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.

Prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibodies in the Mozambican Population: A Cross-Sectional Serologic Study in 3 Cities, July-August 2020.

BACKGROUND: The extent of population exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was uncertain in many African countries during the onset of the pandemic. METHODS: We conducted a cross-sectional study and randomly selected and surveyed general population and occupational groups from 6 July to 24 August 2020, in 3 cities in Mozambique. Anti-SARS-CoV-2-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies were measured using a point-of-care rapid test. The prevalence was weighted for population (by age, sex, and city) and adjusted for test sensitivity and specificity. RESULTS: A total of 21 183 participants, including 11 143 from the general population and 10 040 from occupational groups, were included across all 3 cities. General population seropositivity (IgM or IgG) prevalence was 3.0% (95% confidence interval [CI], 1.0%-6.6%) in Pemba, 2.1% (95% CI, 1.2%-3.3%) in Maputo City, and 0.9% (95% CI, .1%-1.9%) in Quelimane. The prevalence in occupational groups ranged from 2.8% (95% CI, 1.3%-5.2%) to 5.9% (95% CI, 4.3%-8.0%) in Pemba, 0.3% (95% CI, .0%-2.2%) to 4.0% (95% CI, 2.6%-5.7%) in Maputo City, and 0.0% (95% CI, .0%-.7%) to 6.6% (95% CI, 3.8%-10.5%) in Quelimane, and showed variations between the groups tested. CONCLUSIONS: In the first representative COVID-19 serosurveys in Mozambique, in mid-2020, weighted and assay-adjusted seroprevalence in 3 provincial capitals of anti-SARS-CoV-2 ranged from 0.9% to 3.0%, whereas adjusted prevalence in occupational groups ranged from 0.0% to 6.6% with variation between groups. Exposure to SARS-CoV-2 was extensive during the first pandemic wave, and transmission may have been more intense among occupational groups. These data have been of utmost importance to inform public health intervention to control and respond to the pandemic in Mozambique.

Uptake of intermittent preventive treatment and pregnancy outcomes: health facilities and community surveys in Chókwè district, southern Mozambique.

BACKGROUND: Malaria in pregnancy leads to serious adverse effects on the mother and the child and accounts for 75,000-200,000 infant deaths every year. Currently, the World Health Organization recommends intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) at each scheduled antenatal care (ANC) visit. This study aimed to assess IPTp-SP coverage in mothers delivering in health facilities and at the community. In addition, factors associated with low IPTp-SP uptake and malaria adverse outcomes in pregnancy were investigated. METHODS: A community and a health facility-based surveys were conducted in mothers delivering in Chókwè district, southern Mozambique. Social-demographic data, malaria prevention practices and obstetric history were recorded through self-report and antenatal records. For women delivering at health facilities, a clinical examination of mother and child was performed, and malaria infection at delivery was determined by rapid diagnostic test, microscopy, quantitative PCR and placental histology. RESULTS: Of 1141 participants, 46.6, 30.2, 13.5 and 9.6% reported taking ≥ 3, two, one and none SP doses, respectively. Low IPTp uptake (< 3 doses) was associated with non-institutional deliveries (AOR = 2.9, P < 0.001), first ANC visit after week 28 (AOR = 5.4, P < 0.001), low awareness of IPTp-SP (AOR = 1.6, P < 0.002) and having no or only primary education (AOR = 1.3, P = 0.041). The overall prevalence of maternal malaria (peripheral and/or placental) was 16.8% and was higher among women from rural areas compared to those from urban areas (AOR = 1.9, P < 0.001). Younger age (< 20 years; AOR = 1.6, P = 0.042) and living in rural areas (AOR = 1.9, P < 0.001) were predictors of maternal malaria at delivery. Being primigravidae (AOR = 2.2, P = 0.023) and preterm delivery (AOR = 2.6, P < 0.001) predicted low birth weight while younger age was also associated with premature delivery (AOR = 1.4, P = 0.031). CONCLUSION: The coverage for two and ≥ 3 doses of IPTp-SP is moderately higher than estimates from routine health facility records in Gaza province in 2015. However, this is still far below the national target of 80% for ≥ 3 doses. Ongoing campaigns aiming to increase the use of malaria prevention strategies during pregnancy should particularly target rural populations, increasing IPTp-SP knowledge, stimulate early visits to ANC, improve access to health services and the quality of the service provided.

Epidemiology of rotavirus A diarrhea in Chókwè, Southern Mozambique, from February to September, 2011.

Acute diarrhea disease caused by Rotaviruses A (RVA) is still the leading cause of morbidity and mortality in children ≤5 years old in developing countries. An exploratory cross-sectional study was conducted between February and September, 2011 to determine the proportion of acute diarrhea caused by RVA. A total of 254 stool specimens were collected from children ≤5 years old with acute diarrhea, including outpatients (222 children) and inpatients (32 children), in three local health centers in Chókwè District, Gaza Province, South of Mozambique. RVA antigens were detected using enzyme immunoassay (EIA); the RVA G (VP7) and P (VP4) genotypes were determined by RT-PCR or analysis sequencing. Sixty (24%) out of 254 fecal specimens were positive for RVA by EIA; being 58 (97%) from children ≤2 years of age. RVA prevalence peaks in June and July (coldest and drier months) and the G[P] binary combination observed were G12P[8] (57%); G1P[8] (9%); G12P[6] (6%); and 2% for each of the following genotypes: G1P[6], G2P[6] G4P[6], and G9P[8]. Non-Typeable (NT) G and/or P genotypes were observed as follows: G12P [NT] (6%); G1P [NT], G3P[NT] and GNTP[NT] (4%). Considering the different GP combinations, G12 represented 67% of the genotypes. This is the first data showing the diversity of RVA genotypes in Mozambique highlighting the epidemiological importance of these viruses in acute diarrhea cases in children ≤2 years old. In addition, these findings will provide a baseline data before the introduction of the RVA monovalent (Rotarix(®) ) vaccine in the National Immunization Program in September 2015. J. Med. Virol. 88:1751-1758, 2016. © 2016 Wiley Periodicals, Inc.

Frequencies of Cytochrome P450 2B6 and 2C8 Allelic Variants in the Mozambican Population.

BACKGROUND: The cytochrome P450 enzymes (CYP) play an important role in the metabolism of many therapeutic agents. The activities of different enzymes exhibit variability in different populations, which causes variations in drug response or toxicity. The CYP2B6 and CYP2C8 enzymes are encoded by polymorphic genes characterised by different single nucleotide polymorphisms (SNPs). Several of these CYP variants are often associated with slow metabolism phenotypes. This study aimed to analyse the frequencies of allelic variants of CYP2B6 and CYP2C8 in the Mozambican population. METHODS: Using a polymerase chain reaction and restriction fragment length polymorphism assay (PCR-RFLP), the frequencies of the allelic variants of CYP2B6 (c.64C>T, c.516G>T, c.777C>A, c.785A>G, c.1459C>T) and CYP2C8 (c.805A>T, c.416G>A, c.1196A>G, c.792C>G) were determined in 360 Mozambican blood donors. RESULTS: The frequencies of the allelic variants of the CYP2B6 gene were 0.057, 0.426, 0.0, 0.410, and 0.004. For the CYP2C8 gene, the frequencies of the allelic variants were 0.160, 0.048, 0.0, and 0.005. No significant differences were observed between the gender and geographic distribution of volunteers around the country. CONCLUSION: The frequencies of the allelic variants of the CYP2B6 and CYP2C8 genes were found to be homogeneously distributed in the Mozambican population and were comparable to other African populations. Further studies are required to explore the impact of these variants on the clinical response (efficacy and toxicity) of drugs, including antimalarials.

Genomic epidemiology of SARS-CoV-2 during the first four waves in Mozambique.

Mozambique reported the first case of coronavirus disease 2019 (COVID-19) in March 2020 and it has since spread to all provinces in the country. To investigate the introductions and spread of SARS-CoV-2 in Mozambique, 1 142 whole genome sequences sampled within Mozambique were phylogenetically analyzed against a globally representative set, reflecting the first 25 months of the epidemic. The epidemic in the country was marked by four waves of infection, the first associated with B.1 ancestral lineages, while the Beta, Delta, and Omicron Variants of Concern (VOCs) were responsible for most infections and deaths during the second, third, and fourth waves. Large-scale viral exchanges occurred during the latter three waves and were largely attributed to southern African origins. Not only did the country remain vulnerable to the introductions of new variants but these variants continued to evolve within the borders of the country. Due to the Mozambican health system already under constraint, and paucity of data in Mozambique, there is a need to continue to strengthen and support genomic surveillance in the country as VOCs and Variants of interests (VOIs) are often reported from the southern African region.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Prospective surveillance study to detect antimalarial drug resistance, gene deletions of diagnostic relevance and genetic diversity of Plasmodium falciparum in Mozambique: protocol.

INTRODUCTION: Genomic data constitute a valuable adjunct to routine surveillance that can guide programmatic decisions to reduce the burden of infectious diseases. However, genomic capacities remain low in Africa. This study aims to operationalise a functional malaria molecular surveillance system in Mozambique for guiding malaria control and elimination. METHODS AND ANALYSES: This prospective surveillance study seeks to generate Plasmodium falciparum genetic data to (1) monitor molecular markers of drug resistance and deletions in rapid diagnostic test targets; (2) characterise transmission sources in low transmission settings and (3) quantify transmission levels and the effectiveness of antimalarial interventions. The study will take place across 19 districts in nine provinces (Maputo city, Maputo, Gaza, Inhambane, Niassa, Manica, Nampula, Zambézia and Sofala) which span a range of transmission strata, geographies and malaria intervention types. Dried blood spot samples and rapid diagnostic tests will be collected across the study districts in 2022 and 2023 through a combination of dense (all malaria clinical cases) and targeted (a selection of malaria clinical cases) sampling. Pregnant women attending their first antenatal care visit will also be included to assess their value for molecular surveillance. We will use a multiplex amplicon-based next-generation sequencing approach targeting informative single nucleotide polymorphisms, gene deletions and microhaplotypes. Genetic data will be incorporated into epidemiological and transmission models to identify the most informative relationship between genetic features, sources of malaria transmission and programmatic effectiveness of new malaria interventions. Strategic genomic information will be ultimately integrated into the national malaria information and surveillance system to improve the use of the genetic information for programmatic decision-making. ETHICS AND DISSEMINATION: The protocol was reviewed and approved by the institutional (CISM) and national ethics committees of Mozambique (Comité Nacional de Bioética para Saúde) and Spain (Hospital Clinic of Barcelona). Project results will be presented to all stakeholders and published in open-access journals. TRIAL REGISTRATION NUMBER: NCT05306067.

Access to and use of preventive intermittent treatment for Malaria during pregnancy: A qualitative study in the Chókwè district, Southern Mozambique.

BACKGROUND: Malaria remains a significant health problem in Mozambique, particularly in the case of pregnant women and children less than five years old. Intermittent preventive treatment with sulfadoxine-pyrimethamine (IPT-SP) is recommended for preventing malaria in pregnancy (MiP). Despite the widespread use and cost-effectiveness of IPTp-SP, coverage remains low. In this study, we explored factors limiting access to and use of IPTp-SP in a rural part of Mozambique. METHODS AND FINDINGS: We performed a qualitative study using semi-structured interviews to collect data from 46 pregnant women and four health workers in Chókwè, a rural area of southern Mozambique. Data were transcribed, translated where appropriate, manually coded, and the content analyzed according to key themes. The women interviewed were not aware of the risks of MiP or the benefits of its prevention. Delays in accessing antenatal care, irregular attendance of visits, and insufficient time for proper antenatal care counselling by health workers were driving factors for inadequate IPTp delivery. CONCLUSIONS: Pregnant women face substantial barriers in terms of optimal IPTp-SP uptake. Health system barriers and poor awareness of the risks and consequences of MiP and of the measures available for its prevention were identified as the main factors influencing access to and use of IPTp-SP. Implementation of MiP prevention strategies must be improved through intensive community health education and increased access to other sources of information. Better communication between health workers and ANC clients and better knowledge of national ANC and IPTp policies are important.

HIV prevalence and incidence in a cohort of women at higher risk for HIV acquisition in Chókwè, southern Mozambique.

BACKGROUND: Reliable HIV incidence estimates for Mozambique are limited. We conducted a prospective HIV incidence study as part of a clinical research site development initiative in Chókwè district, Gaza Province, southern Mozambique. METHODS: Between June 2010 and October 2012, we recruited women at sites where women at higher risk of HIV infection would likely be found. We enrolled and tested 1,429 sexually active women in the screening phase and 479 uninfected women in the prospective phase. Participants were scheduled for 12+ months follow-up, when they underwent face-to-face interviews, HIV counseling and testing, and pregnancy testing. We observed a total of 373.1 woman-years (WY) of follow-up, with mean (median) of 9.4 (9.7) women-months per participant. RESULTS: The prevalence of HIV was 29.4% (95% confidence interval [CI]: 27.0-31.8%). In multivariable logistic regression analysis, factors that remained significantly associated with prevalent HIV were: older age (OR: 0.6; 95% CI: 0.4-0.7), lower educational level (OR: 0.4; 95% CI: 0.3-0.7), and using hormonal contraception (OR: 0.6; 95% CI: 0.4-0.7) or condoms (OR: 0.5; 95% CI: 0.3-0.7). We observed an HIV incidence rate of 4.6 per 100 WY (95% CI: 2.7, 7.3). The HIV incidence was 4.8 per 100 WY (95% CI: 2.5, 8.3) in women aged 18-24 years, 4.5 per 100 WY (95% CI: 1.2, 11.4) in women aged 25-29 years and 3.2 per 100 WY (95% CI: 0.1, 18.0) in the 30-35 years stratum. None of the demographic factors or time-varying behavioral factors examined was significantly associated with incident HIV infection in bivariable analysis at p ≤ 0.10. CONCLUSIONS: We found a high HIV incidence among sexually active young women in Chókwè, Mozambique. HIV prevention programs should be strengthened in the area, with more comprehensive reproductive health services, regular HIV testing, condom promotion, and messaging about multiple sexual partners.

Frequências alélicas e genotípicas das isoformas CYP2B6 e CYP2C8 na população de Moçambique / Allele and genotype frequencies of CYP2B6 and CYP2C8 isoforms in the population of Mozambique

A malária é uma doença infecciosa que ao lado da AIDS e da tuberculose, constituem três principais causas de mortalidade no mundo, sendo responsáveis por cerca de seis milhões de mortes por ano. De acordo com a OMS, de 300 a 500 milhões de novos episódios de malária e mais de dois milhões de mortes ocorrem por ano, principalmente entre crianças menores de cinco anos na África sub-Sahariana. Devido à indisponibilidade de uma vacina viável contra a malária, uma das principais estratégias para o controle da mesma tem sido o diagnóstico precoce e o tratamento com antimaláricos seguros e eficazes. No entanto, a eficácia deste tratamento é limitada pela emergência de cepas de Plasmódium falciparum (P.falciparum) resistentes aos antimaláricos. Hoje, o conhecimento sobre a contribuição dos fatores genéticos do hospedeiro envolvidos na resposta terapêutica das principais classes de antimaláricos é limitado. Contudo, sabe-se que variações genéticas em genes humanos que codificam para enzimas envolvidas na biotransformação de diferentes fármacos podem contribuir para diferenças interindividuais na resposta terapêutica ou toxidade a várias drogas, estando relacionadas aos desfechos de falha terapêutica e efeitos adversos. As isoenzimas CYP2B e CYP2C8 humanas, ambas envolvidas na metabolização de uma ampla variedade de fármacos, incluindo antimaláricos, são codificados por genes polimórficos, caracterizados por diferentes polimorfismos de base única (SNPs). Algumas destas variantes caracterizam fenótipos de metabolização lenta e possivelmente estão envolvidas na modulação do metabolismo dos antimaláricos. Considerando esta hipótese, este estudo teve como objetivos: (i) análise descritiva das variantes alélicas nos genes CYP2B6 e CYP2C8 em populações moçambicanas provenientes de três regiões distintas e (ii) obter as freqüências genotípicas e haplotípicas dos polimorfismos identificados. O estudo serviu igualmente para fornecermos informações capazes de nortear futuros estudos de associação para avaliar o impacto destes polimorfismos na eficácia terapêutica e/ou eventos adversos. Para atingir esses objetivos, amostras de sangue de 360 doadores de sangue foram coletadas num cartão de filtro “FTA Classic Cards” (Whatman) e genotipadas por PCR-RFLP. As freqüências das variantes alélicas no CYP2B6 foram 0,060 (64C < T), 0,426 (516G < T), 0,00 (777C < A), 0,41 (785A < G) e 0,004 (1459C < T), enquanto a freqüência da variante selvagem foi de 0,183. Para o gene CYP2C8 as freqüências das variantes alélicas foram: 0,048, 0,005, 0,16 e 0,00 para mutações 416G < A, 792C < G, 805A < T e 1196A < G, respectivamente. Não foram encontradas diferenças significativas entre as freqüências das mutações encontradas na variável gênero para os dois genes. Também não foram encontradas diferenças significativas na distribuição das freqüências genotípicas e haplotípicas entre indivíduos das três regiões estudadas, mostrando uma distribuição homogênea das mutações nas populações incluídas neste estudo. Nossos resultados mostram que os polimorfismos presentes nos genes CYP2B6 e CYP2C8, sabidamente envolvidos na alteração do metabolismo de uma variedade de drogas, ocorrem nas populações estudadas, corroborando os resultados reportados em outros estudos realizados em populações africanas. Assim, fazem-se necessários estudos de associação incluindo estes genes para um melhor entendimento sobre a importância destes polimorfismos no tratamento da malária nestas populações.