Plasma from older children in Malawi inhibits Plasmodium falciparum binding in 3D brain microvessels.

A hallmark of cerebral malaria is sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation. Antibodies contribute to malaria immunity, but it remains unclear whether functional antibodies targeting parasite-expressed ligand can block cytoadhesion in the brain. Here, we screened the plasma of older children and young adults in Malawi to characterize the antibody response against the P. falciparum-IE surface and used a bioengineered 3D human brain microvessel model incorporating variable flow dynamics to measure adhesion blocking responses. We found a strong correlation between surface antibody reactivity by flow cytometry and reduced P. falciparum-IE binding in 3D microvessels. Moreover, there was a threshold of surface antibody reactivity necessary to achieve robust inhibitory activity. Our findings provide evidence of the acquisition of adhesion blocking antibodies against cerebral binding variants in people exposed to stable P. falciparum transmission and suggest the quality of the inhibitory response can be influenced by flow dynamics.

Factors associated with malaria vaccine uptake in Nsanje district, Malawi.

BACKGROUND: Malaria remains a significant global health burden affecting millions of people, children under 5 years and pregnant women being most vulnerable. In 2019, the World Health Organization (WHO) endorsed the introduction of RTS,S/AS01 malaria vaccine as Phase IV implementation evaluation in three countries: Malawi, Kenya and Ghana. Acceptability and factors influencing vaccination coverage in implementing areas is relatively unknown. In Malawi, only 60% of children were fully immunized with malaria vaccine in Nsanje district in 2021, which is below 80% WHO target. This study aimed at exploring factors influencing uptake of malaria vaccine and identify approaches to increase vaccination. METHODS: In a cross-sectional study conducted in April-May, 2023, 410 mothers/caregivers with children aged 24-36 months were selected by stratified random sampling and interviewed using a structured questionnaire. Vaccination data was collected from health passports, for those without health passports, data was collected using recall history. Regression analyses were used to test association between independent variables and full uptake of malaria vaccine. RESULTS: Uptake of malaria vaccine was 90.5% for dose 1, but reduced to 87.6%, 69.5% and 41.2% for dose 2, 3, and 4 respectively. Children of caregivers with secondary or upper education and those who attended antenatal clinic four times or more had increased odds of full uptake of malaria vaccine [OR: 2.43, 95%CI 1.08-6.51 and OR: 1.89, 95%CI 1.18-3.02], respectively. Children who ever suffered side-effects following immunization and those who travelled long distances to reach the vaccination centre had reduced odds of full uptake of malaria vaccine [OR: 0.35, 95%CI 0.06-0.25 and OR: 0.30, 95%CI 0.03-0.39] respectively. Only 17% (n = 65) of mothers/caregivers knew the correct schedule for vaccination and 38.5% (n = 158) knew the correct number of doses a child was to receive. CONCLUSION: Only RTS,S dose 1 and 2 uptake met WHO coverage targets. Mothers/caregivers had low level of information regarding malaria vaccine, especially on numbers of doses to be received and dosing schedule. The primary modifiable factor influencing vaccine uptake was mother/caregiver knowledge about the vaccine. Thus, to increase the uptake Nsanje District Health Directorate should strengthen communities' education about malaria vaccine. Programmes to strengthen mother/caregiver knowledge should be included in scale-up of the vaccine in Malawi and across sub-Saharan Africa.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Barriers to accessing malaria treatment amongst school-age children in rural Malawi.

BACKGROUND: Over the last two decades, many countries have moved from malaria control toward malaria elimination. However, some sub-Saharan African countries, like Malawi, have recently seen a reversal in malaria control progress with reported increases in confirmed malaria cases. This may be the result of inadequate access to effective malaria control interventions by key population groups that perpetuate transmission. This study aimed to assess the barriers to accessing malaria treatment among school-aged children (SAC) in Malawi. METHODS: A qualitative study was conducted between September and October 2020, where data were gathered in rural Malawi using free-listing interviews, key-informant interviews, semi-structured interviews and focus group discussions. Purposively sampled participants included SAC, parents of SAC, health workers and key stakeholders at community and district levels. Interviews were digitally recorded and transcribed verbatim. Data were organized using NVivo 12 software and analysed using the thematic method. RESULTS: The study recruited 252 participants, with 156 being SAC, equally divided between boys and girls. Health system barriers to malaria treatment included long waiting hours and queues at clinics, frequent stock-outs of medical supplies, and travel time to the facility. Provider barriers included negative attitude and limited service hours. Individual and cultural barriers included fear of malaria tests and beliefs associating witchcraft as the best treatment for malaria. In addition, COVID-19-related barriers included the inability to follow preventive measures, a shift in focus from malaria to COVID-19, and fear of contracting COVID-19 and/or being tested for COVID-19 at the facility. CONCLUSIONS: This study shows most of the barriers to accessing malaria treatment among SAC are similar to those experienced by other population groups. Furthermore, COVID-19 adversely affected SAC's access to treatment. Interventions that support SAC access to prompt diagnosis and treatment are urgently needed to improve the effective control of malaria.

Genotyping of Anopheles mosquito blood meals reveals nonrandom human host selection: implications for human-to-mosquito Plasmodium falciparum transmission.

BACKGROUND: Control of malaria parasite transmission can be enhanced by understanding which human demographic groups serve as the infectious reservoirs. Because vector biting can be heterogeneous, some infected individuals may contribute more to human-to-mosquito transmission than others. Infection prevalence peaks in school-age children, but it is not known how often they are fed upon. Genotypic profiling of human blood permits identification of individual humans who were bitten. The present investigation used this method to estimate which human demographic groups were most responsible for transmitting malaria parasites to Anopheles mosquitoes. It was hypothesized that school-age children contribute more than other demographic groups to human-to-mosquito malaria transmission. METHODS: In a region of moderate-to-high malaria incidence in southeastern Malawi, randomly selected households were surveyed to collect human demographic information and blood samples. Blood-fed, female Anopheles mosquitoes were sampled indoors from the same houses. Genomic DNA from human blood samples and mosquito blood meals of human origin was genotyped using 24 microsatellite loci. The resultant genotypes were matched to identify which individual humans were sources of blood meals. In addition, Plasmodium falciparum DNA in mosquito abdomens was detected with polymerase chain reaction. The combined results were used to identify which humans were most frequently bitten, and the P. falciparum infection prevalence in mosquitoes that resulted from these blood meals. RESULTS: Anopheles females selected human hosts non-randomly and fed on more than one human in 9% of the blood meals. Few humans contributed most of the blood meals to the Anopheles vector population. Children ≤ 5 years old were under-represented in mosquito blood meals while older males (31-75 years old) were over-represented. However, the largest number of malaria-infected blood meals was from school age children (6-15 years old). CONCLUSIONS: The results support the hypothesis that humans aged 6-15 years are the most important demographic group contributing to the transmission of P. falciparum to the Anopheles mosquito vectors. This conclusion suggests that malaria control and prevention programmes should enhance efforts targeting school-age children and males.

School-Based Malaria Screening and Treatment Reduces Plasmodium falciparum Infection and Anemia Prevalence in Two Transmission Settings in Malawi.

BACKGROUND: In areas highly endemic for malaria, Plasmodium falciparum infection prevalence peaks in school-age children, adversely affecting health and education. School-based intermittent preventive treatment reduces this burden but concerns about cost and widespread use of antimalarial drugs limit enthusiasm for this approach. School-based screening and treatment is an attractive alternative. In a prospective cohort study, we evaluated the impact of school-based screening and treatment on the prevalence of P. falciparum infection and anemia in 2 transmission settings. METHODS: We screened 704 students in 4 Malawian primary schools for P. falciparum infection using rapid diagnostic tests (RDTs), and treated students who tested positive with artemether-lumefantrine. We determined P. falciparum infection by microscopy and quantitative polymerase chain reaction (qPCR), and hemoglobin concentrations over 6 weeks in all students. RESULTS: Prevalence of infection by RDT screening was 37% (9%-64% among schools). An additional 9% of students had infections detected by qPCR. Following the intervention, significant reductions in infections were detected by microscopy (adjusted relative reduction [aRR], 48.8%; P < .0001) and qPCR (aRR, 24.5%; P < .0001), and in anemia prevalence (aRR, 30.8%; P = .003). Intervention impact was reduced by infections not detected by RDT and new infections following treatment. CONCLUSIONS: School-based screening and treatment reduced P. falciparum infection and anemia. This approach could be enhanced by repeating screening, using more-sensitive screening tests, and providing longer-acting drugs. CLINICAL TRIALS REGISTRATION: NCT04858087.

Pregnancy outcomes and risk of placental malaria after artemisinin-based and quinine-based treatment for uncomplicated falciparum malaria in pregnancy: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria in pregnancy, including asymptomatic infection, has a detrimental impact on foetal development. Individual patient data (IPD) meta-analysis was conducted to compare the association between antimalarial treatments and adverse pregnancy outcomes, including placental malaria, accompanied with the gestational age at diagnosis of uncomplicated falciparum malaria infection. METHODS: A systematic review and one-stage IPD meta-analysis of studies assessing the efficacy of artemisinin-based and quinine-based treatments for patent microscopic uncomplicated falciparum malaria infection (hereinafter uncomplicated falciparum malaria) in pregnancy was conducted. The risks of stillbirth (pregnancy loss at ≥ 28.0 weeks of gestation), moderate to late preterm birth (PTB, live birth between 32.0 and < 37.0 weeks), small for gestational age (SGA, birthweight of < 10th percentile), and placental malaria (defined as deposition of malaria pigment in the placenta with or without parasites) after different treatments of uncomplicated falciparum malaria were assessed by mixed-effects logistic regression, using artemether-lumefantrine, the most used antimalarial, as the reference standard. Registration PROSPERO: CRD42018104013. RESULTS: Of the 22 eligible studies (n = 5015), IPD from16 studies were shared, representing 95.0% (n = 4765) of the women enrolled in literature. Malaria treatment in this pooled analysis mostly occurred in the second (68.4%, 3064/4501) or third trimester (31.6%, 1421/4501), with gestational age confirmed by ultrasound in 91.5% (4120/4503). Quinine (n = 184) and five commonly used artemisinin-based combination therapies (ACTs) were included: artemether-lumefantrine (n = 1087), artesunate-amodiaquine (n = 775), artesunate-mefloquine (n = 965), and dihydroartemisinin-piperaquine (n = 837). The overall pooled proportion of stillbirth was 1.1% (84/4361), PTB 10.0% (619/4131), SGA 32.3% (1007/3707), and placental malaria 80.1% (2543/3035), and there were no significant differences of considered outcomes by ACT. Higher parasitaemia before treatment was associated with a higher risk of SGA (adjusted odds ratio [aOR] 1.14 per 10-fold increase, 95% confidence interval [CI] 1.03 to 1.26, p = 0.009) and deposition of malaria pigment in the placenta (aOR 1.67 per 10-fold increase, 95% CI 1.42 to 1.96, p < 0.001). CONCLUSIONS: The risks of stillbirth, PTB, SGA, and placental malaria were not different between the commonly used ACTs. The risk of SGA was high among pregnant women infected with falciparum malaria despite treatment with highly effective drugs. Reduction of malaria-associated adverse birth outcomes requires effective prevention in pregnant women.

Submicroscopic malaria infection is not associated with fever in cross-sectional studies in Malawi.

BACKGROUND: Submicroscopic Plasmodium falciparum infections are widespread in many areas. However, the contribution of these infections to symptomatic malaria is not well understood. This study evaluated whether participants with submicroscopic P. falciparum infections have higher prevalence of fever than uninfected participants in southern Malawi. METHODS: A total of 16,650 children and adults were enrolled in the course of six cross-sectional surveys during the dry season (October-November) and after the rainy season (April-May) between 2012 and 2014 in three districts in southern Malawi. Demographic and socioeconomic data were collected in conjunction with data on clinical histories, use of malaria preventive measures, and anti-malarial medication taken within 2 weeks of the survey. Axillary temperatures were measured, and blood samples were collected for P. falciparum detection by microscopy and PCR. Participants without malaria parasites detected on microscopy but with a positive PCR for P. falciparum were defined as having submicroscopic infection. Fever was defined as having any one of: reported fever in the past 2 weeks, reported fever in the past 48 h, or a temperature of ≥ 37.5 °C measured at the time of interview. RESULTS: Submicroscopic P. falciparum infections and fever were both detected in 9% of the study population. In the final analysis adjusted for clustering within household and enumeration area, having submicroscopic P. falciparum infection was associated with reduced odds of fever in the dry season (odds ratio = 0.52; 95% CI 0.33-0.82); the association in the rainy season did not achieve statistical significance (odds ratio = 1.20; 95% CI 0.91-1.59). The association between submicroscopic infection and fever was consistent across all age groups. When the definition of fever was limited to temperature of ≥ 37.5 °C measured at the time of interview, the association was not statistically significant in either the rainy or dry season. CONCLUSIONS: In this series of cross-sectional studies in southern Malawi, submicroscopic P. falciparum infection was not associated with increased risk of fever. Submicroscopic detection of the malaria parasite is important in efforts to decrease transmission but is not essential for the clinical recognition of malaria disease.

Simulation models predict that school-age children are responsible for most human-to-mosquito Plasmodium falciparum transmission in southern Malawi.

BACKGROUND: Malaria persists in some high-transmission areas despite extensive control efforts. Progress toward elimination may require effective targeting of specific human populations that act as key transmission reservoirs. METHODS: Parameterized using molecular-based Plasmodium falciparum infection data from cross-sectional community studies in southern Malawi, a simulation model was developed to predict the proportions of human-to-mosquito transmission arising from (a) children under 5 years old (U5s), (b) school-age children (SAC, 5-15 years), (c) young adults (16-30 years), and (d) adults > 30 years. The model incorporates mosquito biting heterogeneity and differential infectivity (i.e. probability that a blood-fed mosquito develops oocysts) by age and gametocyte density. RESULTS: The model predicted that SAC were responsible for more than 60% of new mosquito infections in both dry and rainy seasons, even though they comprise only 30% of this southern Malawi population. Young adults were the second largest contributors, while U5s and adults over 30 were each responsible for < 10% of transmission. While the specific predicted values are sensitive to the relative infectiousness of SAC, this group remained the most important contributor to mosquito infections under all realistic estimates. CONCLUSIONS: These results suggest that U5 children play a small role compared to SAC in maintaining P. falciparum transmission in southern Malawi. Models that assume biting homogeneity overestimate the importance of U5s. To reduce transmission, interventions will need to reach more SAC and young adults. This publicly available model can be used by others to estimate age-specific transmission contributions in epidemiologically similar sites with local parameter estimates of P. falciparum prevalence and bed net use.

Insecticide-treated net effectiveness at preventing Plasmodium falciparum infection varies by age and season.

BACKGROUND: After increasing coverage of malaria interventions, malaria prevalence remains high in Malawi. Previous studies focus on the impact of malaria interventions among children under 5 years old. However, in Malawi, the prevalence of infection is highest in school-aged children (SAC), ages 5 to 15 years. This study examined the interaction between age group and insecticide-treated net (ITN) use for preventing individual and community-level infection in Malawi. METHODS: Six cross-sectional surveys were conducted in the rainy and dry seasons in southern Malawi from 2012 to 2014. Data were collected on household ITN usage and demographics. Blood samples for detection of Plasmodium falciparum infection were obtained from all household members present and over 6 months of age. Generalized linear mixed models were used to account for clustering at the household and community level. RESULTS: There were 17,538 observations from six surveys. The association between ITN use and infection varied by season in SAC, but not in other age groups. The adjusted odds ratio (OR) for infection comparing ITN users to non-users among SAC in the rainy season and dry season was 0.78 (95% CI 0.56, 1.10) and 0.51 (0.35, 0.74), respectively. The effect of ITN use did not differ between children under five and adults. Among all non-SACs the OR for infection was 0.78 (0.64, 0.95) in those who used ITNs compared to those that did not. Community net use did not protect against infection. CONCLUSIONS: Protection against infection with ITN use varies by age group and season. Individual estimates of protection are moderate and a community-level effect was not detected. Additional interventions to decrease malaria prevalence are needed in Malawi.

Parasite dynamics in the peripheral blood and the placenta during pregnancy-associated malaria infection.

BACKGROUND: Malaria infections during pregnancy lead to sequestration of parasite infected red blood cells in the placenta. Placental infection can result in adverse outcomes for mothers and infants. Despite many studies, it remains unclear which peripheral blood infections during pregnancy lead to development of placental malaria. Understanding the timing of peripheral infections that lead to placental malaria and the ability of intermittent preventive treatment with sulfadoxine-pyrimethamine (SP-IPT) and artemisinin-based combination therapy to clear infections will enable the rational design of new interventions to decrease the burden of malaria in pregnancy. METHODS: Microsatellite markers were used to genotype peripheral and placental malaria infections in an observational cohort in Blantyre, Malawi. Genotypes were compared to determine the timing of infections that sequester in the placenta. The effects of SP-IPT and artemether-lumefantrine as curative treatment were also evaluated by assessing the occurrence of peripheral infections or matching genotypes between peripheral and placental parasites following treatment. RESULTS: Genotypes from 92 peripheral samples prior to delivery, 26 peripheral samples at delivery, and 29 placental samples were compared. Thirty percent of women with genotyped parasites in their placentas that had peripheral infections detected during pregnancy had matching peripheral-placental genotypes. Matching genotypes were not associated with gestational age and occurred from 13 to 39 weeks. Among women with more than one genotyped peripheral infection during pregnancy, 80 % had persistent infection with the same genotype while the remaining were new infections. Among infections treated with SP or artemether-lumefantrine, 28/84 (33 %) and 9/56 (16 %) had infection detected after treatment, respectively. Recrudescent infections were detected after both treatments and occurred up to 76 days after treatment. Women treated with SP-IPT and artemether-lumefantrine had genotypes matching treated infections detected in the placenta. CONCLUSIONS: Placental malaria can occur at any time during pregnancy. In the context of late enrollment in antenatal care, interventions that protect all women of childbearing age and throughout pregnancy are needed. Currently used medications do not always clear peripheral or placental infections. The ability of anti-malarial drugs to prevent or clear placental infections should be considered in the development of future interventions.

High prevalence of Plasmodium falciparum gametocyte infections in school-age children using molecular detection: patterns and predictors of risk from a cross-sectional study in southern Malawi.

BACKGROUND: In endemic areas, many people experience asymptomatic Plasmodium infections, particularly older children and adults, but their transmission contribution is unknown. Though not the exclusive determinant of infectiousness, transmission from humans to mosquitoes requires blood meals containing gametocytes. Gametocytes often occur at submicroscopic densities, challenging measurement in human populations. More sensitive molecular techniques allow better characterization of gametocyte epidemiologic patterns. METHODS: Approximately 30 households were selected from each of eight sites in southern Malawi during two cross-sectional surveys. Blood was sampled from 623 people during the dry season and 896 the following rainy season. Among people PCR-positive for Plasmodium falciparum, mature gametocytes were detected by qRT-PCR. Regression models evaluated predictors of gametocyte carriage and density in the total population and among those with PCR-positive infections. RESULTS: The prevalence of gametocyte carriage by molecular testing was 3.5% during the dry season and 8.6% during the rainy season, and by microscopy 0.8 and 3.3%, respectively. Nearly half of PCR-positive infections carried gametocytes, regardless of recent symptom status. Among P. falciparum-infected people, only living in unfinished houses and age were significantly associated with gametocyte presence. Infected people in unfinished houses had higher odds of carrying gametocytes (OR 2.24, 95% CI 1.16-4.31), and 31% (95% CI 3-65%) higher gametocyte density than those in finished houses. School-age children (5-15 years), had higher odds than adults (≥16 years) of having gametocytes when infected (OR 2.77, 95% CI 1.47-5.19), but 31% (95% CI 11-47%) lower gametocyte density. Children <5 years did not have significantly higher odds of gametocyte carriage or density when infected than adults. CONCLUSIONS: School-age children frequently carry gametocytes in communities of southern Malawi and represent an under-recognized reservoir of infection. Malaria elimination strategies should address these frequently asymptomatic reservoirs, especially in highly endemic areas. Improved household construction may also reduce the infectious reservoir.

Bed net use among school-aged children after a universal bed net campaign in Malawi.

BACKGROUND: Recent data from Malawi suggest that school-aged children (SAC), aged 5-15 years, have the highest prevalence of Plasmodium falciparum infection among all age groups. They are the least likely group to utilize insecticide-treated nets (ITNs), the most commonly available intervention to prevent malaria in Africa. This study examined the effects of a universal ITN distribution campaign, and their durability over time in SAC in Malawi. This study identified factors that influence net usage among SAC and how these factors changed over time. METHODS: Cross-sectional surveys using cluster random sampling were conducted at the end of each rainy and dry season in southern Malawi from 2012 to 2014; six surveys were done in total. Mass net distribution occurred between the first and second surveys. Data were collected on household and individual net usage as well as demographic information. Statistical analyses used generalized linear mixed models to account for clustering at the household and neighbourhood level. RESULTS: There were 7347 observations from SAC and 14,785 from young children and adults. SAC used nets significantly less frequently than the rest of the population (odds ratio (OR) from 0.14 to 0.38). The most important predictors of net usage among SAC were a lower ratio of people to nets in a household and higher proportion of nets that were hanging at the time of survey. Older SAC (11-15 years) were significantly less likely to use nets than younger SAC (5-10 years) [OR = 0.24 (95 % CI: 0.21, 0.28)]. The universal bed net campaign led to a statistically significant population-wide increase in net use, however net use returned to near baseline within 3 years. CONCLUSIONS: This study suggests that a single universal net distribution campaign, in combination with routine distribution through health clinics is not sufficient to cause a sustained increase in net usage among SAC. Novel approaches to ITN distribution, such as school-based distribution, may be needed to address the high prevalence of infection in SAC.

Pregnant women are a reservoir of malaria transmission in Blantyre, Malawi.

BACKGROUND: During pregnancy, women living in malaria-endemic regions are at increased risk of malaria infection and can harbour chronic placental infections. Intermittent preventive treatment with sulphadoxine-pyrimethamine (SP-IPTp) is administered to reduce malaria morbidity. It was hypothesized that the presence of placental malaria infection and SP-IPTp use would increase the risk of peripheral blood gametocytes, the parasite stage that is transmissible to mosquitoes. This would suggest that pregnant women may be important reservoirs of malaria transmission. METHODS: Light microscopy was used to assess peripheral gametocytaemia in pregnant women enrolled in a longitudinal, observational study in Blantyre, Malawi to determine the association between placental malaria and maternal gametocytaemia. The relationship between SP-IPTp and gametocytaemia was also examined. RESULTS: 2,719 samples from 448 women were analysed and 32 episodes of microscopic gametocytaemia were detected in 27 women. At the time of enrolment 22 of 446 women (4.9%) had gametocytaemia and of the 341 women for whom there was sufficient sampling to analyse infection over the entire course of pregnancy, 27 (7.9%) were gametocytaemic at least once. Gametocytaemia at enrollment was associated with placental malaria, defined as malaria pigment or parasites detected by histology or qPCR, respectively (OR: 32.4, 95% CI: 4.2-250.2), but was not associated with adverse maternal or foetal outcomes. Administration of SP-IPTp did not affect gametocyte clearance or release into peripheral blood. CONCLUSIONS: Gametocytaemia is present in 5% of pregnant women at their first antenatal visit and associated with placental malaria. SP-IPTp does not alter the risk of gametocytaemia. These data suggest that pregnant women are a significant reservoir of gametocyte transmission and should not be overlooked in elimination efforts. Interventions targeting this population would benefit from reaching women prior to first antenatal visit.

Submicroscopic malaria infection during pregnancy and the impact of intermittent preventive treatment.

BACKGROUND: Malaria during pregnancy results in adverse outcomes for mothers and infants. Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (SP) is the primary intervention aimed at reducing malaria infection during pregnancy. Although submicroscopic infection is common during pregnancy and at delivery, its impact throughout pregnancy on the development of placental malaria and adverse pregnancy outcomes has not been clearly established. METHODS: Quantitative PCR was used to detect submicroscopic infections in pregnant women enrolled in an observational study in Blantyre, Malawi to determine their effect on maternal, foetal and placental outcomes. The ability of SP to treat and prevent submicroscopic infections was also assessed. RESULTS: 2,681 samples from 448 women were analysed and 95 submicroscopic infections were detected in 68 women, a rate of 0.6 episodes per person-year of follow-up. Submicroscopic infections were most often detected at enrolment. The majority of women with submicroscopic infections did not have a microscopically detectable infection detected during pregnancy. Submicroscopic infection was associated with placental malaria even after controlling for microscopically detectable infection and was associated with decreased maternal haemoglobin at the time of detection. However, submicroscopic infection was not associated with adverse maternal or foetal outcomes at delivery. One-third of women with evidence of placental malaria did not have documented peripheral infection during pregnancy. SP was moderately effective in treating submicroscopic infections, but did not prevent the development of new submicroscopic infections in the month after administration. CONCLUSIONS: Submicroscopic malaria infection is common and occurs early in pregnancy. SP-IPT can clear some submicroscopic infections but does not prevent new infections after administration. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections.

Comparing approaches for chemoprevention for school-based malaria control in Malawi: an open label, randomized, controlled clinical trial.

Background: School-age children in sub-Saharan Africa suffer an underappreciated burden of malaria which threatens their health and education. To address this problem, we compared the efficacy of two school-based chemoprevention approaches: giving all students intermittent preventive treatment (IPT) or screening and treating only students with detected infections (IST). Methods: In a three-arm, open-label, randomized, controlled trial (NCT05244954) in Malawi, 746 primary school students, aged 5-19 years, were individually randomized within each grade-level to IPT (n = 249), IST with a high-sensitivity rapid diagnostic test (hs-RDT, n = 248), or control (n = 249). At six-week intervals three times within the peak malaria transmission season (February-June 2022) treatment with dihydroartemisinin-piperaquine (DP) was administered to girls <10 years and all boys, and chloroquine to older girls. The primary outcome was Plasmodium falciparum (Pf) infection detected by PCR 6-8 weeks after the final intervention. Secondary outcomes included anaemia, clinical malaria, and scores on tests of attention, literacy, and math. Analysis was by modified intention-to-treat. Findings: Outcomes analyses included 727 (97%) participants. At the end of the study, prevalence of Pf infection was 17% (41/243) in the IPT arm, 24% (58/244) in the IST arm, and 53% (127/240) in the control arm. Compared to controls, IPT and IST reduced the odds of Pf infection (IPT adjusted odds ratio [aOR]: 0.18 (95% CI: 0.11, 0.27); p < 0.0001; IST aOR: 0.27 (95% CI: 0.18, 0.40); p < 0.0001). However, only participants receiving IPT had a lower incidence of clinical malaria (0.19 cases per person per six months (95% CI: 0.14, 0.27) vs 0.56 (95% CI: 0.46, 0.68); incidence rate ratio: 0.38 (95% CI: 0.26, 0.55); p < 0.0001)) and prevalence of anaemia (8% [20/243] vs 15% [36/240]; aOR: 0.49 (95% CI: 0.27, 0.91); p = 0.023) compared to controls. Literacy scores were higher in both intervention arms. No between group differences in tests of attention or math or number of serious adverse events were found. Interpretation: Results support implementation of IST with hs-RDTs or IPT for reduction in the prevalence of infection. Based on reductions in clinical malaria, IPT may provide additional benefits warranting further consideration by school-based malaria chemoprevention programs. Funding: Doris Duke Charitable Foundation Clinical Scientist Development Award 2021191, U.S. NIH K24AI114996 & K23AI135076.

Understanding the Intransigence of Malaria in Malawi.

Despite the scale-up of interventions against malaria over the past decade, this disease remains a leading threat to health in Malawi. To evaluate the epidemiology of both Plasmodium falciparum infection and malaria disease, the Malawi International Center of Excellence for Malaria Research (ICEMR) has developed and implemented diverse and robust surveillance and research projects. Descriptive studies in ICEMR Phase 1 increased our understanding of the declining effectiveness of long-lasting insecticidal nets (LLINs), the role of school-age children in malaria parasite transmission, and the complexity of host-parasite interactions leading to disease. These findings informed the design of ICEMR Phase 2 to test hypotheses about LLIN use and effectiveness, vector resistance to insecticides, demographic targets of malaria control, patterns and causes of asymptomatic to life-threatening disease, and the impacts of RTS,S vaccination plus piperonyl butoxide-treated LLINs on infection and disease in young children. These investigations are helping us to understand mosquito-to-human and human-to-mosquito transmission in the context of Malawi's intransigent malaria problem.

Overall and Gender-Specific Effects of Intermittent Preventive Treatment of Malaria with Artemisinin-Based Combination Therapies among Schoolchildren in Mali: A Three-Group Open Label Randomized Controlled Trial.

Intermittent preventive treatment of malaria among schoolchildren (IPTsc) reduces clinical malaria, asymptomatic parasitemia, and anemia. The effects of IPTsc by gender have not been studied longitudinally. We investigated overall IPTsc efficacy and conducted a secondary analysis to explore gender-specific differences. We enrolled schoolchildren aged 6-13 years in an open-label, rolling-cohort randomized controlled trial between September 2007 and February 2013 in Kolle, Mali. Annually, schoolchildren received two full-treatment courses of sulfadoxine-pyrimethamine (SP) plus artesunate, or amodiaquine (AQ) plus artesunate, or no malaria treatment as control. We used mixed-effects generalized linear models to estimate differences in treatment outcomes across groups with interaction terms to explore gender-specific differences associated with Plasmodium falciparum infection, hemoglobin, and grade point averages (GPA) based on standardized testing. Overall, 305 students contributed 4,564 observations. Compared with the control, SP plus artesunate and AQ plus artesunate reduced the odds of P. falciparum infection (odds ratio [OR]: 0.33, 95% CI: 0.26-0.43; OR: 0.46, 95% CI: 0.36-0.59). We found strong evidence of increased mean hemoglobin concentrations (g/dL) in the SP plus artesunate group versus control (difference +0.37, 95% CI: 0.13-0.58). Collectively, schoolchildren given AQ plus artesunate had higher mean GPA (difference +0.36, 95% CI: 0.02-0.69) relative to control. Schoolgirls, compared with schoolboys, given SP plus artesunate had greater improvement in GPA (+0.50, 95% CI: -0.02 to 1.02 versus -0.27, 95% CI: -0.71 to 0.16); interaction P = 0.048, respectively. The IPTsc decreases P. falciparum infections in schoolchildren. Treatment regimens that include longer-acting drugs may be more effective at decreasing malaria-related anemia and improving educational outcomes as observed among girls in this setting.

Population Attributable Fraction of Anemia Associated with Plasmodium falciparum Infection in Children in Southern Malawi.

Anemia is a leading cause of morbidity in sub-Saharan Africa. The etiologies of anemia are multifactorial, and it is unclear what proportion of anemia is attributable to malaria in children of different ages in Malawi. We evaluated the population attributable fraction (PAF) of anemia due to malaria using multiple cross-sectional surveys in southern Malawi. We found a high prevalence of anemia, with the greatest proportion attributable to malaria among school-age children (5-15 years) in the rainy season (PAF = 18.8% [95% CI: 16.3, 21.0], compared with PAF = 5.2% [95% CI: 4.0, 6.2] among young children pooled across season [< 5 years] and PAF = 9.7% [95% CI: 6.5, 12.4] among school-age children in the dry season). Malaria control interventions will likely lead to decreases in anemia, especially among school-age children.

School-based screening and treatment may reduce P. falciparum transmission.

In areas where malaria remains entrenched, novel transmission-reducing interventions are essential for malaria elimination. We report the impact screening-and-treatment of asymptomatic Malawian schoolchildren (n = 364 in the rainy season and 341 in the dry season) had on gametocyte-the parasite stage responsible for human-to-mosquito transmission-carriage. We used concomitant household-based surveys to predict the potential reduction in transmission in the surrounding community. Among 253 students with P. falciparum infections at screening, 179 (71%) had infections containing gametocytes detected by Pfs25 qRT-PCR. 84% of gametocyte-containing infections were detected by malaria rapid diagnostic test. While the gametocyte prevalence remained constant in untreated children, treatment with artemether-lumefantrine reduced the gametocyte prevalence (p < 0.0001) from 51.8 to 9.7% and geometric mean gametocyte density (p = 0.008) from 0.52 to 0.05 gametocytes/microliter. In community surveys, 46% of all gametocyte-containing infections were in school-age children, who comprised only 35% of the population. Based on these estimates six weeks after the intervention, the gametocyte burden in the community could be reduced by 25-55% depending on the season and the measure used to characterize gametocyte carriage. Thus, school-based interventions to treat asymptomatic infections may be a high-yield approach to not only improve the health of schoolchildren, but also decrease malaria transmission.