Strengthening the perception-assessment tools for dengue prevention: a cross-sectional survey in a temperate region (Madeira, Portugal).

BACKGROUND: Community participation is mandatory in the prevention of Dengue outbreaks. Taking public views into account is crucial to guide more effective planning and quicker community participation in preventing campaigns. This study aims to assess community perceptions of Madeira population in order to explore their involvement in the A. aegypti's control and reinforce health-educational planning. Due to the lack of accurate methodologies for measuring perception, a new tool to assess the community's perceptions was built. METHODS: A cross-sectional survey was performed in the Island's aegypti-infested area, exploring residents' perceptions regarding most critical community behaviour: aegypti-source reduction and their domestic aegypti-breeding sites. A novel tool defining five essential topics which underlie the source reduction's awareness and accession was built, herein called Essential-Perception (EP) analysis. RESULTS: Of 1276 individuals, 1182 completed the questionnaire (92 · 6%). EP-Score analysis revealed that community's perceptions were scarce, inconsistent and possibly incorrect. Most of the population (99 · 6%) did not completely understood the five essential topics explored. An average of 54 · 2% of residents only partially understood each essential topic, revealing inconsistencies in their understanding. Each resident apparently believed in an average of four false assumptions/myths. Significant association (p<0.001) was found between both the EP-Score level and the domestic presence of breeding sites, supporting the validity of this EP-analysis. Aedes aegypti's breeding sites, consisting of décor/leisure containers, presented an atypical pattern of infestation comparing with dengue prone regions. CONCLUSIONS: The studied population was not prepared for being fully engaged in dengue prevention. Evidences suggest that EP-methodology was efficient and accurate in assessing the community perception and its compliance to practices. Moreover, it suggested a list of myths that could persist in the community. This is the first study reporting an aegypti-entomological pattern and community's perception in a developed dengue-prone region. Tailored messages considering findings of this study are recommended to be used in future campaigns in order to more effectively impact the community perception and behaviour.

Finding unveiled: Culex thalassius (Diptera: Culicidae), a new mosquito species emerges in the Cabo Verde archipelago.

Mosquito-borne diseases can pose significant burdens. In many countries, they pose a risk to national economies and the well-being of humans and animals. To mitigate this, mosquito surveillance is crucial to assess the real and potential transmission of mosquito-borne diseases. Between 2020 and 2023, mosquito larvae were collected from both indoor and outdoor breeding sites in urban and rural areas of 4 municipalities of Santiago and Boavista Islands in Cabo Verde. Mosquitoes were identified morphologically and by polymerase chain reaction-based techniques that targeted the mitochondrial cytochrome C oxidase subunit I sequence. During this period, 6,825 breeding sites were assessed, and of 8,094 mosquito specimens screened, 194 specimens of Culex thalassius were identified for the first time in the country in 4 municipalities of Santiago and Boavista Islands. This new finding highlights the importance of including entomological surveillance in health systems. Although this species has only been detected on a few islands, it is important to continuously monitor it to determine its distribution, spread/dispersal, density, and potential involvement in pathogen transmission.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector.

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance (kdr) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

The current and future distribution of the yellow fever mosquito (Aedes aegypti) on Madeira Island.

The Aedes aegypti mosquito is the main vector for several diseases of global importance, such as dengue and yellow fever. This species was first identified on Madeira Island in 2005, and between 2012 and 2013 was responsible for an outbreak of dengue that affected several thousand people. However, the potential distribution of the species on the island remains poorly investigated. Here we assess the suitability of current and future climatic conditions to the species on the island and complement this assessment with estimates of the suitability of land use and human settlement conditions. We used four modelling algorithms (boosted regression trees, generalized additive models, generalized linear models and random forest) and data on the distribution of the species worldwide and across the island. For both climatic and non-climatic factors, suitability estimates predicted the current distribution of the species with good accuracy (mean area under the Receiver Operating Characteristic curve = 0.88 ±0.06, mean true skill statistic = 0.72 ±0.1). Minimum temperature of coldest month was the most influential climatic predictor, while human population density, residential housing density and public spaces were the most influential predictors describing land use and human settlement conditions. Suitable areas under current climates are predicted to occur mainly in the warmer and densely inhabited coastal areas of the southern part of the island, where the species is already established. By mid-century (2041-2060), the extent of climatically suitable areas is expected to increase, mainly towards higher altitudes and in the eastern part of the island. Our work shows that ongoing efforts to monitor and prevent the spread of Ae. aegypti on Madeira Island will have to increasingly consider the effects of climate change.

Simulation models of dengue transmission in Funchal, Madeira Island: Influence of seasonality.

The recent emergence and established presence of Aedes aegypti in the Autonomous Region of Madeira, Portugal, was responsible for the first autochthonous outbreak of dengue in Europe. The island has not reported any dengue cases since the outbreak in 2012. However, there is a high risk that an introduction of the virus would result in another autochthonous outbreak given the presence of the vector and permissive environmental conditions. Understanding the dynamics of a potential epidemic is critical for targeted local control strategies. Here, we adopt a deterministic model for the transmission of dengue in Aedes aegypti mosquitoes. The model integrates empirical and mechanistic parameters for virus transmission, under seasonally varying temperatures for Funchal, Madeira Island. We examine the epidemic dynamics as triggered by the arrival date of an infectious individual; the influence of seasonal temperature mean and variation on the epidemic dynamics; and performed a sensitivity analysis on the following quantities of interest: the epidemic peak size, time to peak, and the final epidemic size. Our results demonstrate the potential for summer and autumn season transmission of dengue, with the arrival date significantly affecting the distribution of the timing and peak size of the epidemic. Late-summer arrivals were more likely to produce large epidemics within a short peak time. Epidemics within this favorable period had an average of 11% of the susceptible population infected at the peak, at an average peak time of 95 days. We also demonstrated that seasonal temperature variation dramatically affects the epidemic dynamics, with warmer starting temperatures producing large epidemics with a short peak time and vice versa. Overall, our quantities of interest were most sensitive to variance in the date of arrival, seasonal temperature, transmission rates, mortality rate, and the mosquito population; the magnitude of sensitivity differs across quantities. Our model could serve as a useful guide in the development of effective local control and mitigation strategies for dengue fever in Madeira Island.

Dengue importation into Europe: A network connectivity-based approach.

The spread of dengue through global human mobility is a major public health concern. A key challenge is understanding the transmission pathways and mediating factors that characterized the patterns of dengue importation into non-endemic areas. Utilizing a network connectivity-based approach, we analyze the importation patterns of dengue fever into European countries. Seven connectivity indices were developed to characterize the role of the air passenger traffic, seasonality, incidence rate, geographical proximity, epidemic vulnerability, and wealth of a source country, in facilitating the transport and importation of dengue fever. We used generalized linear mixed models (GLMMs) to examine the relationship between dengue importation and the connectivity indices while accounting for the air transport network structure. We also incorporated network autocorrelation within a GLMM framework to investigate the propensity of a European country to receive an imported case, by virtue of its position within the air transport network. The connectivity indices and dynamical processes of the air transport network were strong predictors of dengue importation in Europe. With more than 70% of the variation in dengue importation patterns explained. We found that transportation potential was higher for source countries with seasonal dengue activity, high passenger traffic, high incidence rates, high epidemic vulnerability, and in geographical proximity to a destination country in Europe. We also found that position of a European country within the air transport network was a strong predictor of the country's propensity to receive an imported case. Our findings provide evidence that the importation patterns of dengue into Europe can be largely explained by appropriately characterizing the heterogeneities of the source, and topology of the air transport network. This contributes to the foundational framework for building integrated predictive models for bio-surveillance of dengue importation.

Predicting dengue importation into Europe, using machine learning and model-agnostic methods.

The geographical spread of dengue is a global public health concern. This is largely mediated by the importation of dengue from endemic to non-endemic areas via the increasing connectivity of the global air transport network. The dynamic nature and intrinsic heterogeneity of the air transport network make it challenging to predict dengue importation. Here, we explore the capabilities of state-of-the-art machine learning algorithms to predict dengue importation. We trained four machine learning classifiers algorithms, using a 6-year historical dengue importation data for 21 countries in Europe and connectivity indices mediating importation and air transport network centrality measures. Predictive performance for the classifiers was evaluated using the area under the receiving operating characteristic curve, sensitivity, and specificity measures. Finally, we applied practical model-agnostic methods, to provide an in-depth explanation of our optimal model's predictions on a global and local scale. Our best performing model achieved high predictive accuracy, with an area under the receiver operating characteristic score of 0.94 and a maximized sensitivity score of 0.88. The predictor variables identified as most important were the source country's dengue incidence rate, population size, and volume of air passengers. Network centrality measures, describing the positioning of European countries within the air travel network, were also influential to the predictions. We demonstrated the high predictive performance of a machine learning model in predicting dengue importation and the utility of the model-agnostic methods to offer a comprehensive understanding of the reasons behind the predictions. Similar approaches can be utilized in the development of an operational early warning surveillance system for dengue importation.

Insect-specific flaviviruses and densoviruses, suggested to have been transmitted vertically, found in mosquitoes collected in Angola: Genome detection and phylogenetic characterization of viral sequences.

This report describes a survey of RNA and DNA viruses carried out in adult mosquitoes from Angola, raised under laboratory conditions from field-collected immature forms. This viral genomic survey was performed using different sets of primers targeting groups of arboviruses with a considerable impact on human health, including flaviviruses, alphaviruses, and phleboviruses. Furthermore, the viral survey that was performed also included detection of densoviruses. The obtained results did not reveal the presence of recognizable pathogenic arboviruses but allowed the identification of insect-specific flaviviruses from two genetic lineages and a single lineage of brevidensoviruses. These viruses, collectivelly detected in Anopheles sp. and Culex pipiens s.l. mosquitoes, were most probably transmitted vertically.

First dengue virus seroprevalence study on Madeira Island after the 2012 outbreak indicates unreported dengue circulation.

BACKGROUND: In 2012, the first dengue virus outbreak was reported on the Portuguese island of Madeira with 1080 confirmed cases. Dengue virus of serotype 1 (DENV-1), probably imported from Venezuela, caused this outbreak with autochthonous transmission by invasive Aedes aegypti mosquitoes. RESULTS: We investigated the seroprevalence among the population on Madeira Island four years after the outbreak. Study participants (n = 358), representative of the island population regarding their age and gender, were enrolled in 2012 in a cross-sectional study. Dengue antibodies were detected with an in-house enzyme-linked immunosorbent assay (ELISA) using the dimer of domain III (ED3) of the DENV-1 envelope protein as well as commercial Panbio indirect and capture IgG ELISAs. Positive ELISA results were validated with a neutralization test. The overall seroprevalence was found to be 7.8% (28/358) with the in-house ELISA, whereas the commercial DENV indirect ELISA detected IgG antibodies in 8.9% of the individuals (32/358). The results of the foci reduction neutralization test confirmed DENV-1 imported from South America as the causative agent of the 2012 epidemic. Additionally, we found a higher seroprevalence in study participants with an age above 60 years old and probable secondary DENV infected individuals indicating unreported dengue circulation before or after 2012 on Madeira Island. CONCLUSIONS: This study revealed that the number of infections might have been much higher than estimated from only confirmed cases in 2012/2013. These mainly DENV-1 immune individuals are not protected from a secondary DENV infection and the majority of the population of Madeira Island is still naïve for DENV. Surveillance of mosquitoes and arboviruses should be continued on Madeira Island as well as in other European areas where invasive vector mosquitoes are present.

Zika virus threshold determines transmission by European Aedes albopictus mosquitoes.

Since its emergence in Yap Island in 2007, Zika virus (ZIKV) has affected all continents except Europe. Despite the hundreds of cases imported to European countries from ZIKV-infested regions, no local cases have been reported in localities where the ZIKV-competent mosquito Aedes albopictus is well established. Here we analysed the vector competence of European Aedes (aegypti and albopictus) mosquitoes to different genotypes of ZIKV. We demonstrate that Ae. albopictus from France was less susceptible to the Asian ZIKV than to the African ZIKV. Critically we show that effective crossing of anatomical barriers (midgut and salivary glands) after an infectious blood meal depends on a viral load threshold to trigger: (i) viral dissemination from the midgut to infect mosquito internal organs and (ii) viral transmission from the saliva to infect a vertebrate host. A viral load in body ≥4800 viral copies triggered dissemination and ≥12,000 viral copies set out transmission. Only 27.3% and 18.2% of Ae. albopictus Montpellier mosquitoes meet respectively these two criteria. Collectively, these compelling results stress the poor ability of Ae. albopictus to sustain a local transmission of ZIKV in Europe and provide a promising tool to evaluate the risk of ZIKV transmission in future outbreaks.

Potential of Aedes aegypti populations in Madeira Island to transmit dengue and chikungunya viruses.

BACKGROUND: Aedes spp. mosquitoes mainly transmit the arboviruses dengue virus (DENV) and chikungunya virus (CHIKV) in urban areas, causing a severe public health problem. In 2012-2013, a major dengue outbreak occurred on Madeira Island where the mosquito Aedes aegypti was the only vector. Up to now, the competence of Ae. aegypti populations from Madeira to transmit DENV or CHIKV remains unknown. This study aimed to assess experimentally the ability of Ae. aegypti populations from Madeira to transmit these viruses. RESULTS: By orally exposing mosquitoes to CHIKV (NC/2011-568) and DENV-2 (Bangkok), the vector competence of two field-collected Ae. aegypti populations, i.e. Funchal and Paúl do Mar, was evaluated. We found that both populations were similarly infected and ensured the dissemination and transmission of CHIKV at the same rates. With DENV-2, viral dissemination was significantly higher in the Funchal population compared to Paúl do Mar. We found no significant differences in transmission rates between populations. CONCLUSIONS: To our knowledge, this study has demonstrated for the first time the ability of temperate European Ae. aegypti populations from Madeira to transmit DENV and CHIKV. As our results suggest, there is a potential risk for the local transmission of DENV and CHIKV if introduced to Madeira or continental Europe where Aedes albopictus is present. Our results highlight the need for continuing vector surveillance and control on Madeira Island to future-proof the Island against mosquito-borne epidemics.

Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal).

BACKGROUND: Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar. METHODOLOGY/PRINCIPAL FINDINGS: WHO susceptibility bioassays indicated resistance to cyfluthrin, permethrin, fenitrothion and bendiocarb. Use of synergists significantly increased mortality rates, and biochemical assays indicated elevated activities of detoxification enzymes, suggesting the importance of metabolic resistance. Microarray-based transcriptome analysis detected significant upregulation in both populations of nine cytochrome P450 oxidase genes (including four known pyrethroid metabolizing enzymes), the organophosphate metabolizer CCEae3a, Glutathione-S-transferases, and multiple putative cuticle proteins. Genotyping of knockdown resistance loci linked to pyrethroid resistance revealed fixation of the 1534C mutation, and presence with moderate frequencies of the V1016I mutation in each population. CONCLUSIONS/SIGNIFICANCE: Significant resistance to three major insecticide classes (pyrethroid, carbamate and organophosphate) is present in Ae. aegypti from Madeira Island, and appears to be mediated by multiple mechanisms. Implementation of appropriate resistance management strategies including rotation of insecticides with alternative modes of action, and methods other than chemical-based vector control are strongly advised to delay or reverse the spread of resistance and achieve efficient control.

Comparative morphological and molecular analysis confirms the presence of the West Nile virus mosquito vector, Culex univittatus, in the Iberian Peninsula.

BACKGROUND: Culex univittatus and Culex perexiguus mosquitoes (Diptera: Culicidae) are competent arbovirus vectors, but with unclear morphological differentiation. In Europe, and in the Iberian Peninsula in particular, the presence of either or both species is controversial. However, in order to conduct adequate surveillance for arboviruses in this region, it is crucial to clarify whether Cx. univittatus is present or not, as well as to critically assess existing differentiation tools. This study aimed to clarify this situation, by morphological and molecular phylogenetic comparison of Iberian specimens deemed as Cx. univittatus, with others of South African origin, i.e. from the type-locality region. METHODS: Thus, morphological characteristics useful to distinguish both species, such as midfemur pale line, hindfemur R ratio, seta g R1 ratio, seta f shape, length of ventral arm of phalosome and number of setae on IX tergal abdominal segment, were observed. A phylogenetic analysis based on cox1 mtDNA, of which there were no sequences from Cx. univittatus yet available in the GenBank database, was performed. RESULTS: This analysis showed that Iberian and South African specimens are morphologically similar, except for the length of the ventral arm of the phalosome, which was higher in the Iberian specimens. Although the Iberian specimens could not be accurately identified using BOLD Systems, phylogenetic analysis still grouped these closer to South African Cx. univittatus, than to Cx. perexiguus from Turkey and Pakistan, despite the observed segregation of both taxa as two individual monophyletic clusters with shared common ancestry. CONCLUSIONS: This survey demonstrates that the West Nile virus vector Cx. univittatus is present in the Iberian Peninsula.

Impact of a dengue outbreak experience in the preventive perceptions of the community from a temperate region: Madeira Island, Portugal.

The ability to effectively modify behaviours is increasingly relevant to attain and maintain a good health status. Current behaviour-change models and theories present two main approaches for (healthier) decision-making: one analytical/logical, and one experiential/emotional/intuitive. Therefore, to achieve an integral and dynamic understanding of the public perceptions both approaches should be considered: community surveys should measure cognitive understanding of health-risk contexts, and also explore how past experiences affect this understanding. In 2011, community perceptions regarding domestic source reduction were assessed in Madeira Island͘. After Madeira's first dengue outbreak (2012) a unique opportunity to compare perceptions before and after the outbreak-experience occurred. This was the aim of this study, which constituted the first report on the effect of an outbreak experience on community perceptions regarding a specific vector-borne disease. A cross-sectional survey was performed within female residents at the most aegypti-infested areas. Perceptions regarding domestic source reduction were assessed according to the Essential Perception (EP)-analysis tool. A matching process paired individuals from studies performed before and after the outbreak, ensuring homogeneity in six determinant variables. After the outbreak, there were more female residents who assimilated the concepts considered to be essential to understand the proposed behaviour. Nevertheless, no significant difference was observed in the number of female residents who achieved the defined 'minimal understanding''. Moreover, most of the population (95.5%) still believed at least in one of the identified myths. After the outbreak some myths disappeared and others appeared. The present study quantified and explored how the experience of an outbreak influenced the perception regarding a dengue-preventive behaviour. The outbreak experience surprisingly led to the appearance of new myths within the population, apart from the expected increase of relevant concepts' assimilation. Monitoring public perceptions is therefore crucial to make preventing dengue campaigns updated and worthy.

Mosquito fauna on the Cape Verde Islands (West Africa): an update on species distribution and a new finding.

To evaluate the risk of transmission of vector-borne diseases, regular updates of the geographic distribution of insect vectors are required. In the archipelago of Cape Verde, nine mosquito species have been reported. Of these, four are major vectors of diseases that have been present in the archipelago: yellow fever, lymphatic filariasis, malaria and, currently, an outbreak of dengue. In order to assess variation in mosquito biodiversity, we have carried out an update on the distribution of the mosquito species in Cape Verde, based on an enquiry of 26 unpublished technical reports (1983-2006) and on the results of an entomological survey carried out in 2007. Overall, there seems to be a general trend for an expansion of biological diversity in the islands. Mosquito species richness was negatively correlated with the distance of the islands from the mainland but not with the size of the islands. Human- and/or sporadic climatic-mediated events of dispersal may have contributed to a homogenization of species richness regardless of island size but other ecological factors may also have affected the mosquito biogeography in the archipelago. An additional species, Culex perexiguus, was collected for the first time in the archipelago during the 2007 survey.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.