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Abstract Background Rapid diagnostic tests (RDTs) based on the detection of Plasmodium falciparum histidine rich protein 2 (PfHRP2) are widely used for the diagnostic of P. falciparum in Africa. However, deletions of the pfhrp2 and pfhrp3 genes can lead to false negative test results and compromise appropriate case management. Due to the high burden of malaria in Mozambique, it is crucial to monitor the potential emergence of parasites with pfhrp2/3 gene deletions in the country. Methods The presence of pfhrp2/3 deletions was assessed during the 2023 high transmission season in 34 health facilities from 9 districts across 6 provinces in Mozambique. Children between 2 and 10 years of age attending the health facility with fever were tested by both the routine HRP2-RDT and a P. falciparum lactate dehydrogenase (PfLDH)-RDT, and dried blood spots (DBS) were collected from those testing positive by one or both RDTs. DBS from children with a negative HRP2-RDT but positive PfLDH-RDT were tested for the presence of pfhrp2/3 deletions by multiplex real time quantitative polymerase chain reaction (qPCR). Results 3208 children attended the health facilities during the study. 81.6% (2612/3208) participants were positive for at least one malaria RDT and, among them, 0.8% (210/2612) had discrepant RDT results (22 HRP2 − but LDH + and 188 HRP2 + but LDH −). The overall prevalence of suspected false-negative HRP2-RDT results in Mozambique was 0.91% (95% CI 0.58–1.39; 22/2424). pfhrp2/3 gene deletions were confirmed in 4 cases (1 in Nampula and 3 in Inhambane). Therefore, the prevalence of P. falciparum confirmed cases with pfhrp2/3 gene deletions in the six provinces sampled was 0.16% (95% CI 0.15–2.57; 4/2424), being 0.27% (95% CI 0.01–1.75; 1/367) in Nampula and 0.59% (95% CI 0.15–1.88; 3/503) in Inhambane. Conclusion pfhrp2/3 gene deletions were detected in 2 out of 6 provinces surveyed in Mozambique, but at a prevalence far below the 5% threshold recommended for a change in HRP2-based-RDT.

Abstract Routine sampling of pregnant women at first antenatal care (ANC) visits could make Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compare the genetic structure of parasite populations sampled from 289 first ANC users and 93 children from the community in Mozambique between 2015 and 2019. Samples are amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, are consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declines in both populations (p = 0.002-0.007), while for the ANC population, population genetic diversity is also lower (p = 0.0004), and genetic relatedness between infections is higher (p = 0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data.

Abstract Pregnant women attending first antenatal care (ANC) visits represent a promising malaria surveillance target in Sub-Saharan Africa. We assessed the spatio-temporal relationship between malaria trends at ANC (n = 6471) and in children in the community (n = 3933) and at health facilities (n = 15,467) in southern Mozambique (2016–2019). ANC P. falciparum rates detected by quantitative polymerase chain reaction mirrored rates in children, regardless of gravidity and HIV status (Pearson correlation coefficient [PCC] > 0.8, χ²

Abstract 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

Abstract Background Low-density Plasmodium falciparum infections prevail in low transmission settings, where immunity is expected to be minimal, suggesting an immune-independent effect on parasite densities. We aimed to describe parasite densities in pregnancy, and determine how gravidity and antibody-mediated immunity affect these, during a period of declining malaria transmission in southern Mozambique. Methods We documented P. falciparum infections at first antenatal care visits (n = 6471) between November 2016 and October 2019 in Ilha Josina (high-to-moderate transmission area), Manhiça (low transmission area), and Magude (pre-elimination area). Two-way interactions in mixed-effects regression models were used to assess gravidity-dependent differences in quantitative PCR-determined P. falciparum positivity rates (PfPRqPCR) and densities, in the relative proportion of detectable infections (pDi) with current diagnostic tests (≥ 100 parasites/μL) and in antimalarial antibodies. Results PfPRqPCR declined from 28 to 13% in Ilha Josina and from 5–7 to 2% in Magude and Manhiça. In primigravidae, pDi was highest in Ilha Josina at the first study year (p = 0.048), which declined with falling PfPRqPCR (relative change/year: 0.41, 95% CI [0.08; 0.73], p = 0.029), with no differences in antibody levels. Higher parasite densities in primigravidae from Ilha Josina during the first year were accompanied by a larger reduction of maternal hemoglobin levels (− 1.60, 95% CI [− 2.49; − 0.72; p