Detection of acute dengue virus infection, with and without concurrent malaria infection, in a cohort of febrile children in Kenya, 2014-2019, by clinicians or machine learning algorithms.

Poor access to diagnostic testing in resource limited settings restricts surveillance for emerging infections, such as dengue virus (DENV), to clinician suspicion, based on history and exam observations alone. We investigated the ability of machine learning to detect DENV based solely on data available at the clinic visit. We extracted symptom and physical exam data from 6,208 pediatric febrile illness visits to Kenyan public health clinics from 2014-2019 and created a dataset with 113 clinical features. Malaria testing was available at the clinic site. DENV testing was performed afterwards. We randomly sampled 70% of the dataset to develop DENV and malaria prediction models using boosted logistic regression, decision trees and random forests, support vector machines, naïve Bayes, and neural networks with 10-fold cross validation, tuned to maximize accuracy. 30% of the dataset was reserved to validate the models. 485 subjects (7.8%) had DENV, and 3,145 subjects (50.7%) had malaria. 220 (3.5%) subjects had co-infection with both DENV and malaria. In the validation dataset, clinician accuracy for diagnosis of malaria was high (82% accuracy, 85% sensitivity, 80% specificity). Accuracy of the models for predicting malaria diagnosis ranged from 53-69% (35-94% sensitivity, 11-80% specificity). In contrast, clinicians detected only 21 of 145 cases of DENV (80% accuracy, 14% sensitivity, 85% specificity). Of the six models, only logistic regression identified any DENV case (8 cases, 91% accuracy, 5.5% sensitivity, 98% specificity). Without diagnostic testing, interpretation of clinical findings by humans or machines cannot detect DENV at 8% prevalence. Access to point-of-care diagnostic tests must be prioritized to address global inequities in emerging infections surveillance.

Pupal productivity of larval habitats of Aedes aegypti in Msambweni, Kwale County, Kenya.

Aedes aegypti is an important vector of several arboviruses including dengue and chikungunya viruses. Accurate identification of larval habitats of Ae. aegypti is considered an essential step in targeted control. This study determined Ae. aegypti productivity in selected larval habitats in Msambweni, Kwale County, Kenya. Three sequential larval habitat surveys were conducted. The first survey was habitat census (baseline) through which 83 representative larval habitats were identified and selected. The second and third surveys involved estimating daily productivity of the 83 selected larval habitats for 30 consecutive days during a wet and a dry season, respectively. Of 664 larval habitats examined at baseline, 144 larval habitats (21.7%) were found to be infested with Ae. aegypti larvae. At baseline, majority (71%) of the pupae were collected from two (2/6) larval habitat types, tires and pots. Multivariate analysis identified habitat type and the habitat being movable as the predictors for pupal abundance. During the 30-day daily pupal production surveys, only a few of the habitats harbored pupae persistently. Pupae were found in 28% and 12% of the larval habitats during the wet and dry seasons, respectively. In the wet season, drums, tires, and pots were identified as the key habitat types accounting for 85% of all pupae sampled. Three habitats (all drums) accounted for 80% of all the pupae collected in the dry season. Predictors for pupal productivity in the wet season were habitat type, place (whether the habitat is located at the back or front of the house), habitat purpose (use of the water in the habitat), and source of water. Although the multivariate model for habitat type did not converge, habitat type and habitat size were the only significant predictors during the dry season. Drums, pots, and tires were sources of more than 85% of Ae. aegypti pupae, reinforcing the "key container concept." Targeting these three types of habitats makes epidemiological sense, especially during the dry season.

Neurodevelopment in normocephalic children with and without prenatal Zika virus exposure.

OBJECTIVE: Zika virus (ZIKV) targets neural stem cells in the developing brain. However, the majority of ZIKV-exposed children are born without apparent neurological manifestations. It remains unclear if these children were protected from ZIKV neurotropism or if they harbour subtle pathology that is disruptive to brain development. We assess this by comparing neurodevelopmental outcomes in normocephalic ZIKV-exposed children relative to a parallel control group of unexposed controls. DESIGN: Cohort study. SETTING: Public health centres in Grenada, West Indies. PATIENTS: 384 mother-child pairs were enrolled during a period of active ZIKV transmission (April 2016-March 2017) and prospectively followed up to 30 months. Child exposure status was based on laboratory assessment of prenatal and postnatal maternal serum. MAIN OUTCOME MEASURES: The INTERGROWTH-21st Neurodevelopment Assessment (INTER-NDA) package and Cardiff Vision Tests, administered and scored by research staff masked to child's exposure status. RESULTS: A total of 131 normocephalic ZIKV exposed (n=68) and unexposed (n=63) children were assessed between 22 and 30 months of age. Approximately half of these children completed vision testing. There were no group differences in sociodemographics. Deficits in visual acuity (31%) and contrast sensitivity (23%) were apparent in the ZIKV-exposed infants in the absence of cognitive, motor, language or behavioural delays. CONCLUSIONS: Overall neurodevelopment is likely to be unaffected in ZIKV-exposed children with normal head circumference at birth and normal head growth in the first 2 years of life. However, the visual system may be selectively vulnerable, which indicates the need for vision testing by 3 years of age.

Climate predicts geographic and temporal variation in mosquito-borne disease dynamics on two continents.

Climate drives population dynamics through multiple mechanisms, which can lead to seemingly context-dependent effects of climate on natural populations. For climate-sensitive diseases, such as dengue, chikungunya, and Zika, climate appears to have opposing effects in different contexts. Here we show that a model, parameterized with laboratory measured climate-driven mosquito physiology, captures three key epidemic characteristics across ecologically and culturally distinct settings in Ecuador and Kenya: the number, timing, and duration of outbreaks. The model generates a range of disease dynamics consistent with observed Aedes aegypti abundances and laboratory-confirmed arboviral incidence with variable accuracy (28-85% for vectors, 44-88% for incidence). The model predicted vector dynamics better in sites with a smaller proportion of young children in the population, lower mean temperature, and homes with piped water and made of cement. Models with limited calibration that robustly capture climate-virus relationships can help guide intervention efforts and climate change disease projections.

Epilepsy surveillance in normocephalic children with and without prenatal Zika virus exposure.

Children with Congenital Zika Syndrome and microcephaly are at high risk for epilepsy; however, the risk is unclear in normocephalic children with prenatal Zika virus (ZIKV) exposure [Exposed Children (EC)]. In this prospective cohort study, we performed epilepsy screening in normocephalic EC alongside a parallel group of normocephalic unexposed children [Unexposed Children (UC)]. We compared the incidence rate of epilepsy among EC and UC at one year of life to global incidence rates. Pregnant women were recruited from public health centers during the ZIKV outbreak in Grenada, West Indies and assessed for prior ZIKV infection using a plasmonic-gold platform that measures IgG antibodies in serum. Normocephalic children born to mothers with positive ZIKV results during pregnancy were classified as EC and those born to mothers with negative ZIKV results during and after pregnancy were classified as UC. Epilepsy screening procedures included a pediatric epilepsy screening questionnaire and video electroencephalography (vEEG). vEEG was collected using a multi-channel microEEG® system for a minimum of 20 minutes along with video recording of participant behavior time-locked to the EEG. vEEGs were interpreted independently by two pediatric epileptologists, who were blinded to ZIKV status, via telemedicine platform. Positive screening cases were referred to a local pediatrician for an epilepsy diagnostic evaluation. Epilepsy screens were positive in 2/71 EC (IR: 0.028; 95% CI: 0.003-0.098) and 0/71 UC. In both epilepsy-positive cases, questionnaire responses and interictal vEEGs were consistent with focal, rather than generalized, seizures. Both children met criteria for a clinical diagnosis of epilepsy and good seizure control was achieved with carbamazepine. Our results indicate that epilepsy rates are modestly elevated in EC. Given our small sample size, results should be considered preliminary. They support the use of epilepsy screening procedures in larger epidemiological studies of children with congenital ZIKV exposure, even in the absence of microcephaly, and provide guidance for conducting epilepsy surveillance in resource limited settings.

Risk factors for Aedes aegypti household pupal persistence in longitudinal entomological household surveys in urban and rural Kenya.

BACKGROUND: Aedes aegypti is an efficient vector of several arboviruses of public health importance, including Zika and dengue. Currently vector management is the only available avenue for disease control. Development of efficient vector control strategies requires a thorough understanding of vector ecology. In this study, we identified households that are consistently productive for Ae. aegypti pupae and determined the ecological and socio-demographic factors associated with the persistence and abundance of pupae in households in rural and urban Kenya. METHODS: We collected socio-demographic, environmental and entomological data monthly from July 2014 to June 2018 from 80 households across four sites in Kenya. Pupae count data were collected via entomological surveillance of households and paired with socio-demographic and environmental data. We calculated pupal persistence within a household as the number of months of pupal presence within a year. We used spatially explicit generalized additive mixed models (GAMMs) to identify the risk factors for pupal abundance, and a logistic regression to identify the risk factors for pupal persistence in households. RESULTS: The median number of months of pupal presence observed in households was 4 and ranged from 0 to 35 months. We identified pupal persistence in 85 house-years. The strongest risk factors for high pupal abundance were the presence of bushes or tall grass in the peri-domicile area (OR: 1.60, 95% CI: 1.13-2.28), open eaves (OR: 2.57, 95% CI: 1.33-4.95) and high habitat counts (OR: 1.42, 95% CI: 1.21-1.66). The main risk factors for pupal persistence were the presence of bushes or tall grass in the peri-domicile (OR: 4.20, 95% CI: 1.42-12.46) and high number of breeding sites (OR: 2.17, 95% CI: 1.03-4.58). CONCLUSIONS: We observed Ae. aegypti pupal persistence at the household level in urban and rural and in coastal and inland Kenya. High counts of potential breeding containers, vegetation in the peri-domicile area and the presence of eaves were strongly associated with increased risk of pupal persistence and abundance. Targeting households that exhibit pupal persistence alongside the risk factors for pupal abundance in vector control interventions may result in more efficient use of limited resources.

Pre and postnatal exposure to Chikungunya virus does not affect child neurodevelopmental outcomes at two years of age.

BACKGROUND: The 2005-06 chikungunya virus (CHIKV) outbreak in La Réunion suggested that mothers could transmit CHIKV to their neonates while viremic during the intrapartum period, and more than half of the infected neonates showed impaired neurodevelopment at two years of age. However, data sparsity precluded an overview of the developmental impact of vertical infection within the whole prenatal period. OBJECTIVE & METHODS: The current study assessed two-year old children born to mothers who were infected during the 2014 CHIKV outbreak in Grenada to determine the neurodevelopmental impact of perinatal CHIKV infection throughout gestation. Mother and child infection status were confirmed by serologic testing (IgG and IgM) for CHIKV. Cognitive, fine motor, gross motor, language and behavioral outcomes were assessed at two years of age on the INTERGROWTH-21st Neurodevelopment Assessment (INTER-NDA). RESULTS: No differences in neurodevelopmental outcomes were observed between two-year-old children born to mothers infected with CHIKV during gestation (n = 149) and those born to mothers not infected with CHIKV (n = 161). No differences were found in INTER-NDA scores between children infected with CHIKV (n = 47) and children not infected with CHIKV (n = 592). Likewise, there were no differences between children infected with CHIKV post-partum (n = 19) versus children not infected with CHIKV (n = 592). CONCLUSION: Our findings suggest that children exposed and/or infected with CHIKV outside of the intrapartum period experience no significant neurodevelopmental delay at two years of age, as measured by the INTER-NDA, compared to their unexposed and/or uninfected peers. These results complement those of previous studies which showed a neurodevelopmental risk only for children infected during the intrapartum period, while the mother was highly viremic. These results might be reassuring for women of childbearing age and public health officials in CHIKV-endemic regions.

Acceptability and usability of a mobile application for management and surveillance of vector-borne diseases in Colombia: An implementation study.

BACKGROUND: Vector-borne diseases are a public health problem in Colombia, where dengue virus infection is hyperendemic. The introduction of other arboviruses, such as chikungunya and Zika in the last three years, has aggravated the situation. Mobile health (mHealth) offers new strategies for strengthening health care and surveillance systems promoting the collection, delivery, and access of health information to professionals, researchers, and patients. Assessing mobile application performance has been a challenge in low- and middle-income countries due to the difficulty of implementing these technologies in different clinical settings. In this study, we evaluate the usability and acceptability of a mobile application, FeverDX, as a support tool in the management of patients with febrile syndrome and suspected arboviruses infection by general practitioners from Colombia. METHODS: A pilot implementation study was conducted to evaluate the usability and acceptability of FeverDX using the modified version of the Mobile Application Rating Scale (uMARS). The evaluation form included 25 questions regarding quantity and quality of information, engagement, functionality, aesthetics, impact, and acceptability by healthcare workers. Each item uses a 5-point scale (1-Inadequate, 2-Poor, 3-Acceptable, 4-Good, 5-Excellent). A global score was obtained for the evaluation form test by determining the median scores of each subsection. A descriptive statistical analysis of the data obtained was performed. RESULTS: Between December 2016 and January 2017, a total of 20 general practitioners from the Emergency room and hospitalization areas evaluated FeverDX. Less than half (9/20) of the evaluators had a comprehensive knowledge of the Colombian Ministry of Health's guidelines for the diagnosis and management of arboviruses, and evaluators partially (4/9) or completely (5/9) agreed that the content of the application follows the management guidelines. On uMARS scale, FeverDX excelled regarding impact (median 5; IQR = 5-5), functionality (median 5; IQR = 4.8-5), and information and scientific basis (median 4; IQR = 4-4). FeverDX scored well regarding user feedback (median 4; IQR = 4-4.5), design and aesthetics (median 4; IQR = 4-4.3), and subjective assessment of quality (median 4.5; IQR = 4.3-4.8). CONCLUSIONS: FeverDX, a mobile application, is a novel mHealth strategy to strengthen care processes and facilitate the detection and reporting of notifiable surveillance diseases. It could improve adherence to clinical practice guidelines for the management and prevention of prevalent diseases as arboviruses in healthcare settings. Although this pilot study used a small sample size, FeverDx performed adequately in a simulated emergency consultation. Further implementation studies are needed to increase the reliability of mHealth technologies in different scenarios.

Making Pastoralists Count: Geospatial Methods for the Health Surveillance of Nomadic Populations.

Nomadic pastoralists are among the world's hardest-to-reach and least served populations. Pastoralist communities are difficult to capture in household surveys because of factors including their high degree of mobility over remote terrain, fluid domestic arrangements, and cultural barriers. Most surveys use census-based sampling frames which do not accurately capture the demographic and health parameters of nomadic populations. As a result, pastoralists are "invisible" in population data such as the Demographic and Health Surveys (DHS). By combining remote sensing and geospatial analysis, we developed a sampling strategy designed to capture the current distribution of nomadic populations. We then implemented this sampling frame to survey a population of mobile pastoralists in southwest Ethiopia, focusing on maternal and child health (MCH) indicators. Using standardized instruments from DHS questionnaires, we draw comparisons with regional and national data finding disparities with DHS data in core MCH indicators, including vaccination coverage, skilled birth attendance, and nutritional status. Our field validation demonstrates that this method is a logistically feasible alternative to conventional sampling frames and may be used at the population level. Geospatial sampling methods provide cost-affordable and logistically feasible strategies for sampling mobile populations, a crucial first step toward reaching these groups with health services.

Malaria smear positivity among Kenyan children peaks at intermediate temperatures as predicted by ecological models.

BACKGROUND: Ambient temperature is an important determinant of malaria transmission and suitability, affecting the life-cycle of the Plasmodium parasite and Anopheles vector. Early models predicted a thermal malaria transmission optimum of 31 °C, later revised to 25 °C using experimental data from mosquito and parasite biology. However, the link between ambient temperature and human malaria incidence remains poorly resolved. METHODS: To evaluate the relationship between ambient temperature and malaria risk, 5833 febrile children (<18 years-old) with an acute, non-localizing febrile illness were enrolled from four heterogenous outpatient clinic sites in Kenya (Chulaimbo, Kisumu, Msambweni and Ukunda). Thick and thin blood smears were evaluated for the presence of malaria parasites. Daily temperature estimates were obtained from land logger data, and rainfall from National Oceanic and Atmospheric Administration (NOAA)'s Africa Rainfall Climatology (ARC) data. Thirty-day mean temperature and 30-day cumulative rainfall were estimated and each lagged by 30 days, relative to the febrile visit. A generalized linear mixed model was used to assess relationships between malaria smear positivity and predictors including temperature, rainfall, age, sex, mosquito exposure and socioeconomic status. RESULTS: Malaria smear positivity varied between 42-83% across four clinic sites in western and coastal Kenya, with highest smear positivity in the rural, western site. The temperature ranges were cooler in the western sites and warmer in the coastal sites. In multivariate analysis controlling for socioeconomic status, age, sex, rainfall and bednet use, malaria smear positivity peaked near 25 °C at all four sites, as predicted a priori from an ecological model. CONCLUSIONS: This study provides direct field evidence of a unimodal relationship between ambient temperature and human malaria incidence with a peak in malaria transmission occurring at lower temperatures than previously recognized clinically. This nonlinear relationship with an intermediate optimal temperature implies that future climate warming could expand malaria incidence in cooler, highland regions while decreasing incidence in already warm regions with average temperatures above 25 °C. These findings support efforts to further understand the nonlinear association between ambient temperature and vector-borne diseases to better allocate resources and respond to disease threats in a future, warmer world.

Parasitic infections during pregnancy need not affect infant antibody responses to early vaccination against Streptococcus pneumoniae, diphtheria, or Haemophilus influenzae type B.

BACKGROUND: Globally, vaccine-preventable diseases remain a significant cause of early childhood mortality despite concerted efforts to improve vaccine coverage. One reason for impaired protection may be the influence of prenatal exposure to parasitic antigens on the developing immune system. Prior research had shown a decrease in infant vaccine response after in utero parasite exposure among a maternal cohort without aggressive preventive treatment. This study investigated the effect of maternal parasitic infections on infant vaccination in a more recent setting of active anti-parasitic therapy. METHODOLOGY/PRINCIPAL FINDINGS: From 2013-2015, 576 Kenyan women were tested in pregnancy for malaria, soil-transmitted helminths, filaria, and S. haematobium, with both acute and prophylactic antiparasitic therapies given. After birth, 567 infants received 10-valent S. pneumoniae conjugate vaccine and pentavalent vaccine for hepatitis B, pertussis, tetanus, H. influenzae type B (Hib) and C. diphtheriae toxoid (Dp-t) at 6, 10, and 14 weeks. Infant serum samples from birth, 10 and 14 weeks, and every six months until age three years, were analyzed using a multiplex bead assay to quantify IgG for Hib, Dp-t, and the ten pneumococcal serotypes. Antenatal parasitic prevalence was high; 461 women (80%) had at least one and 252 (43.6%) had two or more infections during their pregnancy, with the most common being malaria (44.6%), S. haematobium (43.9%), and hookworm (29.2%). Mixed models comparing influence of infection on antibody concentration revealed no effect of prenatal infection status for most vaccine outcomes. Prevalences of protective antibody concentrations after vaccination were similar among the prenatal exposure groups. CONCLUSIONS/SIGNIFICANCE: These findings are in contrast with results from our prior cohort study performed when preventive anti-parasite treatment was less frequently given. The results suggest that the treatment of maternal infections in pregnancy may be able to moderate the previously observed effect of antenatal maternal infections on infant vaccine responses.

Solid Wastes Provide Breeding Sites, Burrows, and Food for Biological Disease Vectors, and Urban Zoonotic Reservoirs: A Call to Action for Solutions-Based Research.

Background: Infectious disease epidemiology and planetary health literature often cite solid waste and plastic pollution as risk factors for vector-borne diseases and urban zoonoses; however, no rigorous reviews of the risks to human health have been published since 1994. This paper aims to identify research gaps and outline potential solutions to interrupt the vicious cycle of solid wastes; disease vectors and reservoirs; infection and disease; and poverty. Methods: We searched peer-reviewed publications from PubMed, Google Scholar, and Stanford Searchworks, and references from relevant articles using the search terms ("disease" OR "epidemiology") AND ("plastic pollution," "garbage," and "trash," "rubbish," "refuse," OR "solid waste"). Abstracts and reports from meetings were included only when they related directly to previously published work. Only articles published in English, Spanish, or Portuguese through 2018 were included, with a focus on post-1994, after the last comprehensive review was published. Cancer, diabetes, and food chain-specific articles were outside the scope and excluded. After completing the literature review, we further limited the literature to "urban zoonotic and biological vector-borne diseases" or to "zoonotic and biological vector-borne diseases of the urban environment." Results: Urban biological vector-borne diseases, especially Aedes-borne diseases, are associated with solid waste accumulation but vector preferences vary over season and region. Urban zoonosis, especially rodent and canine disease reservoirs, are associated with solid waste in urban settings, especially when garbage accumulates over time, creating burrowing sites and food for reservoirs. Although evidence suggests the link between plastic pollution/solid waste and human disease, measurements are not standardized, confounders are not rigorously controlled, and the quality of evidence varies. Here we propose a framework for solutions-based research in three areas: innovation, education, and policy. Conclusions: Disease epidemics are increasing in scope and scale with urban populations growing, climate change providing newly suitable vector climates, and immunologically naïve populations becoming newly exposed. Sustainable solid waste management is crucial to prevention, specifically in urban environments that favor urban vectors such as Aedes species. We propose that next steps should include more robust epidemiological measurements and propose a framework for solutions-based research.

Neighborhood Violence Impacts Disease Control and Surveillance: Case Study of Cali, Colombia from 2014 to 2016.

Arboviruses are responsible for a large burden of disease globally and are thus subject to intense epidemiological scrutiny. However, a variable notably absent from most epidemiological analyses has been the impact of violence on arboviral transmission and surveillance. Violence impedes surveillance and delivery of health and preventative services and affects an individual's health-related behaviors when survival takes priority. Moreover, low and middle-income countries bear a disproportionately high burden of violence and related health outcomes, including vector borne diseases. To better understand the epidemiology of arboviral outbreaks in Cali, Colombia, we georeferenced chikungunya (CHIKV), dengue (DENV), and Zika (ZIKV) viral cases from The National System of Surveillance in Public Health between October 2014 and April 2016. We extracted homicide data from the municipal monthly reports and kernel density of homicide distribution from IdeasPaz. Crucially, an overall higher risk of homicide is associated with increased risk of reported DENV, lower rates of acute testing, and higher rates of lab versus clinical discordance. In the context of high violence as a potential barrier to access to preventive health services, a community approach to improve health and peace should be considered.

Serological and spatial analysis of alphavirus and flavivirus prevalence and risk factors in a rural community in western Kenya.

Alphaviruses, such as chikungunya virus, and flaviviruses, such as dengue virus, are (re)-emerging arboviruses that are endemic in tropical environments. In Africa, arbovirus infections are often undiagnosed and unreported, with febrile illnesses often assumed to be malaria. This cross-sectional study aimed to characterize the seroprevalence of alphaviruses and flaviviruses among children (ages 5-14, n = 250) and adults (ages 15 ≥ 75, n = 250) in western Kenya. Risk factors for seropositivity were explored using Lasso regression. Overall, 67% of participants showed alphavirus seropositivity (CI95 63%-70%), and 1.6% of participants showed flavivirus seropositivity (CI95 0.7%-3%). Children aged 10-14 were more likely to be seropositive to an alphavirus than adults (p < 0.001), suggesting a recent transmission period. Alphavirus and flavivirus seropositivity was detected in the youngest participants (age 5-9), providing evidence of inter-epidemic transmission. Demographic variables that were significantly different amongst those with previous infection versus those without infection included age, education level, and occupation. Behavioral and environmental variables significantly different amongst those in with previous infection to those without infection included taking animals for grazing, fishing, and recent village flooding. Experience of recent fever was also found to be a significant indicator of infection (p = 0.027). These results confirm alphavirus and flavivirus exposure in western Kenya, while illustrating significantly higher alphavirus transmission compared to previous studies.

Community context and sub-neighborhood scale detail to explain dengue, chikungunya and Zika patterns in Cali, Colombia.

BACKGROUND: Cali, Colombia has experienced chikungunya and Zika outbreaks and hypoendemic dengue. Studies have explained Cali's dengue patterns but lack the sub-neighborhood-scale detail investigated here. METHODS: Spatial-video geonarratives (SVG) with Ministry of Health officials and Community Health Workers were collected in hotspots, providing perspective on perceptions of why dengue, chikungunya and Zika hotspots exist, impediments to control, and social outcomes. Using spatial video and Google Street View, sub-neighborhood features possibly contributing to incidence were mapped to create risk surfaces, later compared with dengue, chikungunya and Zika case data. RESULTS: SVG captured insights in 24 neighborhoods. Trash and water risks in Calipso were mapped using SVG results. Perceived risk factors included proximity to standing water, canals, poverty, invasions, localized violence and military migration. These risks overlapped case density maps and identified areas that are suitable for transmission but are possibly underreporting to the surveillance system. CONCLUSION: Resulting risk maps with local context could be leveraged to increase vector-control efficiency- targeting key areas of environmental risk.

Optimization of a Membrane Feeding Assay for Plasmodium vivax Infection in Anopheles albimanus.

INTRODUCTION: Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity. METHODS/PRINCIPAL FINDINGS: A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy. CONCLUSIONS: We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.