Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation.

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.

The longitudinal interplay between insecure attachment behaviors and psychosocial strengths among children in child welfare services.

Children who have experienced maltreatment are more likely to have disrupted attachments, fewer psychosocial strengths, and poorer long-term psychosocial outcomes. However, few studies have examined the interplay between attachment security and psychosocial strengths among children involved in therapeutic services in the context of the child welfare system. The present longitudinal study examines the insecure attachment behaviors and psychosocial strengths of 555 children referred to the Therapeutic Family Care program (TFCP) in Cobourg, Ontario between 2000 and 2019. The children were assessed by their caregivers on a regular basis using the Assessment Checklist for Children (ACC) and the complementary strengths-focused ACC+ measure. Average age of children at baseline was 9.57 years (SD = 3.51) and 229 (41.26%) were female. We conducted growth curve and random intercepts cross-lagged panel models to test the longitudinal interplay between insecure attachment behaviors and strengths. Results suggest that females' attachment security improved, males' attachment security worsened, and both males and females developed strengths over time. Further, analyses revealed a directional effect, whereby fewer insecure attachment behaviors predicted more psychosocial strengths approximately 6 months later. Implications for attachment-oriented and strengths-based services in the context of child welfare are discussed.

Inferring transmission trees to guide targeting of interventions against visceral leishmaniasis and post-kala-azar dermal leishmaniasis.

Understanding of spatiotemporal transmission of infectious diseases has improved significantly in recent years. Advances in Bayesian inference methods for individual-level geo-located epidemiological data have enabled reconstruction of transmission trees and quantification of disease spread in space and time, while accounting for uncertainty in missing data. However, these methods have rarely been applied to endemic diseases or ones in which asymptomatic infection plays a role, for which additional estimation methods are required. Here, we develop such methods to analyze longitudinal incidence data on visceral leishmaniasis (VL) and its sequela, post-kala-azar dermal leishmaniasis (PKDL), in a highly endemic community in Bangladesh. Incorporating recent data on VL and PKDL infectiousness, we show that while VL cases drive transmission when incidence is high, the contribution of PKDL increases significantly as VL incidence declines (reaching 55% in this setting). Transmission is highly focal: 85% of mean distances from inferred infectors to their secondary VL cases were <300 m, and estimated average times from infector onset to secondary case infection were <4 mo for 88% of VL infectors, but up to 2.9 y for PKDL infectors. Estimated numbers of secondary cases per VL and PKDL case varied from 0 to 6 and were strongly correlated with the infector's duration of symptoms. Counterfactual simulations suggest that prevention of PKDL could have reduced overall VL incidence by up to 25%. These results highlight the need for prompt detection and treatment of PKDL to achieve VL elimination in the Indian subcontinent and provide quantitative estimates to guide spatiotemporally targeted interventions against VL.

Low-cost solution to the grid reliability problem with 100% penetration of intermittent wind, water, and solar for all purposes.

This study addresses the greatest concern facing the large-scale integration of wind, water, and solar (WWS) into a power grid: the high cost of avoiding load loss caused by WWS variability and uncertainty. It uses a new grid integration model and finds low-cost, no-load-loss, nonunique solutions to this problem on electrification of all US energy sectors (electricity, transportation, heating/cooling, and industry) while accounting for wind and solar time series data from a 3D global weather model that simulates extreme events and competition among wind turbines for available kinetic energy. Solutions are obtained by prioritizing storage for heat (in soil and water); cold (in ice and water); and electricity (in phase-change materials, pumped hydro, hydropower, and hydrogen), and using demand response. No natural gas, biofuels, nuclear power, or stationary batteries are needed. The resulting 2050-2055 US electricity social cost for a full system is much less than for fossil fuels. These results hold for many conditions, suggesting that low-cost, reliable 100% WWS systems should work many places worldwide.

Combining indoor and outdoor methods for controlling malaria vectors: an ecological model of endectocide-treated livestock and insecticidal bed nets.

BACKGROUND: Malaria is spread by mosquitoes that are increasingly recognised to have diverse biting behaviours. How a mosquito in a specific environment responds to differing availability of blood-host species is largely unknown and yet critical to vector control efficacy. A parsimonious mathematical model is proposed that accounts for a diverse range of host-biting behaviours and assesses their impact on combining long-lasting insecticidal nets (LLINs) with a novel approach to malaria control: livestock treated with insecticidal compounds ('endectocides') that kill biting mosquitoes. RESULTS: Simulations of a malaria control programme showed marked differences across biting ecologies in the efficacy of both LLINs as a stand-alone tool and the combination of LLINs with endectocide-treated cattle. During the intervals between LLIN mass campaigns, concordant use of endectocides is projected to reduce the bounce-back in malaria prevalence that can occur as LLIN efficacy decays over time, especially if replacement campaigns are delayed. Integrating these approaches can also dramatically improve the attainability of local elimination; endectocidal treatment schedules required to achieve this aim are provided for malaria vectors with different biting ecologies. CONCLUSIONS: Targeting blood-feeding mosquitoes by treating livestock with endectocides offers a potentially useful complement to existing malaria control programmes centred on LLIN distribution. This approach is likely to be effective against vectors with a wide range of host-preferences and biting behaviours, with the exception of species that are so strictly anthropophilic that most blood meals are taken on humans even when humans are much less available than non-human hosts. Identifying this functional relationship in wild mosquito populations and ascertaining the extent to which it differs, within as well as between species, is a critical next step before targets can be set for employing this novel approach and combination.

Incomplete distal renal tubular acidosis from a heterozygous mutation of the V-ATPase B1 subunit.

Congenital distal renal tubular acidosis (RTA) from mutations of the B1 subunit of V-ATPase is considered an autosomal recessive disease. We analyzed a distal RTA kindred with a truncation mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of V-ATPase. All heterozygous carriers in this kindred have normal plasma HCO3- concentrations and thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria were present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also had inappropriate urinary acidification with acute ammonium chloride loading and an impaired urine-blood Pco2 gradient during bicarbonaturia, indicating the presence of a H+ gradient and flux defects. In normal human renal papillae, wild-type B1 is located primarily on the plasma membrane, but papilla from one of the heterozygote who had kidney stones but not nephrocalcinosis showed B1 in both the plasma membrane as well as diffuse intracellular staining. Titration of increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+ pump activity of wild-type B1 in mammalian human embryonic kidney-293 cells and in V-ATPase-deficient Saccharomyces cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of a mutant B1 subunit that cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

Growth rates of black soldier fly larvae fed on fresh human faeces and their implication for improving sanitation.

OBJECTIVES: To determine the capacity of black soldier fly larvae (BSFL) (Hermetia illucens) to convert fresh human faeces into larval biomass under different feeding regimes, and to determine how effective BSFL are as a means of human faecal waste management. METHODS: Black soldier fly larvae were fed fresh human faeces. The frequency of feeding, number of larvae and feeding ratio were altered to determine their effects on larval growth, prepupal weight, waste reduction, bioconversion and feed conversion rate (FCR). RESULTS: The larvae that were fed a single lump amount of faeces developed into significantly larger larvae and prepupae than those fed incrementally every 2 days; however, the development into pre-pupae took longer. The highest waste reduction was found in the group containing the most larvae, with no difference between feeding regimes. At an estimated 90% pupation rate, the highest bioconversion (16-22%) and lowest, most efficient FCR (2.0-3.3) occurred in groups that contained 10 and 100 larvae, when fed both the lump amount and incremental regime. CONCLUSION: The prepupal weight, bioconversion and FCR results surpass those from previous studies into BSFL management of swine, chicken manure and municipal organic waste. This suggests that the use of BSFL could provide a solution to the health problems associated with poor sanitation and inadequate human waste management in developing countries.

From knowledge to action: participant stories of a population health intervention to reduce gender violence and HIV in three southern African countries.

This paper describes implementation research of an intervention in a complex HIV prevention randomised trial in southern Africa. Researchers collected stories of change attributed by 106 community members to an audio-drama edutainment intervention in 41 sites in Botswana, Namibia and Swaziland. The team analysed themes in the stories following a behaviour change model of conscious knowledge, attitudes, subjective norms, intention to change, agency, discussion and action (CASCADA). Storytellers attributed positive changes to the intervention in the areas of gender violence, multiple sexual partners, transactional and intergenerational sex and condom use. Their stories illustrate each of the steps in the CASCADA behaviour change model. As well as supporting an enabling environment for other interventions in the trial, the audio-drama also helped some participants to make personal changes. Collecting and discussing the stories were encouraging for the trial fieldworkers. Documenting the experiences of participants and framing the analysis of stories in an explicit behaviour change model allowed us to reflect on potential mechanisms and pathways through which the intervention impacts on individuals and communities. It helped in the design of the quantitative instruments to measure intermediate outcomes of the trial.

Knockdown resistance mutations in Phlebotomus argentipes sand flies in Bihar, India.

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.

Postintervention Immunological and Entomological Survey of Lymphatic Filariasis in the City of Olinda, Brazil, 2015-2016.

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-to-door 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 Cx. 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.

Modelling spatiotemporal patterns of visceral leishmaniasis incidence in two endemic states in India using environment, bioclimatic and demographic data, 2013-2022.

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.

Exploring networks of complex developmental trauma symptomatology among children and adolescents involved in child welfare.

Background: Clinical presentations of child and adolescent psychopathology can vary systematically for boys and girls. While network analysis is increasingly being applied to explore psychopathology in adults, there is a dearth of network studies considering differences in symptoms for boys and girls, particularly in developmental trauma-related symptomatology. Methods: This study involves rural children (n = 375, 39.47% girls) and adolescents (n = 291, 51.20% girls) involved with child protection services in Ontario, Canada. Caregivers completed the Assessment Checklist for Children or Adolescents within the first 6 months of care. Psychometric network analyses were conducted using subscales for boys and girls. Differences were examined via network comparison permutation tests, moderated network models, and independent t-tests. Results: Attachment-related interpersonal difficulties were the most central nodes in the child and adolescent networks for both boys and girls. Emotional dysregulation also had high strength centrality for adolescents. While network comparison tests found the overall network structures and global network strength to be invariant between boys and girls for children and adolescents, moderated network models and independent t-tests revealed several differences with regards to the expression of specific symptoms. Among children, girls exhibited more indiscriminate and pseudomature interpersonal behaviors, whereas boys expressed significantly more non-reciprocal interpersonal behaviors and self-injury. Adolescent girls exhibited more behavioral dysregulation and suicide discourse in the moderated network model; t-tests also indicated higher levels of emotional dysregulation, negative self-image, and other items considered clinically important complex trauma symptoms (e.g., distrust of adults, confused belonging). Discussion: This study supports evidence of differences in the expression of complex trauma symptomatology for boys and girls. Additionally, girls exhibit more symptoms, in general. Consistent with the transdiagnostic conceptualization of the consequences of developmental trauma, findings demonstrate the primacy of attachment-specific difficulties and emotion dysregulation.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations.

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.

Comparison of collection methods for Phlebotomus argentipes sand flies to use in a molecular xenomonitoring system for the surveillance of visceral leishmaniasis.

BACKGROUND: The 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 FINDINGS: The 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/SIGNIFICANCE: CDC-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.

Dried Blood Spots (DBS): A suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era.

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.

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis.

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.

A Qualitative Risk Assessment for Bluetongue Disease and African Horse Sickness: The Risk of Entry and Exposure at a UK Zoo.

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.

The use of molecular xenomonitoring for surveillance of mosquito-borne diseases.

The scientific community recognizes that molecular xenomonitoring (MX) can allow infected mosquitoes to serve as a proxy for human infection in vector-borne disease surveillance, but developing reliable MX systems for programmatic use has been challenging. The primary aim of this article is to examine the available evidence to recommend how MX can best be used for various purposes. Although much of the literature published within the last 20 years focuses on using MX for lymphatic filariasis elimination, a growing body of evidence supports its use in early warning systems for emerging infectious diseases (EIDs). An MX system design must consider the goal and target (e.g. diseases targeted for elimination versus EIDs), mosquito and pathogen characteristics, and context (e.g. setting and health system). MX is currently used as a 'supplement' to human surveillance and will not be considered as a 'replacement' until the correlation between pathogen-infection rates in human and mosquito populations is better understood. Establishing such relationships may not be feasible in elimination scenarios, due to increasingly dwindling human infection prevalence after successful control, but may still be possible for EIDs and in integrated disease surveillance systems. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.