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Abstract Background Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated. Methods Children aged 6–59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing. Results No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72–76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons. Conclusion In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique.

Abstract Background Artemisinin-based combination therapy (ACT) has been the recommended first-line treatment for uncomplicated malaria in Mozambique since 2006, with artemether–lumefantrine (AL) and amodiaquine–artesunate (AS–AQ) as the first choice. To assess efficacy of currently used ACT, an in vivo therapeutic efficacy study was conducted. Methods The study was conducted in four sentinel sites: Montepuez, Moatize, Mopeia and Massinga. Patients between 6 and 59 months old with uncomplicated Plasmodium falciparum malaria (2000–200,000 parasites/µl) were enrolled between February and September of 2018, assigned to either an AL or AS–AQ treatment arm, and monitored for 28 days. A Bayesian algorithm was applied to differentiate recrudescence from new infection using genotyping data of seven neutral microsatellites. Uncorrected and PCR-corrected efficacy results at day 28 were calculated. Results Totals of 368 and 273 patients were enrolled in the AL and AS–AQ arms, respectively. Of these, 9.5% (35/368) and 5.1% (14/273) were lost to follow-up in the AL and AS–AQ arms, respectively. There were 48 and 3 recurrent malaria infections (late clinical and late parasitological failures) in the AL and AS–AQ arms, respectively. The day 28 uncorrected efficacy was 85.6% (95% confidence interval (CI) 81.3–89.2%) for AL and 98.8% (95% CI 96.7–99.8%) for AS–AQ, whereas day 28 PCR-corrected efficacy was 97.9% (95% CI 95.6–99.2%) for AL and 99.6% (95% CI 97.9–100%) for AS–AQ. Molecular testing confirmed that 87.4% (42/48) and 33.3% (1/3) of participants with a recurrent malaria infection in the AL and AS–AQ arms were new infections; an expected finding in a high malaria transmission area. Adverse events were documented in less than 2% of participants for both drugs. Conclusion Both AL and AS–AQ have therapeutic efficacies well above the 90% WHO recommended threshold and remain well-tolerated in Mozambique. Routine monitoring of therapeutic efficacy should continue to ensure the treatments remain efficacious. Trial registration Clinicaltrials.gov: NCT04370977

Abstract Background While there is increasing evidence on the safety of artemisinin-based combination therapy (ACT) for the case management of malaria in early pregnancy, little is known about the association between exposure to ACT during the first trimester and the effect on fetal growth. Methods Data were analysed from prospective studies of pregnant women enrolled in Mozambique, Burkina Faso and Kenya designed to determine the association between anti-malarial drug exposure in the first trimester and pregnancy outcomes, including low birth weight (LBW) and small for gestational age (SGA). Exposure to anti-malarial drugs was ascertained retrospectively by record linkage using a combination of data collected from antenatal and adult outpatient clinic registries, prescription records and self-reported medication usage by the women. Site-level data synthesis (fixed effects and random effects) was conducted as well as individual-level analysis (fixed effects by site). Results Overall, 1915 newborns were included with 92 and 26 exposed to ACT (artemether–lumefantrine) and quinine, respectively. In Burkina Faso, Mozambique and Kenya at recruitment, the mean age (standard deviation) was 27.1 (6.6), 24.2 (6.2) and 25.7 (6.5) years, and the mean gestational age was 24.0 (6.2), 21.2 (5.7) and 17.9 (10.2) weeks, respectively. The LBW prevalence among newborns born to women exposed to ACT and quinine (QNN) during the first trimester was 10/92 (10.9%) and 7/26 (26.9%), respectively, compared to 9.5% (171/1797) among women unexposed to any anti-malarials during pregnancy. Compared to those unexposed to anti-malarials, ACT and QNN exposed women had the pooled LBW prevalence ratio (PR) of 1.13 (95% confidence interval (CI) 0.62–2.05, p-value 0.700) and 2.03 (95% CI 1.09–3.78, p-value 0.027), respectively. Compared to those unexposed to anti-malarials ACT and QNN-exposed women had the pooled SGA PR of 0.85 (95% CI 0.50–1.44, p-value 0.543) and 1.41 (95% CI 0.71–2.77, p-value 0.322), respectively. Whereas compared to ACT-exposed, the QNN-exposed had a PR of 2.14 (95% CI 0.78–5.89, p-value 0.142) for LBW and 8.60 (95% CI 1.29–57.6, p-value 0.027) for SGA. The level of between sites heterogeneity was moderate to high. Conclusion ACT exposure during the first trimester was not associated with an increased occurrence of LBW or SGA. However, the data suggest a higher prevalence of LBW and SGA for children born to QNN-exposed pregnancies. The findings support the use of ACT (artemether–lumefantrine) for the treatment of uncomplicated malaria during the first trimester of pregnancy.

Abstract Background Poor knowledge on the afebrile Plasmodium falciparum biology limits elimination approaches to target asymptomatic malaria. Therefore, the association of parasite factors involved in cytoadhesion, parasite multiplication and gametocyte maturation with afebrile malaria was assessed. Methods Plasmodium falciparum isolates were collected from febrile (axillary temperature ≥ 37.5 °C or a reported fever in the previous 24 h) and afebrile (fever neither at the visit nor in the previous 24 h) individuals residing in Southern Mozambique. var, PfSir2a and Pfs25 transcript levels were determined by reverse transcriptase quantitative PCRs (RT-qPCRs) and compared among 61 pairs of isolates matched by parasite density, age and year of sample collection. Results The level of varC and PfSir2a transcripts was higher in P. falciparum isolates from afebrile individuals (P ≤ 0.006), while varB and DC8 genes (P ≤ 0.002) were higher in isolates from individuals with febrile infections. After adjusting the analysis by area of residence, doubling the relative transcript unit (RTU) of varC and PfSir2a was associated with a 29.7 (95% CI 4.6–192.3) and 8.5 (95% CI 1.9–32.2) fold increases, respectively, of the odds of being afebrile. In contrast, doubling the RTU of varB and DC8 was associated with a 0.8 (95% CI 0.05–0.6) and 0.2 (95% CI 0.04–0.6) fold changes, respectively, of the odds of being afebrile. No significant differences were found for Pfs25 transcript levels in P. falciparum isolates from afebrile and febrile individuals. Conclusions var and gametocyte-specific transcript patterns in febrile and afebrile infections from southern Mozambique matched by age, parasite density and recruitment period suggest similar transmissibility but differential expression of variant antigens involved in cytoadhesion and immune-evasion.

Abstract Mozambique has historically been one of the countries with the highest malaria burden in the world. Starting in the 1960s, malaria control efforts were intensified in the southern region of the country, especially in Maputo city and Maputo province, to aid regional initiatives aimed to eliminate malaria in South Africa and eSwatini. Despite significant reductions in malaria prevalence, elimination was never achieved. Following the World Health Organization’s renewed vision of a malaria-free-world, and considering the achievements from the past, the Mozambican National Malaria Control Programme (NMCP) embarked on the development and implementation of a strategic plan to accelerate from malaria control to malaria elimination in southern Mozambique. An initial partnership, supported by the Bill and Melinda Gates Foundation and the La Caixa Foundation, led to the creation of the Mozambican Alliance Towards the Elimination of Malaria (MALTEM) and the Malaria Technical and Advisory Committee (MTAC) to promote national ownership and partner coordination to work towards the goal of malaria elimination in local and cross-border initiatives. Surveillance systems to generate epidemiological and entomological intelligence to inform the malaria control strategies were strengthened, and an impact and feasibility assessment of various interventions aimed to interrupt malaria transmission were conducted in Magude district (Maputo Province) through the “Magude Project”. The primary aim of this project was to generate evidence to inform malaria elimination strategies for southern Mozambique. The goal of malaria elimination in areas of low transmission intensity is now included in the national malaria strategic plan for 2017–22 and the NMCP and its partners have started to work towards this goal while evidence continues to be generated to move the national elimination agenda forward.

Abstract Background Insecticide-treated net (ITN) use is crucial for preventing malaria infection. Despite significant improvements in ITN access and use over the past two decades, many malaria-endemic countries in sub-Saharan Africa have not yet reached global targets for universal coverage of ITNs. To reduce the gaps in ITN use, it is important to understand the factors associated with ITN use. The goal of this analysis was to determine the factors associated with ITN use in Manica District, Mozambique. Methods A cross-sectional community-based survey was conducted from October to November 2019. Households were randomly selected, and all members of selected households were eligible to participate. Data on socio-demographic characteristics, housing construction and the ownership, use and characteristics of ITNs were collected using structured questionnaires. Factors independently associated with ITN use were identified using generalized estimating equations multivariate logistic regression. Results Of the 302 households surveyed, 209 (69.2%) owned at least one ITN and 176 (58.3%) had one ITN for every two household members. The multivariate analysis indicated that the odds of ITN use was significantly lower among individuals in households with 3 or more members. However, the odds of ITN use was significantly higher among older age groups, poorer households, and as the number of ITNs in a household increased. Conclusions The findings of this analysis highlight the need for behaviour change communication strategies targeting young people and ITN distribution campaigns targeting larger households to increase ITN ownership, thereby improving ITN use in Manica District.

Abstract Background Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries. Results The qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density. Conclusion This immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.