Multiple Anopheles species complicate downstream analysis and decision-making in a malaria pre-elimination area in southern Mozambique.

BACKGROUND: Different anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni). METHODS: Female anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination. RESULTS: Out of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison. CONCLUSION: The reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species.

An evaluation of LLIN physical integrity and population attitudes towards net use, care and handling during the Magude project in southern Mozambique.

BACKGROUND: The Magude Project assessed the feasibility of eliminating malaria in Magude district, a low transmission setting in southern Mozambique, using a package of interventions, including long-lasting insecticidal nets (LLINs). As the efficacy of LLINs depends in part on their physical integrity, this metric was quantified for Olyset® Nets post mass-distribution, in addition to net use, care and handling practices and other risk factors associated with net physical integrity. METHODS: Nets were collected during a cross-sectional net evaluation, nine months after the Magude project commenced, which was 2 years after the nets were distributed by the National Malaria Control Programme (NMCP). The physical integrity of the nets was assessed by counting and sizing the holes at different positions on each net. A structured questionnaire was administered to assess how the selected net was used and treated (care, wash and repair). Net bio-efficacy was assessed following the standard World Health Organization (WHO) cone bioassay procedures. RESULTS: Out of the 170 Olyset® Nets included in the analysis, 63.5% had been used the night before. The main reason for not using a net was the notion that there were no mosquitoes present. The average number of people using each net was 1.79. Two thirds of the nets had only been washed once or twice since distribution. Most nets (80.9%) were holed and 18% were torn, but none of the risk factors were significantly associated with net integrity, except for presence of mice in the household. Less than half of the participants noticed holes in holed nets, and of those only 38.6% attempted to repair those. None of the six nets that were tested for bio-efficacy passed the WHO threshold of 80% mosquito mortality. CONCLUSION: Overall the majority of Olyset® Nets were in serviceable condition two years post-distribution, but their insecticidal effect may have been lost. This study-together with previous evidence on suboptimal access to and use of LLINs in Magude district-highlights that LLINs as an intervention could have been optimized during the Magude project to achieve maximum intervention impact.

Fine-scale spatial distribution of deltamethrin resistance and population structure of Anopheles funestus and Anopheles arabiensis populations in Southern Mozambique.

BACKGROUND: Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages. METHODS: Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. RESULTS: Phenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy-Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (- 0.00457 to 0.04213), Nm values were high (9.4-71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations. CONCLUSION: Despite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

To spray or target mosquitoes another way: focused entomological intelligence guides the implementation of indoor residual spraying in southern Mozambique.

BACKGROUND: To eliminate malaria in southern Mozambique, the National Malaria Control Programme and its partners are scaling up indoor residual spraying (IRS) activities in two provinces, Gaza and Inhambane. An entomological surveillance planning tool (ESPT) was used to answer the programmatic question of whether IRS would be effective in target geographies, given limited information on local vector bionomics. METHODS: Entomological intelligence was collected in six sentinel sites at the end of the rainy season (April-May 2018) and the beginning of the dry season (June-July 2018). The primary objective was to provide an 'entomological snapshot' by collecting question-based, timely and high-quality data within one single week in each location. Host-seeking behaviour (both indoors and outdoors) was monitored by human-baited tent traps. Indoor resting behaviour was quantified by pyrethrum spray catches and window exit traps. RESULTS: Five different species or species groups were identified: Anopheles funestus sensu lato (s.l.) (66.0%), Anopheles gambiae s.l. (14.0%), Anopheles pharoensis (1.4%), Anopheles tenebrosus (14.1%) and Anopheles ziemanni (4.5%). Anopheles funestus sensu stricto (s.s.) was the major vector among its sibling species, and 1.9% were positive for Plasmodium falciparum infections. Anopheles arabiensis was the most abundant vector species within the An. gambiae complex, but none tested positive for P. falciparum infections. Some An. tenebrosus were positive for P. falciparum (1.3%). When evaluating behaviours that impact IRS efficacy, i.e. endophily, the known primary vector An. funestus s.s., was found to rest indoors-demonstrating at least part of its population will be impacted by the intervention if insecticides are selected to which this vector is susceptible. However, other vector species, including An. gambiae s.l., An. tenebrosus, An. pharoensis and An. ziemanni, showed exophilic and exophagic behaviours in several of the districts surveilled. CONCLUSION: The targeted approach to entomological surveillance was successful in collecting question-based entomological intelligence to inform decision-making about the use of IRS in specific districts. Endophilic An. funestus s.s. was documented as being the most prevalent and primary malaria vector suggesting that IRS can reduce malaria transmission, but the presence of other vector species both indoors and outdoors suggests that alternative vector control interventions that target these gaps in protection may increase the impact of vector control in southern Mozambique.

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa.

BACKGROUND: Vector control tools have contributed significantly to a reduction in malaria burden since 2000, primarily through insecticidal-treated bed nets (ITNs) and indoor residual spraying. In the face of increasing insecticide resistance in key malaria vector species, global progress in malaria control has stalled. Innovative tools, such as dual active ingredient (dual-AI) ITNs that are effective at killing insecticide-resistant mosquitoes have recently been introduced. However, large-scale uptake has been slow for several reasons, including higher costs and limited evidence on their incremental effectiveness and cost-effectiveness. The present report describes the design of several observational studies aimed to determine the effectiveness and cost-effectiveness of dual-AI ITNs, compared to standard pyrethroid-only ITNs, at reducing malaria transmission across a variety of transmission settings. METHODS: Observational pilot studies are ongoing in Burkina Faso, Mozambique, Nigeria, and Rwanda, leveraging dual-AI ITN rollouts nested within the 2019 and 2020 mass distribution campaigns in each country. Enhanced surveillance occurring in select study districts include annual cross-sectional surveys during peak transmission seasons, monthly entomological surveillance, passive case detection using routine health facility surveillance systems, and studies on human behaviour and ITN use patterns. Data will compare changes in malaria transmission and disease burden in districts receiving dual-AI ITNs to similar districts receiving standard pyrethroid-only ITNs over three years. The costs of net distribution will be calculated using the provider perspective including financial and economic costs, and a cost-effectiveness analysis will assess incremental cost-effectiveness ratios for Interceptor® G2, Royal Guard®, and piperonyl butoxide ITNs in comparison to standard pyrethroid-only ITNs, based on incidence rate ratios calculated from routine data. CONCLUSIONS: Evidence of the effectiveness and cost-effectiveness of the dual-AI ITNs from these pilot studies will complement evidence from two contemporary cluster randomized control trials, one in Benin and one in Tanzania, to provide key information to malaria control programmes, policymakers, and donors to help guide decision-making and planning for local malaria control and elimination strategies. Understanding the breadth of contexts where these dual-AI ITNs are most effective and collecting robust information on factors influencing comparative effectiveness could improve uptake and availability and help maximize their impact.

An evaluation of LLIN ownership, access, and use during the Magude project in southern Mozambique.

The Magude Project assessed the feasibly of eliminating malaria in a low transmission setting in southern Mozambique using a package of interventions. This study measured the ownership, access and use of long-lasting insecticide treated nets (LLINs) and inequalities in these indicators across household wealth, size and population subgroups, to understand the protection that LLINs provided during the project. Data were obtained from various household surveys. At least 31% of the nets distributed during the 2014 and 2017 campaigns were lost during the first year post-distribution. Most nets (77.1%) present in the district were Olyset Nets. LLIN access never exceeded 76.3% and use varied seasonally between 40% and 76.4%. LLIN access limited LLIN use during the project, especially during the high transmission season. LLIN ownership, access and use were lower in harder-to-reach localities, in poorer and larger households. Children and women below 30 had poorer access to LLINs than the overall population. Net use was lowest among school-aged children and young adults, especially among young males, and highest in children under 5, pregnant women, in older adults and in households that received indoor residual spraying (IRS). This study revealed that LLIN mass-distribution campaigns alone are not sufficient to achieve the high level of net protection needed during elimination programs and that reviewing the LLIN allocation scheme, top-up distributions and/or community engagement campaigns is needed, also to reduce inequalities in populations' access to LLINs.

Overlaying human and mosquito behavioral data to estimate residual exposure to host-seeking mosquitoes and the protection of bednets in a malaria elimination setting where indoor residual spraying and nets were deployed together.

Characterizing persistent malaria transmission that occurs after the combined deployment of indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) is critical to guide malaria control and elimination efforts. This requires a detailed understanding of both human and vector behaviors at the same temporal and spatial scale. Cross-sectional human behavior evaluations and mosquito collections were performed in parallel in Magude district, Mozambique. Net use and the exact time when participant moved into each of five environments (outdoor, indoor before bed, indoor in bed, indoor after getting up, and outdoor after getting up) were recorded for individuals from three different age groups and both sexes during a dry and a rainy season. Malaria mosquitoes were collected with CDC light traps in combination with collection bottle rotators. The percentage of residual exposure to host-seeking vectors that occurred in each environment was calculated for five local malaria vectors with different biting behaviors, and the actual (at observed levels of LLIN use) and potential (i.e. if all residents had used an LLIN) personal protection conferred by LLINs was estimated. Anopheles arabiensis was responsible for more than 74% of residents' residual exposure to host-seeking vectors during the Magude project. The other four vector species (An. funestus s.s., An. parensis, An. squamosus and An. merus) were responsible for less than 10% each. The personal protection conferred by LLINs prevented only 39.2% of the exposure to host-seeking vectors that survived the implementation of both IRS and LLINs, and it differed significantly across seasons, vector species and age groups. At the observed levels of bednet use, 12.5% of all residual exposure to host-seeking vectors occurred outdoor during the evening, 21.9% indoor before going to bed, almost two thirds (64%) while people were in bed, 1.4% indoors after getting up and 0.2% outdoor after leaving the house. Almost a third of the residual exposure to host-seeking vectors (32.4%) occurred during the low transmission season. The residual bites of An. funestus s.s. and An. parensis outdoors and indoor before bedtime, of An. arabiensis indoors when people are in bed, and of An. squamosus both indoors and outdoors, are likely to have sustained malaria transmission throughout the Magude project. By increasing LLIN use, an additional 24.1% of exposure to the remaining hosts-seeking vectors could have been prevented. Since An. arabiensis, the most abundant vector, feeds primarily while people are in bed, increasing net use and net feeding inhibition (through e.g. community awareness activities and the selection of more effective LLINs) could significantly reduce the exposure to remaining host-seeking mosquitoes. Nonetheless, supplementary interventions aiming to reduce human-vector contact outdoors and/or indoors before people go to bed (e.g. through larval source management, window and eave screening, eave tubes, and spatial repellents) will be needed to reduce residual exposure to the outdoor and early biting An. funestus s.s. and An. parensis.

The realized efficacy of indoor residual spraying campaigns falls quickly below the recommended WHO threshold when coverage, pace of spraying and residual efficacy on different wall types are considered.

Indoor residual spraying (IRS) has been and remains an important malaria control intervention in southern Mozambique, South Africa and Eswatini. A better understanding of the effectiveness of IRS campaigns is critical to guide future elimination efforts. We analyze the three IRS campaigns conducted during a malaria elimination demonstration project in southern Mozambique, the "Magude project", and propose a new method to calculate the efficacy of IRS campaigns adjusting for IRS coverage, pace of house spraying and IRS residual efficacy on different wall types. Anopheles funestus sensu lato (s.l.) and An. gambiae s.l. were susceptible to pirimiphos-methyl and DDT. Anopheles funestus s.l. was resistant to pyrethroids, with 24h post-exposure mortality being lower for An. funestus sensu stricto (s.s.) than for An. parensis (collected indoors). The percentage of structures sprayed was above 90% and percentage of people covered above 86% in all three IRS campaigns. The percentage of households sprayed was above 83% in 2015 and 2016, but not assessed in 2017. Mosquito mortality 24h post-exposure stayed above 80% for 196 days after the 2016 IRS campaign and 222 days after the 2017 campaign and was 1.5 months longer on mud walls than on cement walls. This was extended by up to two months when 120h post-exposure mortality was considered. The district-level realized IRS efficacy was 113 days after the 2016 campaign. While the coverage of IRS campaigns in Magude were high, IRS protection did not remain optimal for the entire high malaria transmissions season. The use of a longer-lasting IRS product could have further supported the interruption of malaria transmission in the district. To better estimate the protection afforded by IRS campaigns, National Malaria Control Programs and partners are encouraged to adjust the calculation of IRS efficacy for IRS coverage, pace of house spraying during the campaign and IRS efficacy on different wall types combined with wall type distribution in the sprayed area.

Physical durability of two types of long-lasting insecticidal nets (LLINs) three years after a mass LLIN distribution campaign in Mozambique, 2008-2011.

We conducted a prospective evaluation to measure the physical durability of two brands of long-lasting insecticidal nets (LLINs) distributed during a campaign in 2008 in Nampula Province, Mozambique. Households with LLINs tagged during the campaign (6,000) were geo-located (34%) and a random sample was selected for each of 3 years of follow-up. The LLINs were evaluated in the field and a laboratory for presence of holes and a proportional hole index (pHI) was calculated following the World Health Organization guidelines. We performed 567 interviews (79.0%) and found 75.3% (72.1-78.4%) of households retained at least one LLIN after 3 years; the most common cause of attrition was damage beyond repair (51.0%). Hole damage was evident after 1 year, and increased by year. Olyset had a significantly greater mean number of holes and pHI compared with PermaNet 2.0 brand (all P values ≤ 0.001). Additional information about LLIN durability is recommended to improve malaria control efforts.