Honey-baited FTA cards in box gravid traps for the assessment of Usutu virus circulation in mosquito populations in Germany.

Usutu virus (USUV) is becoming increasingly important to veterinary and human health in Germany. USUV has been implicated in mass die-off events of birds, especially of blackbirds (Turdus merula), and has experienced significant range expansion in the years since its first detection in 2010. Current detection methods rely primarily on dead bird surveillance or mass mosquito collection using CO2 as the main attractant. Dead bird surveillance can result in detection of disease circulation past the point at which control efforts would be most impactful. Vector surveillance offers the opportunity to detect disease circulation before significant outbreaks occur. However, current methods result in collections of extremely large numbers of predominantly nulliparous female mosquitoes who have not yet taken a blood meal. This study sought to test whether box gravid traps could successfully trap USUV infected gravid Culex mosquitoes, and if viral RNA could be successfully transferred and stabilised on an FTA card. During the month of August 2020, 18 Reiter-Cummings style box gravid traps with honey-baited FTA cards were set in a region of known USUV circulation around the southern border of Hesse, Germany. Four 48-hour trapping rounds were conducted. All mosquitoes and FTA cards were collected and stored during transport to the laboratory on dry ice. Samples and FTA cards were then transferred and stored in a freezer at -5 °C until identification. Identification was carried out on a chill plate before being sent with overnight courier in a styrofoam box with cooling elements for virus detection with a modified generic flavivirus RT-PCR. Mosquitoes were separated into pools by trap, date, species and feeding status. 2003 mosquitoes were caught in four rounds of trapping, 1834 or 88% of which were female Culex mosquitoes used for examination. 13 pools of mosquitoes and four FTA cards tested positive for USUV. No positive FTA cards were found in traps with positive mosquitoes and no positive mosquitoes were found in traps with positive FTA cards. Although fewer FTA cards than expected returned a positive result, this may have been a result of the extreme conditions experienced in the field and highlights the need to establish the temperature and humidity boundaries such a collection method can withstand. Box gravid traps however, provided a highly effective and targeted approach for capturing gravid female Culex mosquitoes, the most appropriate subpopulation for testing for USUV. Additionally, the simplicity and effectiveness of this trapping and surveillance method make it an attractive option for use as an early warning system, including for large scale surveillance programmes.

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 diagnostic and analytic performance of the SD Bioline Dengue Duo test for dengue virus (DENV) infections in an endemic area (Savannakhet province, Lao People's Democratic Republic).

BACKGROUND: Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS: 92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV µ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.

Experimental risk assessment for chikungunya virus transmission based on vector competence, distribution and temperature suitability in Europe, 2018.

BackgroundOver the last decade, the abundant distribution of the Asian tiger mosquito Aedes albopictus in southern Europe and the import of chikungunya virus (CHIKV) by infected travellers has resulted in at least five local outbreaks of chikungunya fever in France and Italy. Considering the ongoing spread of Ae. albopictus to central Europe, we performed an analysis of the Europe-wide spatial risk of CHIKV transmission under different temperature conditions. Methods:Ae. albopictus specimens from Germany and Italy were orally infected with CHIKV from an outbreak in France and kept for two weeks at 18 °C, 21 °C or 24 °C. A salivation assay was conducted to detect infectious CHIKV. Results: Analyses of mosquito saliva for infectious virus particles demonstrated transmission rates (TRs) of > 35%. Highest TRs of 50% for the mosquito population from Germany were detected at 18 °C, while the Italian population had highest TRs of 63% at 18 °C and 21 °C, respectively. Temperature data indicated a potential risk of CHIKV transmission for extended durations, i.e. sufficiently long time periods allowing extrinsic incubation of the virus. This was shown for areas already colonised by Ae. albopictus, as well as for large parts of central Europe that are not colonised. Conclusion: The current risk of CHIKV transmission in Europe is not primarily restricted by temperature, which allows extrinsic incubation of the virus, but rather by the vector distribution. Accordingly, all European countries with established populations of Ae. albopictus should implement respective entomological surveillance and monitoring systems, as basis for suitable control measures.

Control measures following a case of imported Lassa fever from Togo, North Rhine Westphalia, Germany, 2016.

In a patient transferred from Togo to Cologne, Germany, Lassa fever was diagnosed 12 days post mortem. Sixty-two contacts in Cologne were categorised according to the level of exposure, and gradual infection control measures were applied. No clinical signs of Lassa virus infection or Lassa specific antibodies were observed in the 62 contacts. Thirty-three individuals had direct contact to blood, other body fluids or tissue of the patients. Notably, with standard precautions, no transmission occurred between the index patient and healthcare workers. However, one secondary infection occurred in an undertaker exposed to the corpse in Rhineland-Palatinate, who was treated on the isolation unit at the University Hospital of Frankfurt. After German authorities raised an alert regarding the imported Lassa fever case, an American healthcare worker who had cared for the index patient in Togo, and who presented with diarrhoea, vomiting and fever, was placed in isolation and medevacked to the United States. The event and the transmission of Lassa virus infection outside of Africa underlines the need for early diagnosis and use of adequate personal protection equipment (PPE), when highly contagious infections cannot be excluded. It also demonstrates that larger outbreaks can be prevented by infection control measures, including standard PPE.

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection.

BACKGROUND: Since the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important. OBJECTIVES: We evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide. STUDY DESIGN: A panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing. RESULTS: The panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used. CONCLUSION: The external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance.