Larval source management is a key strategy to control the spread of Aedes-borne viral diseases including dengue, Zika, chikungunya, and yellow fever. However, locating potential larval habitats through traditional field methods is challenging and labor-intensive at scale. Here, we demonstrate a scalable, high-resolution drone imagery and supervised machine learning approach to map potential Aedes larval container habitats across Dar es Salaam, Tanzania, a dense urban environment with informal settlements. Local larval surveillance and existing literature confirm epidemiological relevance of buckets and jerry cans, tires, and water tanks as key potential habitats. Drone images revealed rooftop tires, a container type likely overlooked during ground surveillance. We trained a U-Net deep learning model on very-high-resolution drone imagery (3-5 cm resolution) which was manually annotated across 4.6 km2, and applied it to predict containers across 27.27 km2, spanning 20 neighborhoods. The model predicted over 135,000 containers with detection accuracies of 75% for water tanks, 72% for tires, and 54% for buckets. Bucket and tire densities were strongly and positively correlated with population density across neighborhoods, whereas water tank density was not, suggesting the distribution of these container types reflect distinct underlying drivers. This study reveals otherwise difficult-to-observe container types, highlights the abundance and spatial heterogeneity of potential Aedes larval habitats across Dar es Salaam, and demonstrates a scalable approach for improving detection of potential Aedes larval container habitats in a dense urban environment.

Scrub typhus is a common bacterial infection in many parts of Asia. The causative agent, Orientia tsutsugamushi, is transmitted by trombiculid mite (chigger) larvae that require small mammals as maintaining hosts. We studied the prevalence of O. tsutsugamushi infection in mites and small mammals in villages and land surrounding them in South India to determine high-risk settings. We identified 12,431 mite larvae on 883 small mammals, 32% of which were bandicoot rats, 31% black rats, and 31% Asian house shrews. Leptotrombidium imphalum was the most common mite species and the only species associated with O. tsutsugamushi infection (prevalence 3.6%). Orientia infection increased with mite population size on a host. Host numbers, the L. imphalum index, and the prevalence of Orientia infection in chiggers were considerably higher within human settlements than in surrounding fields, suggesting that most human scrub typhus infection occurs inside villages rather than during agricultural work.

Phlebotomus perniciosus is a major sand fly in the Western Mediterranean and a primary vector for Leishmania infantum, causing human and canine visceral leishmaniasis. This species can also transmit phleboviruses, including Toscana virus, associated with meningitis and encephalitis. Despite its importance, genomic resources for P. perniciosus remain limited, with only a low-quality assembly available. A high-quality reference genome is essential to study traits related to vector competence, host preference, and responses to control measures. Here, we present a high-quality de novo assembly using Oxford Nanopore long-read sequencing. We evaluated two assembly strategies, guided and unguided, using Canu and Flye. The guided Canu assembly was optimal, producing a 143 Mb genome with scaffold N50 of 48 Mb and 95% BUSCO completeness. Annotation with RNA-seq and protein homology predicted 12,492 genes (96% BUSCO) and 24% repetitive sequences. The complete mitochondrial genome was also assembled. This robust genome offers a foundation for investigating vector biology and adaptive traits, expanding genomic resources for phlebotomine sand flies and supporting improved leishmaniasis surveillance and control efforts.

Scrub typhus is a mite-borne infection, largely affecting rural populations in many parts of Asia. This cohort study explored socio-demographic, behavioural, and spatial risk factors at different levels of endemicity. 2206 rural residents from 37 villages in Tamil Nadu, South India, underwent a questionnaire survey and blood sampling at baseline and annually over 2 years to detect sero-conversion. Satellite images were used for visual land use classification. Local sero-prevalence was estimated using 5602 baseline blood samples.Two hundred and seventy cases of seroconversions occurred during 3629 person-years (incidence rate 78/1000, 95%CI 67, 91). Older age was associated with scrub typhus in crude but not in multivariable analysis adjusting for socio-economic factors. By contrast, the increased risk in females compared to males (RR 1.4) was unaffected by adjusting for confounders. In multivariable analysis, agricultural and related outdoor activities were only weakly associated with scrub typhus. However, agricultural activities were strongly associated with scrub typhus if local sero-prevalence was low, but not if it was high. Females were at a higher risk than males in high-prevalence areas but not in low-prevalence areas. To conclude, agricultural activities were not strongly associated with scrub typhus. Transmission within human settlements may predominate in highly endemic settings.

BACKGROUND:Hospital studies suggest that scrub typhus is a leading cause of severe undifferentiated fever in regions across Asia where the disease is endemic, but the population-based incidence of infection and illness has been little studied. METHODS:We conducted a population-based cohort study to assess epidemiologic and clinical characteristics of scrub typhus in 37 villages in Tamil Nadu, India, where the disease is highly endemic. Study participants were visited every 6 to 8 weeks over a period of 2 years; a venous blood sample was obtained from those who had had fever since the last visit. A subcohort of participants underwent blood sampling to estimate the incidence of serologically confirmed Orientia tsutsugamushi infection. RESULTS:We systematically assessed 32,279 participants from 7619 households for acute febrile illness. During 54,588 person-years of follow-up, we observed 6175 episodes of fever. A blood sample was obtained in 4474 episodes (72.5%), of which 328 (7.3%) met the clinical case definition of scrub typhus (detection of IgM against O. tsutsugamushi on enzyme-linked immunosorbent assay [ELISA] or detection of O. tsutsugamushi on polymerase-chain-reaction assay). The incidence of clinical infection was 6.0 cases per 1000 person-years (95% confidence interval [CI], 4.8 to 7.5). A total of 71 clinical cases (21.6%) resulted in hospitalization (incidence, 1.3 events per 1000 person-years; 95% CI, 1.0 to 1.7). A total of 29 clinical cases (8.8%) were severe, as indicated by the presence of organ dysfunction or adverse pregnancy outcomes (incidence, 0.5 cases per 1000 person-years; 95% CI, 0.4 to 0.8). Among 2128 participants in the subcohort who provided samples at the beginning and end of a study year, the incidence of seroconversion independent of any symptoms was 81.2 events per 1000 person-years (95% CI, 70.8 to 91.6). The incidence of clinical infection was higher in older age groups than in younger age groups and higher among female participants than among male participants. By contrast, the age-adjusted rate of severe infection was similar among male and female participants. Among 5602 participants assessed at the start of the first year of the study, the seroprevalence of IgG as assessed with ELISA was 42.8% (95% CI, 35.8 to 50.2). IgG seropositivity at the beginning of years 1 or 2 did not protect against clinical illness during the subsequent year but was associated with less severe disease than IgG seronegativity. CONCLUSIONS:We describe the burden of scrub typhus, including the incidence of asymptomatic infection, in a region of Asia where the disease is endemic. (Funded by the U.K. Medical Research Council; ClinicalTrials.gov number, NCT04506944.).

OBJECTIVES:Aedes aegypti is the principal dengue virus (DENV) vector, while Ae. albopictus is often considered to have a negligible role. However, limited field data comparing their involvement in DENV transmission hampers accurate evaluation of current interventions targeting only one species and dengue outbreak risk assessments in non-endemic areas. METHODS:We conducted a systematic review and meta-analysis of studies assessing DENV prevalence in both Ae. albopictus and Ae. aegypti. We searched EMBASE, PubMed, SCIELO, and Global Index Medicus up to 15 September 2023. Risk ratios (RR) were calculated using fixed-effects model in meta-analyses, summarizing prevalences in Ae. albopictus vs Ae. aegypti. RESULTS:Of 5,432 records screened, 36 studies from 14 countries and territories were included (covering 96,884 Ae. aegypti and 106,205 Ae. albopictus mosquitoes). Overall, Ae. albopictus showed a 35% lower DENV prevalence than Ae. aegypti (RR=0.65, 95% CI=0.56-0.75, I²=93%). The difference was more pronounced pre-2000 (63% lower; RR=0.37; CI=0.30, 0.46), but post-2000 data showed no significant difference (2000s: RR=1.17; 95% CI=0.81, 1.69; since 2010 RR=0.86; 95% CI=0.68, 1.07). CONCLUSION:While Ae. aegypti remains the primary vector, recent evidence suggests Ae. albopictus plays a more notable role in DENV transmission than previously thought. Effective vector control strategies should therefore target both species.

Vector-borne diseases (VBDs) in India, which include viral, parasitic, and bacterial infections, present significant public health challenges. These diseases, transmitted by various vectors such as mosquitoes, ticks, and fleas, contribute to high morbidity and mortality rates, particularly in vulnerable populations. Despite existing control measures, these diseases continue to affect millions, driven by factors such as climate change, urbanization, and inadequate disease surveillance. necessitating urgent attention to fill the gaps in various surveillance, treatment, and prevention strategies. There is a critical need for a comprehensive understanding of the epidemiology, transmission dynamics, vector behavior, and environmental factors influencing disease spread. The knowledge of vector biology and ecology, resistance to insecticides, and environmental adaptations are crucial for the development of targeted control strategies. Existing diagnostic tools for VBDs are often inadequate, particularly in resource-limited settings. There is a pressing need for rapid, cost-effective diagnostic methods and novel treatments to address drug resistance. Post-infection syndromes and chronic conditions associated with diseases like dengue and chikungunya contribute to the long-term health impacts of VBDs. There is an urgent need for the development and implementation of vaccines for diseases like dengue and malaria. Continued investment in vaccine research is essential to provide long-term solutions to VBDs along with current vector control measures. An integrated approach that combines biological control, environmental management, and community involvement is necessary to enhance the sustainability and effectiveness of control strategies. Enhancing public awareness about VBDs, their transmission, and preventive measures is vital. Education campaigns should target high-risk populations, focusing on behavioral changes to reduce vector exposure. Promoting interdisciplinary research collaborations among academia, governmental, and Non-Governmental Organizations (NGO) can lead to more holistic approaches in addressing VBDs and strengthening the integration of research findings into public health policy and practice to ensure that evidence-based strategies are implemented at all levels. Establishing sustainable funding mechanisms for research and intervention programs is crucial to maintaining momentum in the fight against VBDs. In summary, addressing the research gaps, enhancing surveillance, and improving treatment and prevention strategies for VBDs in India are imperative for mitigating their impact. By prioritizing these areas, India can develop a more effective and sustainable response to the growing burden of VBDs, ultimately improving public health outcomes including and improving the quality of life.

Abstract Background As cases of visceral leishmaniasis (VL) in India dwindle, there is motivation to monitor elimination progress on a finer geographic scale than sub-district (block). Low-incidence projections across geographically- and demographically- heterogeneous communities are difficult to act upon, and equitable elimination cannot be achieved if local pockets of incidence are overlooked. However, maintaining consistent surveillance at this scale is resource-intensive and not sustainable in the long-term. Methods We analysed VL incidence across 45,000 villages in Bihar state, exploring spatial autocorrelation and associations with local environmental conditions in order to assess the feasibility of inference at this scale. We evaluated a statistical disaggregation approach to infer finer spatial variation from routinely-collected, block-level data, validating against observed village-level incidence. Results This disaggregation approach does not estimate village-level incidence more accurately than a baseline assumption of block-homogeneity. Spatial auto-correlation is evident on a block-level but weak between neighbouring villages within the same block, possibly suggesting that longer-range transmission (e.g., due to population movement) may be an important contributor to village-level heterogeneity. Conclusions Increasing the range of reactive interventions to neighbouring villages may not improve their efficacy in suppressing transmission, but maintaining surveillance and diagnostic capacity in areas distant from recently observed cases - particularly along routes of population movement from endemic regions - could reduce reintroduction risk in currently unaffected villages. The reactive, spatially-targeted approach to VL surveillance limits interpretability of data observed at the village level, and hence the feasibility of routinely drawing and validating inference at this scale.

Abstract Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Abstract Background Vector control based on indoor residual spraying (IRS) is one of the main components of the visceral leishmaniasis (VL) elimination programme in India. Dichlorodiphenyltrichloroethane (DDT) was used for IRS until 2015 and was later replaced by the synthetic pyrethroid alpha-cypermethrin. Both classes of insecticides share the same target site, the voltage-gated sodium channel (Vgsc). As high levels of resistance to DDT have been documented in the local sand fly vector, Phlebotomus argentipes, it is possible that mutations in the Vgsc gene could provide resistance to alpha-cypermethrin, affecting current IRS pyrethroid-based vector control. Methods This study aimed to compare frequencies of knockdown resistance (kdr) mutations in Vgsc between two sprayed and two unsprayed villages in Bihar state, India, which had the highest VL burden of the four endemic states. Across four villages, 350 female P. argentipes were collected as part of a 2019 molecular xenomonitoring study. DNA was extracted and used for sequence analysis of the IIS6 fragment of the Vgsc gene to assess the presence of kdr mutations. Results Mutations were identified at various positions, most frequently at codon 1014, a common site known to be associated with insecticide resistance in mosquitoes and sand flies. Significant inter-village variation was observed, with sand flies from Dharampur, an unsprayed village, showing a significantly higher proportion of wild-type alleles (55.8%) compared with the three other villages (8.5–14.3%). The allele differences observed across the four villages may result from selection pressure caused by previous exposure to DDT. Conclusions While DDT resistance has been reported in Bihar, P. argentipes is still susceptible to pyrethroids. However, the presence of kdr mutations in sand flies could present a threat to IRS used for VL control in endemic villages in India. Continuous surveillance of vector bionomics and insecticide resistance, using bioassays and target genotyping, is required to inform India’s vector control strategies and to ensure the VL elimination target is reached and sustained. Graphical Abstract

Lymphatic filariasis (LF) is a leading cause of disability due to infectious disease worldwide. The Recife Metropolitan Region (RMR) is the only remaining focus of LF in Brazil, where the parasite Wuchereria bancrofti is transmitted solely by the mosquito Culex quinquefasciatus . This study reports the results of transmission assessment surveys and molecular xenomonitoring in the city of Olinda, RMR, after nearly 15 years (2015-2016) of interventions for LF elimination. Participants were screened for W. bancrofti antigen via immunochromatographic card tests (ICT) in: 1) door-todoor surveys conducted for all children aged 5-7 years from 4 out of 17 intervention areas treated with at least five annual doses of mass drug administration (MDA), and 2) a two -stage cluster sampling survey of residents aged 5 years and older in non-MDA areas. Mosquitoes were collected via handheld aspirators in four MDA areas, differentiated by species, sex, and physiological status, pooled into groups of up to 10 blood -fed, semigravid, and gravid mosquitoes, and screened for W. bancrofti infection by real-time quantitative polymerase chain reaction (RT-qPCR). All 1,170 children from MDA areas and the entire population sample of 990 residents in non-MDA areas were ICT negative. In MDA areas, a total of 3,152 female C X . quinquefasciatus mosquitoes in 277 households (range, 0-296 mosquitoes per house) were collected via aspiration. RT-qPCR of 233 pools of mosquitos were negative for W. bancrofti RNA; an independent reference laboratory confirmed these results. These results provide evidence that LF transmission has been halted in this setting.

BACKGROUND:As of 2021, the National Kala-azar Elimination Programme (NKAEP) in India has achieved visceral leishmaniasis (VL) elimination (<1 case / 10,000 population/year per block) in 625 of the 633 endemic blocks (subdistricts) in four states. The programme needs to sustain this achievement and target interventions in the remaining blocks to achieve the WHO 2030 target of VL elimination as a public health problem. An effective tool to analyse programme data and predict/ forecast the spatial and temporal trends of VL incidence, elimination threshold, and risk of resurgence will be of use to the programme management at this juncture. METHODOLOGY/PRINCIPAL FINDINGS:We employed spatiotemporal models incorporating environment, climatic and demographic factors as covariates to describe monthly VL cases for 8-years (2013-2020) in 491 and 27 endemic and non-endemic blocks of Bihar and Jharkhand states. We fitted 37 models of spatial, temporal, and spatiotemporal interaction random effects with covariates to monthly VL cases for 6-years (2013-2018, training data) using Bayesian inference via Integrated Nested Laplace Approximation (INLA) approach. The best-fitting model was selected based on deviance information criterion (DIC) and Watanabe-Akaike Information Criterion (WAIC) and was validated with monthly cases for 2019-2020 (test data). The model could describe observed spatial and temporal patterns of VL incidence in the two states having widely differing incidence trajectories, with >93% and 99% coverage probability (proportion of observations falling inside 95% Bayesian credible interval for the predicted number of VL cases per month) during the training and testing periods. PIT (probability integral transform) histograms confirmed consistency between prediction and observation for the test period. Forecasting for 2021-2023 showed that the annual VL incidence is likely to exceed elimination threshold in 16-18 blocks in 4 districts of Jharkhand and 33-38 blocks in 10 districts of Bihar. The risk of VL in non-endemic neighbouring blocks of both Bihar and Jharkhand are less than 0.5 during the training and test periods, and for 2021-2023, the probability that the risk greater than 1 is negligible (P<0.1). Fitted model showed that VL occurrence was positively associated with mean temperature, minimum temperature, enhanced vegetation index, precipitation, and isothermality, and negatively with maximum temperature, land surface temperature, soil moisture and population density. CONCLUSIONS/SIGNIFICANCE:The spatiotemporal model incorporating environmental, bioclimatic, and demographic factors demonstrated that the KAMIS database of the national programmme can be used for block level predictions of long-term spatial and temporal trends in VL incidence and risk of outbreak / resurgence in endemic and non-endemic settings. The database integrated with the modelling framework and a dashboard facility can facilitate such analysis and predictions. This could aid the programme to monitor progress of VL elimination at least one-year ahead, assess risk of resurgence or outbreak in post-elimination settings, and implement timely and targeted interventions or preventive measures so that the NKAEP meet the target of achieving elimination by 2030.

Abstract Based on a similar exercise carried out by the BOHS in 2020 during the early stages of the COVID-19 pandemic. The BOHS COVID-19 risk matrix was adapted to provide an animal charity with an Avian Influenza (AI) risk management tool. The charity staff handle sick and dead birds in the wild (symptoms of AI may not be immediately apparent) and at their animal treatment and housing centres. This tool guides the employer in determining appropriate control measures to help reduce the risk of their staff, and also the public, from being infected with AI. Although there is currently limited evidence of transference to humans and the risk appears to be low, this could change in the not too distant future. Cross species infections are predicted to increase with the habitat pressures brought about by climate change. Therefore this AI risk matrix has been developed as a precautionary approach. The approach recognises the pitfalls of reliance on PPE alone and endeavours to promote a preventative approach built on good occupational hygiene practice. Given the variety and complexity of the workplace, the AI risk matrix provides an initial reference for control measures, risk evaluation, and grouped job roles to help the employer make balanced decisions and decide what are the reasonable precautions. Acknowledgements- Authors of the BOHS COVID-19 risk matrix Kelvin Williams BSc(Hons) DipOH CFFOH Dr Mangala Patil-Mead

Abstract Background Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. Results Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%. Conclusions This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

International laws for commercial aviation have achieved an exceptional degree of harmonisation and greatly improved passenger safety. Yet, despite much international guidance, enforceable laws for public health protection in aviation are mainly the responsibility of national authorities. As a result, public health laws may be incoherent, in conflict with other countries and/or based on disputed scientific evidence. The COVID-19 pandemic has highlighted the responsibility of airlines and regulatory authorities to protect not only air passengers but also populations in destination countries. While the greatest risk to global public health is the potential spread of disease by infected passengers or vectors, lesser-known risks include food contamination, inadequate sanitary facilities and poor air quality within the cabin. In preparedness for inevitable future disease outbreaks and pandemics, an urgent review of international law as it applies to public health in commercial aviation is needed, with greater investment in scientific research to enable more accurate and effective risk assessment and management, supported by enforceable laws and clear responsibility.

BACKGROUND:Serum or whole blood collection, processing, transport and storage still present significant challenges in low resource settings where mass surveillance is required to sustain disease elimination. Therefore, in this study, we explored the diagnostic efficacy of dried blood spots (DBS) as a minimally invasive and potentially cost-effective alternative sampling technique to whole blood sampling procedures for subsequent detection of Leishmania donovani antibodies or DNA. METHODOLOGY AND PRINCIPAL FINDINGS:Archived serum, DNA samples from whole blood of visceral leishmaniasis (VL) cases and healthy controls, and DBS from corresponding cases and controls, were used. Both molecular and serological assays were optimized to detect L. donovani antibodies or DNA in DBS elute and results were compared against those obtained with whole blood. Serological assays (both rK28 ELISA and rK39 ELISA) of DBS samples showed sensitivity and specificity of 100% and had excellent agreement with results from whole blood samples (kappa value ranged from 0.98-1). Bland-Altman analysis of OD values from rK28-ELISA with DBS elute and patients' serum showed an excellent agreement (ICC = 0.9) whereas a good agreement (ICC = 0.8) was observed in the case of rK39-ELISA. However, qPCR and RPA of DBS samples had a diminished sensitivity of 76% and 68%, respectively, and poor agreement was observed with the whole blood samples. CONCLUSION:Our results demonstrate that DBS offer excellent diagnostic efficiency for serological assays and represent a viable alternative to whole blood sampling procedures.

BackgroundThe kala-azar elimination programme has resulted in a significant reduction in visceral leishmaniasis (VL) cases across the Indian Subcontinent. To detect any resurgence of transmission, a sensitive cost-effective surveillance system is required. Molecular xenomonitoring (MX), detection of pathogen DNA/RNA in vectors, provides a proxy of human infection in the lymphatic filariasis elimination programme. To determine whether MX can be used for VL surveillance in a low transmission setting, large numbers of the sand fly vector Phlebotomus argentipes are required. This study will determine the best method for capturing P. argentipes females for MX.Methodology/principal findingsThe field study was performed in two programmatic and two non-programmatic villages in Bihar, India. A total of 48 households (12/village) were recruited. Centers for Disease Control and Prevention light traps (CDC-LTs) were compared with Improved Prokopack (PKP) and mechanical vacuum aspirators (MVA) using standardised methods. Four 12x12 Latin squares, 576 collections, were attempted (12/house, 144/village,192/method). Molecular analyses of collections were conducted to confirm identification of P. argentipes and to detect human and Leishmania DNA. Operational factors, such as time burden, acceptance to householders and RNA preservation, were also considered. A total of 562 collections (97.7%) were completed with 6,809 sand flies captured. Females comprised 49.0% of captures, of which 1,934 (57.9%) were identified as P. argentipes. CDC-LTs collected 4.04 times more P. argentipes females than MVA and 3.62 times more than PKP (p<0.0001 for each). Of 21,735 mosquitoes in the same collections, no significant differences between collection methods were observed. CDC-LTs took less time to install and collect than to perform aspirations and their greater yield compensated for increased sorting time. No significant differences in Leishmania RNA detection and quantitation between methods were observed in experimentally infected sand flies maintained in conditions simulating field conditions. CDC-LTs were favoured by householders.Conclusions/significanceCDC-LTs are the most useful collection tool of those tested for MX surveillance since they collected higher numbers of P. argentipes females without compromising mosquito captures or the preservation of RNA. However, capture rates are still low.

A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45–98.75%), 90% (95% CI: 78.19–96.67%), and 88% (95% CI: 75.69–95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45–98.75%) and 92.00% (80.77–97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919–0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) cause economically important diseases that are currently exotic to the United Kingdom (UK), but have significant potential for introduction and onward transmission. Given the susceptibility of animals kept in zoo collections to vector-borne diseases, a qualitative risk assessment for the introduction of BTV and AHSV to ZSL London Zoo was performed. Risk pathways for each virus were identified and assessed using published literature, animal import data and outputs from epidemiological models. Direct imports of infected animals, as well as wind-borne infected Culicoides, were considered as routes of incursion. The proximity of ongoing disease events in mainland Europe and proven capability of transmission to the UK places ZSL London Zoo at higher risk of BTV release and exposure (estimated as low to medium) than AHSV (estimated as very low to low). The recent long-range expansion of AHSV into Thailand from southern Africa highlights the need for vector competence studies of Palearctic Culicoides for AHSV to assess the risk of transmission in this region.