First Detection and Molecular Analysis of Leishmania infantum DNA in Sand Flies of Kosovo.

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum.

Seroprevalence of canine leishmaniosis in asymptomatic dogs in Kosovo.

Canine leishmaniosis (CanL) is a neglected zoonotic disease caused by Leishmania spp. Leishmania infantum is the species responsible for the zoonotic form of the disease where dogs are reservoir hosts. This study aimed to determine the seroprevalence of CanL in asymptomatic dogs in Kosovo. Blood samples were collected from 285 dogs in all seven regions in Kosovo (35-50 samples per region) from summer 2021 to spring 2022. Sera were tested using enzyme-linked immunosorbent assay (ELISA), and the presence of anti-Leishmania IgG was confirmed by an indirect fluorescent antibody test (IFAT). The true overall seroprevalence of CanL of asymptomatic dogs in Kosovo with ELISA was 4.21% (95% CI: 2.42-7.21) while with IFAT was 3.51% (95% CI: 1.92-6.34). The highest rates were found in the Prishtina region to be 8.0% (4/50) by ELISA and 6.0% (3/50) by IFAT, and in the Mitrovica region, the prevalence was 0% (0/40). There were no significant differences among the different regions, gender, age, health status, and breed. These findings highlight the presence of CanL in most regions of Kosovo and underline the veterinary relevance of clinically asymptomatic dogs infected with Leishmania.

Serologic and Genomic Investigation of West Nile Virus in Kosovo.

The prevalence of West Nile virus (WNV) is increasing across Europe, with cases emerging in previously unaffected countries. Kosovo is situated in a WNV-endemic region where the seroepidemiological data on WNV in humans remains absent. To address this issue, we have conducted a seroepidemiological investigation of 453 randomly selected sera from a hospital in Kosovo, revealing a 1.55% anti-WNV IgG seroprevalence. Comparative and phylogeographic analyses of the WNV genomes obtained by sequencing archived samples from patients with West Nile fever indicate at least two recent and distinct introductions of WNV lineage 2 into Kosovo from neighboring countries. These findings confirm the eco-epidemiological status of WNV in southeast Europe, where long- and short-range dispersion of lineage 2 strains contributes to a wider circulation via central Europe. Our results suggest an increasing risk for WNV spreading in Kosovo, underscoring the need for an integrated national surveillance program targeting vectors and avian populations for early epidemic detection, as well as the screening of blood donors to gauge the impact of virus circulation on the human population.

Assessment of expertise in morphological identification of mosquito species (Diptera, Culicidae) using photomicrographs.

Accurate identification of insect species is an indispensable and challenging requirement for every entomologist, particularly if the species is involved in disease outbreaks. The European MediLabSecure project designed an identification (ID) exercise available to any willing participant with the aim of assessing and improving knowledge in mosquito taxonomy. The exercise was based on high-definition photomicrographs of mosquitoes (26 adult females and 12 larvae) collected from the western Palaearctic. Sixty-five responses from Europe, North Africa and the Middle East were usable. The study demonstrated that the responders were better at identifying females (82% correct responses) than larvae (63%). When the responders reported that they were sure of the accuracy of their ID, the success rate of ID increased (92% for females and 88% for larvae). The top three tools used for ID were MosKeyTool (72% of responders), the ID key following Becker et al. [2010. Mosquitoes and their control, 2nd edn. Berlin: Springer] (38%), and the CD-ROM of Schaffner et al. [2001. Les moustiques d'Europe: logiciel d'identification et d'enseignement - The mosquitoes of Europe: an identification and training programme. Montpellier: IRD; EID] (32%), while other tools were used by less than 10% of responders. Responders reporting the identification of mosquitoes using the MosKeyTool were significantly better (80% correct responses) than non-MosKeyTool users (69%). Most responders (63%) used more than one ID tool. The feedback from responders in this study was positive, with the exercise being perceived as halfway between educational training and a fun quiz. It raised the importance of further expanding training in mosquito ID for better preparedness of mosquito surveillance and control programmes.

Title: Évaluation de l'expertise en identification morphologique des espèces de moustiques (Diptera, Culicidae) à l'aide de photomicrographies. Abstract: L'identification précise des espèces d'insectes est une exigence indispensable et difficile pour tout entomologiste, en particulier si l'espèce est impliquée dans des épidémies. Le projet européen MediLabSecure a conçu un exercice d'identification (ID) accessible à tout participant volontaire dans le but d'évaluer et d'améliorer les connaissances en taxonomie des moustiques. L'exercice était basé sur des photomicrographies haute définition de moustiques (26 femelles adultes et 12 larves) prélevées dans le Paléarctique occidental. Soixante-cinq réponses d'Europe, d'Afrique du Nord et du Moyen-Orient ont été utilisables. L'étude a démontré que les répondants étaient meilleurs pour identifier les femelles (82 % de réponses correctes) que les larves (63 %). Lorsque les répondants ont déclaré être sûrs de l'exactitude de leur ID, le taux de réussite de l'identification était meilleur (92 % pour les femelles et 88 % pour les larves). Les trois principaux outils utilisés pour les ID étaient MosKeyTool (72 % des répondants), la clé d'identification du livre de Becker et al. (38%) et le CD-ROM de Schaffner et al. (32 %), tandis que d'autres outils étaient utilisés par moins de 10 % des répondants. Les répondants déclarant identifier des moustiques à l'aide de MosKeyTool étaient significativement meilleurs (80 % de réponses correctes) que les non-utilisateurs de MosKeyTool (69 %). La plupart des répondants (63 %) ont utilisé plus d'un outil d'identification. Les commentaires des répondants de cette étude ont été positifs, l'exercice étant perçu comme à mi-chemin entre une formation pédagogique et un quiz amusant. Il a souligné l'importance d'étendre la formation complémentaire à l'identification des moustiques pour une meilleure préparation des programmes de surveillance et de contrôle des moustiques.

The Asian tiger mosquito Aedes albopictus (Skuse) in Kosovo: First record.

The Asian tiger mosquito, Aedes albopictus, is an invasive mosquito species that is considered a potential vector of about 22 arboviruses, among which dengue, chikungunya and Zika. Here we report the first record of Aedes albopictus in the territory of the Republic of Kosovo. The first finding, in July 2020, was driven by a photo of an adult mosquito published by a citizen in social media. The subsequent field investigation in July 2020 confirmed the presence of adult mosquitoes by human landing catch and collection of eggs in ovitraps at the village Zhur. Monitoring was performed for 7 weeks with ovitraps and BG-Sentinel adult traps at 36 sampling stations from 7 localities, in the Southern part of Kosovo, on the border with Albania. Fifty-two out of 81 ovitrap samples resulted positive for the presence of Ae. albopictus. A total of 2,711 eggs are collected in 22 out 36 stations and a total of 78 adults from 4 out 4 stations with BG Sentinel trap and 15 adults with handled electric aspirator. Our finding shows that the tiger mosquito is expanding its geographical range in the Balkans, southeastern Europe.

Hyalomma aegyptium the dominant hard tick in tortoises Tesdudo hermanni boettgeri found in different regions of Albania.

The purpose of this study was to estimate the level of hard tick infestation in the tortoise subspecies Testudo hermanni boettgeri living within the free-range hills and mountains of four regions of central and south Albania. In addition, this study showed the morphological differences of infested and non-infested tortoises in several geographic locations where tortoises are known to be infested with Hyalomma aegyptium, a natural carrier of different zoonotic pathogens. Thirty-six of 145 (24.8%) examined Hermann's tortoises were found to be infested with hard ticks. After the tortoises were carefully captured and controlled, a total of 67 H. aegyptium were collected: 47 in Berati, 11 in Ballshi and 9 in Saranda. None of the 40 tortoises in the Tirana region were found to be infested with ticks. All ticks were identified as H. aegyptium adults. The highest prevalence of tick infestation was in the Berati region at 49.1%, followed by the Ballshi and Saranda region by 24% and 12%, respectively. The mean infestation intensity was 1.86 H. aegyptium per Hermann's tortoise, and it was found that H. aegyptium are less common in large Hermann's tortoises. The number of H. aegyptium male ticks was negatively correlated with the body dimensions of Hermann's tortoises. Hyalomma aegyptium is the most prevalent tick in Hermann's tortoises in three regions of south Albania, and with a typical three-host life cycle in different wild and domestic animals, they may be a vector of zoonotic pathogens. Furthermore, other studies should be conducted to detect the presence of zoonotic pathogens in ticks from these regions and to estimate the risk of transmission in animals and humans.

First evidence of Schmallenberg virus infection in domestic ruminants in Kosovo and Albania.

Schmallenberg virus (SBV), a novel Orthobunyavirus, emerged in European domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. Here, a total of 364 bovine, ovine and caprine serum samples were collected in Kosovo and Albania between May 2014 and August 2016 and analyzed for the presence of anti­SBV antibodies. Sera were tested using an enzyme­linked immunosorbent assay (ELISA), and 48 ELISA­positive samples were subsequently analyzed by serum neutralization test (SNT). The overall percentage of ELISA positive results was 17.9%; 23.1% (53/229) was the prevalence observed in Kosovo (cattle 45.5%, sheep 19.2% and goat 6.8%), while 8.9% (12/135) was that observed in Albania (cattle 11.1%, sheep 0% and goat 20.0%). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested. This is the first study reporting SBV circulation in domestic ruminants in Kosovo and Albania, with indication that this virus has been present in Kosovo and Albania at least since 2014 without being detected.

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary. A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: µ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection. In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection. Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.

First Serological Evidence of West Nile Virus Among Equines and Birds in Kosovo, 2018-2019.

This study was conducted to assess the presence of West Nile virus (WNV) in Kosovo by serological testing of apparently healthy local horses and free-range chicken, and it attempted to detect viral nucleic acid in birds and mosquitoes. Between January 2018 and June 2019, 260 equine serum samples were collected, additionally 580 adult mosquitoes (53 pools) were grouped in for genera, including Culex spp. (226 individuals; 26 pools), Aedes spp. (136 individuals; 16 pools), Anopheles spp. (184 individuals; 7 pools), and Culiseta spp. (34 individuals; 4 pools). Fifty domestic birds and 51 wild birds were collected from different regions of Kosovo. Equine and domestic bird serum samples were tested by flavivirus IgG enzyme-linked immunosorbent assay (ELISA), while mosquitoes and bird viscera were tested for WNV RNA by RT-qPCR. All ELISA-positive results were confirmed by plaque reduction neutralization test (PRNT) and eight by virus neutralization test. WNV antibodies were present in 27 out of 260 equine sera (10.38%) and one out of 50 samples in domestic birds by ELISA and PRNT. Eight of 27 positive equine serum samples with high titer neutralized WNV, but not Usutu virus. No WNV RNA was detected in birds or mosquitoes. The occurrence of WNV antibodies in local equines from all regions of Kosovo indicates that the virus is circulating within the country. Public health authorities should therefore plan a risk assessment and disease control program.

Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions.

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.

External quality assessment of Rift Valley fever diagnosis in 17 veterinary laboratories of the Mediterranean and Black Sea regions.

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples.

First serological evidence of infections with selected vector-borne pathogens in dogs in Kosovo.

Canine vector-borne pathogens are a group of widespread microorganisms and nematodes transmitted by arthropods that do not only impact dog health but may also pose a health risk to humans as many of them are zoonotic. As no data exist on the presence of canine vector-borne pathogens in Kosovo, we here present a first study on the seroprevalence of selected vector-borne pathogens, the dog heartworm Dirofilaria immitis and the bacteria Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi sensu lato. The study was carried out from July to October 2019 in all regions of Kosovo and included 149 clinically healthy dogs (84 owned, 40 sheltered and 25 free-ranging). Blood samples collected from each individual were tested using a commercially available rapid multiplex enzyme-linked immunosorbent assay. In total, 37.6% of the examined dogs were seropositive for one or more of the target pathogens. Most frequently, antibodies were found against Anaplasma spp. (24.8% of the dogs tested), followed by antigen detection of D. immitis (14.8%) and antibodies of B. burgdorferi s.l. (1.3%). The lowest antibody prevalence (0.7%) showed E. canis where only one dog was found positive. This preliminary study demonstrates the circulation of several zoonotic vector-borne pathogens in natural cycles involving dogs in Kosovo. It should trigger studies on infection prevalences in humans and initiate vector surveillance programmes in order to manage and control transmission and the diseases associated with the pathogens.

Serological and clinical survey of Canine Leishmaniasis in Southwestern Region of Kosovo.

Canine Leishmaniasis caused by Leishmania infantum is endemic in Mediterranean countries. The disease represents an important public health issue in Kosovo in which the autochthonous cases by humans were reported in the last years. The objective of this study was to survey the serological and clinical data of Canine Leishmaniasis in dogs in southwestern region of Kosovo. The blood samples were collected from stray dogs in four municipalities, respectively Prizren, Gjakova, Rahovec and Deçan. The total 125 samples were collected between April/September 2016. Antibodies of Leishmania infantum were detected by competitive enzyme-linked immunosorbent assay (ELISA IgG). Out of total 125 serum samples, 23 dogs (18.4%) were positive for Leishmania infantum. In three of 23 infected dogs (13%) were shown the clinical signs of Canine Leishmaniasis as skin and ocular lesions, decreased appetite, lameness, diarrhea, lethargy and progressive weight loss. The highest infection rate was detected in Prizren municipality (21.6%), followed by Gjakova (17.6%), Rahovec (17.1%) and Deçan (7.8%). The present study confirmed that the Leishmaniasis is endemic in southwestern part of Kosovo and emphasized the need to establish the surveillance and control of this disease.

Immunoglobulin-like Domain of HsFcµR as a Capture Molecule for Detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-Specific IgM Antibodies.

BACKGROUND: The cellular surface molecule HsTOSO/FAIM3/HsFcµR has been identified as an IgM-specific Fc receptor expressed on lymphocytes. Here, we show that its extracellular immunoglobulin-like domain (HsFcµR-Igl) specifically binds to IgM/antigen immune complexes (ICs) and exploit this property for the development of novel detection systems for IgM antibodies directed against Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV). METHODS: His-tagged HsFcµR-Igl was expressed in Escherichia coli and purified by affinity chromatography, oxidative refolding, and size-exclusion chromatography. Specific binding of HsFcµR-Igl to IgM/antigen ICs was confirmed, and 2 prototypic ELISAs for the detection of anti-CCHFV and anti-ZIKV IgM antibodies were developed. Thereby, patient sera and virus-specific recombinant antigens directly labeled with horseradish peroxidase (HRP) were coincubated on HsFcµR-Igl-coated ELISA plates. Bound ICs were quantified by measuring turnover of a chromogenic HRP substrate. RESULTS: Assay validation was performed using paired serum samples from 15 Kosovar patients with a PCR-confirmed CCHFV infection and 28 Brazilian patients with a PCR-confirmed ZIKV infection, along with a panel of a priori CCHFV/ZIKV-IgM-negative serum samples. Both ELISAs were highly reproducible. Sensitivity and specificity were comparable with or even exceeded in-house gold standard testing and commercial kits. Furthermore, latex beads coated with HsFcµR-Igl aggregated upon coincubation with an IgM-positive serum and HRP-labeled antigen but not with either component alone, revealing a potential for use of HsFcµR-Igl as a capture molecule in aggregation-based rapid tests. CONCLUSIONS: Recombinant HsFcµR-Igl is a versatile capture molecule for IgM/antigen ICs of human and animal origin and can be applied for the development of both plate- and bead-based serological tests.

Crimean-Congo Hemorrhagic Fever, Kosovo, 2013-2016.

During 2013-2016, a total of 32 patients were treated for Crimean-Congo hemorrhagic fever in Prishtina, Kosovo; 11 died. In the 11 patients who died, findings included viral loads >1 × 108.5/mL, lactate dehydrogenase >2,700 U/mL, bleeding, and impaired consciousness. Ribavirin therapy had no noticeable effect in this small patient sample.

Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network.

BACKGROUND: Identification of vectors is of prime importance in the field of medical entomology for both operational and research purposes. An external quality assessment of mosquito identification capacities was carried out within the MediLabSecure Network, which is composed of laboratories located in 19 countries close to the European Union around the Mediterranean and Black seas. METHODS: A set of blind samples consisting of 7 or 8 adult mosquitoes and 4 larvae was given to each participant laboratory. In all, 138 adult mosquitoes and 76 larvae of different species were distributed for genus and species identification. RESULTS: All identifications were exclusively morphology based. Overall, 81% of identifications were correct at the genus level, 64% at the species level. The results were highly varied among the 19 participating laboratories. The levels of correct identifications were: 100% (three laboratories), 90-95% (four laboratories), 50-75% (six laboratories) and < 50% (six laboratories). CONCLUSIONS: This evaluation showed the need to maintain efforts in capacity building and quality control in the field of medical entomology and, more specifically, in the morphological identification of the Culicidae.

Sensitive and specific detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-Specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in µ-capture and IgG immune complex (IC) ELISA tests.

As the most widespread tick-borne arbovirus causing infections in numerous countries in Asia, Africa and Europe, Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) was included in the WHO priority list of emerging pathogens needing urgent Research & Development attention. To ensure preparedness for potential future outbreak scenarios, reliable diagnostic tools for identification of acute cases as well as for performance of seroprevalence studies are necessary. Here, the CCHFV ortholog of the major bunyavirus antigen, the nucleoprotein (NP), was recombinantly expressed in E.coli, purified and directly labeled with horseradish peroxidase (HRP). Employing this antigen, two serological tests, a µ-capture ELISA for the detection of CCHFV-specific IgM antibodies (BLACKBOX CCHFV IgM) and an IgG immune complex (IC) ELISA for the detection of CCHFV-specific IgG antibodies (BLACKBOX CCHFV IgG), were developed. Test performance was evaluated and compared with both in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available ELISA tests (VectoCrimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples collected in Kosovo during the years 2013-2016 from 15 patients with an acute, RT-PCR-confirmed CCHFV infection, and 12 follow-up sera of the same patients collected approximately one year after having overcome the infection. Reliably detecting IgM antibodies in all acute phase sera collected later than day 4 after onset of symptoms, both IgM ELISAs displayed excellent diagnostic and analytical sensitivity (100%, 95% confidence interval (CI): 85.2%-100.0%). While both IgG ELISAs readily detected the high IgG titers present in convalescent patients approximately one year after having overcome the infection (sensitivity 100%, 95% CI: 73.5%-100.0%), the newly developed BLACKBOX CCHFV IgG ELISA was superior to the commercial IgG ELISA in detecting the rising IgG titers during the acute phase of the disease. While all samples collected between day 11 and 19 after onset of symptoms tested positive in both the in-house gold standard IIFT and the BLACKBOX CCHFV IgG ELISA (sensitivity 100%, 95% CI: 71.5%-100.0%), only 27% (95% CI: 6.0%-61.0%) of those samples were tested positive in the commercial IgG ELISA. No false positive signals were observed in either IgM/IgG ELISA when analyzing a priori CCHFV IgM/IgG negative serum samples from healthy blood donors, malaria patients and flavivirus infected patients as well as CCHFV IgM/IgG IIFT negative serum samples from healthy Kosovar blood donors (for BLACKBOX CCHFV IgM/IgG: n = 218, 100% specificity, 95% CI: 98.3%-100.0%, for VectoCrimean-CHF-IgM/IgG: n = 113, 100% specificity, 95% CI: 96.8%-100.0%).

Crimean-Congo Hemorrhagic Fever Virus and Borrelia burgdorferi sensu lato in Ticks from Kosovo and Albania.

Tick-borne diseases pose a serious threat to human health in South-Eastern Europe, including Kosovo. While Crimean-Congo hemorrhagic fever (CCHF) is a well-known emerging infection in this area, there are no accurate data on Lyme borreliosis and tick-borne encephalitis (TBE). Therefore, we sampled and tested 795 ticks. Ixodes ricinus (n = 218), Dermacentor marginatus (n = 98), and Haemaphysalis spp. (n = 24) were collected from the environment by flagging (all from Kosovo), while Hyalomma marginatum (n = 199 from Kosovo, all from Kosovo) and Rhipicephalus bursa (n = 130, 126 from Albania) could be collected only by removal from animal pasture and domestic ruminants. Ticks were collected in the years 2014/2015 and tested for viral RNA of CCHF and TBE viruses, as well as for DNA of Borrelia burgdorferi sensu lato by real-time PCR. In Kosovo, nine ticks were positive for RNA of Crimean-Congo hemorrhagic fever virus and seven for DNA of B. burgdorferi s. l. None of the ticks tested positive for TBEV. CCHF virus was detected in one H. marginatum male specimen collected while feeding on grazing cattle from the Prizren region and in eight R. bursa specimens (five females and three males collected while feeding on grazing sheep and cattle) from the Prishtina region (Kosovo). B. burgdorferi s. l. was detected in seven questing ticks (four male and one female D. marginatus, two I. ricinus one female and one male) from the Mitrovica region (Kosovo). Our study confirmed that CCHF virus is circulating in Kosovo mainly in H. marginatum and R. bursa in the central areas of the country. B. burgdorferi s. l. was found in its major European host tick, I. ricinus, but also in D. marginatus, in the north of the Kosovo. In order to prevent the spread of these diseases and better control of the tick-borne infections, an improved vector surveillance and testing of ticks for the presence of pathogens needs to be established.

High Rates of Neutralizing Antibodies to Toscana and Sandfly Fever Sicilian Viruses in Livestock, Kosovo.

Toscana and sandfly fever Sicilian viruses (TOSV and SFSV, respectively), both transmitted by sand flies, are prominent human pathogens in the Old World. Of 1,086 serum samples collected from cattle and sheep during 2013 in various regions of Kosovo (Balkan Peninsula), 4.7% and 53.4% had neutralizing antibodies against TOSV and SFSV, respectively.

Surveillance of Arthropod-Borne Viruses and Their Vectors in the Mediterranean and Black Sea Regions Within the MediLabSecure Network.

PURPOSE OF REVIEW: Arboviruses, viruses transmitted by arthropods such as mosquitoes, ticks, sandflies, and fleas are a significant threat to public health because of their epidemic and zoonotic potential. The geographical distribution of mosquito-borne diseases such as West Nile (WN), Rift Valley fever (RVF), Dengue, Chikungunya, and Zika has expanded over the last decades. Countries of the Mediterranean and Black Sea regions are not spared. Outbreaks of WN are repeatedly reported in the Mediterranean basin. Human cases of RVF were reported at the southern borders of the Maghreb region. For this reason, establishing the basis for the research to understand the potential for the future emergence of these and other arboviruses and their expansion into new geographic areas became a public health priority. In this context, the European network "MediLabSecure" gathering laboratories in 19 non-EU countries from the Mediterranean and Black Sea regions seeks to improve the surveillance (of animals, humans, and vectors) by reinforcing capacity building and harmonizing national surveillance systems to address this important human and veterinary health issue. The aim of this review is to give an exhaustive overview of arboviruses and their vectors in the region. RECENT FINDINGS: The data presented underline the importance of surveillance in the implementation of more adapted control strategies to combat vector-borne diseases. Partner laboratories within the MediLabSecure network present a wide range of infrastructures and have benefited from different training programs. SUMMARY: Although reporting of arboviral presence is not carried out in a systematic manner, the expansion of the area where arboviruses are present cannot be disputed. This reinforces the need for increasing surveillance capacity building in this region to prevent future emergences.