Assessing the histidine-rich protein 2/3 gene deletion in Plasmodium falciparum isolates from Burkina Faso.

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.

Plasmodium falciparum drug resistance-associated mutations in isolates from children living in endemic areas of Burkina Faso.

BACKGROUND: Artemisinin-based combinations therapy (ACT) is the current frontline curative therapy for uncomplicated malaria in Burkina Faso. Sulfadoxine-pyrimethamine (SP) is used for the preventive treatment of pregnant women (IPTp), while SP plus amodiaquine (SP-AQ) is recommended for children under five in seasonal malaria chemoprevention (SMC). This study aimed to assess the proportions of mutations in the P. falciparum multidrug-resistance 1 (Pfmdr1), P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum dihydrofolate reductase (pfdhfr), and P. falciparum dihydropteroate synthase (pfdhps), genes from isolates collected during household surveys in Burkina Faso. METHODS: Dried blood spots from Plasmodium falciparum-positive cases at three sites (Orodara, Gaoua, and Banfora) collected during the peak of transmission were analysed for mutations in Pfcrt (codons 72-76, 93, 97, 145, 218, 343, 350 and 353), Pfmdr-1 (codons 86, 184, 1034, 1042 and 1246) dhfr (codons 51, 59, 108, 164) and dhps (at codons 431, 436, 437, 540, 581, 613) genes using deep sequencing of multiplexed Polymerase chaine reaction (PCR) amplicons. RESULTS: Of the 377 samples analysed, 346 (91.7%), 369 (97.9%), 368 (97.6%), and 374 (99.2%) were successfully sequenced for Pfcrt, Pfmdr-1, dhfr, and dhps, respectively. Most of the samples had a Pfcrt wild-type allele (89.3%). The 76T mutation was below 10%. The most frequent Pfmdr-1 mutation was detected at codon 184 (Y > F, 30.9%). The single mutant genotype (NFSND) predominated (66.7%), followed by the wild-type genotype (NYSND, 30.4%). The highest dhfr mutations were observed at codon 59R (69.8%), followed by codons 51I (66.6%) and 108 N (14.7%). The double mutant genotype (ACIRSI) predominated (52.4%). For mutation in the dhps gene, the highest frequency was observed at codon 437 K (89.3%), followed by codons 436 A (61.2%), and 613 S (14.4%). The double mutant genotype (IAKKAA) and the single mutant genotype (ISKKAA) were predominant (37.7% and 37.2%, respectively). The most frequent dhfr/dhps haplotypes were the triple mutant ACIRSI/IAKKAA (23%), the wild-type ACNCSI/ISKKAA (19%) and the double mutant ACIRSI/ISKKAA (14%). A septuple mutant ACIRNI/VAKKGA was observed in 2 isolates from Gaoua (0.5%). CONCLUSION: The efficacy of ACT partner drugs and drugs used in IPTp and SMC does not appear to be affected by the low proportion of highly resistant mutants observed in this study. Continued monitoring, including molecular surveillance, is critical for decision-making on effective treatment policy in Burkina Faso.

Using antenatal care as a platform for malaria surveillance data collection: study protocol.

BACKGROUND: While many malaria-endemic countries have health management information systems that can measure and report malaria trends in a timely manner, these routine systems have limitations. Periodic community cross-sectional household surveys are used to estimate malaria prevalence and intervention coverage but lack geographic granularity and are resource intensive. Incorporating malaria testing for all women at their first antenatal care (ANC) visit (i.e., ANC1) could provide a more timely and granular source of data for monitoring trends in malaria burden and intervention coverage. This article describes a protocol designed to assess if ANC-based surveillance could be a pragmatic tool to monitor malaria. METHODS: This is an observational, cross-sectional study conducted in Benin, Burkina Faso, Mozambique, Nigeria, Tanzania, and Zambia. Pregnant women attending ANC1 in selected health facilities will be tested for malaria infection by rapid diagnostic test and administered a brief questionnaire to capture key indicators of malaria control intervention coverage and care-seeking behaviour. In each location, contemporaneous cross-sectional household surveys will be leveraged to assess correlations between estimates obtained using each method, and the use of ANC data as a tool to track trends in malaria burden and intervention coverage will be validated. RESULTS: This study will assess malaria prevalence at ANC1 aggregated at health facility and district levels, and by gravidity relative to current pregnancy (i.e., gravida 1, gravida 2, and gravida 3 +). ANC1 malaria prevalence will be presented as monthly trends. Additionally, correlation between ANC1 and household survey-derived estimates of malaria prevalence, bed net ownership and use, and care-seeking will be assessed. CONCLUSION: ANC1-based surveillance has the potential to provide a cost-effective, localized measure of malaria prevalence that is representative of the general population and useful for tracking monthly changes in parasite prevalence, as well as providing population-representative estimates of intervention coverage and care-seeking behavior. This study will evaluate the representativeness of these measures and collect information on operational feasibility, usefulness for programmatic decision-making, and potential for scale-up of malaria ANC1 surveillance.

Randomized, open-label, phase 2a study to evaluate the contribution of artefenomel to the clinical and parasiticidal activity of artefenomel plus ferroquine in African patients with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The contribution of artefenomel to the clinical and parasiticidal activity of ferroquine and artefenomel in combination in uncomplicated Plasmodium falciparum malaria was investigated. METHODS: This Phase 2a, randomized, open-label, parallel-group study was conducted from 11th September 2018 to 6th November 2019 across seven centres in Benin, Burkina Faso, Gabon, Kenya, and Uganda. Patients aged ≥ 14-69 years with microscopically confirmed infection (≥ 3000 to ≤ 50,000 parasites/µL blood) were randomized 1:1:1:1 to 400 mg ferroquine, or 400 mg ferroquine plus artefenomel 300, 600, or 1000 mg, administered as a single oral dose. The primary efficacy analysis was a logistic regression evaluating the contribution of artefenomel exposure to Day 28 PCR-adjusted adequate clinical and parasitological response (ACPR). Safety was also evaluated. RESULTS: The randomized population included 140 patients. For the primary analysis in the pharmacokinetic/pharmacodynamic efficacy population (N = 121), the contribution of artefenomel AUC0-∞ to Day 28 PCR-adjusted ACPR was not demonstrated when accounting for ferroquine AUC0-d28, baseline parasitaemia, and other model covariates: odds ratio 1.1 (95% CI 0.98, 1.2; P = 0.245). In the per-protocol population, Day 28 PCR-adjusted ACPR was 80.8% (21/26; 95% CI 60.6, 93.4) with ferroquine alone and 90.3% (28/31; 95% CI 74.2, 98.0), 90.9% (30/33; 95% CI 75.7, 98.1) and 87.1% (27/31; 95% CI 70.2, 96.4) with 300, 600, and 1000 mg artefenomel, respectively. Median time to parasite clearance (Kaplan-Meier) was 56.1 h with ferroquine, more rapid with artefenomel, but similar for all doses (30.0 h). There were no deaths. Adverse events (AEs) of any cause occurred in 51.4% (18/35) of patients with ferroquine 400 mg alone, and 58.3% (21/36), 66.7% (24/36), and 72.7% (24/33) with 300, 600, and 1000 mg artefenomel, respectively. All AEs were of mild-to-moderate severity, and consistent with the known profiles of the compounds. Vomiting was the most reported AE. There were no cases of QTcF prolongation ≥ 500 ms or > 60 ms from baseline. CONCLUSION: The contribution of artefenomel exposure to the clinical and parasitological activity of ferroquine/artefenomel could not be demonstrated in this study. Parasite clearance was faster with ferroquine/artefenomel versus ferroquine alone. All treatments were well tolerated. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03660839 (7 September, 2018).

Effects of the free healthcare policy on health services' usage by children under 5 years in Burkina Faso: a controlled interrupted time-series analysis.

OBJECTIVES: This study aimed to analyse, at national level, the effects of the free healthcare policy for children on the use of health services by children under five in Burkina Faso. We hypothesised that this policy has led to an immediate and sustained increase in the use of health services for these children in the country. SETTING: We conducted a controlled interrupted time series. Monthly data at district level, spanning from January 2013 to December 2018 and corresponding to 72 monthly data points (39 before and 33 after), were extracted from the Burkina Faso National Health Information System. The analysed dataset included data from all the 70 health districts of the country. PARTICIPANTS: The study consisted of aggregated data from children under five as the target for the policy with children aged between 5 and 14 years old as control group. INTERVENTION: The intervention was the introduction of the free healthcare policy for women and children under 5 years from April 2016. OUTCOME: The primary outcome was the monthly mean rate of health services visits by children. RESULTS: Among the children under five, the rate of visits increased of 57% (incidence rate ratio (IRR)=1.57; 95% CI 1.2 to 2.0) in the month immediately following the launching of the free healthcare policy. An increase in the rate of health facility visits of 1% (IRR=1.01; 95% CI 1.0 to 1.1) per month was also noted during postintervention. Compared with the control group, we observed an increase in the rate of visits of 2.5% (IRR=1.025; 95% CI 1.023 to 1.026) per month. CONCLUSION: Findings suggest that the free healthcare policy increased the use of health facilities for care in Burkina Faso immediately after the implementation of the policy with a small increase in the rate overtime. Strategies to maintain the policy effect over time are necessary.

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa.

BACKGROUND: Vector control tools have contributed significantly to a reduction in malaria burden since 2000, primarily through insecticidal-treated bed nets (ITNs) and indoor residual spraying. In the face of increasing insecticide resistance in key malaria vector species, global progress in malaria control has stalled. Innovative tools, such as dual active ingredient (dual-AI) ITNs that are effective at killing insecticide-resistant mosquitoes have recently been introduced. However, large-scale uptake has been slow for several reasons, including higher costs and limited evidence on their incremental effectiveness and cost-effectiveness. The present report describes the design of several observational studies aimed to determine the effectiveness and cost-effectiveness of dual-AI ITNs, compared to standard pyrethroid-only ITNs, at reducing malaria transmission across a variety of transmission settings. METHODS: Observational pilot studies are ongoing in Burkina Faso, Mozambique, Nigeria, and Rwanda, leveraging dual-AI ITN rollouts nested within the 2019 and 2020 mass distribution campaigns in each country. Enhanced surveillance occurring in select study districts include annual cross-sectional surveys during peak transmission seasons, monthly entomological surveillance, passive case detection using routine health facility surveillance systems, and studies on human behaviour and ITN use patterns. Data will compare changes in malaria transmission and disease burden in districts receiving dual-AI ITNs to similar districts receiving standard pyrethroid-only ITNs over three years. The costs of net distribution will be calculated using the provider perspective including financial and economic costs, and a cost-effectiveness analysis will assess incremental cost-effectiveness ratios for Interceptor® G2, Royal Guard®, and piperonyl butoxide ITNs in comparison to standard pyrethroid-only ITNs, based on incidence rate ratios calculated from routine data. CONCLUSIONS: Evidence of the effectiveness and cost-effectiveness of the dual-AI ITNs from these pilot studies will complement evidence from two contemporary cluster randomized control trials, one in Benin and one in Tanzania, to provide key information to malaria control programmes, policymakers, and donors to help guide decision-making and planning for local malaria control and elimination strategies. Understanding the breadth of contexts where these dual-AI ITNs are most effective and collecting robust information on factors influencing comparative effectiveness could improve uptake and availability and help maximize their impact.

Plasmodium falciparum Gametocyte Density and Infectivity in Peripheral Blood and Skin Tissue of Naturally Infected Parasite Carriers in Burkina Faso.

BACKGROUND: Plasmodium falciparum transmission depends on mature gametocytes that can be ingested by mosquitoes taking a blood meal on human skin. Although gametocyte skin sequestration has long been hypothesized as important contributor to efficient malaria transmission, this has never been formally tested. METHODS: In naturally infected gametocyte carriers from Burkina Faso, we assessed infectivity to mosquitoes by direct skin feeding and membrane feeding. We directly quantified male and female gametocytes and asexual parasites in finger-prick and venous blood samples, skin biopsy samples, and in of mosquitoes that fed on venous blood or directly on skin. Gametocytes were visualized in skin tissue with confocal microscopy. RESULTS: Although more mosquitoes became infected when feeding directly on skin then when feeding on venous blood (odds ratio, 2.01; 95% confidence interval, 1.21-3.33; P = .007), concentrations of gametocytes were not higher in the subdermal skin vasculature than in other blood compartments; only sparse gametocytes were observed in skin tissue. DISCUSSION: Our data strongly suggest that there is no significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes and can be used to target and monitor malaria elimination initiatives.

Plasmodium falciparum malaria in children aged 0-2 years: the role of foetal haemoglobin and maternal antibodies to two asexual malaria vaccine candidates (MSP3 and GLURP).

BACKGROUND: Children below six months are reported to be less susceptible to clinical malaria. Maternally derived antibodies and foetal haemoglobin are important putative protective factors. We examined antibodies to Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate-rich protein (GLURP), in children in their first two years of life in Burkina Faso and their risk of malaria. METHODS: A cohort of 140 infants aged between four and six weeks was recruited in a stable transmission area of south-western Burkina Faso and monitored for 24 months by active and passive surveillance. Malaria infections were detected by examining blood smears using light microscopy. Enzyme-linked immunosorbent assay was used to quantify total Immunoglobulin G to Plasmodium falciparum antigens MSP3 and two regions of GLURP (R0 and R2) on blood samples collected at baseline, three, six, nine, 12, 18 and 24 months. Foetal haemoglobin and variant haemoglobin fractions were measured at the baseline visit using high pressure liquid chromatography. RESULTS: A total of 79.6% of children experienced one or more episodes of febrile malaria during monitoring. Antibody titres to MSP3 were prospectively associated with an increased risk of malaria while antibody responses to GLURP (R0 and R2) did not alter the risk. Antibody titres to MSP3 were higher among children in areas of high malaria risk. Foetal haemoglobin was associated with delayed first episode of febrile malaria and haemoglobin CC type was associated with reduced incidence of febrile malaria. CONCLUSIONS: We did not find any evidence of association between titres of antibodies to MSP3, GLURP-R0 or GLURP-R2 as measured by enzyme-linked immunosorbent assay and early protection against malaria, although anti-MSP3 antibody titres may reflect increased exposure to malaria and therefore greater risk. Foetal haemoglobin was associated with protection against febrile malaria despite the study limitations and its role is therefore worthy further investigation.