Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps.

BACKGROUND: For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. METHODS: In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. RESULTS: A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. CONCLUSION: The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory.

Assessment of Immunoglobulin M Enzyme-Linked Immunosorbent Assay Ratios to Identify West Nile Virus and St. Louis Encephalitis Virus Infections During Concurrent Outbreaks of West Nile Virus and St. Louis Encephalitis Virus Diseases, Arizona 2015.

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are closely related mosquito-borne flaviviruses that cause clinical disease ranging from febrile illness to encephalitis. The standard for serological diagnosis is immunoglobulin M (IgM) testing followed by confirmatory plaque reduction neutralization test (PRNT) to differentiate the infecting virus. However, the PRNT is time-consuming and requires manipulation of live virus. During concurrent WNV and SLEV outbreaks in Arizona in 2015, we assessed use of a diagnostic algorithm to simplify testing. It incorporated WNV and SLEV ratios based on positive-to-negative (P/N) values derived from the IgM antibody-capture enzyme-linked immunosorbent assay. We compared each sample's ratio-based result with the confirmed WNV or SLEV sample result indicated by PRNT or PCR testing. We analyzed data from 70 patients with 77 serum and cerebrospinal fluid samples, including 53 patients with confirmed WNV infection and 17 patients with confirmed SLEV infection. Both WNV and SLEV ratios had specificity ≥95%, indicating a high likelihood that each ratio was correctly identifying the infecting virus. The SLEV ratio sensitivity of 30% was much lower than the WNV ratio sensitivity of 91%, likely because of higher cross-reactivity of SLEV antibodies and generation of lower P/N values. The standard for serological diagnosis of WNV and SLEV infections remains IgM testing followed by PRNT. However, these results suggest the ratios could potentially be used as part of a diagnostic algorithm in outbreaks to substantially reduce the need for PRNTs.

The direct healthcare costs attributable to West Nile virus illness in Ontario, Canada: a population-based cohort study using laboratory and health administrative data.

BACKGROUND: West Nile virus (WNV) is a mosquito-borne flavivirus, first detected in the Western Hemisphere in 1999 and spread across North America over the next decade. Though endemic in the most populous areas of North America, few studies have estimated the healthcare costs associated with WNV. The objective of this study was to determine direct healthcare costs attributable to WNV illness in Ontario, Canada. METHODS: We conducted a cost-of-illness study on incident laboratory confirmed and probable WNV infected subjects identified from the provincial laboratory database from Jan 1, 2002 through Dec 31, 2012. Infected subjects were linked to health administrative data and matched to uninfected subjects. We used phase-of-care methods to calculate costs for 3 phases of illness: acute infection, continuing care, and final care prior to death. Mean 10-day attributable costs were reported in 2014 Canadian dollars, per capita. Sensitivity analysis was conducted to test the impact of WNV neurologic syndromes on healthcare costs. RESULTS: One thousand five hundred fifty-one laboratory confirmed and probable WNV infected subjects were ascertained; 1540 (99.3%) were matched to uninfected subjects. Mean age of WNV infected subjects was 49.1 ± 18.4 years, 50.5% were female. Mean costs attributable to WNV were $1177 (95% CI: $1001, $1352) for acute infection, $180 (95% CI: $122, $238) for continuing care, $11,614 (95% CI: $5916, $17,313) for final care - acute death, and $3199 (95% CI: $1770, $4627) for final care - late death. Expected 1-year costs were $13,648, adjusted for survival. Three hundred seventeen infected subjects were diagnosed with at least one neurologic syndrome and greatest healthcare costs in acute infection were associated with encephalitis ($4710, 95% CI: $3770, $5650). CONCLUSIONS: WNV is associated with increased healthcare resource utilization across all phases of care. High-quality studies are needed to understand the health system impact of vector-borne diseases and evaluate the cost effectiveness of novel WNV interventions.

Environmental and Sociological Factors Associated with the Incidence of West Nile Virus Cases in the Northern San Joaquin Valley of California, 2011-2015.

Environmental and socioeconomic risk factors associated with the incidence of human West Nile virus (WNV) cases were investigated in the Northern San Joaquin Valley region of California, a largely rural area. The study included human WNV cases from the years 2011 to 2015 in the three-county area of San Joaquin, Stanislaus, and Merced Counties, and used census tracts as the unit of analysis. Environmental factors included temperature, precipitation, and WNV-positive mosquito pools. Socioeconomic variables included age, housing age, housing foreclosures, median income, and ethnicity. Chi-square independence tests were used to examine whether each variable was associated with the incidence of WNV cases using data from the three counties combined. In addition, negative binomial regression revealed that the environmental factors of temperature and precipitation were the strongest predictors of the incidence of human WNV cases, while the socioeconomic factor of ethnicity was a significant predictor as well, and is a factor to consider in prevention efforts. Source reduction of mosquito breeding sites and targeted prevention and education remain key in reducing the risk associated with WNV.

A spatio-temporal individual-based network framework for West Nile virus in the USA: Spreading pattern of West Nile virus.

West Nile virus (WNV)-a mosquito-borne arbovirus-entered the USA through New York City in 1999 and spread to the contiguous USA within three years while transitioning from epidemic outbreaks to endemic transmission. The virus is transmitted by vector competent mosquitoes and maintained in the avian populations. WNV spatial distribution is mainly determined by the movement of residential and migratory avian populations. We developed an individual-level heterogeneous network framework across the USA with the goal of understanding the long-range spatial distribution of WNV. To this end, we proposed three distance dispersal kernels model: 1) exponential-short-range dispersal, 2) power-law-long-range dispersal in all directions, and 3) power-law biased by flyway direction -long-range dispersal only along established migratory routes. To select the appropriate dispersal kernel we used the human case data and adopted a model selection framework based on approximate Bayesian computation with sequential Monte Carlo sampling (ABC-SMC). From estimated parameters, we find that the power-law biased by flyway direction kernel is the best kernel to fit WNV human case data, supporting the hypothesis of long-range WNV transmission is mainly along the migratory bird flyways. Through extensive simulation from 2014 to 2016, we proposed and tested hypothetical mitigation strategies and found that mosquito population reduction in the infected states and neighboring states is potentially cost-effective.

Determining the rate of underrecognition of West Nile virus neurologic disease in the province of Quebec in 2012.

BACKGROUND: During a major outbreak of West Nile virus (WNV) infection in the province of Quebec in 2012, public health authorities (PHAs) suspected underrecognition of West Nile neurologic disease (WNND). With data on acute infections detected in blood donors, an estimate of the degree of underrecognition was produced. STUDY DESIGN AND METHODS: All 2012 donors were tested for WNV infection with the use of reverse transcription-polymerase chain reaction (RT-PCR). With the number of cases detected, the number of donors tested, our estimate of the duration of viremia, an estimate of the population at risk, and the ratio of WNND to total cases, an expected number of WNND cases was calculated. A Monte Carlo simulation was used to estimate the range of several of these variables. RESULTS: Seventeen RT-PCR-positive donors were found among 52,309 donations tested. In the base case, the total number of cases was 16,095 and the expected number of WNND cases was 115. In the Monte Carlo simulation, the mean number of expected WNND cases was 136, and the median was 129. Since only 85 cases were reported to PHAs, it is estimated that between 26 and 37.5% of cases occurring in the province went undetected. CONCLUSION: The observation that close to one-third of cases of WNND went undetected because of the omission of appropriate laboratory testing indicates the need for improvement in the investigation of acute neurologic syndrome of suspected infectious etiology in Québec.

Early detection of West Nile virus in France: quantitative assessment of syndromic surveillance system using nervous signs in horses.

West Nile virus (WNV) is a growing public health concern in Europe and there is a need to develop more efficient early detection systems. Nervous signs in horses are considered to be an early indicator of WNV and, using them in a syndromic surveillance system, might be relevant. In our study, we assessed whether or not data collected by the passive French surveillance system for the surveillance of equine diseases can be used routinely for the detection of WNV. We tested several pre-processing methods and detection algorithms based on regression. We evaluated system performances using simulated and authentic data and compared them to those of the surveillance system currently in place. Our results show that the current detection algorithm provided similar performances to those tested using simulated and real data. However, regression models can be easily and better adapted to surveillance objectives. The detection performances obtained were compatible with the early detection of WNV outbreaks in France (i.e. sensitivity 98%, specificity >94%, timeliness 2·5 weeks and around four false alarms per year) but further work is needed to determine the most suitable alarm threshold for WNV surveillance in France using cost-efficiency analysis.

Managing mosquito spaces: Citizen self-governance of disease vectors in a desert landscape.

Public health agencies' strategies to control disease vectors have increasingly included "soft" mosquito management programs that depend on citizen education and changing homeowner behaviors. In an effort to understand public responses to such campaigns, this research assesses the case of Tucson, Arizona, where West Nile virus presents a serious health risk and where management efforts have focused on public responsibility for mosquito control. Using surveys, interviews, and focus groups, we conclude that citizens have internalized responsibilities for mosquito management but also expect public management of parks and waterways while tending to reject the state's interference with privately owned parcels. Resident preferences for individualized mosquito management hinge on the belief that mosquito-borne diseases are not a large threat, a pervasive distrust of state management, and a fear of the assumed use of aerial pesticides by state managers. Opinions on who is responsible for mosquitoes hinge on both perceptions of mosquito ecology and territorial boundaries, with implications for future disease outbreaks.

Climatic, ecological, and socioeconomic factors associated with West Nile virus incidence in Atlanta, Georgia, U.S.A.

The integrated effects of the many risk factors associated with West Nile virus (WNV) incidence are complex and not well understood. We studied an array of risk factors in and around Atlanta, GA, that have been shown to be linked with WNV in other locations. This array was comprehensive and included climate and meteorological metrics, vegetation characteristics, land use / land cover analyses, and socioeconomic factors. Data on mosquito abundance and WNV mosquito infection rates were obtained for 58 sites and covered 2009-2011, a period following the combined storm water - sewer overflow remediation in that city. Risk factors were compared to mosquito abundance and the WNV vector index (VI) using regression analyses individually and in combination. Lagged climate variables, including soil moisture and temperature, were significantly correlated (positively) with vector index as were forest patch size and percent pine composition of patches (both negatively). Socioeconomic factors that were most highly correlated (positively) with the VI included the proportion of low income households and homes built before 1960 and housing density. The model selected through stepwise regression that related risk factors to the VI included (in the order of decreasing influence) proportion of houses built before 1960, percent of pine in patches, and proportion of low income households.

Estimating the economic impact of a possible equine and human epidemic of West Nile virus infection in Belgium.

This study aimed at estimating, in a prospective scenario, the potential economic impact of a possible epidemic of WNV infection in Belgium, based on 2012 values for the equine and human health sectors, in order to increase preparedness and help decision-makers. Modelling of risk areas, based on the habitat suitable for Culex pipiens, the main vector of the virus, allowed us to determine equine and human populations at risk. Characteristics of the different clinical forms of the disease based on past epidemics in Europe allowed morbidity among horses and humans to be estimated. The main costs for the equine sector were vaccination and replacement value of dead or euthanised horses. The choice of the vaccination strategy would have important consequences in terms of cost. Vaccination of the country's whole population of horses, based on a worst-case scenario, would cost more than EUR 30 million; for areas at risk, the cost would be around EUR 16-17 million. Regarding the impact on human health, short-term costs and socio-economic losses were estimated for patients who developed the neuroinvasive form of the disease, as no vaccine is available yet for humans. Hospital charges of around EUR 3,600 for a case of West Nile neuroinvasive disease and EUR 4,500 for a case of acute flaccid paralysis would be the major financial consequence of an epidemic of West Nile virus infection in humans in Belgium.

Comparison of the efficiency and cost of West Nile virus surveillance methods in California.

Surveillance systems for West Nile virus (WNV) combine several methods to determine the location and timing of viral amplification. The value of each surveillance method must be measured against its efficiency and costs to optimize integrated vector management and suppress WNV transmission to the human population. Here we extend previous comparisons of WNV surveillance methods by equitably comparing the most common methods after standardization on the basis of spatial sampling density and costs, and by estimating optimal levels of sampling effort for mosquito traps and sentinel chicken flocks. In general, testing for evidence of viral RNA in mosquitoes and public-reported dead birds resulted in detection of WNV approximately 2-5 weeks earlier than serological monitoring of sentinel chickens at equal spatial sampling density. For a fixed cost, testing of dead birds reported by the public was found to be the most cost effective of the methods, yielding the highest number of positive results per $1000. Increased spatial density of mosquito trapping was associated with more precise estimates of WNV infection prevalence in mosquitoes. Our findings also suggested that the most common chicken flock size of 10 birds could be reduced to six to seven without substantial reductions in timeliness or sensitivity. We conclude that a surveillance system that uses the testing of dead birds reported by the public complemented by strategically timed mosquito and chicken sampling as agency resources allow would detect viral activity efficiently in terms of effort and costs, so long as susceptible bird species that experience a high mortality rate from infection with WNV, such as corvids, are present in the area.

Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies.

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for 'Imported' Viral Diseases (ENIVD).

A novel system for rapid and cost-effective production of detection and diagnostic reagents of West Nile virus in plants.

The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses.

West Nile virus: the Italian national transplant network reaction to an alert in the north-eastern region, Italy 2011.

We report four cases of West Nile virus (WNV) transmission following a single multiorgan donation in north-eastern Italy. The transmissions were promptly detected by local transplant centres. The donor had been tested for WNV by nucleic acid amplification test (NAT) prior to transplantation and was negative. There were no detected errors in the nationally implemented WNV safety protocols.

Second external quality assessment of the molecular diagnostic of West Nile virus: are there improvements towards the detection of WNV?

BACKGROUND: WNV epidemics occur worldwide, new WNV isolates were isolated in southern-east Europe belonging to WNV lineage 2. A first international proficiency study on WNV indicted that some laboratories were not able to detect WNV lineage 2 virus genome by their PCR diagnostic assays. Therefore an actual External Quality Assessment with both virus lineages was performed to monitor the improvements in molecular diagnostics. OBJECTIVES: To asses the proficiency of laboratories to detect West Nile virus with molecular diagnostic tests. STUDY DESIGN: A test panel of different WNV isolates and virus dilutions was given to 26 laboratories to test the samples with their routine diagnostic methods. RESULTS: Twenty-one participating laboratories provided 28 data set results. WNV lineage 1 was detected with high overall efficiency of 92% (67.9-100%) but two different WNV lineage 2 strains were detected at lower rates (mean = 73%, 67.9-75%) by the different PCR assays. 93% of the laboratories were able to detect a WNV lineage 1 with a concentration of 1.2×10(4)copies/ml but the detection rate was decreased to 68% for 1.2×10(3)copies/ml. One laboratory generated false-positive result from the non-virus control samples and 29% of the datasets showed false-positive results for non-WNV flavivirus samples. CONCLUSIONS: The WNV EQA showed an improved proficiency of laboratories as compared to the first EQA. However, the data suggest that problems in the detection of both lineages were still present since the first proficiency test was performed in 2006. Further proceedings versus the detection of both lineages are needed particularly for in-house assays.

West Nile virus in Europe: a comparison of surveillance system designs in a changing epidemiological context.

Current knowledge suggests that there is a low-level and recurrent circulation of West Nile virus (WNV) in Europe, with sporadic human and/or equines cases. However, recent events indicate that this picture is changing, raising the possibility that Europe could experience a modification in the virus' circulation patterns. We used an existing model of WNV circulation between Southern Europe and West Africa to estimate the sample size of equivalent West Nile surveillance systems, either passive (based upon horse populations and sentinel veterinarians) or active (sentinel horses, sentinel chickens, or WNV genome detection in trapped mosquito pools). The costs and calendar day of first detection of these different surveillance systems were compared under three different epidemiological scenarios: very low level circulation, low level recurrent circulation, and epidemic situation. The passive surveillance of 1000 horses by specialized veterinarian clinics appeared to be the most cost-effective system in the current European context, and estimated median dates of first detection appeared consistent with recent field observations. Our results can be used to optimize surveillance designs for different epidemiological requirements.

Economic conditions predict prevalence of West Nile virus.

Understanding the conditions underlying the proliferation of infectious diseases is crucial for mitigating future outbreaks. Since its arrival in North America in 1999, West Nile virus (WNV) has led to population-wide declines of bird species, morbidity and mortality of humans, and expenditures of millions of dollars on treatment and control. To understand the environmental conditions that best explain and predict WNV prevalence, we employed recently developed spatial modeling techniques in a recognized WNV hotspot, Orange County, California. Our models explained 85-95% of the variation of WNV prevalence in mosquito vectors, and WNV presence in secondary human hosts. Prevalence in both vectors and humans was best explained by economic variables, specifically per capita income, and by anthropogenic characteristics of the environment, particularly human population and neglected swimming pool density. While previous studies have shown associations between anthropogenic change and pathogen presence, results show that poorer economic conditions may act as a direct surrogate for environmental characteristics related to WNV prevalence. Low-income areas may be associated with higher prevalence for a number of reasons, including variations in property upkeep, microhabitat conditions conducive to viral amplification in both vectors and hosts, host community composition, and human behavioral responses related to differences in education or political participation. Results emphasize the importance and utility of including economic variables in mapping spatial risk assessments of disease.

A method to increase efficiency in testing pooled field-collected mosquitoes.

Testing field-caught mosquito collections can result in thousands of pools, and testing pools of 50 mosquitoes each can be both time consuming and cost prohibitive. Consequently, we have developed an alternative approach to testing mosquito pools for arboviruses, utilizing a superpool strategy. When mosquito samples are processed for extraction of viral RNA and subsequent virus testing via quantitative real-time polymerase chain reaction, each pool is tested individually. Using the method described here, 0.025 ml from each of 10 pools is combined into a superpool for RNA extraction and testing. When a virus-positive superpool sample is found, each of the original 10 pools that constitute this sample is tested individually in order to find the specific positive sample. By retesting the original samples after the initial superpool screen, we are still able to obtain reliable estimates for minimum infection rates or maximum likelihood estimations. To test this principle, we created controlled mosquito pools of known titer and subjected them to our superpool process. We were able to detect our entire range of laboratory-created pools as being West Nile virus (WNV) positive. In 2005, field surveillance efforts from our laboratory resulted in over 4,000 mosquito pools tested, with 8 resulting WNV-positive samples. We found that all of these field samples were detected as WNV positive using the superpool method and contained calculated virus titers from < 0.1 to 4.1 log10 plaque-forming units/ml WNV, indicating that the limit of superpool detection of WNV is below this point. These results reveal that the superpool method could be accurately used to detect WNV in field-collected specimens.

Blood screening for west nile virus: the cost-effectiveness of a real-time, trigger-based strategy.

Previous studies have demonstrated that universal blood screening for West Nile virus is not cost-effective. A newly proposed, real-time, trigger-based screening strategy was analyzed and was also shown to be not cost-effective. These results were highly sensitive to pricing of screening assays.