Selective whole-genome sequencing of Plasmodium parasites directly from blood samples by nanopore adaptive sampling.

IMPORTANCE: Malaria is caused by parasites of the genus Plasmodium, and reached a global disease burden of 247 million cases in 2021. To study drug resistance mutations and parasite population dynamics, whole-genome sequencing of patient blood samples is commonly performed. However, the predominance of human DNA in these samples imposes the need for time-consuming laboratory procedures to enrich Plasmodium DNA. We used the Oxford Nanopore Technologies' adaptive sampling feature to circumvent this problem and enrich Plasmodium reads directly during the sequencing run. We demonstrate that adaptive nanopore sequencing efficiently enriches Plasmodium reads, which simplifies and shortens the timeline from blood collection to parasite sequencing. In addition, we show that the obtained data can be used for monitoring genetic markers, or to generate nearly complete genomes. Finally, owing to its inherent mobility, this technology can be easily applied on-site in endemic areas where patients would benefit the most from genomic surveillance.

Failure of artemether-lumefantrine therapy in travellers returning to Belgium with Plasmodium falciparum malaria: an observational case series with genomic analysis.

BACKGROUND: Failure of artemisinin-based combination therapy is increasingly reported in patients with Plasmodium falciparum malaria in sub-Saharan Africa. We aimed to describe the clinical and genomic characteristics of recent cases of P. falciparum malaria failing artemether-lumefantrine in Belgium. METHODS: Travel-related cases of malaria confirmed at the national reference laboratory of the Institute of Tropical Medicine, Antwerp, Belgium, were reviewed. All cases for which attending clinicians reported persistence (beyond Day 3 post-treatment initiation, i.e. early failure) or recrudescence (from Day 7 to 42, i.e. late failure) of P. falciparum parasites despite adequate drug intake were analysed. Both initial and persistent/recurrent samples were submitted to next generation sequencing to investigate resistance-conferring mutations. RESULTS: From July 2022 to June 2023, eight P. falciparum cases of failure with artemether-lumefantrine therapy were reported (early failure = 1; late failure = 7). All travellers were returning from sub-Saharan Africa, most (6/8) after a trip to visit friends and relatives. PfKelch13 (PF3D7_1343700) mutations associated with resistance to artemisinin were found in two travellers returning from East Africa, including the validated marker R561H in the patient with early failure and the candidate marker A675V in a patient with late failure. Additional mutations were detected that could contribute to decreased susceptibility to artemisinin in another three cases, lumefantrine in six cases and proguanil in all eight participants. Various regimens were used to treat the persistent/recrudescent cases, with favourable outcome. CONCLUSION: Within a 12-month period, we investigated eight travellers returning from sub-Saharan Africa with P. falciparum malaria and in whom artemether-lumefantrine failure was documented. Mutations conferring resistance to antimalarials were found in all analysed blood samples, especially against lumefantrine and proguanil, but also artemisinin. There is a pressing need for systematic genomic surveillance of resistance to antimalarials in international travellers with P. falciparum malaria, especially those experiencing treatment failure.

Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000-2016).

Emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACT) is a major challenge for Greater Mekong Subregion countries in their goal to eliminate malaria by 2030. Tools to efficiently monitor drug resistance beyond resource-demanding therapeutic efficacy studies are necessary. A custom multiplex amplicon sequencing assay based on Illumina technology was designed to target the marker of partial resistance to artemisinin (K13), five candidate modulators of artemisinin resistance, the marker of resistance to chloroquine (crt), and four neutral microsatellite loci. The assay was used to genotype 635 P. falciparum-positive blood samples collected across seven provinces of Vietnam and one of Cambodia between 2000 and 2016. Markers of resistance to artemisinin partner-drugs piperaquine (copy number of plasmepsin-2) and mefloquine (copy number of multidrug-resistance 1) were determined by qPCR. Parasite population structure was further assessed using a 101-SNP barcode. Validated mutations of artemisinin partial resistance in K13 were found in 48.1% of samples, first detection was in 2000, and by 2015 prevalence overcame > 50% in Central Highlands and Binh Phuoc province. K13-C580Y variant became predominant country-wide, quickly replacing an outbreak of K13-I543T in Central Highlands. Mutations in candidate artemisinin resistance modulator genes paralleled the trends of K13 mutants, whereas resistance to piperaquine and mefloquine remained low (≈ 10%) by 2015-2016. Genomic tools applied to malaria surveillance generate comprehensive information on dynamics of drug resistance and population structure and reflect drug efficacy profiles from in vivo studies.

Correction: Performance of PfHRP2­RDT for malaria diagnosis during the first year of life in a high malaria transmission area in Burkina Faso.

[This corrects the article DOI: 10.1007/s12639-023-01566-x.].

Performance of PfHRP2-RDT for malaria diagnosis during the first year of life in a high malaria transmission area in Burkina Faso.

In this study, we evaluated the performance of a P. falciparum Histidine Rich Protein 2 (PfHRP2)-based rapid diagnostic test (RDT) used for malaria case detection (SD-Bioline malaria RDT P.f®) along with light microscopy (LM) against qPCR among children during the first year of life in a high and seasonal malaria transmission area in Burkina Faso. A total of 723 suspected malaria cases (including multiple episodes) that occurred among 414 children participating in a birth-cohort study were included in the present analysis. Factors including age at the time of malaria screening, transmission season and parasite densities were investigated for their potential influence in the performance of the RDT. Clinical malaria cases as detected by RDT, LM and qPCR were 63.8%, 41.5% and 49.8%, respectively. Compared with qPCR, RDT had a false-positive results rate of 26.7%, resulting in an overall accuracy of 79.9% with a sensitivity of 93%, a specificity of 66.1%, a Positive Predictive Value of 73.3% and a Negative Predictive Value of 91.6%. Its specificity differed significantly between high and low transmission seasons (53.7% vs 79.8%; P < 0.001) and decreased with increasing age (80.6-62%; P for trend = 0.024). The overall accuracy of LM was 91.1% and its performance was not significantly influenced by transmission season or age. These findings highlight the need to adapt malaria diagnostic tools recommendations to face the challenge of adequate malaria detection in this population group living in high burden and seasonal malaria transmission settings.

Molecular Surveillance of Malaria Using the PF AmpliSeq Custom Assay for Plasmodium falciparum Parasites from Dried Blood Spot DNA Isolates from Peru.

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

Malaria Molecular Surveillance in the Peruvian Amazon with a Novel Highly Multiplexed Plasmodium falciparum AmpliSeq Assay.

Molecular surveillance for malaria has great potential to support national malaria control programs (NMCPs). To bridge the gap between research and implementation, several applications (use cases) have been identified to align research, technology development, and public health efforts. For implementation at NMCPs, there is an urgent need for feasible and cost-effective tools. We designed a new highly multiplexed deep sequencing assay (Pf AmpliSeq), which is compatible with benchtop sequencers, that allows high-accuracy sequencing with higher coverage and lower cost than whole-genome sequencing (WGS), targeting genomic regions of interest. The novelty of the assay is its high number of targets multiplexed into one easy workflow, combining population genetic markers with 13 nearly full-length resistance genes, which is applicable for many different use cases. We provide the first proof of principle for hrp2 and hrp3 deletion detection using amplicon sequencing. Initial sequence data processing can be performed automatically, and subsequent variant analysis requires minimal bioinformatic skills using any tabulated data analysis program. The assay was validated using a retrospective sample collection (n = 254) from the Peruvian Amazon between 2003 and 2018. By combining phenotypic markers and a within-country 28-single-nucleotide-polymorphism (SNP) barcode, we were able to distinguish different lineages with multiple resistance haplotypes (in dhfr, dhps, crt and mdr1) and hrp2 and hrp3 deletions, which have been increasing in recent years. We found no evidence to suggest the emergence of artemisinin (ART) resistance in Peru. These findings indicate a parasite population that is under drug pressure but is susceptible to current antimalarials and demonstrate the added value of a highly multiplexed molecular tool to inform malaria strategies and surveillance systems. IMPORTANCE While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance. In addition, there is a lack of standardized laboratory protocols and automated analysis pipelines to generate reproducible and timely results useful for relevant stakeholders. With our standardized laboratory and bioinformatic workflow, malaria genetic surveillance data can be readily generated by surveillance researchers and malaria control programs in countries of endemicity, increasing ownership and ensuring timely results for informed decision- and policy-making.

Plasmodium falciparum sexual conversion rates can be affected by artemisinin-based treatment in naturally infected malaria patients.

BACKGROUND: Artemisinins (ART) are the key component of the frontline antimalarial treatment, but their impact on Plasmodium falciparum sexual conversion rates in natural malaria infections remains unknown. This is an important knowledge gap because sexual conversion rates determine the relative parasite investment between maintaining infection in the same human host and transmission to mosquitoes. METHODS: The primary outcome of this study was to assess the impact of ART-based treatment on sexual conversion rates by comparing the relative transcript levels of pfap2-g and other sexual ring biomarkers (SRBs) before and after treatment. We analysed samples from previously existing cohorts in Vietnam, Burkina Faso and Mozambique (in total, n=109) collected before treatment and at 12 h intervals after treatment. As a secondary objective, we investigated factors that may influence the effect of treatment on sexual conversion rates. FINDINGS: In the majority of infections from the African cohorts, but not from Vietnam, we observed increased expression of pfap2-g and other SRBs after treatment. Estimated parasite age at the time of treatment was negatively correlated with the increase in pfap2-g transcript levels, suggesting that younger parasites are less susceptible to stimulation of sexual conversion. INTERPRETATION: We observed enhanced expression of SRBs after ART-based treatment in many patients, which suggests that in natural malaria infections sexual conversion rates can be altered by treatment. ART-based treatment reduces the potential of a treated individual to transmit the disease, but we hypothesise that under some circumstances this reduction may be attenuated by ART-enhanced sexual conversion. FUNDING: Spanish Agencia Estatal de Investigación (AEI), European Regional Development Fund (ERDF, European Union), Belgium Development Cooperation (DGD), Canadian University Health Network (UHN), TransGlobalHealth-Erasmus Mundus (European Union).

Novel highly-multiplexed AmpliSeq targeted assay for Plasmodium vivax genetic surveillance use cases at multiple geographical scales.

Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax. Several genetic surveillance applications ('use cases') have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP within-country barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/µL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33-SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy.

High Proportion of Genome-Wide Homology and Increased Pretreatment pvcrt Levels in Plasmodium vivax Late Recurrences: a Chloroquine Therapeutic Efficacy Study.

Chloroquine (CQ) is the first-line treatment for Plasmodium vivax malaria in most countries where malaria is endemic. Monitoring P. vivax CQ resistance (CQR) is critical but remains challenged by the difficulty to distinguish real treatment failure from reinfection or liver relapse. The therapeutic efficacy of CQ against uncomplicated P. vivax malaria was evaluated in Gia Lai Province, Vietnam. Sixty-seven patients were enrolled and followed for 42 days using microscopy and quantitative PCR. Adequate clinical and parasitological response (ACPR) was 100% (66/66) on day 28 but 75.4% (49/65) on day 42. Eighteen recurrences (27.7%) were detected, with a median time to recurrence of 42 days (interquartile range [IQR], 35 to 42) and blood CQ concentration of <100 ng/ml. Primary infections leading to recurrence occurred in younger individuals (median age for ACPR = 25 years [IQR, 20 to 28]; recurrences = 18 [16 to 21]; P = 0.002) had a longer parasite clearance time (PCT for ACPR = 47.5 h [IQR, 36.2 to 59.8 h]; recurrences = 54.2 [48.4 to 62.0]; P = 0.035) and higher pvcrt gene expression (median relative expression ratio for ACPR = 0.09 [IQR, 0.05 to 0.22]; recurrences = 0.20 [0.15 to 0.56]; P = 0.002), but showed no differences in ex vivo CQ sensitivity. Parasite genotyping by microsatellites, single nucleotide polymorphism (SNP) barcoding, and whole-genome sequencing (WGS) identified a majority of homologous recurrences, with 80% (8/10) showing >98% identity by descent to paired day 0 samples. This study shows that CQ remained largely efficacious to treat P. vivax in Gia Lai; i.e., recurrences occurred late (>day 28) and in the presence of low blood CQ concentrations. However, the combination of both WGS and gene expression analysis (pvcrt) data with clinical data (PCT) allowed us to identify potential emergence of low-grade CQR, which should be closely monitored. (This study has been registered at ClinicalTrials.gov under identifier NCT02610686.).

Genetic variation in the immune system and malaria susceptibility in infants: a nested case-control study in Nanoro, Burkina Faso.

BACKGROUND: Genetic polymorphisms in the human immune system modulate susceptibility to malaria. However, there is a paucity of data on the contribution of immunogenetic variants to malaria susceptibility in infants, who present differential biological features related to the immaturity of their adaptive immune system, the protective effect of maternal antibodies and fetal haemoglobin. This study investigated the association between genetic variation in innate immune response genes and malaria susceptibility during the first year of life in 656 infants from a birth cohort survey performed in Nanoro, Burkina Faso. METHODS: Seventeen single nucleotide polymorphisms (SNPs) in 11 genes of the immune system previously associated with different malaria phenotypes were genotyped using TaqMan allelic hybridization assays in a Fluidigm platform. Plasmodium falciparum infection and clinical disease were documented by active and passive case detection. Case-control association analyses for both alleles and genotypes were carried out using univariate and multivariate logistic regression. For cytokines showing significant SNP associations in multivariate analyses, cord blood supernatant concentrations were measured by quantitative suspension array technology (Luminex). RESULTS: Genetic variants in IL-1ß (rs1143634) and FcγRIIA/CD32 (rs1801274)-both in allelic, dominant and co-dominant models-were significantly associated with protection from both P. falciparum infection and clinical malaria. Furthermore, heterozygote individuals with rs1801274 SNP in FcγRIIA/CD32 showed higher IL-1RA levels compared to wild-type homozygotes (P = 0.024), a cytokine whose production is promoted by the binding of IgG immune complexes to Fcγ receptors on effector immune cells. CONCLUSIONS: These findings indicate that genetic polymorphisms in genes driving innate immune responses are associated to malaria susceptibility during the first year of life, possibly by modulating production of inflammatory mediators.

Efficacy of dihydroartemisinin/piperaquine and artesunate monotherapy for the treatment of uncomplicated Plasmodium falciparum malaria in Central Vietnam.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have significantly contributed to reduce Plasmodium falciparum malaria burden in Vietnam, but their efficacy is challenged by treatment failure of dihydroartemisinin/piperaquine ACT in Southern provinces. OBJECTIVES: To assess the efficacy of dihydroartemisinin/piperaquine for uncomplicated P. falciparum malaria in Gia Lai, Central Vietnam, and determine parasite resistance to artemisinin (ClinicalTrials.gov identifier NCT02604966). METHODS: Sixty patients received either dihydroartemisinin/piperaquine (4 mg/kg/day, 3 days; n = 33) or artesunate monotherapy (4 mg/kg/day, 3 days; n = 27) followed by dihydroartemisinin/piperaquine (AS + DHA/PPQ). Clinical phenotypes were determined during a 42 day follow-up and analysed together with ex vivo susceptibility to antimalarials and molecular markers of drug resistance. RESULTS: Day 3 positivity rate was significantly higher in the AS + DHA/PPQ arm compared with dihydroartemisinin/piperaquine (70.4% versus 39.4%, P = 0.016). Parasite clearance time was 95.2 h (AS + DHA/PPQ) versus 71.9 h (dihydroartemisinin/piperaquine, P = 0.063) and parasite clearance half-life was 7.4 h (AS + DHA/PPQ) versus 7.0 h (dihydroartemisinin/piperaquine, P = 0.140). Adequate clinical and parasitological response at Day 42 was 100% in both arms. By RT-qPCR, 36% (19/53) patients remained positive until Day 7. No recurrences were detected. kelch13 artemisinin resistance mutations were found in 87% (39/45) of isolates and 50% (20/40) were KEL1/C580Y. The piperaquine resistance marker plasmepsin-2 was duplicated in 10.4% (5/48). Isolates from Day 3-positive patients (n = 18) had higher ex vivo survival rates to artemisinin compounds (P < 0.048) and prevalence of kelch13 mutations (P = 0.005) than Day 3-negative patients (n = 5). The WHO definition of artemisinin resistance was fulfilled in 60% (24/40) of cases. CONCLUSIONS: Although dihydroartemisinin/piperaquine remained effective to treat P. falciparum, the high Day 3 positivity rate and prevalence of KEL1 strains calls for continuous monitoring of dihydroartemisinin/piperaquine efficacy in Central Vietnam.

Imported malaria and artemisinin-based combination therapy failure in travellers returning to Belgium: A retrospective study.

BACKGROUND: Malaria (Plasmodium spp) remains a top cause of travel-associated morbidity among European residents. Here, we describe recent trends of imported malaria to Belgium and characterize the first cases of P.falciparum failure to artemisinin-based combination therapy (ACT). METHODS: National surveillance data and registers from national reference laboratory were used to investigate malaria cases and ACT failures in the past 20 years. Recurrent infections were confirmed by pfmsp genotyping and polymorphisms in drug resistance-associated genes pfk13, pfcrt, pfmdr1, pfpm2, pfap2mu and pfubp1 were determined by sequencing or quantitative PCR. RESULTS: Annual malaria cases steadily increased in the last decade, reaching 428 in 2017 (all species). An estimated 15% of P.falciparum cases were severe. Between 2014 and 2017, 727 P.falciparum cases were reported and six non-immune travellers presented late recurrence. Five had hyperparasitaemia and/or signs of severe malaria at initial consultation. No mutations in ACT drug resistance markers were detected, although pfcrt-pfmdr1 haplotypes associated with lumefantrine tolerance were common. CONCLUSIONS: The upward trend in imported malaria, the substantial proportion of severe cases and the emergence of ACT failures are sources of concern, although late failures were infrequent. Genetic analysis did not support parasitological resistance to ACT, suggesting prospective pharmacokinetic studies should assess adequacy of partner drug dosage and duration of treatment in non-immune populations.

A pilot evaluation of alternative procedures to simplify LAMP-based malaria diagnosis in field conditions.

Highly-sensitive and field-friendly diagnostic tools are needed for accurate detection of low-density malaria infections. Although loop-mediated isothermal amplification (LAMP) fulfills these conditions, operational challenges are still encountered during pilot population screenings in remote settings when employing Loopamp™ MALARIA Pan/Pf detection kit (Eiken Chemical Co.). This study evaluates different procedures for the simplification of sample preparation and result reading steps of current LAMP protocols. The reference 'Boil & Spin' (B&S) pre-amplification procedure was compared to three alternative methods, along with a colorimetric staining protocol based on malachite green. Results suggested that the B&S supernatant transference step may be omitted without an impact on test performance, even when colorimetry was incorporated to facilitate results visualization. Procedures skipping centrifugation and/or heat-incubation were proved to be compatible with LAMP-based malaria DNA detection, but resulted in a low-to-moderate decrease in sensitivity and ambiguous result interpretation for the most straightforward protocol. Nevertheless, all simplified LAMP methods could still reach lower limits of detection than the currently used tools for malaria mass-screening (i.e. microscopy and rapid tests), indicating that these alternative strategies may deserve further consideration. This evaluation, therefore, demonstrates the feasibility of skipping some of the main procedural bottlenecks of LAMP-malaria protocols, a much-needed achievement to make point-of-care implementation of molecular diagnostics a reality.

Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes.

During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection.

Malaria incidence and prevalence during the first year of life in Nanoro, Burkina Faso: a birth-cohort study.

BACKGROUND: Infants are thought to be protected against malaria during the first months of life mainly due to passage of maternal antibodies. However, in high transmission settings, malaria in early infancy is not uncommon and susceptibility to the infections varies between individuals. This study aimed to determine malaria morbidity and infection during early childhood in rural Burkina Faso. METHODS: Malariometric indices were determined over 1-year follow-up in a birth cohort of 734 infants living in Nanoro health district. Clinical malaria episodes were determined by passive case detection at peripheral health centres while asymptomatic malaria infections were identified during  4 cross-sectional surveys at 3, 6, 9 and 12 months of age. Plasmodium falciparum infections were detected by rapid diagnostic test and/or light microscopy (LM) and quantitative PCR (qPCR). RESULTS: In total, 717 clinical episodes were diagnosed by qPCR over 8335.18 person-months at risk. The overall malaria incidence was 1.03 per child-year and increased from 0.27 per child-year at 0-3 months of age to 1.92 per child-year at 9-12 months of age. Some 59% of children experienced at least one clinical episode with a median survival time estimated at 9.9 months, while 20% of infants experienced the first episode before 6 months of age. The majority of the clinical episodes were attributable to microscopic parasitaemia (84.2%), and there was a positive correlation between parasite density and age (Spearman's rho = 0.30; P < 0.0001). Prevalence of asymptomatic infections was similar at 3, 6 and 9 months of age (17.7-20.1%) and nearly 1.6 times higher at 12 months (31.3%). Importantly, gametocyte prevalence among the LM-positive study population was 6.7%, but increased to 10% among asymptomatic infections. In addition, 46% of asymptomatic infections were only detected by qPCR suggesting that infants below 1 year are a potential reservoir for sustaining malaria transmission. Both symptomatic and asymptomatic infections showed marked seasonal distribution with the highest transmission period (July to December) accounting for about 89 and 77% of those infections, respectively. CONCLUSIONS: These findings indicate high and marked age and seasonal-dependency of malaria infections and disease during the first year of life in Nanoro, calling for intensified efforts to control malaria in rural Burkina Faso.

Uptake of intermittent preventive treatment and pregnancy outcomes: health facilities and community surveys in Chókwè district, southern Mozambique.

BACKGROUND: Malaria in pregnancy leads to serious adverse effects on the mother and the child and accounts for 75,000-200,000 infant deaths every year. Currently, the World Health Organization recommends intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) at each scheduled antenatal care (ANC) visit. This study aimed to assess IPTp-SP coverage in mothers delivering in health facilities and at the community. In addition, factors associated with low IPTp-SP uptake and malaria adverse outcomes in pregnancy were investigated. METHODS: A community and a health facility-based surveys were conducted in mothers delivering in Chókwè district, southern Mozambique. Social-demographic data, malaria prevention practices and obstetric history were recorded through self-report and antenatal records. For women delivering at health facilities, a clinical examination of mother and child was performed, and malaria infection at delivery was determined by rapid diagnostic test, microscopy, quantitative PCR and placental histology. RESULTS: Of 1141 participants, 46.6, 30.2, 13.5 and 9.6% reported taking ≥ 3, two, one and none SP doses, respectively. Low IPTp uptake (< 3 doses) was associated with non-institutional deliveries (AOR = 2.9, P < 0.001), first ANC visit after week 28 (AOR = 5.4, P < 0.001), low awareness of IPTp-SP (AOR = 1.6, P < 0.002) and having no or only primary education (AOR = 1.3, P = 0.041). The overall prevalence of maternal malaria (peripheral and/or placental) was 16.8% and was higher among women from rural areas compared to those from urban areas (AOR = 1.9, P < 0.001). Younger age (< 20 years; AOR = 1.6, P = 0.042) and living in rural areas (AOR = 1.9, P < 0.001) were predictors of maternal malaria at delivery. Being primigravidae (AOR = 2.2, P = 0.023) and preterm delivery (AOR = 2.6, P < 0.001) predicted low birth weight while younger age was also associated with premature delivery (AOR = 1.4, P = 0.031). CONCLUSION: The coverage for two and ≥ 3 doses of IPTp-SP is moderately higher than estimates from routine health facility records in Gaza province in 2015. However, this is still far below the national target of 80% for ≥ 3 doses. Ongoing campaigns aiming to increase the use of malaria prevention strategies during pregnancy should particularly target rural populations, increasing IPTp-SP knowledge, stimulate early visits to ANC, improve access to health services and the quality of the service provided.

Uptake of intermittent preventive treatment and pregnancy outcomes: health facilities and community surveys in Chókwè district, southern Mozambique

Malaria in pregnancy leads to serious adverse effects on the mother and the child and accounts for 75,000-200,000 infant deaths every year. Currently, the World Health Organization recommends intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) at each scheduled antenatal care (ANC) visit. This study aimed to assess IPTp-SP coverage in mothers delivering in health facilities and at the community. In addition, factors associated with low IPTp-SP uptake and malaria adverse outcomes in pregnancy were investigated. Methods: A community and a health facility-based surveys were conducted in mothers delivering in Chókwè district, southern Mozambique. Social-demographic data, malaria prevention practices and obstetric history were recorded through self-report and antenatal records. For women delivering at health facilities, a clinical examination of mother and child was performed, and malaria infection at delivery was determined by rapid diagnostic test, microscopy, quantitative PCR and placental histology. Results: Of 1141 participants, 46.6, 30.2, 13.5 and 9.6% reported taking ≥ 3, two, one and none SP doses, respectively. Low IPTp uptake (< 3 doses) was associated with non-institutional deliveries (AOR = 2.9, P < 0.001), first ANC visit after week 28 (AOR = 5.4, P < 0.001), low awareness of IPTp-SP (AOR = 1.6, P < 0.002) and having no or only primary education (AOR = 1.3, P = 0.041). The overall prevalence of maternal malaria (peripheral and/or placental) was 16.8% and was higher among women from rural areas compared to those from urban areas (AOR = 1.9, P < 0.001). Younger age (< 20 years; AOR = 1.6, P = 0.042) and living in rural areas (AOR = 1.9, P < 0.001) were predictors of maternal malaria at delivery. Being primigravidae (AOR = 2.2, P = 0.023) and preterm delivery (AOR = 2.6, P < 0.001) predicted low birth weight while younger age was also associated with premature delivery (AOR = 1.4, P = 0.031). Conclusion: The coverage for two and ≥ 3 doses of IPTp-SP is moderately higher than estimates from routine health facility records in Gaza province in 2015. However, this is still far below the national target of 80% for ≥ 3 doses. Ongoing campaigns aiming to increase the use of malaria prevention strategies during pregnancy should particularly target rural populations, increasing IPTp-SP knowledge, stimulate early visits to ANC, improve access to health services and the quality of the service provided.

Expression of the Plasmodium falciparum Clonally Variant clag3 Genes in Human Infections.

Background: Many genes of the malaria parasite Plasmodium falciparum show clonally variant expression regulated at the epigenetic level. These genes participate in fundamental host-parasite interactions and contribute to adaptive processes. However, little is known about their expression patterns during human infections. A peculiar case of clonally variant genes are the 2 nearly identical clag3 genes, clag3.1 and clag3.2, which mediate nutrient uptake and are linked to resistance to some toxic compounds. Methods: We developed a procedure to characterize the expression of clag3 genes in naturally infected patients and in experimentally infected human volunteers. Results: We provide the first description of clag3 expression during human infections, which revealed mutually exclusive expression and identified the gene predominantly expressed. Adaptation to culture conditions or selection with a toxic compound resulted in isolate-dependent changes in clag3 expression. We also found that clag3 expression patterns were reset during transmission stages. Conclusions: Different environment conditions select for parasites with different clag3 expression patterns, implying functional differences between the proteins encoded. The epigenetic memory is likely erased before parasites start infection of a new human host. Altogether, our findings support the idea that clonally variant genes facilitate the adaptation of parasite populations to changing conditions through bet-hedging strategies.