Insecticide susceptibility status of Anopheles gambiae mosquitoes and the effect of pre-exposure to a piperonyl butoxide (PBO) synergist on resistance to deltamethrin in northern Namibia.

BACKGROUND: Pyrethroid-based indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been employed as key vector control measures against malaria in Namibia. However, pyrethroid resistance in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the World Health Organization (WHO) recommends the use of piperonyl butoxide (PBO) LLINs in areas where pyrethroid resistance is confirmed to be mediated by mixed function oxidase (MFO). METHODS: This study assessed the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. Additionally, the study explored the effect of piperonyl butoxide (PBO) synergist by sequentially exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin (0.05%), and PBO alone. The Anopheles mosquitoes were further identified morphologically and molecularly. RESULTS: The findings revealed that An. gambiae sensu stricto (s.s.) (62%) was more prevalent than Anopheles arabiensis (38%). The WHO tube bioassays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79, 86, and 67%, respectively. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality). Notably, there was reduced susceptibility to DDT 4% in both An. gambiae s.s. and An. arabiensis from the Kavango West region. Subsequently, a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis in Oshana (84.4%) and Oshikoto (73.6%), and 0.42% of Anopheles quadriannulatus in Oshana. Non-amplifiers were also present (15.2% in Oshana; 26.4% in Oshikoto). Deltamethrin resistance with less than 95% mortality, was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. Following pre-exposure to the PBO synergist, susceptibility to deltamethrin was fully restored with 100.0% mortality at all sites in 2020 and 2021. CONCLUSIONS: Pyrethroid resistance has been identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions, indicating potential challenges for pyrethroid-based IRS and LLINs. Consequently, the data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region.

Effectiveness of reactive focal mass drug administration and reactive focal vector control to reduce malaria transmission in the low malaria-endemic setting of Namibia: a cluster-randomised controlled, open-label, two-by-two factorial design trial.

BACKGROUND: In low malaria-endemic settings, screening and treatment of individuals in close proximity to index cases, also known as reactive case detection (RACD), is practised for surveillance and response. However, other approaches could be more effective for reducing transmission. We aimed to evaluate the effectiveness of reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) in the low malaria-endemic setting of Zambezi (Namibia). METHODS: We did a cluster-randomised controlled, open-label trial using a two-by-two factorial design of 56 enumeration area clusters in the low malaria-endemic setting of Zambezi (Namibia). We randomly assigned these clusters using restricted randomisation to four groups: RACD only, rfMDA only, RAVC plus RACD, or rfMDA plus RAVC. RACD involved rapid diagnostic testing and treatment with artemether-lumefantrine and single-dose primaquine, rfMDA involved presumptive treatment with artemether-lumefantrine, and RAVC involved indoor residual spraying with pirimiphos-methyl. Interventions were administered within 500 m of index cases. To evaluate the effectiveness of interventions targeting the parasite reservoir in humans (rfMDA vs RACD), in mosquitoes (RAVC vs no RAVC), and in both humans and mosquitoes (rfMDA plus RAVC vs RACD only), an intention-to-treat analysis was done. For each of the three comparisons, the primary outcome was the cumulative incidence of locally acquired malaria cases. This trial is registered with ClinicalTrials.gov, number NCT02610400. FINDINGS: Between Jan 1, 2017, and Dec 31, 2017, 55 enumeration area clusters had 1118 eligible index cases that led to 342 interventions covering 8948 individuals. The cumulative incidence of locally acquired malaria was 30·8 per 1000 person-years (95% CI 12·8-48·7) in the clusters that received rfMDA versus 38·3 per 1000 person-years (23·0-53·6) in the clusters that received RACD; 30·2 per 1000 person-years (15·0-45·5) in the clusters that received RAVC versus 38·9 per 1000 person-years (20·7-57·1) in the clusters that did not receive RAVC; and 25·0 per 1000 person-years (5·2-44·7) in the clusters that received rfMDA plus RAVC versus 41·4 per 1000 person-years (21·5-61·2) in the clusters that received RACD only. After adjusting for imbalances in baseline and implementation factors, the incidence of malaria was lower in clusters receiving rfMDA than in those receiving RACD (adjusted incidence rate ratio 0·52 [95% CI 0·16-0·88], p=0·009), lower in clusters receiving RAVC than in those that did not (0·48 [0·16-0·80], p=0·002), and lower in clusters that received rfMDA plus RAVC than in those receiving RACD only (0·26 [0·10-0·68], p=0·006). No serious adverse events were reported. INTERPRETATION: In a low malaria-endemic setting, rfMDA and RAVC, implemented alone and in combination, reduced malaria transmission and should be considered as alternatives to RACD for elimination of malaria. FUNDING: Novartis Foundation, Bill & Melinda Gates Foundation, and Horchow Family Fund.

Community acceptance of reactive focal mass drug administration and reactive focal vector control using indoor residual spraying, a mixed-methods study in Zambezi region, Namibia.

BACKGROUND: In Namibia, as in many malaria elimination settings, reactive case detection (RACD), or malaria testing and treatment around index cases, is a standard intervention. Reactive focal mass drug administration (rfMDA), or treatment without testing, and reactive focal vector control (RAVC) in the form of indoor residual spraying, are alternative or adjunctive interventions, but there are limited data regarding their community acceptability. METHODS: A parent trial aimed to compare the effectiveness of rfMDA versus RACD, RAVC versus no RAVC, and rfMDA + RAVC versus RACD only. To assess acceptability of these interventions, a mixed-methods study was conducted using key informant interviews (KIIs) and focus group discussions (FGDs) in three rounds (pre-trial and in years 1 and 2 of the trial), and an endline survey. RESULTS: In total, 17 KIIs, 49 FGDs were conducted with 449 people over three annual rounds of qualitative data collection. Pre-trial, community members more accurately predicted the level of community acceptability than key stakeholders. Throughout the trial, key participant motivators included: malaria risk perception, access to free community-based healthcare and IRS, and community education by respectful study teams. RACD or rfMDA were offered to 1372 and 8948 individuals in years 1 and 2, respectively, and refusal rates were low (< 2%). RAVC was offered to few households (n = 72) in year 1. In year 2, RAVC was offered to more households (n = 944) and refusals were < 1%. In the endline survey, 94.3% of 2147 respondents said they would participate in the same intervention again. CONCLUSIONS: Communities found both reactive focal interventions and their combination highly acceptable. Engaging communities and centering and incorporating their perspectives and experiences during design, implementation, and evaluation of this community-based intervention was critical for optimizing study engagement.

Children with Plasmodium vivax infection previously observed in Namibia, were Duffy negative and carried a c.136G > A mutation.

BACKGROUND: In a previous study, using a molecular approach, we reported the presence of P. vivax in Namibia. Here, we have extended our investigation to the Duffy antigen genetic profile of individuals of the same cohort with and without Plasmodium infections. METHODS: Participants with P. vivax (n = 3), P. falciparum (n = 23) mono-infections and co-infections of P. vivax/P. falciparum (n = 4), and P. falciparum/P. ovale (n = 3) were selected from seven regions. Participants with similar age but without any Plasmodium infections (n = 47) were also selected from all the regions. Duffy allelic profile was examined using standard PCR followed by sequencing of amplified products. Sequenced samples were also examined for the presence or absence of G125A mutation in codon 42, exon 2. RESULTS: All individuals tested carried the - 67 T > C mutation. However, while all P. vivax infected participants carried the c.G125A mutation, 7/28 P. falciparum infected participants and 9/41 of uninfected participants did not have the c.G125A mutation. The exon 2 region surrounding codon 42, had a c.136G > A mutation that was present in all P. vivax infections. The odds ratio for lack of this mutation with P. vivax infections was (OR 0.015, 95% CI 0.001-0.176; p = 0.001). CONCLUSION: We conclude that P. vivax infections previously reported in Namibia, occurred in Duffy negative participants, carrying the G125A mutation in codon 42. The role of the additional mutation c.136 G > A in exon 2 in P. vivax infections, will require further investigations.

District-level approach for tailoring and targeting interventions: a new path for malaria control and elimination.

Despite huge investments and implementation of effective interventions for malaria, progress has stalled, with transmission being increasingly localized among difficult-to-reach populations and outdoor-biting vectors. Targeting difficult pockets of transmission will require the development of tailored and targeted approaches suited to local context, drawing from insights close to the frontlines. Districts are best placed to develop tailored, locally appropriate approaches. We propose a reorganization of how malaria services are delivered. Firstly, enabling district health officers to serve as conduits between technical experts in national malaria control programmes and local community leaders with knowledge specific to local, at-risk populations; secondly, empowering district health teams to make malaria control decisions. This is a radical shift that requires the national programme to cede some control. Shifting towards a district or provincial level approach will necessitate deliberate planning, and repeated, careful assessment, starting with piloting and learning through experience. Donors will need to alter current practice, allowing for flexible funding to be controlled at sub-national levels, and to mix finances between case management, vector control and surveillance, monitoring and evaluation. System-wide changes proposed are challenging but may be necessary to overcome stalled progress in malaria control and elimination and introduce targeted interventions tailored to the needs of diverse malaria affected populations.

Subpatent malaria in a low transmission African setting: a cross-sectional study using rapid diagnostic testing (RDT) and loop-mediated isothermal amplification (LAMP) from Zambezi region, Namibia.

BACKGROUND: Subpatent malaria infections, or low-density infections below the detection threshold of microscopy or standard rapid diagnostic testing (RDT), can perpetuate persistent transmission and, therefore, may be a barrier for countries like Namibia that are pursuing malaria elimination. This potential burden in Namibia has not been well characterized. METHODS: Using a two-stage cluster sampling, cross-sectional design, subjects of all age were enrolled during the end of the 2015 malaria transmission season in Zambezi region, located in northeast Namibia. Malaria RDTs were performed with subsequent gold standard testing by loop-mediated isothermal amplification (LAMP) using dried blood spots. Infection prevalence was measured and the diagnostic accuracy of RDT calculated. Relationships between recent fever, demographics, epidemiological factors, and infection were assessed. RESULTS: Prevalence of Plasmodium falciparum malaria infection was low: 0.8% (16/1919) by RDT and 2.2% (43/1919) by LAMP. All but one LAMP-positive infection was RDT-negative. Using LAMP as gold standard, the sensitivity and specificity of RDT were 2.3% and 99.2%, respectively. Compared to LAMP-negative infections, a higher portion LAMP-positive infections were associated with fever (45.2% vs. 30.4%, p = 0.04), though 55% of infections were not associated with fever. Agricultural occupations and cattle herding were significantly associated with LAMP-detectable infection (Adjusted ORs 5.02, 95% CI 1.77-14.23, and 11.82, 95% CI 1.06-131.81, respectively), while gender, travel, bed net use, and indoor residual spray coverage were not. CONCLUSIONS: This study presents results from the first large-scale malaria cross-sectional survey from Namibia using molecular testing to characterize subpatent infections. Findings suggest that fever history and standard RDTs are not useful to address this burden. Achievement of malaria elimination may require active case detection using more sensitive point-of-care diagnostics or presumptive treatment and targeted to high-risk groups.

Malaria risk in young male travellers but local transmission persists: a case-control study in low transmission Namibia.

BACKGROUND: A key component of malaria elimination campaigns is the identification and targeting of high risk populations. To characterize high risk populations in north central Namibia, a prospective health facility-based case-control study was conducted from December 2012-July 2014. Cases (n = 107) were all patients presenting to any of the 46 health clinics located in the study districts with a confirmed Plasmodium infection by multi-species rapid diagnostic test (RDT). Population controls (n = 679) for each district were RDT negative individuals residing within a household that was randomly selected from a census listing using a two-stage sampling procedure. Demographic, travel, socio-economic, behavioural, climate and vegetation data were also collected. Spatial patterns of malaria risk were analysed. Multivariate logistic regression was used to identify risk factors for malaria. RESULTS: Malaria risk was observed to cluster along the border with Angola, and travel patterns among cases were comparatively restricted to northern Namibia and Angola. Travel to Angola was associated with excessive risk of malaria in males (OR 43.58 95% CI 2.12-896), but there was no corresponding risk associated with travel by females. This is the first study to reveal that gender can modify the effect of travel on risk of malaria. Amongst non-travellers, male gender was also associated with a higher risk of malaria compared with females (OR 1.95 95% CI 1.25-3.04). Other strong risk factors were sleeping away from the household the previous night, lower socioeconomic status, living in an area with moderate vegetation around their house, experiencing moderate rainfall in the month prior to diagnosis and living <15 km from the Angolan border. CONCLUSIONS: These findings highlight the critical need to target malaria interventions to young male travellers, who have a disproportionate risk of malaria in northern Namibia, to coordinate cross-border regional malaria prevention initiatives and to scale up coverage of prevention measures such as indoor residual spraying and long-lasting insecticide nets in high risk areas if malaria elimination is to be realized.

Integrating rapid risk mapping and mobile phone call record data for strategic malaria elimination planning.

BACKGROUND: As successful malaria control programmes re-orientate towards elimination, the identification of transmission foci, targeting of attack measures to high-risk areas and management of importation risk become high priorities. When resources are limited and transmission is varying seasonally, approaches that can rapidly prioritize areas for surveillance and control can be valuable, and the most appropriate attack measure for a particular location is likely to differ depending on whether it exports or imports malaria infections. METHODS/RESULTS: Here, using the example of Namibia, a method for targeting of interventions using surveillance data, satellite imagery, and mobile phone call records to support elimination planning is described. One year of aggregated movement patterns for over a million people across Namibia are analyzed, and linked with case-based risk maps built on satellite imagery. By combining case-data and movement, the way human population movements connect transmission risk areas is demonstrated. Communities that were strongly connected by relatively higher levels of movement were then identified, and net export and import of travellers and infection risks by region were quantified. These maps can aid the design of targeted interventions to maximally reduce the number of cases exported to other regions while employing appropriate interventions to manage risk in places that import them. CONCLUSIONS: The approaches presented can be rapidly updated and used to identify where active surveillance for both local and imported cases should be increased, which regions would benefit from coordinating efforts, and how spatially progressive elimination plans can be designed. With improvements in surveillance systems linked to improved diagnosis of malaria, detailed satellite imagery being readily available and mobile phone usage data continually being collected by network providers, the potential exists to make operational use of such valuable, complimentary and contemporary datasets on an ongoing basis in infectious disease control and elimination.

Namibia's path toward malaria elimination: a case study of malaria strategies and costs along the northern border.

BACKGROUND: Low malaria transmission in Namibia suggests that elimination is possible, but the risk of imported malaria from Angola remains a challenge. This case study reviews the early transition of a program shift from malaria control to elimination in three northern regions of Namibia that comprise the Trans-Kunene Malaria Initiative (TKMI): Kunene, Omusati, and Ohangwena. METHODS: Thirty-four key informant interviews were conducted and epidemiological and intervention data were assembled for 1995 to 2013. Malaria expenditure records were collected for each region for 2009, 2010, and 2011, representing the start of the transition from control to elimination. Interviews and expenditure data were analyzed across activity and expenditure type. RESULTS: Incidence has declined in all regions since 2004; cases are concentrated in the border zone. Expenditures in the three study regions have declined, from an average of $6.10 per person at risk per year in 2009 to an average of $3.61 in 2011. The proportion of spending allocated for diagnosis and treatment declined while that for vector control increased. Indoor residual spraying is the main intervention, but coverage varies, related to acceptability, mobility, accessibility, insecticide stockouts and staff shortages. Bed net distribution was scaled up beginning in 2005, assisted by NGO partners in later years, but coverage was highly variable. Distribution of rapid diagnostic tests in 2005 resulted in more accurate diagnosis and can help explain the large decline in cases beginning in 2006; however, challenges in personnel training and supervision remained during the expenditure study period of 2009 to 2011. CONCLUSIONS: In addition to allocating sufficient human resources to vector control activities, developing a greater emphasis on surveillance will be central to the ongoing program shift from control to elimination, particularly in light of the malaria importation challenges experienced in the northern border regions. While overall program resources may continue on a downward trajectory, the program will be well positioned to actively eliminate the remaining foci of malaria if greater resources are allocated toward surveillance efforts.

The receptive versus current risks of Plasmodium falciparum transmission in northern Namibia: implications for elimination.

BACKGROUND: Countries aiming for malaria elimination need to define their malariogenic potential, of which measures of both receptive and current transmission are major components. As Namibia pursues malaria elimination, the importation risks due to cross-border human population movements with higher risk neighboring countries has been identified as a major challenge. Here we used historical and contemporary Plasmodium falciparum prevalence data for Namibia to estimate receptive and current levels of malaria risk in nine northern regions. We explore the potential of these risk maps to support decision-making for malaria elimination in Namibia. METHODS: Age-corrected geocoded community P. falciparum rate PfPR2-10 data from the period 1967-1992 (n = 3,260) and 2009 (n = 120) were modeled separately within a Bayesian model-based geostatistical (MBG) framework. A full Bayesian space-time MBG model was implemented using the 1967-1992 data to make predictions for every five years from 1969 to 1989. These maps were used to compute the maximum mean PfPR2-10 at 5 x 5 km locations in the northern regions of Namibia to estimate receptivity. A separate spatial Bayesian MBG was fitted to the 2009 data to predict current risk of malaria at similar spatial resolution. Using a high-resolution population map for Namibia, population at risk by receptive and current endemicity by region and population adjusted PfPR2-10 by health district were computed. Validations of predictions were undertaken separately for the historical and current risk models. RESULTS: Highest receptive risks were observed in the northern regions of Caprivi, Kavango and Ohangwena along the border with Angola and Zambia. Relative to the receptive risks, over 90% of the 1.4 million people across the nine regions of northern Namibia appear to have transitioned to a lower endemic class by 2009. The biggest transition appeared to have occurred in areas of highest receptive risks. Of the 23 health districts, 12 had receptive PAPfPR2-10 risks of 5% to 18% and accounted for 57% of the population in the north. Current PAPfPR2-10 risks was largely <5% across the study area. CONCLUSIONS: The comparison of receptive and current malaria risks in the northern regions of Namibia show health districts that are most at risk of importation due to their proximity to the relatively higher transmission northern neighbouring countries, higher population and modeled receptivity. These health districts should be prioritized as the cross-border control initiatives are rolled out.

Baseline characterization of entomological drivers of malaria transmission in Namibia: a targeted operational entomological surveillance strategy.

BACKGROUND: Namibia's focus on the elimination of malaria requires an evidence-based strategy directed at understanding and targeting the entomological drivers of malaria transmission. In 2018 and 2019, the Namibia National Vector-borne Diseases Control Program (NVDCP) implemented baseline entomological surveillance based on a question-based approach outlined in the Entomological Surveillance Planning Tool (ESPT). In the present study, we report on the findings of the ESPT-based NVDCP on baseline vector species composition and bionomic traits in malaria endemic regions in northern Namibia, which has the aim of generating an evidence base for programmatic decision-making. METHODS: Nine representative sentinel sites were included in the 2018 entomological surveillance program (Kunene, Omusati, Oshana, Ohangwena, Oshikoto, Otjozondjupa, Kavango West, Kavango East and Zambezi); the number was reduced to four sites in 2019 due to limited funding (Ohangwena, Kavango West, Kavango East, and Zambezi). In the 2018 baseline collections, multiple sampling methods (human landing catches, pyrethroid spray catches, U.S. Centers for Disease Control and Prevention light traps [CDC-LTs], resting boxes [RBs] and larval sampling) were utilized to evaluate indoor/outdoor human biting rates, resting behaviors and insecticide resistance (IR). CDC-LTs and RBs were not used in 2019 due to low and non-representative sampling efficacies. RESULTS: Overall, molecular evidence demonstrated the presence of three primary mosquito vectors, namely Anopheles arabiensis, rediscovered Anopheles gambiae sensu stricto and Anopheles funestus sensu stricto, alongside Anopheles squamosus and members of the Anopheles coustani complex. Vectors were found to bite throughout the night (1800 hours 0600 hours) both indoors and outdoors, with An. arabiensis having the highest biting rates outdoors. Low numbers of indoor resting Anopheles point to possible low indoor residual spraying (IRS) efficacy-with An. arabiensis found to be the major vector species resting indoors. The IR tests demonstrated varying country-wide resistance levels to the insecticide deltamethrin, with the resistance levels confirmed to have increased in 2019, evidence that impacts national programmatic decision-making. Vectors demonstrated susceptibility to the insecticides dichlorodiphenyltrichloroethane, bendiocarb and Actellic 300CS in 2018, with mosquitoes from only one site (Kavango West) demonstrating possible resistance to DDT. Targeted and question-based entomological surveillance enabled a rapid and focused evidence base to be built, showing where and when humans were being bitten and providing entomological data on long-lasting insecticidal nets, IRS efficacy and insecticide resistance, which the Ministry of Health and Social Services-Namibia can use to further build a monitoring and evaluation framework for understanding the drivers of transmission. CONCLUSION: Identification and characterization of species-specific bionomic traits allows for an understanding of where and when vector human contact may occur as well as the potential impact of interventions. Low indoor resting rates as well as the presence of insecticide resistance (and the increase in its frequency) point to the need for mosquito-behavior-directed and appropriate interventions as well as the requirement for a resistance mitigation strategy. The ESPT-based question- and minimal essential indicator-based operational research strategy provides programs with directed and focused data for facilitating decision-making while requiring limited funding and capacity.

Cost and cost effectiveness of reactive case detection (RACD), reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) to reduce malaria in the low endemic setting of Namibia: an analysis alongside a 2×2 factorial design cluster randomised controlled trial.

OBJECTIVES: To estimate the cost and cost effectiveness of reactive case detection (RACD), reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) to reduce malaria in a low endemic setting. SETTING: The study was part of a 2×2 factorial design cluster randomised controlled trial within the catchment area of 11 primary health facilities in Zambezi, Namibia. PARTICIPANTS: Cost and outcome data were collected from the trial, which included 8948 community members that received interventions due to their residence within 500 m of malaria index cases. OUTCOME MEASURES: The primary outcome was incremental cost effectiveness ratio (ICER) per in incident case averted. ICER per prevalent case and per disability-adjusted life years (DALY) averted were secondary outcomes, as were per unit interventions costs and personnel time. Outcomes were compared as: (1) rfMDA versus RACD, (2) RAVC versus no RAVC and (3) rfMDA+RAVC versus RACD only. RESULTS: rfMDA cost 1.1× more than RACD, and RAVC cost 1.7× more than no RAVC. Relative to RACD only, the cost of rfMDA+RAVC was double ($3082 vs $1553 per event). The ICERs for rfMDA versus RACD, RAVC versus no RAVC and rfMDA+RAVC versus RACD only were $114, $1472 and $842, per incident case averted, respectively. Using prevalent infections and DALYs as outcomes, trends were similar. The median personnel time to implement rfMDA was 20% lower than for RACD (30 vs 38 min per person). The median personnel time for RAVC was 34 min per structure sprayed. CONCLUSION: Implemented alone or in combination, rfMDA and RAVC were cost effective in reducing malaria incidence and prevalence despite higher implementation costs in the intervention compared with control arms. Compared with RACD, rfMDA was time saving. Cost and time requirements for the combined intervention could be decreased by implementing rfMDA and RAVC simultaneously by a single team. TRIAL REGISTRATION NUMBER: NCT02610400; Post-results.

Human and vector behaviors determine exposure to Anopheles in Namibia.

BACKGROUND: Although the Republic of Namibia has significantly reduced malaria transmission, regular outbreaks and persistent transmission impede progress towards elimination. Towards an understanding of the protective efficacy, as well as gaps in protection, associated with long-lasting insecticidal nets (LLINs), human and Anopheles behaviors were evaluated in parallel in three malaria endemic regions, Kavango East, Ohangwena and Zambezi, using the Entomological Surveillance Planning Tool to answer the question: where and when are humans being exposed to bites of Anopheles mosquitoes? METHODS: Surveillance activities were conducted during the malaria transmission season in March 2018 for eight consecutive nights. Four sentinel structures per site were selected, and human landing catches and human behavior observations were consented to for a total of 32 collection nights per site. The selected structures were representative of local constructions (with respect to building materials and size) and were at least 100 m from each other. For each house where human landing catches were undertaken, a two-person team collected mosquitoes from 1800 to 0600 hours. RESULTS: Surveillance revealed the presence of the primary vectors Anopheles arabiensis, Anopheles gambiae sensu stricto (s.s.) and Anopheles funestus s.s., along with secondary vectors (Anopheles coustani sensu lato and Anopheles squamosus), with both indoor and outdoor biting behaviors based on the site. Site-specific human behaviors considerably increased human exposure to vector biting. The interaction between local human behaviors (spatial and temporal presence alongside LLIN use) and vector behaviors (spatial and temporal host seeking), and also species composition, dictated where and when exposure to infectious bites occurred, and showed that exposure was primarily indoors in Kavango East (78.6%) and outdoors in Ohangwena (66.7%) and Zambezi (81.4%). Human behavior-adjusted exposure was significantly different from raw vector biting rate. CONCLUSIONS: Increased LLIN use may significantly increase protection and reduce exposure to malaria, but may not be enough to eliminate the disease, as gaps in protection will remain both indoors (when people are awake and not using LLINs) and outdoors. Alternative interventions are required to address these exposure gaps. Focused and question-based operational entomological surveillance together with human behavioral observations may considerably improve our understanding of transmission dynamics as well as intervention efficacy and gaps in protection.

Serological evaluation of the effectiveness of reactive focal mass drug administration and reactive vector control to reduce malaria transmission in Zambezi Region, Namibia: Results from a secondary analysis of a cluster randomised trial.

BACKGROUND: Due to challenges in measuring changes in malaria at low transmission, serology is increasingly being used to complement clinical and parasitological surveillance. Longitudinal studies have shown that serological markers, such as Etramp5.Ag1, can reflect spatio-temporal differences in malaria transmission. However, these markers have yet to be used as endpoints in intervention trials. METHODS: Based on data from a 2017 cluster randomised trial conducted in Zambezi Region, Namibia, evaluating the effectiveness of reactive focal mass drug administration (rfMDA) and reactive vector control (RAVC), this study conducted a secondary analysis comparing antibody responses between intervention arms as trial endpoints. Antibody responses were measured on a multiplex immunoassay, using a panel of eight serological markers of Plasmodium falciparum infection - Etramp5.Ag1, GEXP18, HSP40.Ag1, Rh2.2030, EBA175, PfMSP119, PfAMA1, and PfGLURP.R2. FINDINGS: Reductions in sero-prevalence to antigens Etramp.Ag1, PfMSP119, Rh2.2030, and PfAMA1 were observed in study arms combining rfMDA and RAVC, but only effects for Etramp5.Ag1 were statistically significant. Etramp5.Ag1 sero-prevalence was significantly lower in all intervention arms. Compared to the reference arms, adjusted prevalence ratio (aPR) for Etramp5.Ag1 was 0.78 (95%CI 0.65 - 0.91, p = 0.0007) in the rfMDA arms and 0.79 (95%CI 0.67 - 0.92, p = 0.001) in the RAVC arms. For the combined rfMDA plus RAVC intervention, aPR was 0.59 (95%CI 0.46 - 0.76, p < 0.0001). Significant reductions were also observed based on continuous antibody responses. Sero-prevalence as an endpoint was found to achieve higher study power (99.9% power to detect a 50% reduction in prevalence) compared to quantitative polymerase chain reaction (qPCR) prevalence (72.9% power to detect a 50% reduction in prevalence). INTERPRETATION: While the observed relative reduction in qPCR prevalence in the study was greater than serology, the use of serological endpoints to evaluate trial outcomes measured effect size with improved precision and study power. Serology has clear application in cluster randomised trials, particularly in settings where measuring clinical incidence or infection is less reliable due to seasonal fluctuations, limitations in health care seeking, or incomplete testing and reporting. FUNDING: This study was supported by Novartis Foundation (A122666), the Bill & Melinda Gates Foundation (OPP1160129), and the Horchow Family Fund (5,300,375,400).

Malaria risk factors in northern Namibia: The importance of occupation, age and mobility in characterizing high-risk populations.

In areas of low and unstable transmission, malaria cases occur in populations with lower access to malaria services and interventions, and in groups with specific malaria risk exposures often away from the household. In support of the Namibian National Vector Borne Disease Program's drive to better target interventions based upon risk, we implemented a health facility-based case control study aimed to identify risk factors for symptomatic malaria in Zambezi Region, northern Namibia. A total of 770 febrile individuals reporting to 6 health facilities and testing positive by rapid diagnostic test (RDT) between February 2015 and April 2016 were recruited as cases; 641 febrile individuals testing negative by RDT at the same health facilities through June 2016 were recruited as controls. Data on socio-demographics, housing construction, overnight travel, use of malaria prevention and outdoor behaviors at night were collected through interview and recorded on a tablet-based questionnaire. Remotely-sensed environmental data were extracted for geo-located village residence locations. Multivariable logistic regression was conducted to identify risk factors and latent class analyses (LCA) used to identify and characterize high-risk subgroups. The majority of participants (87% of cases and 69% of controls) were recruited during the 2016 transmission season, an outbreak year in Southern Africa. After adjustment, cases were more likely to be cattle herders (Adjusted Odds Ratio (aOR): 4.46 95%CI 1.05-18.96), members of the police or other security personnel (aOR: 4.60 95%CI: 1.16-18.16), and pensioners/unemployed persons (aOR: 2.25 95%CI 1.24-4.08), compared to agricultural workers (most common category). Children (aOR 2.28 95%CI 1.13-4.59) and self-identified students were at higher risk of malaria (aOR: 4.32 95%CI 2.31-8.10). Other actionable risk factors for malaria included housing and behavioral characteristics, including traditional home construction and sleeping in an open structure (versus modern structure: aOR: 2.01 95%CI 1.45-2.79 and aOR: 4.76 95%CI: 2.14-10.57); cross border travel in the prior 30 days (aOR: 10.55 95%CI 2.94-37.84); and outdoor agricultural work at night (aOR: 2.09 95%CI 1.12-3.87). Malaria preventive activities were all protective and included personal use of an insecticide treated net (ITN) (aOR: 0.61 95%CI 0.42-0.87), adequate household ITN coverage (aOR: 0.63 95%CI 0.42-0.94), and household indoor residual spraying (IRS) in the past year (versus never sprayed: (aOR: 0.63 95%CI 0.44-0.90). A number of environmental factors were associated with increased risk of malaria, including lower temperatures, higher rainfall and increased vegetation for the 30 days prior to diagnosis and residing more than 5 minutes from a health facility. LCA identified six classes of cases, with class membership strongly correlated with occupation, age and select behavioral risk factors. Use of ITNs and IRS coverage was similarly low across classes. For malaria elimination these high-risk groups will need targeted and tailored intervention strategies, for example, by implementing alternative delivery methods of interventions through schools and worksites, as well as the use of specific interventions that address outdoor transmission.

Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria.

BACKGROUND: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution. METHODS AND FINDINGS: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations. CONCLUSIONS: Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.

Molecular detection of P. vivax and P. ovale foci of infection in asymptomatic and symptomatic children in Northern Namibia.

BACKGROUND: Knowledge of the foci of Plasmodium species infections is critical for a country with an elimination agenda. Namibia is targeting malaria elimination by 2020. To support decision making regarding targeted intervention, we examined for the first time, the foci of Plasmodium species infections and regional prevalence in northern Namibia, using nested and quantitative polymerase chain reaction (PCR) methods. METHODS: We used cross-sectional multi-staged sampling to select 952 children below 9 years old from schools and clinics in seven districts in northern Namibia, to assess the presence of Plasmodium species. RESULTS: The median participant age was 6 years (25-75%ile 4-8 y). Participants had a median hemoglobin of 12.0 g/dL (25-75%ile 11.1-12.7 g/dL), although 21% of the cohort was anemic, with anemia being severer in the younger population (p<0.002). Most of children with Plasmodium infection were asymptomatic (63.4%), presenting a challenge for elimination. The respective parasite prevalence for Plasmodium falciparum (Pf), Plasmodium vivax (Pv) and Plasmodium ovale curtisi (Po) were (4.41%, 0.84% and 0.31%); with Kavango East and West (10.4%, 6.19%) and Ohangwena (4.5%) having the most prevalence. Pv was localized in Ohangwena, Omusati and Oshana, while Po was found in Kavango. All children with Pv/Pf coinfections in Ohangwena, had previously visited Angola, affirming that perennial migrations are risks for importation of Plasmodium species. The mean hemoglobin was lower in those with Plasmodium infection compared to those without (0.96 g/dL less, 95%CI 0.40-1.52 g/dL less, p = 0.0009) indicating that quasi-endemicity exists in the low transmission setting. CONCLUSIONS: We conclude that Pv and Po species are present in northern Namibia. Additionally, the higher number of asymptomatic infections present challenges to the efforts at elimination for the country. Careful planning, coordination with neighboring Angola and execution of targeted active intervention, will be required for a successful elimination agenda.

Glucose-6-phosphate dehydrogenase deficiency genotypes and allele frequencies in the Kavango and Zambezi regions of northern Namibia.

BACKGROUND: Namibia has made significant gains in the fight against malaria, with a target of elimination by 2023. We examined the genotype and allele frequencies of glucose-6-phosphate dehydrogenase (G6PD) deficiency to inform decisions on primaquine use, as we recently detected clusters of Plasmodium ovale curtisi in Kavango. METHODS: A multistaged cross-sectional sampling method was used to enrol 212 children 2-9 y of age from schools and clinics in the Okavango and Zambezi regions of northern Namibia. Genotypes for the 202 G→A and 376 A→G mutations were assigned by polymerase chain reaction restriction fragment length polymorphism. RESULTS: Of the 212 subjects enrolled, genotypes were available for 210, made up of 61 males and 149 females. G6PD-deficient males (hemizygotes) and females (homozygotes) constituted 3.27% (2/61) and 0.0% (0/149), respectively. Female heterozygotes (AA- and BA-) constituted 10.07% (15/149), while G6PD wild-type males (with A or B haplotype) and females (with AA, BB or AB haplotypes) consisted of 96.72% (59/61) and 89.93% (134/149), respectively. The A-, A and B allele frequencies were 0.0474, 0.3036 and 0.6490, respectively. Hardy-Weinberg equilibrium tests for female genotype frequencies did not show deviation (p=0.29). CONCLUSIONS: The frequency of G6PD deficiency alleles in males in the Kavango and Zambezi regions of northern Namibia constitute 3.27%, a first report to inform policy on primaquine role out.

Study protocol for a cluster randomised controlled factorial design trial to assess the effectiveness and feasibility of reactive focal mass drug administration and vector control to reduce malaria transmission in the low endemic setting of Namibia.

INTRODUCTION: To interrupt malaria transmission, strategies must target the parasite reservoir in both humans and mosquitos. Testing of community members linked to an index case, termed reactive case detection (RACD), is commonly implemented in low transmission areas, though its impact may be limited by the sensitivity of current diagnostics. Indoor residual spraying (IRS) before malaria season is a cornerstone of vector control efforts. Despite their implementation in Namibia, a country approaching elimination, these methods have been met with recent plateaus in transmission reduction. This study evaluates the effectiveness and feasibility of two new targeted strategies, reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) in Namibia. METHODS AND ANALYSIS: This is an open-label cluster randomised controlled trial with 2×2 factorial design. The interventions include: rfMDA (presumptive treatment with artemether-lumefantrine (AL)) versus RACD (rapid diagnostic testing and treatment using AL) and RAVC (IRS with Acellic 300CS) versus no RAVC. Factorial design also enables comparison of the combined rfMDA+RAVC intervention to RACD. Participants living in 56 enumeration areas will be randomised to one of four arms: rfMDA, rfMDA+RAVC, RACD or RACD+RAVC. These interventions, triggered by index cases detected at health facilities, will be targeted to individuals residing within 500 m of an index. The primary outcome is cumulative incidence of locally acquired malaria detected at health facilities over 1 year. Secondary outcomes include seroprevalence, infection prevalence, intervention coverage, safety, acceptability, adherence, cost and cost-effectiveness. ETHICS AND DISSEMINATION: Findings will be reported on clinicaltrials.gov, in peer-reviewed publications and through stakeholder meetings with MoHSS and community leaders in Namibia. TRIAL REGISTRATION NUMBER: NCT02610400; Pre-results.

Spatial clustering of patent and sub-patent malaria infections in northern Namibia: Implications for surveillance and response strategies for elimination.

BACKGROUND: Reactive case detection (RACD) around passively detected malaria cases is a strategy to identify and treat hotspots of malaria transmission. This study investigated the unproven assumption on which this approach is based, that in low transmission settings, infections cluster over small scales. METHODS: A prospective case-control study was conducted between January 2013 and August 2014 in Ohangwena and Omusati regions in north central Namibia. Patients attending health facilities who tested positive by malaria rapid diagnostic test (RDT) (index cases) were traced back to their home. All occupants of index case households (n = 116 households) and surrounding households (n = 225) were screened for Plasmodium infection with a rapid diagnostic test (RDT) and loop mediated isothermal amplification (LAMP) and interviewed to identify risk factors. A comparison group of 286 randomly-selected control households was also screened, to compare infection levels of RACD and non-RACD households and their neighbours. Logistic regression was used to investigate spatial clustering of patent and sub-patent infections around index cases and to identify potential risk factors that would inform screening approaches and identify risk groups. Estimates of the impact of RACD on onward transmission to mosquitoes was made using previously published figures of infection rates. RESULTS: Prevalence of Plasmodium falciparum infection by LAMP was 3.4%, 1.4% and 0.4% in index-case households, neighbors of index case households and control households respectively; adjusted odds ratio 6.1 [95%CI 1.9-19.5] comparing case households versus control households. Using data from Engela, neighbors of cases had higher odds of infection [adjusted OR 5.0 95%CI 1.3-18.9] compared to control households. All infections identified by RDTs were afebrile and RDTs identified only a small proportion of infections in case (n = 7; 17%) and control (0%) neighborhoods. Based on published estimates of patent and sub-patent infectiousness, these results suggest that infections missed by RDTs during RACD would allow 50-71% of infections to mosquitoes to occur in this setting. CONCLUSION: Malaria infections cluster around passively detected cases. The majority of infections are asymptomatic and of densities below the limit of detection of current RDTs. RACD using standard RDTs are unlikely to detect enough malaria infections to dramatically reduce transmission. In low transmission settings such as Namibia more sensitive field diagnostics or forms of focal presumptive treatment should be tested as strategies to reduce malaria transmission.