Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants

Abstract

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

Keywords: IgG antibody; Rift Valley fever virus; diagnostic accuracy; domestic ruminants; enzyme-linked immunosorbent assay; recombinant nucleocapsid; validation.

Figure 1

Optimization of cut-offs for the Rift Valley fever IgG indirect ELISA based on recombinant nucleocapsid antigen in sheep (A), goats (B), and cattle (C) using the two-graph receiver operating characteristic analysis (TG-ROC). The insertion point of the sensitivity (Se, smooth line) and specificity (Sp, dashed line) graphs represents a cut-off PP value (31.23, 26.57, and 30.46, respectively) at which the highest and equivalent test parameters (Se = Sp) are achieved at 95% accuracy level. Using the misclassification cost term (MCT) option of the TG-ROC, at these cut-off values, the overall misclassification costs in sheep (A1), goats (B1), and cattle (C1) become minimal (0.0045, 0.0054, and 0.0625, respectively) under the assumption of 50% disease prevalence and equal costs of false-positive and false-negative results. The two MCT curves represent values based on non-parametric (smooth line) or parametric (dashed line) estimates of Se and Sp derived from datasets in field-collected sera.

Figure 2

Linear correlation analysis of the non-parametric (smooth line) sensitivity (Se) vs. parametric (dashed line) sensitivity (Se*) in sheep (A), goats (B), and cattle (C), and the non-parametric specificity (Sp) vs. parametric specificity (Sp*) in sheep (A1), goats (B1) and cattle (C1).

Figure 3

The effect of different I-ELISA cut-off values on the categorization between sera from RVF-endemic countries tested positive or negative in the virus neutralization test. Distribution of IgG I-ELISA PP values in sera tested positive in VNT (grey area): (A) sheep (n = 275), (B) goats (n = 369), and (C) cattle (n = 356). Distribution of IgG I-ELISA PP values in sera tested negative in VNT (dark area): (A) sheep (n = 1874), (B) goats (n = 2072), and (C) cattle (n = 2864) in the VNT. Sera ordered according to ELISA PP values. Vertical lines indicate the ELISA cut-off values determined by the TG-ROC analysis (solid line), and as mean plus three (dotted line) and two (slashed line) standard deviations observed in the VNT-negative sera.

Figure 3

The effect of different I-ELISA cut-off values on the categorization between sera from RVF-endemic countries tested positive or negative in the virus neutralization test. Distribution of IgG I-ELISA PP values in sera tested positive in VNT (grey area): (A) sheep (n = 275), (B) goats (n = 369), and (C) cattle (n = 356). Distribution of IgG I-ELISA PP values in sera tested negative in VNT (dark area): (A) sheep (n = 1874), (B) goats (n = 2072), and (C) cattle (n = 2864) in the VNT. Sera ordered according to ELISA PP values. Vertical lines indicate the ELISA cut-off values determined by the TG-ROC analysis (solid line), and as mean plus three (dotted line) and two (slashed line) standard deviations observed in the VNT-negative sera.

Figure 4

The effect of different I-ELISA cut-off values on the categorization of sera from RVF-non-endemic countries tested negative in the virus neutralization test. Distribution of IgG I-ELISA PP values (grey area) in (A) sheep (n = 1493), (B) goats (n = 560), and (C) cattle (n = 955). Sera ordered according to ELISA PP values. Vertical lines indicate the ELISA cut-off values determined by the TG-ROC analysis (solid line) in ruminant serum panels from RVF-endemic countries and as mean plus three (dotted line) and as mean plus two (slashed line) standard deviations observed in the VNT-negative serum panels from RVF-free countries.

Figure 4

The effect of different I-ELISA cut-off values on the categorization of sera from RVF-non-endemic countries tested negative in the virus neutralization test. Distribution of IgG I-ELISA PP values (grey area) in (A) sheep (n = 1493), (B) goats (n = 560), and (C) cattle (n = 955). Sera ordered according to ELISA PP values. Vertical lines indicate the ELISA cut-off values determined by the TG-ROC analysis (solid line) in ruminant serum panels from RVF-endemic countries and as mean plus three (dotted line) and as mean plus two (slashed line) standard deviations observed in the VNT-negative serum panels from RVF-free countries.

Figure 5

Dose-response kinetics of a positive RVFV IgG sheep (A), goat (B), and cattle (C) serum before and after different inactivation procedures measured by recombinant nucleocapsid I-ELISA. * Percent positivity of internal positive control serum.