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Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys., (© 2023. The Author(s).)

Background: Controlled infection studies in malaria-naive adults suggest increased vaccine efficacy for fractional-dose versus full-dose regimens of RTS,S/AS01. We report first results of an ongoing trial assessing different fractional-dose regimens in children, in natural exposure settings., Methods: This open-label, phase 2b, randomised controlled trial is conducted at the Malaria Research Center, Agogo, Ashanti Region (Ghana), and the Kenya Medical Research Institute and the US Centers for Disease Control and Prevention site in Siaya County (Kenya). We enrolled children aged 5-17 months without serious acute or chronic illness who had previously received three doses of diphtheria, tetanus, pertussis, and hepatitis B vaccine and at least three doses of oral polio vaccine. Children were randomly assigned (1:1:1:1:1) using a web-based randomisation system with a minimisation procedure accounting for centre to receive rabies control vaccine (M012 schedule) or two full doses of RTS,S/AS01 E at month 0 and month 1, followed by either full doses at months 2 and 20 (group R012-20 [standard regimen]), full doses at months 2, 14, 26, and 38 (R012-14), fractional doses at months 2, 14, 26, and 38 (Fx012-14), or fractional doses at months 7, 20, and 32 (Fx017-20). The fractional doses were administered as one fifth (0·1 mL) of the full RTS,S dose (0·5 mL) after reconstitution. All vaccines were administered by intramuscular injection in the left deltoid. The primary outcome was occurrence of clinical malaria cases from month 2·5 until month 14 for the Fx012-14 group versus the pooled R012-14 and R012-20 groups in the per-protocol set. We assessed incremental vaccine efficacy of the Fx012-14 group versus the pooled R012-14 and R012-20 group over 12 months after dose three. Safety was assessed in all children who received at least one vaccine dose. This trial is registered with ClinicalTrials.gov, NCT03276962., Findings: Between Sept 28, 2017, and Sept 25, 2018, 2157 children were enrolled, of whom 1609 were randomly assigned to a treatment group (322 to each RTS,S/AS01 E group and 321 to the rabies vaccine control group). 1500 children received at least one study vaccine dose and the per-protocol set comprised 1332 children. Over 12 months after dose three, the incremental vaccine efficacy in the Fx012-14 group versus the pooled R012-14 and R12-20 groups was -21% (95% CI -57 to 7; p=0·15). Up to month 21, serious adverse events occurred in 48 (16%) of 298 children in the R012-20 group, 45 (15%) of 294 in the R012-14 group, 47 (15%) of 304 in the Fx012-14 group, 62 (20%) of 311 in the Fx017-20 group, and 71 (24%) of 293 in the control group, with no safety signals observed., Interpretation: The Fx012-14 regimen was not superior to the standard regimen over 12 months after dose three. All RTS,S/AS01 E regimens provided substantial, similar protection against clinical malaria, suggesting potential flexibility in the recommended dosing regimen and schedule. This, and the effect of annual boosters, will be further evaluated through 50 months of follow-up., Funding: GlaxoSmithKline Biologicals; PATH's Malaria Vaccine Initiative., Competing Interests: Declaration of interests The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention. OO-A, LS, ML, DM, ABo, FR, and EJ are employees of the GSK group of companies. OO-A, LS, DM, FR, and EJ have restricted shares in the GSK group of companies. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Intermittent preventive treatment of malaria in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a proven strategy to protect infants against malaria. Sierra Leone is the first country to implement IPTi nationwide. IPTi implementation was evaluated in Kambia, one of two initial pilot districts, to assess quality and coverage of IPTi services., Methods: This mixed-methods evaluation had two phases, conducted 3 (phase 1) and 15-17 months (phase 2) after IPTi implementation. Methods included: assessments of 18 health facilities (HF), including register data abstraction (phases 1 and 2); a knowledge, attitudes and practices survey with 20 health workers (HWs) in phase 1; second-generation sequencing of SP resistance markers (pre-IPTi and phase 2); and a cluster-sample household survey among caregivers of children aged 3-15 months (phase 2). IPTi and vaccination coverage from the household survey were calculated from child health cards and maternal recall and weighted for the complex sampling design. Interrupted time series analysis using a Poisson regression model was used to assess changes in malaria cases at HF before and after IPTi implementation., Results: Most HWs (19/20) interviewed had been trained on IPTi; 16/19 reported feeling well prepared to administer it. Nearly all HFs (17/18 in phase 1; 18/18 in phase 2) had SP for IPTi in stock. The proportion of parasite alleles with dhps K540E mutations increased but remained below the 50% WHO-recommended threshold for IPTi (4.1% pre-IPTi [95%CI 2-7%]; 11% post-IPTi [95%CI 8-15%], p < 0.01). From the household survey, 299/459 (67.4%) children ≥ 10 weeks old received the first dose of IPTi (versus 80.4% for second pentavalent vaccine, given simultaneously); 274/444 (62.5%) children ≥ 14 weeks old received the second IPTi dose (versus 65.4% for third pentavalent vaccine); and 83/217 (36.4%) children ≥ 9 months old received the third IPTi dose (versus 52.2% for first measles vaccine dose). HF register data indicated no change in confirmed malaria cases among infants after IPTi implementation., Conclusions: Kambia district was able to scale up IPTi swiftly and provide necessary health systems support. The gaps between IPTi and childhood vaccine coverage need to be further investigated and addressed to optimize the success of the national IPTi programme.

Background: Malaria data reported through Mozambique's routine health information system are used to guide the implementation of prevention and control activities. Although previous studies have identified issues with the quality of aggregated data reported from public health facilities in the country, no studies have evaluated the quality of routine indicators recorded in health facility registries. This study addresses this issue by comparing indicators calculated from data from exit interviews and re-examinations of patients with data based on registry records from health facilities in order to measure the quality of registry data and data reporting in three provinces in Mozambique., Methods: Data were collected from 1,840 outpatients from 117 health facilities in Maputo, Zambézia, and Cabo Delgado Provinces interviewed and examined as part of a malaria-specific health facility survey. Key indicators based on exit interview / re-examination data were compared to the same indicators based on records from health facility registries. Multivariable regression was performed to identify factors associated with indicators matching in re-examination / exit interview data and health facility registries. Aggregated indicators abstracted from facility registries were compared to those reported through the routine health management information system (HMIS) for the same time period., Results: Sensitivity of exit interview / re-examination data compared with those recorded in facility registries was low for all indicators in all facilities. The lowest sensitivities were in Maputo, where the sensitivity for recording negative RDT results was 9.7%. The highest sensitivity was for recording positive RDT results in Cabo Delgado, at 75%. Multivariable analysis of factors associated with agreement between gold standard and registry data showed patients were less likely to be asked about having a fever in the triage ward in Maputo and Cabo Delgado (adjusted Odds Ratio 0.75 and 0.39 respectively), and in the outpatient ward in Cabo Delgado (aOR = 0.37), compared with the emergency department. Patients with positive RDT were also more likely to have RDT results recorded in all three provinces when patients had been managed according to national treatment guidelines during initial examination. Comparison of retrospective data abstracted from facility registries to HMIS data showed discrepancies in all three provinces. The proportion of outpatient cases with suspected and confirmed malaria were similar in registry and HMIS data across all provinces (a relatively low difference between registry and HMIS data of 3% in Maputo and Zambézia), though the total number of all-cause outpatient cases was consistently higher in the HMIS. The largest difference was in Maputo, where a total of 87,992 all-cause outpatient cases were reported in HMIS, compared with a total of 42,431 abstracted from facility registries., Conclusion: This study shows that care should be taken in interpreting trends based solely on routine data due to data quality issues, though the discrepancy in all-cause outpatient cases may be indicative that register availability and storage are important factors. As such, simple steps such as providing consistent access and storage of registers that include reporting of patient fever symptoms might improve the quality of routine data recorded at health facilities., Competing Interests: The authors have declared that no competing interests exist.

Malaria is a leading cause of morbidity and mortality among children under five years of age, with most cases occurring in Sub-Saharan Africa. Children in this age group in Africa are at greatest risk worldwide for developmental deficits. There are research gaps in quantifying the risks of mild malaria cases, understanding the pathways linking malaria infection and poor child development, and evaluating the impact of malaria on the development of children under five years. We analyzed the association between malaria infection and gross motor, communication, and personal social development in 592 children age 24 months in rural, western Kenya as part of the WASH Benefits environmental enteric dysfunction sub-study. Eighteen percent of children had malaria, 20% were at risk for gross motor delay, 21% were at risk for communication delay, and 23% were at risk for personal social delay. Having a positive malaria test was associated with increased risk for gross motor, communication, and personal social delay while adjusting for child characteristics, household demographics, study cluster, and intervention treatment arm. Mediation analyses suggested that anemia was a significant mediator in the pathway between malaria infection and risk for gross motor, communication, and personal social development delays. The proportion of the total effect of malaria on the risk of developmental delay that is mediated by anemia across the subscales was small (ranging from 9% of the effect on gross motor development to 16% of the effect on communication development mediated by anemia). Overall, malaria may be associated with short-term developmental delays during a vulnerable period of early life. Therefore, preventative malaria measures and immediate treatment are imperative for children's optimal development, particularly in light of projections of continued high malaria transmission in Kenya and Africa.

Background: Data on congenital cytomegalovirus (CMV) infection in Africa are limited., Objective: To describe the prevalence of congenital CMV infection in a population with high prevalence of maternal HIV and malaria infection in western Kenya., Study Design: We screened newborns for CMV by polymerase chain reaction assay of saliva swabs and dried blood spots (DBS), and assessed maternal CMV immunoglobulin G (IgG) status by testing serum eluted from newborn's DBS. We calculated adjusted prevalence ratios (aPRs) using log-binomial regression models., Results: Among 1066 mothers, 210 (19·7%) had HIV infection and 207 (19·4%) had malaria infection; 33 (3·1%) mothers had both. Maternal CMV IgG prevalence was 93·1% (95% confidence interval [CI]: 88·3%-96·0%). Among 1078 newborns (12 sets of twins), 39 (3·6%, 95% CI: 2·7-4·9%) were CMV positive. The prevalence of congenital CMV infection by maternal HIV and malaria infection status was 5·0% (95% CI: 2·7-9·2%) for HIV only, 5·1% (95% CI: 2·7-9·4%) for malaria only, 8·8 (95% CI: 3·1-23·0) for HIV and malaria co-infection, and 2·6% (95% CI: 1·7-4·1%) for none. Congenital CMV infection was independently associated with maternal HIV infection (aPR=2·1; 95% CI: 1·0-4·2), adjusting for maternal age, parity, and malaria infection., Conclusions: The prevalence of congenital CMV infection was higher than the 0·2-0·7% in developed countries. Maternal HIV infection may increase the risk of congenital CMV infection, but the role of maternal malaria on intrauterine transmission of CMV remains unclear., (Published by Elsevier B.V.)

Background: Fever associated with malaria is the leading cause of health care-seeking in Mozambique, yet there is limited evidence on the quality of malaria case management. This study evaluated the quality of malaria service provision offered in public health facilities in Mozambique., Methods: A cross-sectional assessment was conducted in April-May 2018 in three provinces of Mozambique: Maputo Province (low malaria burden), Cabo Delgado (high), and Zambézia (high). The study included all secondary and tertiary facilities and a random sample of primary facilities in each province. Data collection included exit interviews and re-examinations of 20 randomly selected outpatient service patients, interviews with up to five health care providers and the health facility director, a stockroom inventory and routine data abstraction., Results: A total of 319 health care providers and 1840 patients from 117 health facilities were included. Of these, 1325 patients (72%) had suspected malaria (fever/history of fever) and 550 (30%) had febrile, confirmed malaria with the highest burden in Cabo Delgado (43%), followed by Zambézia (34%) and Maputo Province (2%). Appropriate management of malaria cases, defined as testing malaria suspects and treating confirmed cases with the correct dose of anti-malarial, was highest in Zambézia and Cabo Delgado where 52% (95% CI 42-62) and 49% (42-57) of febrile malaria cases were appropriately managed, respectively. Only 14% (5-34) of febrile cases in Maputo Province were appropriately managed. The biggest gap in the malaria case management pathway was failure to test febrile patients, with only 46% of patients with this indication tested for malaria in Maputo Province. Additionally, anti-malarial treatment of patients with a negative malaria test result was common, ranging from 8% (2-23) in Maputo Province to 22% (14-32) of patients with a negative test in Zambézia. Only 58-62% of patients prescribed an anti-malarial correctly recited dosing instructions. Provider training and malaria knowledge was low outside of Zambézia and supervision rates were low in all provinces. Factors associated with correct case management varied by province and included patient age, facility type, treatment and testing availability, supervision, and training., Conclusion: These findings underscore the need to strengthen provider testing of all patients with fever, provider adherence to negative test results, and effective counselling of patients across epidemiological settings in Mozambique.

Background: Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays., Methods/principal Findings: We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff., Conclusions/significance: Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets.

Background: High levels of patient adherence to antimalarial treatment are important in ensuring drug effectiveness. To achieve this goal, it is important to understand levels of patient adherence, and the range of study designs and methodological challenges involved in measuring adherence and interpreting results. Since antimalarial adherence was reviewed in 2004, there has been a major expansion in the use of artemisinin-based combination therapies (ACTs) in the public sector, as well as initiatives to make them more widely accessible through community health workers and private retailers. These changes and the large number of recent adherence studies raise the need for an updated review on this topic., Objective: We conducted a systematic review of studies reporting quantitative results on patient adherence to antimalarials obtained for treatment., Results: The 55 studies identified reported extensive variation in patient adherence to antimalarials, with many studies reporting very high adherence (90-100%) and others finding adherence of less than 50%. We identified five overarching approaches to assessing adherence based on the definition of adherence and the methods used to measure it. Overall, there was no clear pattern in adherence results by approach. However, adherence tended to be higher among studies where informed consent was collected at the time of obtaining the drug, where patient consultations were directly observed by research staff, and where a diagnostic test was obtained., Conclusion: Variations in reported adherence may reflect factors related to patient characteristics and the nature of their consultation with the provider, as well as methodological variations such as interaction between the research team and patients before and during the treatment. Future studies can benefit from an awareness of the impact of study procedures on adherence outcomes, and the identification of improved measurement methods less dependent on self-report.