Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique.

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.

Readiness of Mozambique Health Facilities to Address Undernutrition and Diarrhea in Children under Five: Indicators from 2018 and 2021 Survey Data.

The World Health Organization's systems framework shows that service delivery is key to addressing pressing health needs. Inadequate healthcare and the lack of healthcare services are factors associated with undernutrition and diarrhea in children under five, two health conditions with high morbi-mortality rates in Mozambique. The aim of the analysis was to determine the readiness score of nutrition and diarrhea services for children under five and the influence of malaria and HIV (Human Immunodeficiency Virus) service readiness on the readiness of these two services. A total of 1644 public health facilities in Mozambique were included from the 2018 Service Availability and Readiness Assessment. Additionally, a cross-sectional study was conducted to determine the availability and readiness scores of nutrition services in 2021 in five referral health facilities. The availability of nutrition and diarrhea services for children is low in Mozambique, with both scoring below 75%. Major unavailability was observed for human resources, guidelines, and training dimensions. Diarrhea (median (IQ): 72.2% (66.7 to 83.3)) and nutrition service readiness (median (IQ): 57.1% (52.4 to 57.1)) scores were significantly different (p < 0.001), while it is desirable for both services to be comprehensively ready. Nutrition services are positively associated with diarrhea service readiness and both services are associated with malaria and HIV service readiness (p < 0.05). None of the health facilities had all tracer items available and none of the facilities were considered ready (100%). There is a persisting need to invest comprehensively in readiness dimensions, within and across child health services.

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.

Genetic Characterisation of South African and Mozambican Bovine Rotaviruses Reveals a Typical Bovine-like Artiodactyl Constellation Derived through Multiple Reassortment Events.

This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bovine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evidence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also emphasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity.

Rotavirus A infection in pre- and post-vaccine period: Risk factors, genotypes distribution by vaccination status and age of children in Nampula Province, Northern Mozambique (2015-2019).

Mozambique introduced the monovalent rotavirus vaccine (Rotarix®, GSK Biologicals, Rixensart, Belgium) in September 2015. Previous analysis, showed that Nampula province continues reporting a high frequency of Rotavirus A (RVA) infection and the emergence of G9P[6], G9P[4] and G3P[4] genotypes. This analysis aimed to determine the RVA frequency; risk factors; genotype distribution by vaccination status and age between pre- and post-vaccine periods in children under-five years old with diarrhea in Nampula. A cross-sectional, hospital-based surveillance study was conducted in the Hospital Central de Nampula in Mozambique. Socio-demographic and clinical data were collected to assess factors related to RVA infection in both periods. Stool specimens were screened to detect RVA by ELISA, and positive samples were genotyped. Between 2015 (pre-vaccine period) and 2016-2019 (post-vaccine period), 614 stool specimens were collected and tested for RVA in which 34.9% (67/192) were positive in pre-vaccine period and 21.8% (92/422) in post-vaccine (p = 0.001). In the post-vaccine period, age, year, and contact with different animal species (chicken, duck, or multiple animals) were associated with RVA infection. RVA infection was higher in children partially vaccinated (40.7%, 11/27) followed by the fully vaccinated (29.3%, 56/191) and the unvaccinated (15.3%, 21/137) (p = 0.002). G1P[8] and G9P[4] were common in vaccinated children less than 12 months. The present analysis showed that RVA infection reduced slightly in the post-vaccine period, with a high proportion of infection and genotype diversity in children, under 12 months of age, vaccinated. Further research on factors associated with RVA infection on vaccinated compared to unvaccinated children and vaccination optimization should be done.

Rotavirus A infection in children under five years old with a double health problem: undernutrition and diarrhoea - a cross-sectional study in four provinces of Mozambique.

BACKGROUND: Mozambique has a high burden of group A rotavirus (RVA) infection and chronic undernutrition. This study aimed to determine the frequency and potential risk factors for RVA infection in undernourished children under 5 years old with diarrhoea in Mozambique. METHODS: The analysis was conducted using data from March 2015 to December 2017, regarding children under 5 years old with at least one type of undernutrition. Anthropometric measures were used to calculate indices of weight-for-age, weight-for-height and height-for-age through the Z-Scores. RVA results were extracted from the National Diarrhoea Surveillance database. Descriptive statistics, chi-square test was used for qualitative variables and organized in contingency tables and 95% Confidence Intervals (CI) were considered for the calculation of RVA infection proportion and in the multiple logistic regression models to estimate the adjusted odds ratios (AOR). RESULTS: Of the 842 undernourished children included in the analysis, 27.2% (95% CI: 24.3-30.3%) were positive for RVA. The rate of RVA infection was 42.7% (95% CI: 38.0-47.5%) in the pre-vaccine period, with great reduction to 12.2% (95% CI: 9.4-15.6%) in the post-vaccine period. Most of the RVA undernourished children had severe wasting (33.3%) and severe stunting (32.0%). The risk of infection was significantly high in children from 0 to 11 months (p-value < 0.001) when compared to the age group of 24-59 months. A higher proportion of RVA infection was detected in households with five or more members (p-value = 0.029). Similar proportions of RVA were observed in children fed only by breast milk (34.9%) and breast milk with formula (35.6%). A higher proportion of undernourished HIV-positive children co-infected with RVA (7.4%) was observed. CONCLUSIONS: The frequency of RVA infection in undernourished children declined following the introduction of the vaccine in Mozambique. Beyond the temporal variation, Maputo province, age and crowded households were also associated to RVA infection. A high proportion of RVA infection was observed in children with severe wasting and a triple burden of disease: undernutrition, RVA and HIV, highlighting the need to conduct follow-up studies to understand the long-term impact of these conditions on children's development.

Prevalence and genome characterization of porcine rotavirus A in southern Mozambique.

Rotavirus A (RVA) is an important pathogen causing gastroenteritis in many species, including humans and pigs. The objective of this study was to determine the prevalence of RVA in pigs from smallholdings and commercial farms in southern Mozambique and characterize the complete genomes of selected strains. RVA was detected at a rate of 11.8% (n = 288), of which 7.6% was detected at commercial farms and 4.2% at smallholdings. The whole genomes of eight rotavirus strains were determined using an Illumina MiSeq platform. Seven displayed a G9P[13] and one a G4P[6] genotype combination, all with a typical porcine backbone (I1/5-R1-C1-M1-A1/8-N1-T1/7-E1-H1). Phylogenetic analysis indicated that the seven G9P[13] strains were in fact one strain that circulated on a commercial pig farm. The genome segments of this strain clustered with diverse segments of human and porcine RVA strains from various Asian countries. Analysis of the G4P[6] strain revealed four distinct genome segments (VP2, VP4, VP6 and VP7) and five genome segments closely related to South African porcine rotavirus strains (NSP1, NSP3, NSP4, NSP5 and VP1). These results suggest that both the G4P[6] and the G9P[13] strains possibly emerged through multiple reassortment events. The presence of these strains on the commercial farms and smallholdings calls for a more in-depth surveillance of rotavirus in Mozambique.

Whole Genome Characterization and Evolutionary Analysis of G1P[8] Rotavirus A Strains during the Pre- and Post-Vaccine Periods in Mozambique (2012-2017).

Mozambique introduced the Rotarix® vaccine (GSK Biologicals, Rixensart, Belgium) into the National Immunization Program in September 2015. Although G1P[8] was one of the most prevalent genotypes between 2012 and 2017 in Mozambique, no complete genomes had been sequenced to date. Here we report whole genome sequence analysis for 36 G1P[8] strains using an Illumina MiSeq platform. All strains exhibited a Wa-like genetic backbone (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Phylogenetic analysis showed that most of the Mozambican strains clustered closely together in a conserved clade for the entire genome. No distinct clustering for pre- and post-vaccine strains were observed. These findings may suggest no selective pressure by the introduction of the Rotarix® vaccine in 2015. Two strains (HJM1646 and HGM0544) showed varied clustering for the entire genome, suggesting reassortment, whereas a further strain obtained from a rural area (MAN0033) clustered separately for all gene segments. Bayesian analysis for the VP7 and VP4 encoding gene segments supported the phylogenetic analysis and indicated a possible introduction from India around 2011.7 and 2013.0 for the main Mozambican clade. Continued monitoring of rotavirus strains in the post-vaccine period is required to fully understand the impact of vaccine introduction on the diversity and evolution of rotavirus strains.

Molecular Epidemiology of Rotavirus A Strains Pre- and Post-Vaccine (Rotarix®) Introduction in Mozambique, 2012-2019: Emergence of Genotypes G3P[4] and G3P[8].

Group A rotavirus (RVA) remains the most important etiological agent associated with severe acute diarrhea in children. Rotarix® monovalent vaccine was introduced into Mozambique's Expanded Program on Immunization in September 2015. In the present study, we report the diversity and prevalence of rotavirus genotypes, pre- (2012-2015) and post-vaccine (2016-2019) introduction in Mozambique, among diarrheic children less than five years of age. Genotyping data were analyzed for five sentinel sites for the periods indicated. The primary sentinel site, Mavalane General Hospital (HGM), was analyzed for the period 2012-2019, and for all five sites (country-wide analyses), 2015-2019. During the pre-vaccine period, G9P[8] was the most predominant genotype for both HGM (28.5%) and the country-wide analysis (46.0%). However, in the post-vaccine period, G9P[8] was significantly reduced. Instead, G3P[8] was the most common genotype at HGM, while G1P[8] predominated country-wide. Genotypes G9P[4] and G9P[6] were detected for the first time, and the emergence of G3P[8] and G3P[4] genotypes were observed during the post-vaccine period. The distribution and prevalence of rotavirus genotypes were distinct in pre- and post-vaccination periods, while uncommon genotypes were also detected in the post-vaccine period. These observations support the need for continued country-wide surveillance to monitor changes in strain diversity, due to possible vaccine pressure, and consequently, the effect on vaccine effectiveness.