Understanding malaria transmission in Angola: first detection of Plasmodium malariae in Anopheles spp. and the role of secondary vectors in the Balombo region, Benguela Province

Background: Angola is one of the most affected countries in sub-Saharan Africa and was off-track to meet the WHO Global Technical Strategy targeting of a 75% reduction in malaria incidence and mortality by 2025. Recent studies highlighted major knowledge gaps regarding malaria vectors in Angola, particularly secondary vectors. A malaria control project initiated in 2007 in Balombo (Benguela Province, central Angola) has monitored malaria dynamics for 18 years, revealing increased Plasmodium prevalence and more frequent detection of non-falciparum species, mainly Plasmodium malariae (in co-infection with P. falciparum) in villagers of the Balombo region. This study aimed to characterize Anopheles populations in this region by assessing species composition, behavioural patterns, and infection rate. And to a less extent, to compare the performance of two indoor and outdoor mosquito trapping methods. Methods: Surveys were conducted during the rainy (December 2023 and 2024) and dry seasons (July 2024) in villages around the town of Balombo. Adult mosquitoes were collected indoors and outdoors using CDC Light Traps and BG-Pro traps in randomly selected houses. Female Anopheles were morphologically and molecularly identified. Primary and secondary vectors were screened for Plasmodium spp. using qPCR-high resolution melting, and positive samples were sequenced. Results: A strong seasonal effect was observed in Anopheles populations, with higher abundance, diversity, and Plasmodium- infection rates during the rainy season. An. funestus remained the main vector, but Plasmodium spp. was also detected in five other species: An. arabiensis, An. gambiae, An. marshallii, An. rufipes, and An. maculipalpis. This is the first report of P. malariae in Anopheles mosquitoes and the first mention of the role of secondary vectors in malaria transmission in Benguela Province. Conclusion: This study provides a thorough and updated inventory of Anopheles species in the Balombo region. Molecular biology confirmed the presence of P. falciparum and P. malariae in several species of Anopheles underscoring the existence of a complex transmission system in villages of Balombo. Infected exophagic and exophilic Anopheles species, exhibiting opportunistic behaviour, likely contribute, as secondary vectors, to the increase of Plasmodiumspp. transmission in the study sites. Therefore, confirming the need to implement complementary interventions targeting outdoor malaria transmission.