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

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%).

Diagnostic Testing for Crimean-Congo Hemorrhagic Fever

“…These methods were replaced by ELISA and immunofluorescence assays due to improved reliability and sensitivity compared with older techniques (10,68,69). Many in-house ELISAs have been described utilizing either heat-inactivated CCHFV prepared from suckling mouse brain or, more commonly, recombinant CCHFV nucleocapsid protein as the target antigen (10,41,(70)(71)(72)(73)(74)(75)(76)(77)(78). Several commercial ELISA kits are available for research use, but none are approved for human clinical diagnostic testing.…”

Advanced Immunotechnological Methods for Detection and Diagnosis of Viral Infections: Current Applications and Future Challenges

“…As a part of most advanced immunotechniques, an ultrasensitive colorimetric assay called magnetic nano(e)zyme-linked immunosorbent assay (MagLISA) was developed, wherein silica-shelled magnetic nanobeads (MagNBs) and gold nanoparticles were pooled to monitor influenza A virus up to femtogram per milliliter concentration (Oh et al 2018). Sensitive and specific detection of Crimean-Congo hemorrhagic fever virus (CCHFV) was developed employing specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests (Emmerich et al 2018). Recently, truncated forms of HeV and NiV (Hendra and Nipah virus) G proteins as well as full-length NiV nucleocapsid (N) protein were used for detection of Hendra and Nipah virus specific antibodies in pigs.…”

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests

“…Ellipticity values (mdeg) were normalized to mean molar ellipticity per residue (MRE). Thermal data was analysed using Global3 (Applied Photophysics), and secondary structure content predicted using BeStSel [14].…”

“…RT-PCR methods are the current gold standard for detection of CCHFV during the viremic phase, offering sensitive and highly specific results [12]. An antigen-detection ELISA test (VectoCrimea-CHF-antigen, VectorBest) is commercially available, but most available immunoassays are based on detection of CCHFV-specific IgG or IgM [13][14][15][16][17]. Such tests are normally used in animal serosurveys [16,18] or during late phases of disease but, in severe patients cases, the production of antibodies is normally delayed, low, or even absent [19].…”