Abstract
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%).
Highlights
Being endemic in a variety of countries in Asia, Africa, the Middle East and Southeastern Europe [1], the Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family: Nairoviridae, genus: Orthonairovirus; [2]) is the geographically most widespread tick-borne pathogen [3,4].An infection with this zoonotic virus occurs either by tick bite or contact with body fluids or tissues of viremic humans or animals [5]
Due to the severity of the disease caused by this bunyavirus, the lack of specific prophylactic and therapeutic measures and the infection’s epidemic potential, CCHFV was included in the WHO priority list of diseases needing urgent R&D attention, in particular the development and improvement of diagnostic tools
While both IgM Enzyme-linked immunosorbent assays (ELISAs), like the CCHFV IgM IIFT, detected CCHFV-specific IgM antibodies in all sera collected during the acute phase of the disease on day 5 after onset of symptoms or later, the BLACKBOX CCHFV IgG ELISA and the CCHFV IgG IIFT were found to be significantly more sensitive than the VectoCrimean-CHF-IgG ELISA in detecting the rising IgG antibody titers in samples collected between days 11 and 19 after onset of symptoms
Summary
Being endemic in a variety of countries in Asia, Africa, the Middle East and Southeastern Europe [1], the Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family: Nairoviridae, genus: Orthonairovirus; [2]) is the geographically most widespread tick-borne pathogen [3,4]. An infection with this zoonotic virus occurs either by tick bite or contact with body fluids or tissues of viremic humans or animals [5]. No CCHFV-specific humoral immune response is observable in the majority of fatal cases [7,8]
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