Micro-heterogeneity of transmission shapes submicroscopic malaria carriage in coastal Tanzania.

BACKGROUND: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low density infections are often submicroscopic, not detected by rapid diagnostic tests (RDTs) or microscopy, but detectable by PCR. METHODS: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over four weeks. RESULTS: Among 6,076 participants, P. falciparum prevalence by RDT, microscopy, and PCR was 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female gender, dry season months, screened windows, and bednet use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over four weeks, 23% (61/266) of submicroscopic carriers became RDT-positive, with half exhibiting symptoms, while half (133/266) were no longer parasitemic at the end of four weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence. CONCLUSIONS: Micro-heterogeneity in transmission observed at the village level appears to impact both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Leech, potato, and tomato carboxypeptidase inhibitors against Anopheles stephensi carboxypeptidase B1 and B2.

Carboxypeptidase B (CPB) in Anopheles spp. breaks down blood and releases free amino acids, which promote Plasmodium sexual development in the mosquito midgut. Our goal was to computationally assess the inhibitory effectiveness of carboxypeptidase inhibitors obtained from tomato, potato (CPiSt), and leech against the Anopheles stephensi CPBAs1 and CPBAs2 enzymes. The tertiary structures of CPB inhibitors were predicted and their interaction mode with CPBAs1 and CPBAs2 were examined using molecular docking. Next, this data was compared with four licensed medications that are known to reduce the Anopheles' CPB activity. Molecular dynamics simulations were used to evaluate the stability of complexes containing CPiSt and its mutant form. Both CPiSt and its mutant form showed promise as possible candidates for further evaluations in the paratransgenesis technique for malaria control, based on the similar bindings of CPiSt and CPiSt-Mut to the active sites of CPBAs1 and CPBAs2, as well as their binding affinity in comparison to the drugs.

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission.

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane-associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localizes at the cytoplasmic periphery and concavin(-) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(-) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(-) sporozoites are transmitted. Strikingly, motile concavin(-) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Livestock keeping, mosquitoes and community viewpoints: a mixed methods assessment of relationships between livestock management, malaria vector biting risk and community perspectives in rural Tanzania.

BACKGROUND: Livestock keeping is one of the potential factors related to malaria transmission. To date, the impact of livestock keeping on malaria transmission remains inconclusive, as some studies suggest a zooprophylactic effect while others indicate a zoopotentiation effect. This study assessed the impact of livestock management on malaria transmission risks in rural Tanzania. Additionally, the study explored the knowledge and perceptions of residents about the relationships between livestock keeping and malaria transmission risks in a selected village. METHODS: In a longitudinal entomological study in Minepa village, South Eastern Tanzania, 40 households were randomly selected (20 with livestock, 20 without). Weekly mosquito collection was performed from January to April 2023. Indoor and outdoor collections used CDC-Light traps, Prokopack aspirators, human-baited double-net traps, and resting buckets. A subsample of mosquitoes was analysed using PCR and ELISA for mosquito species identification and blood meal detection. Livestock's impact on mosquito density was assessed using negative binomial GLMMs. Additionally, in-depth interviews explored community knowledge and perceptions of the relationship between livestock keeping and malaria transmission risks. RESULTS: A total of 48,677 female Anopheles mosquitoes were collected. Out of these, 89% were Anopheles gambiae sensu lato (s.l.) while other species were Anopheles funestus s.l., Anopheles pharoensis, Anopheles coustani, and Anopheles squamosus. The findings revealed a statistically significant increase in the overall number of An. gambiae s.l. outdoors (RR = 1.181, 95%CI 1.050-1.862, p = 0.043). Also, there was an increase of the mean number of An. funestus s.l. mosquitoes collected in households with livestock indoors (RR = 2.866, 95%CI: 1.471-5.582, p = 0.002) and outdoors (RR = 1.579,95%CI 1.080-2.865, p = 0.023). The human blood index of Anopheles arabiensis mosquitoes from houses with livestock was less than those without livestock (OR = 0.149, 95%CI 0.110-0.178, p < 0.001). The majority of participants in the in-depth interviews reported a perceived high density of mosquitoes in houses with livestock compared to houses without livestock. CONCLUSION: Despite the potential for zooprophylaxis, this study indicates a higher malaria transmission risk in livestock-keeping communities. It is crucial to prioritize and implement targeted interventions to control vector populations within these communities. Furthermore, it is important to enhance community education and awareness regarding covariates such as livestock that influence malaria transmission.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Modeling the Impact of a Highly Potent Plasmodium falciparum Transmission-Blocking Monoclonal Antibody in Areas of Seasonal Malaria Transmission.

Transmission-blocking interventions can play an important role in combating malaria worldwide. Recently, a highly potent Plasmodium falciparum transmission-blocking monoclonal antibody (TB31F) was demonstrated to be safe and efficacious in malaria-naive volunteers. Here we predict the potential public health impact of large-scale implementation of TB31F alongside existing interventions. We developed a pharmaco-epidemiological model, tailored to 2 settings of differing transmission intensity with already established insecticide-treated nets and seasonal malaria chemoprevention interventions. Community-wide annual administration (at 80% coverage) of TB31F over a 3-year period was predicted to reduce clinical incidence by 54% (381 cases averted per 1000 people per year) in a high-transmission seasonal setting, and 74% (157 cases averted per 1000 people per year) in a low-transmission seasonal setting. Targeting school-aged children gave the largest reduction in terms of cases averted per dose. An annual administration of the transmission-blocking monoclonal antibody TB31F may be an effective intervention against malaria in seasonal malaria settings.

High vector diversity and malaria transmission dynamics in five sentinel sites in Cameroon.

BACKGROUND: Malaria remains one of the main causes of morbidity and mortality in Cameroon. To inform vector control intervention decision making, malaria vector surveillance was conducted monthly from October 2018 to September 2020 in five selected sentinel sites (Gounougou and Simatou in the North, and Bonabéri, Mangoum and Nyabessang in the South). METHODS: Human landing catches (HLCs), U.S. Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSCs) were used to assess vector density, species composition, human biting rate (HBR), endophagic index, indoor resting density (IRD), parity, sporozoite infection rates, entomological inoculation rate (EIR), and Anopheles vectorial capacity. RESULTS: A total of 139,322 Anopheles mosquitoes from 18 species (or 21 including identified sub-species) were collected across all sites. Out of the 18 species, 12 were malaria vectors including Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l.., Anopheles nili, Anopheles moucheti, Anopheles paludis, Anopheles demeilloni, Anopheles. pharoensis, Anopheles ziemanni, Anopheles multicinctus, Anopheles tenebrosus, Anopheles rufipes, and Anopheles marshallii. Anopheles gambiae s.l. remains the major malaria vector (71% of the total Anopheles) collected, though An. moucheti and An. paludis had the highest sporozoite rates in Nyabessang. The mean indoor HBR of Anopheles ranged from 11.0 bites/human/night (b/h/n) in Bonabéri to 104.0 b/h/n in Simatou, while outdoors, it varied from 24.2 b/h/n in Mangoum to 98.7 b/h/n in Simatou. Anopheles gambiae s.l. and An. moucheti were actively biting until at least 8:00 a.m. The mean Anopheles IRD was 17.1 females/room, and the parity rate was 68.9%. The mean EIRs for each site were 55.4 infective bites/human/month (ib/h/m) in Gounougou, 99.0 ib/h/m in Simatou, 51.2 ib/h/m in Mangoum, 24.4 ib/h/m in Nyabessang, and 18.1 ib/h/m in Bonabéri. Anopheles gambiae s.l. was confirmed as the main malaria vector with the highest vectorial capacity in all sites based on sporozoite rate, except in Nyabessang. CONCLUSION: These findings highlight the high malaria transmission occurring in Cameroon and will support the National Malaria Control Program to design evidence-based malaria vector control strategies, and deployment of effective and integrated vector control interventions to reduce malaria transmission and burden in Cameroon, where several Anopheles species could potentially maintain year-round transmission.

Socio-demographic trends in malaria knowledge and implications for behaviour change interventions in Zanzibar.

BACKGROUND: Zanzibar is among the few places within East Africa that have documented a significant reduction of malaria morbidity and mortality. Despite tremendous gains over the past decade, malaria transmission still persists in Zanzibar. This study aimed at understanding levels of malaria knowledge to provide recommendations that can be used to reinforce and scale up targeted malaria social and behaviour change interventions. METHODS: A descriptive cross-sectional survey was conducted through an administered questionnaire to 431 households selected randomly. The interviewees were the heads of household or representative adults above 18 years. This study investigated the levels of knowledge about the causes, symptoms, and prevention of malaria in areas with high (> 1.9 per 1000) and low (< 1 per 1000) incidence of local malaria cases. The Principal Component Analysis (PCA) was used to compute the composite variable of each category. Descriptive statistics were calculated to understand variables of interest between low and high transmission areas. Multinomial logistic regression model was used to compare knowledge on malaria based on key variables. RESULTS: A total of 431 heads of households were interviewed. Respondent age, education level, and wealth status were significantly associated with variations in level of malaria knowledge. Old age was found to be significantly associated with low knowledge of malaria (P < 0.001). The majority of study participants who had secondary and higher education levels had good knowledge of malaria (P < 0.006). Participants characterized as middle-income had good knowledge compared to those characterized as low-income (P < 0.001). CONCLUSION: The study identified existing gaps in malaria knowledge in low and high transmission areas. Low levels of malaria knowledge were documented among elderly and populations with lower education and income levels. There is a need to extend mobilization, advocacy, and expand channels of communication to reach all community members. The reported gaps in knowledge are important to consider when designing strategies to engage communities in malaria elimination in Zanzibar. Tailored social and behavioural change interventions aiming to increase malaria knowledge could enhance the uptake of malaria prevention services in the community.

Utilization of insecticide-treated nets and associated factors among childbearing women in Northern Nigeria.

BACKGROUND: Studies have explored the correlates of insecticide-treated nets in Nigeria. The few studies that focused on Northern Nigeria mostly examined individual correlates, but largely ignored the community correlates. Also, the persistence of armed insurgencies in the region calls for more research attention. This study examines the utilization and the associated individual and community factors of insecticide-treated nets in Northern Nigeria. METHODS: The study adopted a cross-sectional design. Data were extracted from the 2021 Nigeria Malaria Indicator Survey (NMIS). A weighted sample size of 6873 women was analysed. The outcome variable was the utilization of insecticide-treated nets. The explanatory variables selected at the individual/household level were maternal age, maternal education, parity, religion, sex of head of household, household wealth, and household size. The variables selected at the community level were the type of place of residence, geo-political zone of residence, the proportion of children under five who slept under a bed net, the proportion of women aged 15-49 who heard malaria media messages, and the community literacy level. Two variables, namely, the number of mosquito bed nets in the household, and the number of rooms used for sleeping were included for statistical control. Three multilevel mixed-effect regression models were fitted. RESULTS: The majority of childbearing women (71.8%) utilized insecticide-treated nets. Parity and household size were the significant individual/household characteristics associated with the utilization of insecticide-treated nets. The proportion of under-five children in the community who slept under mosquito bed nets, and the geopolitical zone of residence were significant community correlates of the use of insecticide-treated nets. In addition, the number of rooms for sleeping, and the number of mosquito bed nets in the households were significantly associated with the utilization of insecticide-treated nets. CONCLUSION: Parity, household size, number of sleeping rooms, number of treated bed nets, geo-political zone of residence, and proportion of under-five children sleeping under bed nets are important associated factors of the utilization of insecticide-treated nets in Northern Nigeria. Existing malaria preventive initiatives should be strengthened to target these characteristics.

Clinical isolates of uncomplicated falciparum malaria from high and low malaria transmission areas show distinct pfcrt and pfmdr1 polymorphisms in western Ethiopia.

BACKGROUND: Pfcrt gene has been associated with chloroquine resistance and the pfmdr1 gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether-lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria, pfcrt haplotype, and pfmdr1 single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission. METHODS: 230 microscopically confirmed P. falciparum isolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes and pfmdr1 SNPs. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A P-value of less or equal to 0.05 was considered significant. RESULTS: Of the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM for pfcrt haplotype, pfmdr1-86, pfmdr1-184, pfmdr1-1042 and pfmdr1-1246, respectively. The mutant pfcrt haplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively. Plasmodium falciparum with chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4, P = 0.00). Pfmdr1-N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at the pfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codons N86Y-Y184F-D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution of pfmdr1 SNPs, haplotypes, and CNV between the two study sites (P > 0.05). CONCLUSION: Plasmodium falciparum with the pfcrt wild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y-Y184F-D1246Y. A continuous investigation is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT.

Anopheles vector distribution and malaria transmission dynamics in Gbêkê region, central Côte d'Ivoire.

BACKGROUND: A better understanding of vector distribution and malaria transmission dynamics at a local scale is essential for implementing and evaluating effectiveness of vector control strategies. Through the data gathered in the framework of a cluster randomized controlled trial (CRT) evaluating the In2Care (Wageningen, Netherlands) Eave Tubes strategy, the distribution of the Anopheles vector, their biting behaviour and malaria transmission dynamics were investigated in Gbêkê region, central Côte d'Ivoire. METHODS: From May 2017 to April 2019, adult mosquitoes were collected monthly using human landing catches (HLC) in twenty villages in Gbêkê region. Mosquito species wereidentified morphologically. Monthly entomological inoculation rates (EIR) were estimated by combining the HLC data with mosquito sporozoite infection rates measured in a subset of Anopheles vectors using PCR. Finally, biting rate and EIR fluctuations were fit to local rainfall data to investigate the seasonal determinants of mosquito abundance and malaria transmission in this region. RESULTS: Overall, Anopheles gambiae, Anopheles funestus, and Anopheles nili were the three vector complexes found infected in the Gbêkê region, but there was a variation in Anopheles vector composition between villages. Anopheles gambiae was the predominant malaria vector responsible for 84.8% of Plasmodium parasite transmission in the area. An unprotected individual living in Gbêkê region received an average of 260 [222-298], 43.5 [35.8-51.29] and 3.02 [1.96-4] infected bites per year from An. gambiae, An. funestus and An. nili, respectively. Vector abundance and malaria transmission dynamics varied significantly between seasons and the highest biting rate and EIRs occurred in the months of heavy rainfall. However, mosquitoes infected with malaria parasites remained present in the dry season, despite the low density of mosquito populations. CONCLUSION: These results demonstrate that the intensity of malaria transmission is extremely high in Gbêkê region, especially during the rainy season. The study highlights the risk factors of transmission that could negatively impact current interventions that target indoor control, as well as the urgent need for additional vector control tools to target the population of malaria vectors in Gbêkê region and reduce the burden of the disease.

Stakeholder perspectives on a door-to-door intervention to increase community engagement for malaria elimination in Zanzibar.

BACKGROUND: Malaria remains a major public health problem in sub-Saharan Africa. The 2021 World Health Organization (WHO) World Malaria Report indicates a slowing in the decline of malaria incidence since 2015. Malaria prevalence in Zanzibar has been maintained at less than 1% since 2010, however from 2018 to 2021, the annual number of reported malaria cases has gradually increased from 4106 to 9290. Community engagement has been emphasized by the WHO for reducing malaria transmission. To better understand the potential for a door-to-door approach for malaria, a three-month pilot programme was carried out. This qualitative study aimed at understanding stakeholder experiences with the pilot programme and considerations for its implementation. METHODS: Through multistage sampling, four shehias (wards-the lowest administrative structure) with comparatively high (> 1.9 per 1000) and four with low (< 1 per 1000) incidence of local malaria cases were selected and involved in a door-to-door pilot intervention. The qualitative study was conducted after the pilot intervention and employed focus group discussions and in-depth interviews. All field notes were written on paper and audiotaped using digital audio-recorders. Summaries were developed by integrating field notes with reviews of recordings; themes were developed based on the topics identified a priori. Responses for each theme were summarized using an iterative process. RESULTS: Most community members reported high levels of acceptance of door-to-door interventions. Some factors that might affect implementation of door-to-door include, low risk perception of the disease, local beliefs and practice, lack of initiative from the programme level to involve communities, and political instability during the election period. All Community Health Volunteers (CHVs) recommended this approach for community engagement, however, ensuring adequate resources was identified as a key factor for ensuring its sustainability. CONCLUSION: The door-to-door intervention was perceived as helpful for promoting community engagement. There are several factors to consider including ensuring that CHVs are provided with adequate education, regular supervision, and have access to essential resources. Community leaders should be fully involved in choosing CHVs that are acceptable to the community. To ensure sustainability, the government should allocate sufficient resources and improve coordination systems.

Exploring activities and behaviours potentially increases school-age children's vulnerability to malaria infections in south-eastern Tanzania.

BACKGROUND: Strengthening malaria control activities in Tanzania has dramatically declined human malaria infections. However, there is an increasing epidemiological shift in the burden on school-age children. The underlying causes for such an epidemiological shift remain unknown in this context. This study explored activities and behaviours that could increase the vulnerability of school-age children to transmission risk to provide insight into protection gap with existing interventions and opportunities for supplementary interventions. METHODS: This cross-sectional study conducted twenty-four focus group discussions (FGDs) in three districts of Rufiji, Kibiti and Kilwa in south-eastern Tanzania. Sixteen FGDs worked with school-age children (13 to 18 years) separating girls and boys and eight FGDs with their parents in mixed-gender groups. A total of 205 community members participated in FGDs across the study area. Of them, 72 participants were parents, while 133 were school-age children (65 boys and 68 girls). RESULTS: Routine domestic activities such as fetching water, washing kitchen utensils, cooking, and recreational activities such as playing and watching television and studying were the reported activities that kept school-age children outdoors early evening to night hours (between 18:00 and 23:00). Likewise, the social and cultural events including initiation ceremonies and livelihood activities also kept this age group outdoors from late evening to early night and sometimes past midnight hours. Parents migrating to farms from December to June, leaving behind school-age children unsupervised affecting their net use behaviour plus spending more time outdoors at night, and the behaviour of children sprawling legs and hands while sleeping inside treated bed nets were identified as potential risks to infectious mosquito bites. CONCLUSION: The risky activities, behaviours, and social events mostly occurring outdoors might increase school-age children's vulnerability to malaria infections. The findings provide preliminary insight on potential risk factors for persisting transmission. Further studies to quantify the risk behaviour and activities are recommended to establish the magnitude and anticipated impact on supplementary control strategies to control infection in school-age children.

Implementation strategies for the introduction of the RTS,S/AS01 (RTS,S) malaria vaccine in countries with areas of highly seasonal transmission: workshop meeting report.

A workshop on implementation strategies for the introduction of the RTS,S/AS01 (RTS,S) malaria vaccine in countries with areas of highly seasonal transmission, was held as a hybrid meeting in Dakar, Senegal, and online, 23-25 January 2023. Delegates from Expanded Programmes on Immunization (EPI) and National Malaria Control Programmes (NMCPs) from 13 African countries, and representatives from key stakeholders participated. RTS,S is the first malaria vaccine to be recommended by the World Health Organization (WHO). The recommendation followed pilot implementation of the vaccine in Ghana, Kenya and Malawi, which showed that introduction of the vaccine was highly effective at scale, and was associated with a 30% reduction in hospital admissions with severe malaria in age groups eligible to have received the vaccine and no evidence of the safety signals that had been observed in the phase 3 trial. Clinical trials in Mali and Burkina Faso, showed that in children receiving Seasonal Malaria Chemoprevention (SMC), providing the vaccine just prior to high transmission seasons, matching the period of highest efficacy to the peak transmission season, resulted in substantial reduction in the incidence of clinical malaria and of severe malaria. While SMC has been successfully scaled-up despite the challenges of delivery, there is no established platform for seasonal vaccine delivery and no real-world experience. The objectives of this workshop were, therefore, to share experiences from countries that have introduced the RTS,S vaccine in routine child vaccination programmes, with SMC-implementing countries as they consider malaria vaccine introduction, and to explore implementation strategies in countries with seasonal transmission and where EPI coverage may be low especially in the second year of life. Practical implementation challenges, lessons learned for vaccine introduction, and research questions, towards facilitating the introduction of the RTS,S (and other malaria vaccines) in countries with seasonal malaria transmission were discussed.

Mosquito bites and stage-specific antibody responses against Plasmodium falciparum in southern Ghana.

BACKGROUND: The human host elicits specific immune responses after exposure to various life stages of the malaria parasite as well as components of mosquito saliva injected into the host during a mosquito bite. This study describes differences in IgG responses against antigens derived from the sporozoite (PfCSP), asexual stage parasite (PfEBA175) and the gametocyte (Pfs230), in addition to an Anopheles gambiae salivary gland antigen (gSG6-P1), in two communities in Ghana with similar blood stage malaria parasite prevalence. METHODS: This study used archived plasma samples collected from an earlier cross-sectional study that enrolled volunteers aged from 6 months to 70 years from Simiw, peri-urban community (N = 347) and Obom, rural community (N = 291). An archived thick and thin blood smear for microscopy was used for the estimation of Plasmodium parasite density and species and DNA extraction from blood spots and P. falciparum confirmation was performed using PCR. This study used the stored plasma samples to determine IgG antibody levels to P. falciparum and Anopheles salivary antigens using indirect ELISA. RESULTS: Individuals from Simiw had significantly higher levels of IgG against mosquito gSG6-P1 [median (95%CI)] [2.590 (2.452-2.783) ng/mL] compared to those from Obom [2.119 (1.957-2.345) ng/mL], p < 0.0001. Both IgG responses against Pfs230proC (p = 0.0006), and PfCSP (p = 0.002) were significantly lower in volunteers from Simiw compared to the participants from Obom. The seroprevalence of PfEBA-175.5R (p = 0.8613), gSG6-P1 (p = 0.0704), PfCSP (p = 0.7798) IgG were all similar in Obom and Simiw. However, Pfs230 seroprevalence was significantly higher at Obom compared to Simiw (p = 0.0006). Spearman correlation analysis showed no significant association between IgG responses against gSG6-P1, PfCSP, Pfs230proC and PfEBA-175.5R and parasite density at both Obom and Simiw (p > 0.05). CONCLUSION: In conclusion, the study showed that participants from Simiw had higher concentrations of circulating gSG6-P1 IgG antibodies but lower concentrations of P. falciparum antibodies, PfCSP IgG and Pfs230proC IgG compared to participants from Obom.

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers.

BACKGROUND: Sickle cell trait (SCT) refers to the carriage of one abnormal copy of the ß-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas. METHODS: The study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source. RESULTS: Among the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29-1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype. CONCLUSION: Plasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite's enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance.

BACKGROUND: The impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos' propensity to transmit Plasmodium spp. METHODS: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes. RESULTS: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1-5.2), p = 0.03; IRR = 2.5, 95% CI (1.2-5.8), p = 0.01 respectively). CONCLUSION: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda.

Achieving malaria elimination requires a better understanding of the transmissibility of human infections in different transmission settings. This study aimed to characterize the human infectious reservoir in a high endemicity setting in eastern Uganda, using gametocyte quantification and mosquito feeding assays. In asymptomatic infections, gametocyte densities were positively associated with the proportion of infected mosquitoes (ß = 1.60; 95% CI, 1.32-1.92; P < .0001). Combining transmissibility and abundance in the population, symptomatic and asymptomatic infections were estimated to contribute to 5.3% and 94.7% of the infectious reservoir, respectively. School-aged children (5-15 years old) contributed to 50.4% of transmission events and were important drivers of malaria transmission.