Biomarkers of Inflammation among Patients with COVID-19: A Single-Centre Prospective Study from Prishtina, Kosovo.

SARS-CoV-2 infection involves the phase of viral replication and inflammatory response predicting the severity of COVID-19. The aim of the study was to analyze the association between IL-6 and hematological and inflammatory parameters and outcomes of patients with COVID-19. Plasma interleukin 6 (IL-6) levels and other inflammatory and hematological parameters were analyzed in 86 adult patients diagnosed with SARS-CoV-2 infection in Kosovo. The median age of patients was 61.50 (49.75-67.25) years. Over half of patients were categorised as severe (58%) and had comorbidities (69%) with hypertension being the most common. The overall mortality rate was 4.7%. The distribution of biochemical parameters across disease severity groups was significantly different for C-reactive protein (CRP), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), white blood cells (WBC), and granulocytes with higher median values in more severe and critically ill patients whereas lower percentage of lymphocytes, monocytes, and platelet count in severe and critically ill patients. IL-6 levels were increased in 63% of patients with significant differences in the distribution across the following groups; age, disease severity, hospitalisation status, pulmonary infiltrates, oxygen therapy, and hypertension status. IL-6 significantly correlated with CRP, LDH, CK, ESR, and percentages of granulocytes. IL-6 and other inflammatory and hematological parameters were strongly associated with disease severity and may predict the outcome of the SARS-CoV-2 infection.

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.

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.

Viral metagenomics, genetic and evolutionary characteristics of Crimean-Congo hemorrhagic fever orthonairovirus in humans, Kosovo.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a tick-borne virus which causes severe disease in humans with fatality cases up to 30%. We investigated the genetic and evolutionary characteristics of CCHFV in Kosovo, in particular in humans and found that different virus variants of genotype V circulate, with Turkey as a possible origin for the progenitor of southern European CCHF outbreaks. Phylogenetic analyses also revealed a single introduction event and in situ evolution of CCHFV in this country. The viral metagenomics revealed a more abundant virome in the fatal CCHF cases and the presence of a novel tick-borne segmented RNA virus belonging to the recently discovered Jingmenvirus group which raises questions about the potential pathogenic effect of this novel virus on human and animal health.

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%).

The epidemiology of bacterial meningitis in Kosovo.

INTRODUCTION: The purpose of this study was to present the epidemiologic features of bacterial meningitis in the developing country of Kosovo. METHODOLOGY: Data were collected from active surveillance of bacterial meningitis cases treated at the University Clinical Center of Kosovo in the years 2000 (first post-war year) and 2010. RESULTS: Meningitis cases in 2000 compared with 2010 showed a 35.5% decline in incidence (from 4.8 to 3.1 cases per 100,000 population) and a decrease in the case fatality rate from 10% to 5%. In children, there was a lower mortality rate (5% versus 2%) and a lower incidence of neurological complications (13% versus 16%) as compared to adults (32% versus 10% and 16% versus 35%, respectively). Neisseria meningitidis was the most common pathogen of bacterial meningitis in both study periods. Bacterial meningitis was most prevalent in the pediatric population, and showed an increase in the median age, from three years in 2000 to seven years in 2010. A steady number of bacterial meningitis cases in adults throughout last decade (around 20 cases per year) was recorded. CONCLUSIONS: During the last decade, gradual changes have been observed in the epidemiology of bacterial meningitis that are unrelated to the introduction of new vaccines, but are partly due to the improvement of living conditions.

Acute arthritis in crimean-congo hemorrhagic Fever.

Crimean-Congo hemorrhagic fever is a severe viral disease caused by a Nairovirus. An atypical manifestation in the form of acute arthritis was found in a confirmed Crimean-Congo hemorrhagic fever virus Kosova-Hoti strain positive patient. Acute arthritis in Crimean-Congo hemorrhagic fever (CCHF) may be as a result of immune mechanisms or the bleeding disorder underlying CCHF.