Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay.

While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs (pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 (pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.

Anti-malarial resistance in Mozambique: Absence of Plasmodium falciparum Kelch 13 (K13) propeller domain polymorphisms associated with resistance to artemisinins.

BACKGROUND: Malaria remains one of the most serious public health problems in sub-Saharan Africa and Mozambique is the world's fourth largest contributor, with 4.7% of disease cases and 3.6% of total deaths due to malaria. Its control relies on the fight against the vector and treatment of confirmed cases with anti-malarial drugs. Molecular surveillance is an important tool for monitoring the spread of anti-malarial drug resistance. METHODS: A cross-sectional study recruited 450 participants with malaria infection detected by Rapid Diagnostic Tests, from three different study sites (Niassa, Manica and Maputo) between April and August 2021. Correspondent blood samples were collected on filter paper (Whatman® FTA® cards), parasite DNA extracted and pfk13 gene sequenced using Sanger method. SIFT software (Sorting Intolerant From Tolerant) was used, predict whether an amino acid substitution affects protein function. RESULTS: No pfkelch13-mediated artemisinin resistance gene mutation was detected in this study settings. However, non-synonymous mutations were detected at prevalence of 10.2%, 6% and 5% in Niassa, Manica and Maputo, respectively. Most (56.3%) of the reported non-synonymous mutations were due to substitution at the first base of the codon, 25% at the second base and 18.8% at the third base. Additionally, 50% of non-synonymous mutations showed a SIFTscore bellow cut off value of 0.05, therefore, they were predicted to be deleterious. CONCLUSION: These results do not show an emergence of artemisinin resistance cases in Mozambique. However, the increased number of novel non-synonymous mutations highlights the relevance of increasing the number of studies focused on the molecular surveillance of artemisinin resistance markers, for its early detection.

Imported Malaria in Portugal: Prevalence of Polymorphisms in the Anti-Malarial Drug Resistance Genes pfmdr1 and pfk13.

Malaria is one of the 'big three' killer infectious diseases, alongside tuberculosis and HIV. In non-endemic areas, malaria may occur in travelers who have recently been to or visited endemic regions. The number of imported malaria cases in Portugal has increased in recent years, mostly due to the close relationship with the community of Portuguese language countries. Samples were collected from malaria-infected patients attending Centro Hospitalar Lisboa Ocidental (CHLO) or the outpatient clinic of Instituto de Higiene e Medicina Tropical (IHMT-NOVA) between March 2014 and May 2021. Molecular characterization of Plasmodium falciparum pfk13 and pfmdr1 genes was performed. We analyzed 232 imported malaria cases. The majority (68.53%) of the patients came from Angola and only three patients travelled to a non-African country; one to Brazil and two to Indonesia. P. falciparum was diagnosed in 81.47% of the cases, P. malariae in 7.33%, P. ovale 6.47% and 1.72% carried P. vivax. No mutations were detected in pfk13. Regarding pfmdr1, the wild-type haplotype (N86/Y184/D1246) was also the most prevalent (64.71%) and N86/184F/D1246 was detected in 26.47% of the cases. The typical imported malaria case was middle-aged male, traveling from Angola, infected with P. falciparum carrying wild type pfmdr1 and pfk13. Our study highlights the need for constant surveillance of malaria parasites imported into Portugal as an important pillar of public health.