for treating uncomplicated P. falciparum malaria. They may be used for treatment at health facilities and at the community level, and for mass drug administration campaigns.

Title: Efficacité thérapeutique et sécurité de l'artésunate + amodiaquine et de l'artéméther + luméfantrine pour le traitement du paludisme simple à Plasmodium falciparum chez les enfants sur la côte sud-est pluvieuse de Madagascar. Abstract: Le paludisme demeure un problème majeur de santé publique à Madagascar notamment dans les régions côtières. Nous avons réalisé une étude multisite, randomisée, contrôlée, en groupes parallèles sur la combinaison thérapeutique à base des dérivés d'artémisinine (CTA) à Mananjary et Farafangana, deux localités sur la côte sud-est pluvieuse de Madagascar, de mars au septembre 2018. L'efficacité et la sécurité de l'artésunate + amodiaquine (ASAQ) et de l'artéméther + luméfantrine (AL) ont été évaluées selon le protocole de l'OMS avec un suivi de 28 jours. Des enfants âgés de 6 mois à 14 ans souffrant de paludisme non compliqué à Plasmodium falciparum ont été randomisés (1:1) pour recevoir ASAQ ou AL pendant trois jours. 347/352 (98,5 %) des patients randomisés ont pu être suivis jusqu'au jour 28. Le taux de réponse clinique et parasitologique adéquate (RCPA) était de 100 % (95 % CI : 98,8 ­ 100 %) dans le bras thérapeutique ASAQ et de 96 % (95 % CI : 93,1 ­ 98,9 %) dans le bras thérapeutique AL (population per protocole). Cependant, après correction par PCR, le taux de RCPA était de 97,7 % (95 % CI : 95,4 ­ 100 %) dans le bras thérapeutique AL. Deux cas de recrudescence et trois cas de réinfections ont été observées. Des effets indésirables légers et modérés, notamment des troubles gastro-intestinaux et/ou nerveux, ont été rapportés chez 11,9 % (42/352) des patients. Nos résultats démontrent que l'ASAQ et l'AL sont sûrs et efficaces pour le traitement du paludisme non compliqué à P. falciparum. Ces deux CTA peuvent par conséquent être utilisés pour traiter le paludisme dans les centres de santé et au niveau communautaire, et aussi pendant les campagnes de traitement de masse.

Proactive community case management decreased malaria prevalence in rural Madagascar: results from a cluster randomized trial.

BACKGROUND: Malaria remains a leading cause of morbidity and mortality worldwide, with progress in malaria control stalling in recent years. Proactive community case management (pro-CCM) has been shown to increase access to diagnosis and treatment and reduce malaria burden. However, lack of experimental evidence may hinder the wider adoption of this intervention. We conducted a cluster randomized community intervention trial to assess the efficacy of pro-CCM at decreasing malaria prevalence in rural endemic areas of Madagascar. METHODS: Twenty-two fokontany (smallest administrative unit) of the Mananjary district in southeast Madagascar were selected and randomized 1:1 to pro-CCM (intervention) or conventional integrated community case management (iCCM). Residents of all ages in the intervention arm were visited by a community health worker every 2 weeks from March to October 2017 and screened for fever; those with fever were tested by a rapid diagnostic test (RDT) and treated if positive. Malaria prevalence was assessed using RDTs on all consenting study area residents prior to and following the intervention. Hemoglobin was measured among women of reproductive age. Intervention impact was assessed via difference-in-differences analyses using logistic regressions in generalized estimating equations. RESULTS: A total of 27,087 and 20,475 individuals participated at baseline and endline, respectively. Malaria prevalence decreased from 8.0 to 5.4% in the intervention arm for individuals of all ages and from 6.8 to 5.7% in the control arm. Pro-CCM was associated with a significant reduction in the odds of malaria positivity in children less than 15 years (OR = 0.59; 95% CI [0.38-0.91]), but not in older age groups. There was no impact on anemia among women of reproductive age. CONCLUSION: This trial suggests that pro-CCM approaches could help reduce malaria burden in rural endemic areas of low- and middle-income countries, but their impact may be limited to younger age groups with the highest malaria burden. TRIAL REGISTRATION: NCT05223933. Registered on February 4, 2022.

Identification of factors associated with residual malaria transmission using school-based serological surveys in settings pursuing elimination.

BACKGROUND: Targeted research on residual malaria transmission is important to improve strategies in settings pursuing elimination, where transmission reductions prove challenging. This study aimed to detect and characterize spatial heterogeneity and factors associated with Plasmodium falciparum infections and exposure, P. falciparum apical membrane antigen 1 (PfAMA1) antibody (Ab) response, in the Central Highlands of Madagascar (CHL). METHODS: From May to July 2014, a cross-sectional school-based survey was carried out in 182 fokontany (villages) within 7 health districts of the CHL. Rapid diagnostic tests (RDTs) and a bead-based immunoassay including PfAMA1 antigen biomarker were used to estimate malaria prevalence and seroprevalence, respectively. Local Moran's I index was used to detect spatial "hotspots". Remotely sensed environmental data-temperature, vegetation indices, land covers, and elevation-were used in multivariable mixed-effects logistic regression models to characterize factors associated with malaria infection and cumulative exposure. RESULTS: Among 6,293 school-children ages 2-14 years surveyed, RDT prevalence was low at 0.8% (95% CI 0.6-1.1%), while PfAMA1 Ab seroprevalence was 7.0% (95% CI 6.4-7.7%). Hotspots of PfAMA1 Ab seroprevalence were observed in two districts (Ankazobe and Mandoto). Seroprevalence increased for children living > 5 km from a health centre (adjusted odds ratio (OR) = 1.6, 95% CI 1.2-2.2), and for those experiencing a fever episode in the previous 2 weeks (OR 1.7, 95% CI 1.2-2.4), but decreased at higher elevation (for each 100-m increase, OR = 0.7, 95% CI 0.6-0.8). A clear age pattern was observed whereby children 9-10 years old had an OR of 1.8 (95% CI 1.2-2.4), children 11-12 years an OR of 3.7 (95% CI 2.8-5.0), and children 13-14 years an OR of 5.7 (95% CI 4.0-8.0) for seropositivity, compared with younger children (2-8 years). CONCLUSION: The use of serology in this study provided a better understanding of malaria hotspots and associated factors, revealing a pattern of higher transmission linked to geographical barriers in health care access. The integration of antibody-assays into existing surveillance activities could improve exposure assessment, and may help to monitor the effectiveness of malaria control efforts and adapt elimination interventions.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Parasites and diet as main drivers of the Malagasy gut microbiome richness and function.

Interactions between the prokaryotic microbiome and eukaryotic parasites in the vertebrate gut may affect overall host health and disease. While intertropical areas exhibit a high rate of parasites carriers, such interactions are understudied in these populations. Our objectives were to (1) describe the gut microbiome of individuals living in Madagascar, (2) identify potential associations between bacterial taxa and parasites colonizing the digestive tract and (3) highlight main determinants of the gut microbiota composition in this developing country. Metadata (socioeconomic, diet, clinical) and fecal samples were collected from 219 volunteers from North-West Madagascar (Mahajanga). Fecal microbiome was assessed through 16S rRNA gene sequencing and metabolomics, and related to dietary habits and parasites carriage. We highlight important Malagasy gut microbiome peculiarities. Out of three detected enterotypes, only one is similar to that observed in Westernized countries (Ruminococcus-driven). Functions associated with the two others (Clostridium sensu stricto-driven and Escherichia/Shigella-driven) are mostly directed toward amino acids biosynthesis and degradation, respectively. Diet and protozoan carriage were the main drivers of microbiota composition. High protozoan carriage was associated with higher diversity, richness and microbial functionalities. The gut microbiome of Malagasy strongly differs from that of Westernized countries. Asymptomatic protozoan carriage and dietary habits are the external factors with the deepest impact on gut microbiome. Further studies are needed to understand whether gut microbial richness constitute a predilection niche for protozoans colonization, due to their gazing features, or whether the parasites themselves induce a higher bacterial richness.

Diversity, distribution, and drivers of Polychromophilus infection in Malagasy bats.

BACKGROUND: Numerous studies have been undertaken to advance knowledge of apicomplexan parasites infecting vertebrates, including humans. Of these parasites, the genus Plasmodium has been most extensively studied because of the socio-economic and public health impacts of malaria. In non-human vertebrates, studies on malaria or malaria-like parasite groups have been conducted but information is far from complete. In Madagascar, recent studies on bat blood parasites indicate that three chiropteran families (Miniopteridae, Rhinonycteridae, and Vespertilionidae) are infected by the genus Polychromophilus with pronounced host specificity: Miniopterus spp. (Miniopteridae) harbour Polychromophilus melanipherus and Myotis goudoti (Vespertilionidae) is infected by Polychromophilus murinus. However, most of the individuals analysed in previous studies were sampled on the western and central portions of the island. The aims of this study are (1) to add new information on bat blood parasites in eastern Madagascar, and (2) to highlight biotic and abiotic variables driving prevalence across the island. METHODS: Fieldworks were undertaken from 2014 to 2016 in four sites in the eastern portion of Madagascar to capture bats and collect biological samples. Morphological and molecular techniques were used to identify the presence of haemosporidian parasites. Further, a MaxEnt modelling was undertaken using data from Polychromophilus melanipherus to identify variables influencing the presence of this parasite RESULTS: In total, 222 individual bats belonging to 17 species and seven families were analysed. Polychromophilus infections were identified in two families: Miniopteridae and Vespertilionidae. Molecular data showed that Polychromophilus spp. parasitizing Malagasy bats form a monophyletic group composed of three distinct clades displaying marked host specificity. In addition to P. melanipherus and P. murinus, hosted by Miniopterus spp. and Myotis goudoti, respectively, a novel Polychromophilus lineage was identified from a single individual of Scotophilus robustus. Based on the present study and the literature, different biotic and abiotic factors are shown to influence Polychromophilus infection in bats, which are correlated based on MaxEnt modelling. CONCLUSIONS: The present study improves current knowledge on Polychromophilus blood parasites infecting Malagasy bats and confirms the existence of a novel Polychromophilus lineage in Scotophilus bats. Additional studies are needed to obtain additional material of this novel lineage to resolve its taxonomic relationship with known members of the genus. Further, the transmission mode of Polychromophilus in bats as well as its potential effect on bat populations should be investigated to complement the results provided by MaxEnt modelling and eventually provide a comprehensive picture of the biology of host-parasite interactions.

School-Based Serosurveys to Assess the Validity of Using Routine Health Facility Data to Target Malaria Interventions in the Central Highlands of Madagascar.

BACKGROUND: In low-malaria-transmission areas of Madagascar, annual parasite incidence (API) from routine data has been used to target indoor residual spraying at subdistrict commune level. To assess validity of this approach, we conducted school-based serological surveys and health facility (HF) data quality assessments in 7 districts to compare API to gold-standard commune-level serological measures. METHODS: At 2 primary schools in each of 93 communes, 60 students were randomly selected with parents and teachers. Capillary blood was drawn for rapid diagnostic tests (RDTs) and serology. Multiplex bead-based immunoassays to detect antibodies to 5 Plasmodium falciparum antigens were conducted, and finite mixture models used to characterize seronegative and seropositive populations. Reversible catalytic models generated commune-level annual seroconversion rates (SCRs). HF register data were abstracted to assess completeness and accuracy. RESULTS: RDT positivity from 12 770 samples was 0.5%. Seroprevalence to tested antigens ranged from 17.9% (MSP-1) to 59.7% (PF13). Median commune-level SCR was 0.0108 (range, 0.001-0.075). Compared to SCRs, API identified 71% (95% confidence interval, 51%-87%) of the 30% highest-transmission communes; sensitivity declined at lower levels. Routine data accuracy did not substantially affect API performance. CONCLUSIONS: API performs reasonably well at identifying higher-transmission communes but sensitivity declined at lower transmission levels.

Targeted Next Generation Sequencing for malaria research in Africa: current status and outlook.

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed.

Major subpopulations of Plasmodium falciparum in sub-Saharan Africa.

Understanding genomic variation and population structure of Plasmodium falciparum across Africa is necessary to sustain progress toward malaria elimination. Genome clustering of 2263 P. falciparum isolates from 24 malaria-endemic settings in 15 African countries identified major western, central, and eastern ancestries, plus a highly divergent Ethiopian population. Ancestry aligned to these regional blocs, overlapping with both the parasite's origin and with historical human migration. The parasite populations are interbred and shared genomic haplotypes, especially across drug resistance loci, which showed the strongest recent identity-by-descent between populations. A recent signature of selection on chromosome 12 with candidate resistance loci against artemisinin derivatives was evident in Ghana and Malawi. Such selection and the emerging substructure may affect treatment-based intervention strategies against P. falciparum malaria.

Herbal Remedies to Treat Malaria in Madagascar: Hype and Hope.

On the island of Madagascar, prior to the arrival of the Europeans, some pathologies including malaria, locally known as tazo (fever), were already described. As part of the Malagasy traditional knowledge, traditional medicine mainly based on the use of herbal remedies is part of the malaria treatment still today. Across the country, hundreds of plants are identified as antimalarial, and some compounds from plants show interesting in vitro activities against human Plasmodium. However, it has become clear that most of the antimalarial herbal remedies traditionally used are not efficient antimalarials. In order to identify authentic antimalarial herbal remedies, methodical approaches should range from plant selection to biological screening. In this paper, we share our point of view based on our experience on antimalarial plants in Madagascar.

Evidence of Insecticide Resistance to Pyrethroids and Bendiocarb in Anopheles funestus from Tsararano, Marovoay District, Madagascar.

INTRODUCTION: In Madagascar, malaria control relies on the countrywide use of long lasting insecticide treated bed nets (LLINs) and on indoor residual spraying (IRS) in the central highland area as well as a small area on the eastern coast. We tested insecticide resistance mechanisms of Anopheles funestus from Tsararano, a malaria endemic village in the coastal health district of Marovoay. METHODS: Insecticide susceptibility bioassays were done in July 2017 on first-generation Anopheles funestus (F1) to assess (i) the susceptibility to permethrin (0.05%), deltamethrin (0.05%), DDT (4%), malathion (5%), fenitrothion (1%), and bendiocarb (0.1%); (ii) the effect of preexposure to the piperonyl butoxide (PBO) synergist; and (iii) the enzymatic activities of cytochrome P450, esterases, and glutathione S-transferases (GST). RESULTS: Our results demonstrated that An. funestus was phenotypically resistant to pyrethroids and bendiocarb, with a mortality rate (MR) of 33.6% (95%CI: 24.5-43.7%) and 86% (95%CI: 77.6-92.1%), respectively. In contrast, An. funestus were 100% susceptible to DDT and organophosphates (malathion and fenitrothion). Preexposure of An. funestus to PBO synergist significantly restored the susceptibility to bendiocarb (MR=100%) and increased the MR in the pyrethroid group, from 96% (95%CI: 90.0-98.9%) to 100% for deltamethrin and permethrin, respectively (χ 2 = 43, df = 3, P< 0.0001). Enzymatic activities of cytochrome P450 and α-esterases were significantly elevated among An. funestus compared with the IPM reference strain (Mann-Whitney U= 30, P<0.0001; U = 145.5, P <0.0001, respectively). No significant differences of ß-esterases activities compared to the IPM reference strain were observed (Mann-Whitney U = 392.5, P = 0.08). CONCLUSION: In Tsararano, despite the absence of an IRS programme, there is evidence of high levels of insecticide resistance to pyrethroids and bendiocarb in An.

Human intestinal parasites in Mahajanga, Madagascar: The kingdom of the protozoa.

INTRODUCTION: Intestinal parasitic infections are a major public health problem in inter-tropical areas. The aim of our study was to describe the situation in Mahajanga, Madagascar with a particular focus on two protozoa, Dientamoeba fragilis and Blastocystis sp. METHODS: This was a prospective study from February to June 2015. Stool samples from symptomatic hospitalized patients and asymptomatic volunteers were submitted to microscopy and molecular assays in order to detect parasites. RESULTS: A wide panel of intestinal parasites were identified among the 265 included subjects, protozoa being the most prevalent with 72.8% whereas the prevalence of helminths and microsporidia was of 7.9% and 4.5%, respectively. Blastocystis sp. was the most prevalent protozoa (64.5% of the entire cohort) followed by various amoebas (35.5%) and flagellates (27,5%). We only detected subtypes 1, 2 and 3 of Blastocystis sp. Among the patients positive for D. fragilis (9.4%), 23 carried genotype 1 and 1 genotype 2. For the first time, we detected in 4 human stools the DNA of a recently described protozoon, Simplicimonas similis. Interestingly, subjects living in urban areas harbored significantly more different parasitic species than subjects living in rural areas with a correlation between sanitary level of neighborhood and protozoan infection. However, there was no difference in prevalence of digestive symptoms between parasite-free and parasite-infected subjects, except for Giardia intestinalis which had more symptomatic carriers. DISCUSSION: Our study reveals a high overall parasite prevalence, similar to what had been found in 2003 in the same city and to other prevalence studies conducted in Africa. The poor access of the population to sanitary infrastructures may explain this result. Data from our study provide valuable key for sanitation programs and prevention of fecal-related infectious diseases.

Evaluating Effectiveness of Mass and Continuous Long-lasting Insecticidal Net Distributions Over Time in Madagascar: A Sentinel Surveillance Based Epidemiological Study.

BACKGROUND: The reduction of global malaria burden over the past 15 years is much attributed to the expansion of mass distribution campaigns (MDCs) of long-lasting insecticidal nets (LLIN). In Madagascar, two LUN MDCs were implemented and one district also benefited from a community-based continuous distribution (CB-CD). Malaria incidence dropped but eventually rebounded after a decade. METHODS: Data from a sentinel surveillance network over the 2009-2015 period was analyzed. Alerts were defined as w eekly number o f malaria cases exceeding the 90th percentile value for three consecutive weeks. Statistical analyses assessed the temporal relationship between LLIN MDCs and (i) number of malaria cases and (ii) malaria alerts detected, and (iii) the effect of a combination of MDCs and a CB-CD in Toamasina District. FINDINGS: Analyses showed an increase of 13.6 points and 21.4 points in the percentile value of weekly malaria cases during the second and the third year following the MDC of LLINs respectively. The percentage of alert-free sentinel sites was 98.2% during the first year after LLIN MDC, 56.7% during the second year and 31.5% during the third year. The number of weekly malaria cases decreased by 14% during the CB-CD in Toamasina District. In contrast, sites without continuous distribution had a 12% increase of malaria cases. INTERPRETATION: These findings support the malaria-preventive effectiveness of MDCs in Madagascar but highlight their limited duration when not followed by continuous distribution. The resulting policy implications are crucial to sustain reductions in malaria burden in high transmission settings.

Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors.

BACKGROUND: Bats are home to diverse haemosporidian parasites namely Plasmodium and Plasmodium-related. While information is available at a worldwide level, haemosporidian infection in bats from Madagascar is still scarce and recent changes in the taxonomy of the island's bat fauna, particularly the description of several new species, require a reassessment of previously described patterns, including blood parasite ecology and vectorial transmission. METHODS: A sample representing seven of the nine known bat families and 31 of the 46 currently recognized taxa from Madagascar and collected in the western and central portions of the island were screened by PCR for the presence of Polychromophilus. In addition, Nycteribiidae flies parasitizing Miniopteridae and Vespertilionidae were screened for parasites with the aim to better understand aspects of vector transmission. Phylogenetic reconstruction using the mitochondrial cytochrome b encoding gene was used in a Bayesian analysis to examine the relationship between Polychromophilus recovered from Malagasy bats and those identified elsewhere. RESULTS: Polychromophilus infection was restricted to Miniopterus spp. (Miniopteridae), Myotis goudoti (Vespertilionidae), and Paratriaenops furculus (Rhinonycteridae), with an overall infection rate of 13.5%. Polychromophilus melanipherus was found infecting Miniopterus spp. and P. furculus, whereas Polychromophilus murinus was only recovered from M. goudoti. These two protozoan parasites species were also detected in bat flies species known to parasitize Miniopterus spp. and M. goudoti, respectively. Generalized linear model analyses were conducted to elucidate the effect of species and sex on haemoparasites infection in Miniopterus spp., which revealed that males have higher risk of infection than females and prevalence differed according to the considered Miniopterus host. Molecular screening of nycteribiid flies revealed three positive species for Polychromophilus spp., including Penicillidia sp. (cf. fulvida), Penicillidia leptothrinax, and Nycteribia stylidiopsis. These three fly species are known to parasitize Miniopterus spp. and M. goudoti and should be considered as potential vectors of Polychromophilus spp. CONCLUSION: Phylogenetic analyses demonstrated the existence of at least four distinct clades within the genus Polychromophilus, two of which were documented in the present study. The screening of nycteribiid flies overlaid on the highly diversified genus Miniopterus, provides considerable insight into parasite transmission, with bat infection being associated with their roosting behaviour and the occurrence of specific arthropod vectors.

Study on causes of fever in primary healthcare center uncovers pathogens of public health concern in Madagascar.

BACKGROUND: The increasing use of malaria diagnostic tests reveals a growing proportion of patients with fever but no malaria. Clinicians and health care workers in low-income countries have few tests to diagnose causes of fever other than malaria although several diseases share common symptoms. We propose here to assess etiologies of fever in Madagascar to ultimately improve management of febrile cases. METHODOLOGY: Consenting febrile outpatients aged 6 months and older were recruited in 21 selected sentinel sites throughout Madagascar from April 2014 to September 2015. Standard clinical examinations were performed, and blood and upper respiratory specimens were taken for rapid diagnostic tests and molecular assays for 36 pathogens of interest for Madagascar in terms of public health, regardless of clinical status. PRINCIPAL FINDINGS: A total of 682 febrile patients were enrolled. We detected at least one pathogen in 40.5% (276/682) of patients and 6.2% (42/682) with co-infections. Among all tested patients, 26.5% (181/682) had at least one viral infection, 17.0% (116/682) had malaria and 1.0% (7/682) presented a bacterial or a mycobacterial infection. None or very few of the highly prevalent infectious agents in Eastern Africa and Asia were detected in this study, such as zoonotic bacteria or arboviral infections. CONCLUSIONS: These results raise questions about etiologies of fever in Malagasy communities. Nevertheless, we noted that viral infections and malaria still represent a significant proportion of causes of febrile illnesses. Interestingly our study allowed the detection of pathogens of public health interest such as Rift Valley Fever Virus but also the first case of laboratory-confirmed leptospirosis infection in Madagascar.

Spatio-temporal mapping of Madagascar's Malaria Indicator Survey results to assess Plasmodium falciparum endemicity trends between 2011 and 2016.

BACKGROUND: Reliable measures of disease burden over time are necessary to evaluate the impact of interventions and assess sub-national trends in the distribution of infection. Three Malaria Indicator Surveys (MISs) have been conducted in Madagascar since 2011. They provide a valuable resource to assess changes in burden that is complementary to the country's routine case reporting system. METHODS: A Bayesian geostatistical spatio-temporal model was developed in an integrated nested Laplace approximation framework to map the prevalence of Plasmodium falciparum malaria infection among children from 6 to 59 months in age across Madagascar for 2011, 2013 and 2016 based on the MIS datasets. The model was informed by a suite of environmental and socio-demographic covariates known to influence infection prevalence. Spatio-temporal trends were quantified across the country. RESULTS: Despite a relatively small decrease between 2013 and 2016, the prevalence of malaria infection has increased substantially in all areas of Madagascar since 2011. In 2011, almost half (42.3%) of the country's population lived in areas of very low malaria risk (<1% parasite prevalence), but by 2016, this had dropped to only 26.7% of the population. Meanwhile, the population in high transmission areas (prevalence >20%) increased from only 2.2% in 2011 to 9.2% in 2016. A comparison of the model-based estimates with the raw MIS results indicates there was an underestimation of the situation in 2016, since the raw figures likely associated with survey timings were delayed until after the peak transmission season. CONCLUSIONS: Malaria remains an important health problem in Madagascar. The monthly and annual prevalence maps developed here provide a way to evaluate the magnitude of change over time, taking into account variability in survey input data. These methods can contribute to monitoring sub-national trends of malaria prevalence in Madagascar as the country aims for geographically progressive elimination.

The protective effectiveness of control interventions for malaria prevention: a systematic review of the literature.

Background: Thanks to a considerable increase in funding, malaria control interventions (MCI) whose efficacy had been demonstrated by controlled trials have been largely scaled up during the last decade. Nevertheless, it was not systematically investigated whether this efficacy had been preserved once deployed on the field. Therefore, we sought the literature to assess the disparities between efficacy and effectiveness and the effort to measure the protective effectiveness (PE) of MCI. Methods: The PubMed database was searched for references with keywords related to malaria, to control interventions for prevention and to study designs that allow for the measure of the PE against parasitemia or against clinical outcomes. Results: Our search retrieved 1423 references, and 162 articles were included in the review. Publications were scarce before the year 2000 but dramatically increased afterwards. Bed nets was the MCI most studied (82.1%). The study design most used was a cross-sectional study (65.4%). Two thirds (67.3%) were conducted at the district level or below, and the majority (56.8%) included only children even if the MCI didn't target only children. Not all studies demonstrated a significant PE from exposure to MCI: 60.6% of studies evaluating bed nets, 50.0% of those evaluating indoor residual spraying, and 4/8 showed an added PE of using both interventions as compared with one only; this proportion was 62.5% for intermittent preventive treatment of pregnant women, and 20.0% for domestic use of insecticides. Conclusions: This review identified numerous local findings of low, non-significant PE -or even the absence of a protective effect provided by these MCIs. The identification of such failures in the effectiveness of MCIs advocates for the investigation of the causes of the problem found. Ideal evaluations of the PE of MCIs should incorporate both a large representativeness and an evaluation of the PE stratified by subpopulations.

Analysing trends and forecasting malaria epidemics in Madagascar using a sentinel surveillance network: a web-based application.

BACKGROUND: The use of a malaria early warning system (MEWS) to trigger prompt public health interventions is a key step in adding value to the epidemiological data routinely collected by sentinel surveillance systems. METHODS: This study describes a system using various epidemic thresholds and a forecasting component with the support of new technologies to improve the performance of a sentinel MEWS. Malaria-related data from 21 sentinel sites collected by Short Message Service are automatically analysed to detect malaria trends and malaria outbreak alerts with automated feedback reports. RESULTS: Roll Back Malaria partners can, through a user-friendly web-based tool, visualize potential outbreaks and generate a forecasting model. The system already demonstrated its ability to detect malaria outbreaks in Madagascar in 2014. CONCLUSION: This approach aims to maximize the usefulness of a sentinel surveillance system to predict and detect epidemics in limited-resource environments.

Post-deployment effectiveness of malaria control interventions on Plasmodium infections in Madagascar: a comprehensive phase IV assessment.

BACKGROUND: Because international funding for malaria control is plateauing, affected countries that receive foreign funding are expected to maintain a constant budget while continuing to reduce Plasmodium transmission. To investigate the appropriateness of a malaria control policy in Madagascar, the effectiveness of all currently deployed malaria control interventions (MCIs) was measured. METHODS: A nationwide cross-sectional survey was conducted in 2012-2013 at 62 sites throughout Madagascar. A total of 15,746 individuals of all ages were tested for Plasmodium infection using rapid diagnostic tests and were interviewed about their use of long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), intermittent preventive treatment of pregnant women (IPTp), and exposure to information, education and communication (IEC) campaigns. The association between Plasmodium infection and MCI exposure was calculated using multivariate multilevel models, and the protective effectiveness (PE) of an intervention was defined as one minus the odds ratio of this association. RESULTS: The individual PE of regular LLIN use was high and significant (41 %, 95 % confidence interval [CI] 23-54), whereas its community PE was not. The PE of IRS at the household level was significant in one transmission pattern only (44 %, 95 % CI 11-65), and the community PE with high IRS coverage (>75 %) was high and significant overall (78 %, 95 % CI 44-91). Using LLINs after IRS increased the PE, and the reciprocal was also true. The maternal PE of IPTp was high but non-significant (65 %, 95 % CI -32 to 91). The PE of IEC was low, non-significant and restricted to certain areas (24 %, 95 % CI -34 to 57). CONCLUSIONS: This snapshot of the effectiveness of MCIs confirms that integrated vector control is required in malaria control policies in Madagascar and suggests combining MCIs when one is questionable. Policymakers should consider the local effectiveness of all deployed MCIs through a similar phase IV assessment.

Genomic analysis of local variation and recent evolution in Plasmodium vivax.

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.