Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia

Abstract

Serological diagnosis of equine infectious anaemia virus (EIAV) infections has depended mainly on the agar gel immunodiffusion test (AGIDT). This study documents the presence of EIAV genetic sequences in a number of persistently infected horses and mules whose serums were interpreted as negative/equivocal on AGIDT, but positive on more than one ELISA test and in immunoblot tests. Strategies designed to take advantage of the combined strengths of the ELISA and AGIDT are shown effective in a national surveillance program for EIA in Italy where 17 per cent (25/149) of the equids considered to be infected with EIAV on combined/ comparative serological data had reactions in the AGIDT that were interpreted as negative or equivocal. These data document the benefits of using a three-tiered laboratory system for the diagnosis of EIA. Although the ELISA-first strategy introduces some confusing results, the discovery of up to 20 per cent more cases of EIA makes it compelling. In our opinion, it is better and more defensible to find two samples in 1000 with resolvable but falsely positive ELISA tests for EIA than to release two to three horses in 10,000 with falsely negative test results for EIA (the rates seen in the Italian surveillance presented here).

Validation according to OIE criteria of a monoclonal, recombinant p26–based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies

“…In the CRAIE laboratory, the adoption of the cELISA as a screening test in routine testing, generated 182 sera (0.19%) with nonspecific reactions, as confirmed by AGID and immunoblot, out of a total of 96,468 samples examined. 13 For this, the screening method has an apparent DSp of 99.81%, higher than that estimated in its validation. The discrepancy between the values of specificity could be ascribed to the great divergence in the number of samples analyzed for the test validation compared with those processed during surveillance.…”

“…Furthermore, this discrepancy clearly demonstrates that the cELISA possesses a DSe and DSp higher than that of the AGID test (47 samples were cELISA-positive, AGID-negative, immunoblot-positive). 13 Regarding the hypothesis that there could have been an overestimation of the sensitivity in the validation process, the performance of the cELISA was superior to that of AGID test, as confirmed by the fact that cELISA used in a 3-tier system detected 17% more cases than if AGID alone was used 13 and, until now, no AGID-positive sample reacted negatively when examined by the cELISA. Because PPV of the test decreases when the prevalence is low, as is the case in Italy, where the national prevalence is ~0.14% as estimated by CRAIE from surveillance activities, the cELISA is still capable of detecting more cases than if the AGID test was used as the screening test.…”

“…A second cutoff value of PI was introduced at 30%, creating a range within which samples were defined as equivocal. This was established on the basis of the PI of samples of 5 EIAV-infected animals, as confirmed by PCR and immunoblot, 13,18 which was 31-47% in various runs. At 30% of PI, sensitivity was still 100% (CI: 91.6-100%) and specificity was 44.12% (CI: 38.3-50.1%).…”

“…A possible reduction of specificity was considered acceptable, as the test was to be used in the aforementioned 3-tier system that would use other assays, namely AGID and immunoblot, which would compensate for the low specificity. 13 Receiver operating characteristic (ROC) curve analysis was employed for the determination of the cutoff values using the percent inhibition (PI) results of panel 1 (sera analyzed by immunoblot), and the statistical analysis was performed using commercial software.…”

“…10 The Italian National Reference Center for EIA (CRAIE; Rome Italy) developed and validated a monoclonal antibody (mAb)-based semiliquid phase competitive enzyme-linked immunosorbent assay (cELISA), using the major core protein (p26) as a recombinant antigen expressed in Escherichia coli. 13 The aim was to employ the method as a screening test for the detection of antibodies against EIAV in horse sera examined during surveillance programs.…”