Community participatory mapping of malaria mosquito breeding sites in Mozambique.

BACKGROUND: The community involvement and the people's knowledge allow detailed information about the distribution, location, and identification of mosquito breeding-sites. Information which is fundamental for their efficient management and elimination. Since participatory mapping has proven to be an effective tool to identify health determinants, the study aimed to apply the methodology to identify and map potential mosquito breeding-sites in Tambai, Nhamatanda, Mozambique. METHODS: A study was conducted using an open-question guide. Discussions were held with 94 participants within ten focus groups, selected in collaboration with local community leaders. A thematic content analysis was performed. Descriptive statistics were used to characterize sociodemographic data. Geographic Positioning System (GPS) was used to compare and map potential breeding-sites. Children under 5 years of age who tested positive for malaria, were georeferenced to the maps. RESULTS: Participants were aware of causes and transmission of malaria, no major differences between groups were observed regarding knowledge and identification of principal potential breeding sites. Gender and age determined specific information, number, and diversity of identified potential breeding sites. A total of 125 potential breeding-sites (36 permanent and 89 temporary) were mapped. CONCLUSIONS: Several potential mosquito breeding-sites were identified, located throughout the community, often near house conglomerates and malaria cases. Community participatory mapping could be used to identify potential mosquito breeding-sites by the national malaria control programmes to establish an efficient larval surveillance system, while improving community engagement and control strategies. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT04419766.

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.

Characterisation of human exposure to nocturnal biting by malaria and arbovirus vectors in a rural community in Chókwè district, southern Mozambique.

Background: Understanding the magnitude of human exposure to mosquito biting is fundamental to reduce pathogen transmission. Here we report on a study quantifying the levels of mosquitoes attacking humans throughout the night in a rural area of Southern Mozambique. Methods: Surveys were carried out in Massavasse village, southern Mozambique. The abundance and composition of host-seeking mosquito communities at night were assessed by human-landing catches (HLC) at one-hour intervals. Periods when people were located predominantly outdoors or indoors were used to estimate the amount of residents' exposure to mosquito bites in either location, to explore the potential impact a bed net could have had in reducing biting by each vector species. Results: A total of 69,758 host-seeking female mosquitoes comprising 23 species in four genera were collected. The exposure to biting by virtually all vector species was consistently high outdoors, typically at early evening and morning, with exception of An. gambiae s.l which was likely of biting a person with nearly same intensity indoors and outdoors throughout the night. Bed nets use could have reduced biting by An. gambiae s.l (dominated by An. arabiensis), Ma. africana, Ma. uniformis, Cx. pipiens, Cx. antennatus, and Cx. poicilipes by 53%, 47%, 46%, 38%, 31%, and 28% respectively, compared to non-users. Conversely, a bed net user would have had little protection against An. pharoensis, An. ziemanni, An. tenebrosus, and Cx. tritaeniorhynchus biting exposures. Conclusions: This study showed that Massavasse residents were exposed to high levels of outdoor biting by malaria and arbovirus vectors that abound in the village. The findings help to identify entomological drivers of persistent malaria transmission in Mozambique and identify a wide range of arbovirus vectors nocturnally active in rural areas, many with outbreak potential. The study highlights the need for a surveillance system for monitoring arboviral diseases vectors in Mozambique.

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.

The Shockwè trap: a human-baited exposure-free device for surveillance and behaviour studies of anthropophilic vectors.

Background: The human biting rate (MBR) and entomological inoculation rate (EIR) are common parameters routinely used to measure the risk of malaria transmission. Both parameters can be estimated using human landing catches (HLC). Although it is considered the gold-standard, HLC puts collectors at higher risk of infection with mosquito-transmitted pathogens. Methods: A novel exposure-free host-seeking mosquito electrocution trap, the Shockwè trap (SHK), was developed and its efficiency for monitoring mosquito community composition and abundance was compared with human landing catches (HLC) as the gold-standard. Field experiments were performed in Massavasse village, southern Mozambique. Simultaneous indoor and outdoor collections of nocturnal host-seeking mosquitoes were carried out using the SHK and HLC methods. The relative sampling efficiency of SHK was estimated as the ratio of the numbers of mosquitoes caught in SHK compared HLC. Proportionality and density-dependence between SHK and HLC catches were estimated by mean of Bayesian regression approaches. Results: A total of 69,758 and 27,359 host-seeking mosquitoes comprising nineteen species and four genera, were collected by HLC and SHK respectively. In general, SHK and HLC sampled similar numbers of mosquito species, with the exceptions of the least common species Aedes sudanensis, Ae. subargenteus, and Coquillettidia versicolor that were caught only by HLC. The relative sampling efficiency and proportionality between SHK and matched HLC catches varied greatly between species and collection site. However, all mosquitoes collected by SHK were unfed, confirming the Shockwè trap design's performance and reliability as a successful mosquito exposure free sampling approach. Conclusions: Results demonstrate that SHK is a safe and reliable human-exposure free device for monitoring the occurrence of a wide range of mosquito, including major malaria and arboviruses vector species. However, improvements are needed to increase its sampling efficiency for less abundant mosquito species.

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.

Correlation of textile 'resistance to damage' scores with actual physical survival of long-lasting insecticidal nets in the field.

BACKGROUND: Attempts have been made to link procurement of long-lasting insecticidal nets (LLIN) not only to the price but also the expected performance of the product. However, to date it has not been possible to identify a specific textile characteristic that predicts physical durability in the field. The recently developed resistance to damage (RD) score could provide such a metric. This study uses pooled data from durability monitoring to explore the usefulness of the RD methodology. METHODS: Data from standardized, 3-year, prospective LLIN durability monitoring for six LLIN brands in 10 locations and four countries involving 4672 campaign LLIN were linked to the RD scores of the respective LLIN brands. The RD score is a single quantitative metric based on a suite of standardized textile tests which in turn build on the mechanisms of damage to a mosquito net. Potential RD values range from 0 to 100 where 100 represents optimal resistance to expected day-to-day stress during reasonable net use. Survival analysis was set so that risk of failure only started when nets were first hung. Cox regression was applied to explore RD effects on physical survival adjusting for known net use environment variables. RESULTS: In a bivariate analysis RD scores showed a linear relationship with physical integrity suggesting that the proportion of LLIN with moderate damage decreased by 3%-points for each 10-point increase of the RD score (p = 0.02, R2 = 0.65). Full adjustment for net care and handling behaviours as well as other relevant determinants and the country of study showed that increasing RD score by 10 points resulted in a 36% reduction of risk of failure to survive in serviceable condition (p < 0.0001). LLINs with RD scores above 50 had an additional useful life of 7 months. CONCLUSIONS: This study provides proof of principle that the RD metric can predict physical durability of LLIN products in the field and could be used to assess new products and guide manufacturers in creating improved products. However, additional validation from other field data, particularly for next generation LLIN, will be required before the RD score can be included in procurement decisions for LLINs.

Variation of physical durability between LLIN products and net use environments: summary of findings from four African countries.

BACKGROUND: Physical durability of long-lasting-insecticidal nets (LLIN) is an important aspect of the effectiveness of LLIN as a malaria prevention tool, but there is limited data on performance across locations and products. This secondary analysis of data from the VectorWorks project from 10 sites in four African countries involving six LLIN brands provides such data. METHODS: A total of 4672 campaign nets from 1976 households were recruited into prospective cohort studies 2-6 months after distribution through campaigns and followed for 3 years in Mozambique, Nigeria, DRC and Zanzibar, Tanzania. LLIN products included two 100 denier polyester LLIN (DawaPlus® 2.0, PermaNet® 2.0) distributed in five sites and four 150 denier polyethylene LLIN (Royal Sentry®, MAGNet®, DuraNet©, Olyset™ Net) distributed in five sites. Primary outcome was LLIN survival in serviceable condition and median survival in years. Net use environment and net care variables were collected during four household surveys. Determinants of physical durability were explored by survival analysis and Cox regression models with risk of failure starting with the first hanging of the net. RESULTS: Definite outcomes for physical durability were obtained for 75% of study nets. After 31 to 37 months survival in serviceable condition varied between sites by 63 percentage-points, from 17 to 80%. Median survival varied by 3.7 years, from 1.6 to 5.3 years. Similar magnitude of variation was seen for polyethylene and polyester LLIN and for the same brand. Cox regression showed increasing net care attitude in combination with exposure to net related messages to be the strongest explanatory variable of survival. However, differences between countries also remained significant. In contrast, no difference was seen for LLIN material types. CONCLUSIONS: Variation in net use environment and net care is the main reason for differences in the physical durability of LLIN products in different locations. While some of these factors have been identified to work across countries, other factors remain poorly defined and further investigation is needed in this area. Grouping LLIN brands by similar textile characteristics, such as material or yarn strength, is insufficient to distinguish LLIN product performance suggesting a more differentiated, composite metric is needed.

First confirmed occurrence of the yellow fever virus and dengue virus vector Aedes (Stegomyia) luteocephalus (Newstead, 1907) in Mozambique.

BACKGROUND: Mozambique, same as many other tropical countries, is at high risk of arthropod-borne virus (arbovirus) diseases and recently two dengue virus (DENV) outbreaks occurred in the northern part of the country. The occurrence of some important vector species, such as Aedes (Stegomyia) aegypti (Linnaeus) and Ae. (Stg.) albopictus (Skuse), besides several other sylvatic vectors, have been reported in the country, which may indicate that the transmission of some arboviruses of public health importance may involve multiple-vector systems. Therefore, knowing the occurrence and distribution of existing and the new important vectors species, is crucial for devising systematic transmission surveillance and vector control approaches. The aim of this study was to map the occurrence and distribution of mosquito species with potential for transmitting arboviruses of human and veterinary relevance in Niassa Province, Northern Mozambique. METHODS: Field entomological surveys were undertaken in April 2016 in Lago District, Niassa Province, northern Mozambique. Breeding sites of mosquitoes were inspected and immature stages were collected and reared into adult. Mosquitoes in the adult stages were morphologically identified using taxonomic keys. Morphological identification of Aedes (Stegomyia) luteocephalus (Newstead) were later confirmed using dissected male genitalia and molecular based on the phylogenetic analyses of the sequenced barcode (cox1 mtDNA) gene. RESULTS: A total of 92 mosquito larvae collected developed into adults. Of these, 16 (17.39%) were morphologically identified as Ae. luteocephalus. The remaining specimens belonged to Ae. (Stg.) aegypti (n = 4, 4.35%), Ae. (Aedimorphus) vittatus (n = 24, 26.09%), Anopheles garnhami (n = 1, 1.09%), Culex (Culiciomyia) nebulosus (n = 28, 30.43%), Eretmapodites subsimplicipes (n = 18, 19.57%) and Toxorhynchites brevipalpis (n = 1, 1.09%), taxa already known to the country. Male genitalia and phylogenetic analyses confirmed the identity of Ae. luteocephalus specimens collected in this study. CONCLUSIONS: To our knowledge, this is the first detection of Ae. luteocephalus in Mozambican territory, a vector species of yellow fever virus (YFV), Zika virus (ZIKV) and dengue virus (DENV) in Africa. Further studies are encouraged to investigate the role of Ae. luteocephalus in the transmission of arboviral diseases in Mozambique.

Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

A survey of RNA viruses in mosquitoes from Mozambique reveals novel genetic lineages of flaviviruses and phenuiviruses, as well as frequent flavivirus-like viral DNA forms in Mansonia.

BACKGROUND: Mosquito-borne diseases involving arboviruses represent expanding threats to sub-Saharan Africa imposing as considerable burden to human and veterinary public health. In Mozambique over one hundred species of potential arbovirus mosquito vectors have been identified, although their precise role in maintaining such viruses in circulation in the country remains to be elucidated. The aim of this study was to screen for the presence of flaviviruses, alphaviruses and bunyaviruses in mosquitoes from different regions of Mozambique. RESULTS: Our survey analyzed 14,519 mosquitoes, and the results obtained revealed genetically distinct insect-specific flaviviruses, detected in multiple species of mosquitoes from different genera. In addition, smaller flavivirus-like NS5 sequences, frequently detected in Mansonia seemed to correspond to defective viral sequences, present as viral DNA forms. Furthermore, three lineages of putative members of the Phenuiviridae family were also detected, two of which apparently corresponding to novel viral genetic lineages. CONCLUSION: This study reports for the first-time novel insect-specific flaviviruses and novel phenuiviruses, as well as frequent flavivirus-like viral DNA forms in several widely known vector species. This unique work represents recent investigation of virus screening conducted in mosquitoes from Mozambique and an important contribution to inform the establishment of a vector control program for arbovirus in the country and in the region.

Monitoring the durability of the long-lasting insecticidal nets MAGNet and Royal Sentry in three ecological zones of Mozambique.

BACKGROUND: Malaria prevention with long-lasting insecticidal nets (LLINs) has seen a tremendous scale-up in sub-Saharan Africa in the last decade. To sustain this success, it is important to understand how long LLINs remain in the households and continue to protect net users, which is termed durability. This information is needed to decide the appropriate timing of LLIN distribution and also to identify product(s) that may be underperforming relative to expectations. Following guidance from the U.S. President's Malaria Initiative, durability monitoring of polyethylene 150-denier LLIN (Royal Sentry® and MAGNet®) distributed during a 2017 mass campaign in Mozambique was implemented in three ecologically different sites: Inhambane, Tete, and Nampula. METHODS: This was a prospective cohort study in which representative samples of households from each district were recruited at baseline, 1 to 6 months after the mass campaign. All campaign LLINs in these households were labelled and followed up over a period of 36 months. The primary outcome was the "proportion of LLINs surviving in serviceable condition" based on attrition and integrity measures and the median survival in years. The outcome for insecticidal durability was determined by bio-assay from subsamples of campaign LLINs. RESULTS: A total of 998 households (98% of target) and 1998 campaign LLIN (85% of target) were included in the study. Definite outcomes could be determined for 80% of the cohort LLIN in Inhambane, 45% in Tete, and 72% in Nampula. The highest all-cause attrition was seen in Nampula with 74% followed by Inhambane at 56% and Tete at 50%. Overall, only 2% of campaign LLINs were used for other purposes. Estimated survival in serviceable condition of campaign LLINs after 36 months was 57% in Inhambane, 43% in Tete, and 33% in Nampula, corresponding to median survival of 3.0, 2.8, and 2.4 years, respectively. Factors that were associated with better survival were exposure to social and behavioural change communication, a positive net care attitude, and folding up the net during the day. Larger household size negatively impacted survival. Insecticidal performance was optimal up to 24 months follow-up, but declined at 36 months when only 3% of samples showed optimal effectiveness in Inhambane, 11% in Tete and 29% in Nampula. However, 96% of LLIN still had minimal effectiveness at 36 months. CONCLUSIONS: Differences in median survival could be attributed at least in part to household environment and net care and repair behaviours. This means that in two of the three sites the assumption of a three-year cycle of campaign distributions holds, while in the Nampula site either continuous distribution channels could be expanded or more intense or targeted social and behaviour change activities to encourage net care and retention could be considered.

Distribution and breeding sites of Aedes aegypti and Aedes albopictus in 32 urban/peri-urban districts of Mozambique: implication for assessing the risk of arbovirus outbreaks.

BACKGROUND: Aedes-borne arboviruses have emerged as an important public health problem worldwide and, in Mozambique, the number of cases and its geographical spread have been growing. However, information on the occurrence, distribution and ecology of Aedes aegypti and Ae. albopictus mosquitoes remain poorly known in the country. METHODS: Between March and April 2016, a cross-sectional study was conducted in 32 districts in Mozambique to determine the distribution and breeding sites of Ae. aegypti and Ae. albopictus. Larvae and pupae were collected from a total of 2,807 water-holding containers using pipette, dipper, funnel and sweeping procedures, depending on the container type and location. Both outdoor and indoor water-holding containers were inspected. The immature forms were reared to adults and the identifications of the mosquito species was carried out with a stereomicroscope using a taxonomic key. RESULTS: Aedes aegypti was found in every district sampled, while Ae. albopictus was only found in Moatize district, situated in Tete Province in the central part of the country. Six hundred and twenty-eight of 2,807 (22.4%) containers were positive for Ae. aegypti but only one (0.03%) was positive for Ae. albopictus. The Container Index (CI) of Aedes was highest in densely populated suburban areas of the central region (260/604; 43.0%), followed by suburban areas in northern areas (228/617; 36.9%) whilst the lowest proportion was found in urbanized southern areas (140/1586; 8.8%). The highest CI of Aedes was found in used tires (448/1268; 35.3%), cement tanks (20/62; 32.3%) and drums (21/95; 22.1%). CONCLUSION: Data from our study showed that Ae. aegypti is present nation-wide, since it occurred in every sampled district, whilst Ae. albopictus had a limited distribution. Therefore, the risk of transmission of dengue and chikungunya is likely to have been underestimated in Mozambique. This study highlights the need for the establishment of a national entomological surveillance program for Aedes spp. in Mozambique in order to gain a better understanding about vector bionomics and to support the development of informed effective vector control strategies.

Discovery of Novel Viruses in Mosquitoes from the Zambezi Valley of Mozambique.

Mosquitoes carry a wide variety of viruses that can cause vector-borne infectious diseases and affect both human and veterinary public health. Although Mozambique can be considered a hot spot for emerging infectious diseases due to factors such as a rich vector population and a close vector/human/wildlife interface, the viral flora in mosquitoes have not previously been investigated. In this study, viral metagenomics was employed to analyze the viral communities in Culex and Mansonia mosquitoes in the Zambezia province of Mozambique. Among the 1.7 and 2.6 million sequences produced from the Culex and Mansonia samples, respectively, 3269 and 983 reads were classified as viral sequences. Viruses belonging to the Flaviviridae, Rhabdoviridae and Iflaviridae families were detected, and different unclassified single- and double-stranded RNA viruses were also identified. A near complete genome of a flavivirus, tentatively named Cuacua virus, was obtained from the Mansonia mosquitoes. Phylogenetic analysis of this flavivirus, using the NS5 amino acid sequence, showed that it grouped with 'insect-specific' viruses and was most closely related to Nakiwogo virus previously identified in Uganda. Both mosquito genera had viral sequences related to Rhabdoviruses, and these were most closely related to Culex tritaeniorhynchus rhabdovirus (CTRV). The results from this study suggest that several viruses specific for insects belonging to, for example, the Flaviviridae and Rhabdoviridae families, as well as a number of unclassified RNA viruses, are present in mosquitoes in Mozambique.

The Asian tiger hunts in Maputo city--the first confirmed report of Aedes (Stegomyia) albopictus (Skuse, 1895) in Mozambique.

BACKGROUND: Increasing evidence suggests that dengue fever is endemic in Mozambique. Larvae of both the Afrotropical vector Aedes aegypti and its subspecies, Ae. aegypti formosus, have been reported from three provinces in Mozambique, two of which recently experienced dengue outbreaks. Despite reports of the invasive Oriental vector Ae. albopictus on the islands in the Mozambique Channel and nearby Indian Ocean, the species has not yet been reported in Mozambique. FINDINGS: Four host-seeking female mosquitoes, collected biting the authors in an urban neighbourhood of Maputo City in the late afternoon of 6 December, 2015, are herein morphologically confirmed as Ae. albopictus. CONCLUSION: This is the first report confirming the occurrence in Mozambique of Ae. albopictus, an invasive species and an important vector of human arboviruses. In view of its potential role as a vector of dengue, Chikungunya and Zika viruses, studies are urgently needed to assess the geographical expansion and relative abundance of these important vectors to better understand the potential transmission impact of arboviruses that are efficiently transmitted and globally spread by these vectors.

Clinical and Epidemiological Characterization of the First Recognized Outbreak of Dengue Virus-Type 2 in Mozambique, 2014.

Since the first reported epidemic of dengue in Pemba, the capital of Cabo Delgado province, in 1984-1985, no further cases have been reported in Mozambique. In March 2014, the Provincial Health Directorate of Cabo Delgado reported a suspected dengue outbreak in Pemba, associated with a recent increase in the frequency of patients with nonmalarial febrile illness. An investigation conducted between March and June detected a total of 193 clinically suspected dengue patients in Pemba and Nampula, the capital of neighboring Nampula Province. Dengue virus-type 2 (DENV-2) was detected by reverse transcriptase polymerase chain reaction in sera from three patients, and 97 others were classified as probable cases based on the presence of DENV nonstructural protein 1 antigen or anti-DENV immunoglobulin M antibody. Entomological investigations demonstrated the presence of Aedes aegypti mosquitos in both Pemba and Nampula cities.

Bio-efficacy of new long-lasting insecticide-treated bed nets against Anopheles funestus and Anopheles gambiae from central and northern Mozambique.

BACKGROUND: Long-lasting insecticide-treated nets (LLINs) are one of the main methods used for controlling malaria transmission in Mozambique. The proliferation of several types of LLINs and the re-emergence of insecticide resistance in the local vector populations poses challenges to the local malaria control programme on selecting suitable insecticide-based vector control products. Therefore, this study evaluated the insecticide susceptibility and bio-efficacy of selected new LLINs against wild populations of Anopheles funestus sensu lato and A. gambiae s.l. from Northern and Central Mozambique. The study also investigated whether the insecticide contents on the LINNs fabrics were within the WHOPES recommended target range. METHODS: The susceptibility of 2-5 day old wild female A. funestus and A. gambiae sensu stricto against the major classes of insecticides used for vector control, viz: deltamethrin (0.05 %), permethrin (0.75 %), propoxur (0.1 %), bendiocarb (0.1 %) and DDT (4 %), was determined using WHO cylinder susceptibility tests. WHO cone bioassays were conducted to determine the bio-efficacy of both pyrethroid-only LLINs (Olyset(®), Permanet 2.0(®), NetProtect(®) and Interceptor(®)) and, Permanet 3.0(®) a combination LLIN against A. funestus s.s, from Balama, Mocuba and Milange districts, respectively. The bio-efficacy of LLINs against the insectary-susceptible A. arabiensis (Durban strain) was assessed, as well. Untreated bed net swatches were used as negative controls. Chemical analyses, by high performance liquid chromatography, were undertaken to assess whether the insecticide contents on the LLINs fabrics fell within recommended target dose ranges. The frequency of kdr gene mutations was determined from a random sample of A. gambiae s.s. from both WHO susceptibility and cone bioassay experiments. RESULTS: Anopheles funestus from Balama district showed resistance to deltamethrin and possible resistance to permethrin, propoxur and bendiocarb, whilst A. gambiae from Mocuba district was susceptible to deltamethrin, bendiocarb and propoxur. There were no kdr mutants found in the sample of 256 A. gambiae tested. Overall, 186 LLIN swatches were tested. Mosquitoes exposed to Olyset(®) had the lowest knockdown (±standard error) and mortality rate (±standard error) in all studied sites regardless of vectors species tested. Permanet 3.0 showed the highest bio-efficacy independent of vector species tested and level of insecticide resistance detected. All types of LLINs effectively killed susceptible A. arabiensis Durban strain. The insecticide content of Olyset(®) and Permanet 2.0(®) was higher than the target dose but NetProtect(®) had a lower insecticide content than the target dose. CONCLUSION: The study shows evidence of considerable heterogeneity in both insecticide susceptibility and the level of bio-efficacy of commonly available types of LLINs against wild A. funestus and A. gambiae from Balama, Mocuba and Milange districts, located in north and centre of Mozambique. The findings suggest that vector control approaches combining different types of insecticides might help to tackle the apparent problem of pyrethroid resistance in the vector populations from these three sites. Results from bioassays on laboratory-susceptible A. arabiensis strongly suggest that LLINs can offer some protection against susceptible malaria vectors.

Long-lasting insecticidal nets no longer effectively kill the highly resistant Anopheles funestus of southern Mozambique.

BACKGROUND: Chemical insecticides are crucial to malaria control and elimination programmes. The frontline vector control interventions depend mainly on pyrethroids; all long-lasting insecticidal nets (LLINs) and more than 80% of indoor residual spraying (IRS) campaigns use chemicals from this class. This extensive use of pyrethroids imposes a strong selection pressure for resistance in mosquito populations, and so continuous resistance monitoring and evaluation are important. As pyrethroids have also been used for many years in the Manhiça District, an area in southern Mozambique with perennial malaria transmission, an assessment of their efficacy against the local malaria vectors was conducted. METHODS: Female offspring of wild-caught Anopheles funestus s.s. females were exposed to deltamethrin, lambda-cyhalothrin and permethrin using the World Health Organization (WHO) insecticide-resistance monitoring protocols. The 3-min WHO cone bioassay was used to evaluate the effectiveness of the bed nets distributed or available for purchase in the area (Olyset, permethrin LLIN; PermaNet 2.0, deltamethrin LLIN) against An. funestus. Mosquitoes were also exposed to PermaNet 2.0 for up to 8 h in time-exposure assays. RESULTS: Resistance to pyrethroids in An. funestus s.s. was extremely high, much higher than reported in 2002 and 2009. No exposure killed more than 25.8% of the mosquitoes tested (average mortality, deltamethrin: 6.4%; lambda-cyhalothrin: 5.1%; permethrin: 19.1%). There was no significant difference in the mortality generated by 3-min exposure to any net (Olyset: 9.3% mortality, PermaNet 2.0: 6.0%, untreated: 2.0%; p = 0.2). Six hours of exposure were required to kill 50% of the An. funestus s.s. on PermaNet 2.0. CONCLUSIONS: Anopheles funestus s.s. in Manhiça is extremely resistant to pyrethroids, and this area is clearly a pyrethroid-resistance hotspot. This could severely undermine vector control in this district if no appropriate countermeasures are undertaken. The National Malaria Control Programme (NMCP) of Mozambique is currently improving its resistance monitoring programme, to design and scale up new management strategies. These actions are urgently needed, as the goal of the NMCP and its partners is to reach elimination in southern Mozambique by 2020.

Abundant Aedes (Stegomyia) aegypti aegypti mosquitoes in the 2014 dengue outbreak area of Mozambique.

In early 2014, dengue cases were reported from northern Mozambique, 30 years after the last outbreak. We identified potential dengue vector species in three northern towns, Pemba, Nampula and Nacala, and one southern town, Maputo, during the outbreak in April 2014. A major dengue vector species, Aedes (Stegomyia) aegypti, was found in all these towns. The dominant vector subspecies in the northern towns was Aedes aegypti aegypti, while Ae. aegypti formosus was dominant in Maputo. Considering the high proportion of Ae. aegypti aegypti and its high vector competence, the findings from this study suggest that Ae. aegypti aegypti was responsible for the outbreak in northern Mozambique.