COVID-19 cross-border case and contact tracing activities - experiences and lessons learnt, Germany, April-December 2020.

BACKGROUND: Interruption of transmission chains has been crucial in the COVID-19 response. The Emergency Operations Centre (EOC) at the Robert Koch Institute (RKI) coordinated cross-border case and contact tracing activities at the national level by sharing data with German public health authorities (PHA) and other countries. Data on these activities were not collected in the national surveillance system, and thus were challenging to quantify. Our aim was to describe cross-border COVID-19 case and contact tracing activities including lessons learnt by PHA to adapt the procedures accordingly. METHODS: Case and contact tracing events were recorded using unique identifiers. We collected data on cases, contacts, dates of exposure and/or SARS-CoV-2 positive test results and exposure setting. We performed descriptive analyses of events from 06.04.-31.12.2020. We conducted interviews with PHA to understand experiences and lessons learnt, applying a thematic approach for qualitative analysis. RESULTS: From 06.04.-31.12.2020 data on 7,527 cross-border COVID-19 case and contact tracing activities were collected. Germany initiated communication 5,200 times, and other countries 2,327 times. Communication from other countries was most frequently initiated by Austria (n = 1,184, 50.9%), Switzerland (n = 338, 14.5%), and the Netherlands (n = 168, 7.2%). Overall, 3,719 events (49.4%) included information on 5,757 cases (median 1, range: 1-42), and 4,114 events (54.7%) included information on 13,737 contacts (median: 1, range: 1-1,872). The setting of exposure was communicated for 2,247 of the events (54.6%), and most frequently included private gatherings (35.2%), flights (24.1%) and work-related meetings (20.3%). The median time delay between exposure date and contact information receipt at RKI was five days. Delay between positive test result and case information receipt was three days. Main challenges identified through five interviews were missing data or delayed accessibility particularly from flights, and lack of clear and easy to use communication channels. More and better trained staff were mentioned as ideas for improving future pandemic response preparedness. CONCLUSION: Cross-border case and contact tracing data can supplement routine surveillance but are challenging to measure. We need improved systems for cross-border event management, by improving training and communication channels, that will help strengthen monitoring activities to better guide public health decision-making and secure a good future pandemic response.

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey.

BACKGROUND: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. RESULTS: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as "Ochlerotatus caspius flavivirus Turkey", was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. CONCLUSIONS: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.

Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey.

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses from mosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed a monophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored.

Evaluation of different serological diagnostic methods for tick-borne encephalitis virus: enzyme-linked immunosorbent, immunofluorescence, and neutralization assay.

Tick-borne encephalitis (TBE) is a zoonotic disease, transmitted mainly by the bite of ticks. The TBE virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus and is able to cause meningoencephalitis. For serological TBEV detection, the neutralization test (NT) is the most specific assay available. Different NT protocols are used in the laboratories, and until now the performance of these NTs has never been tested in an external quality assessment (EQA). In this EQA, we compared the results of eight European laboratories in detecting 17 samples (11 TBEV positive, five flavivirus cross reactive, and one negative sample) by NT. Furthermore, 14 of these EQA samples and 15 additional samples were tested in different commercial assays: 15 immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and an immunofluorescence assay (IFA). Four laboratories showed a good NT EQA performance, whereas four laboratories had some sensitivity problems. Additionally, two of these laboratories showed a lack in specificity, misidentifying a dengue-positive sample as TBEV positive. The comparison of the commercial ELISAs revealed a high sensitivity in all assays, but as expected for IgG, the ELISAs showed a high degree of flavivirus cross reactivity. The assessment of Vienna Units in some of the ELISAs revealed deviations in the standards used by the different companies. Therefore, these standards should be revised. Generally, in this EQA, we found that reliable NT protocols are used in most of the laboratories, and the evaluation of the IgG ELISAs and the IFA showed a good agreement.

Multicentre evaluation of central nervous system infections due to Flavi and Phleboviruses in Turkey.

OBJECTIVES: Flavi- and Phleboviruses associated with central nervous system (CNS) infections including West Nile Virus (WNV), Tick-borne Encephalitis Virus (TBEV) and Toscana Virus (TOSV) cause significant morbidity and mortality in humans. In this study, the impact of these agents have been investigated in CNS infections at referral hospitals in two provinces in Turkey, where circulation of these viruses have previously been recognized. METHODS: In the study, 258 samples from 126 individuals from Ankara and 113 samples from 108 individuals from Izmir provinces collected in 2010 were included. Viral RNAs were investigated by multiple genus and strain specific primers. Commercial serological assays were employed in screening and reactive results were evaluated with additional assays and by plaque reduction neutralization assay. RESULTS: Two cases of WNV CNS infections, 14 cases of TOSV infections and one TBEV-exposed individual were identified via serological testing. WNV infections in 61 and 56-year old individuals from Ankara presented with fever and encephalitis without skin rash and residual neurologic damage. TOSV-associated cases from both provinces mainly displayed signs of meningitis. TOSV exposure was documented for the first time from Izmir. CONCLUSIONS: WNV, TBEV and TOSV infections must be considered in cases of meningoencephalitis of unknown etiology in Turkey.

Performance of various commercial assays for the detection of Toscana virus antibodies.

INTRODUCTION: Sandfly fever virus (SFV) serotypes sandfly fever Naples virus, sandfly fever Sicilian virus, and sandfly fever Cyprus virus cause febrile diseases, whereas Toscana virus (TOSV) is responsible for aseptic meningoencephalitis. Diagnosis and surveillance of TOSV depend heavily on virus serology, and various commercial assays utilizing various antigen sources and formats have been available. The aim of this study was to perform comparative evaluation of commercially available serological assays for anti-TOSV immunoglobulins. MATERIALS AND METHODS: A collection of 120 sera from healthy blood donors from an endemic region, previously identified to be reactive for antibodies against various SFV serotypes by indirect immunofluorescence test (IIFT), was reevaluated for IgG/IgM via IIFT, enzyme-linked immunosorbent assay, and an immunoblot assay manufactured by Euroimmun, Diesse, and Mikrogen, respectively. Virus neutralization test (VNT) was performed for 99 sera using standard TOSV, sandfly fever Sicilian virus, and sandfly fever Naples virus strains. RESULTS: A total of 89 samples (74.2%) were reactive for TOSV IgG in at least one of the commercial assays, and 31 samples (31.3%) were reactive in VNT for various SFV serotypes. Average percentage agreements among commercial assays and between VNT and the commercial assays were noted as 57.8% and 62.6%, respectively. No significant correlation between assay results and VNT titers was observed. SFV IgM antibodies were detected in a total of eight samples (6.7%) via IIFT, which were nonreactive in enzyme-linked immunosorbent assay and VNT. DISCUSSION: Commercial diagnostic immunoassays displayed slight to fair agreement for TOSV IgG as assessed via kappa and percentage agreement values. The results could only be confirmed via virus neutralization in a portion of the samples, and overall agreement between the commercial assays and VNT was slight. Commercial assays such as immunoblot can be used in addition to VNT for confirmation of TOSV exposure.

Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination.

A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.

[Investigation of dengue virus and yellow fever virus seropositivities in blood donors from Central/Northern Anatolia, Turkey]. / Orta/Kuzey Anadolu Bölgesi kan donörlerinde Dengue virusu ve sari humma virusu seropozitifliginin arastirilmasi

Dengue virus (DENV) and yellow fever virus (YFV) are two of the globally prevalent vector-borne flaviviruses. Data on these viruses from Turkey is limited to a single study originating from the western, Aegean region of Turkey, where evidence for DENV exposure had been confirmed in residents and presence of hemagglutination inhibiting antibodies against YFV had been revealed. The aim of this study was to investigate the rates of seropositivity of DENV and YFV in blood donors from Central/Northern Anatolia, Turkey, for the demonstration of possible human exposure. Serum samples were collected by the Turkish Red Crescent Middle Anatolia Regional Blood Center from donation sites at Ankara, Konya, Eskisehir and Zonguldak provinces and included in the study after informed consent. Ankara is the capital and second most-populated city in Turkey. All samples were previously evaluated for West Nile and tick-borne encephalitis virus antibodies and found to be negative. A total of 2435 and 1502 sera have been evaluated for IgG antibodies against DENV and YFV, respectively. Commercial enzymelinked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFTs) were applied (Euroimmun, Germany) for DENV/YFV IgG surveillance. DENV IgG reactive sera were further evaluated for IgM by ELISA and a commercial mosaic IIFT to determine DENV subtypes. IgM positive samples were also analyzed by a commercial NS1 antigen detection assay (Bio-Rad Laboratories, France). YFV IgG reactive samples were evaluated by IIFT for IgM and via mosaic IIFT and antibody specificity were confirmed by plaque reduction neutralization test (PRNT). Anti-DENV IgGs were demonstrated in repeated assays in 0.9% (21/2435) of the sera. In two samples with borderline IgG results, presence of DENV IgM was detected, one of which was also borderline positive for DENV NS1 antigen. In 14.3% (3/21) of the IgG reactive sera, mosaic IIFT was evaluated as positive and displayed prominent reactivity for DENV-2 in all samples. From five donors with DENV reactivity, new samples were obtained after at least six months which revealed the continuing presence of DENV IgG activity in four. One sample which was initially positive for IgM, borderline for NS1 antigen and borderline for IgG was observed to be positive for IgG and negative for IgM in redonation. IIFT results in three redonation samples also indicated reactivity for DENV-1 and DENV-2 subtypes. Anti-YFV IgGs were detected in 0.6% (9/1502) of the sera. YFV IgM could not be demonstrated in any of the IgG reactive samples and PRNT was evaluated as negative. In conclusion, evidence for DENV exposure, presumably to DENV-2, was identified in residents from Central Anatolian provinces of Ankara and Konya for the first time, however, seroreactivity detected against YFV could not be confirmed by PRNT. These findings indicated that DENV or an antigenically-similar flavivirus was probably present in the study region and sporadic human exposure might have occurred.

Immunogenicity and safety of yellow fever vaccination for 102 HIV-infected patients.

BACKGROUND: Yellow fever vaccine (17DV) has been investigated incompletely in human immunodeficiency virus (HIV)-infected patients, and adequate immunogenicity and safety are of concern in this population. METHODS: In the Swiss HIV Cohort Study, we identified 102 patients who received 17DV while they were HIV infected. We analyzed neutralization titers (NTs) after 17DV administration using the plaque reduction neutralization test. NTs of 1:>or=10 were defined as reactive, and those of 1:<10 were defined as nonreactive, which was considered to be nonprotective. The results were compared with data for HIV-uninfected individuals. Serious adverse events were defined as hospitalization or death within 6 weeks after receipt of 17DV. RESULTS: At the time of 17DV administration, the median CD4 cell count was 537 cells/mm(3) (range, 11-1730 cells/mm(3)), and the HIV RNA level was undetectable in 41 of 102 HIV-infected patients. During the first year after vaccination, fewer HIV-infected patients (65 [83%] of 78; P = .01) than HIV-uninfected patients revealed reactive NTs, and their NTs were significantly lower (P < .001) than in HIV-uninfected individuals. Eleven patients with initially reactive NTs lost these reactive NTs

Evaluation of the first commercial chikungunya virus indirect immunofluorescence test.

The chikungunya virus (CHIKV), an arbovirus of the genus Alphavirus, family Togaviridae, is mainly transmitted by Aedes mosquitoes. It causes an acute infection, characterized by high fever, polyarthralgia and rash and was responsible for a major outbreak which started in 2005 and spread over many islands of the south western Indian Ocean before it hit the Indian subcontinent. As nucleic acid amplification can be used only during the viremic period, serological tests are most widely used for the diagnosis of CHIKV infections. CHIKV IgM and IgG antibodies can be detected as soon as 3-6 days after clinical onset, respectively. Presently only in-house ELISA and immunofluorescence tests exist for analysing the CHIKV specific immune response. The first commercial indirect immunofluorescence test (IIFT) (EUROIMMUN AG, Lüebeck, Germany) was evaluated using two sera panels of patients from La Reunion and travellers returning with CHIKV infections from the Indian Ocean region. The IgM IIFT shows a specificity of 98.3% and a sensitivity of 96.9%. The specificity and sensitivity for the IgG IIFT are 100.0% and 95.4%, respectively. This commercial IIFT is a valuable tool for the diagnosis of CHIKV infections and antibody seroprevalence studies.