Development and diagnostic evaluation of a reverse passive latex agglutination test for rapid field detection of...

Lumpy skin disease (LSD) is a vector-borne viral disease of cattle that causes significant production losses. Caused by Lumpy skin disease virus (LSDV), it has recently emerged as a Transboundary Animal Disease (TAD), creating a need for rapid and sensitive diagnostic assays to facilitate disease surveillance and control programs. In this study, a reverse passive latex agglutination test (RPLAT) was developed and validated for the rapid detection of lumpy skin disease virus (LSDV) antigen. Polystyrene Latex beads were coated with hyperimmune serum raised against LSDV in Guinea pig, and assay conditions were optimized for detecting antigen in vitro. We used 173 field samples, including nasal swabs, scabs/skin lesions, and arthropod vectors that were tested alongside PCR as the reference standard. Using receiver operating characteristic curve (ROC) analysis, we determined the assay sensitivity to be 93.4% and specificity to be 94.4% at the optimal cutoff value. The assay area under the curve (AUC) was calculated to be 0.936 (95% CI: 0.889-0.968). Real-time PCR analysis of representative RPLAT-positive samples and all RPLAT-negative samples demonstrated high concordance between the two assays, with 65 of 67 representative RPLAT-positive samples confirmed by qPCR. Optimal detection was observed for samples taken at a late stage of infection, which would correlate with higher antigen amounts during infection. We did not detect any cross-reactivity to other bovine pathogens tested, indicating high analytical specificity of the assay. Results were interpretable within 5 min, require limited equipment, and are visually interpreted. Due to the preliminary nature of this study and constraints in sample availability and resources, extensive inter- and intra-laboratory validation was not performed and will be addressed in future studies. Overall, the study demonstrates the preliminary feasibility of a rapid antigen detection assay for field-level screening of LSDV.