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%)

Inter-laboratory variation of BLACKBOX CCHFV IgG ELISA.

<p>Inter-laboratory variation of BLACKBOX CCHFV IgG ELISA.</p

Analysis of CCHF patient samples: Raw data (OD450 – OD620), dependence from day after onset.

<p><b>(A) BLACKBOX CCHFV IgM ELISA. (B) BLACKBOX CCHFV IgG ELISA. (C) VectoCrimean-CHF-IgM ELISA. (D) VectoCrimean-CHF-IgG ELISA.</b> Paired serum samples from 15 CCHF patients were classified according to their time point of collection (days after onset of symptoms). Filled circles indicate PCR positive serum samples. Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006366#pntd.0006366.s003" target="_blank">S2 Fig</a>).</p

Analysis of paired CCHF patient samples: Raw data (OD420 – OD620).

<p>A serum panel consisting of 30 paired serum samples from 15 CCHF patients, serum samples from 12 CCHF patients collected approximately one year after recovery from CCHFV infection, a set of a priori CCHFV IgM/IgG negative serum samples (neg) ((A), (B): n = 120; (C), (D): n = 15, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006366#pntd.0006366.s002" target="_blank">S1 Fig</a>), and 98 CCHFV IgM/IgG IIFT negative sera from healthy blood donors from Kosovo (HD K) were analyzed with the <b>BLACKBOX CCHFV IgM ELISA (A)</b>, the <b>BLACKBOX CCHFV IgG ELISA (B),</b> the <b>VectoCrimean-CHF-IgM ELISA (C)</b> and the <b>VectoCrimean-CHF-IgG ELISA (D).</b> Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006366#pntd.0006366.s003" target="_blank">S2 Fig</a>). Filled circles indicate PCR positive serum samples.</p