Diagnostic validity of molecular diagnostic assays for white spot syndrome virus at different severity grades

The high virulence of the white spot syndrome virus (WSSV) in acute infections and the lack of effective treatments underscore the necessity for a rapid, accurate, and efficient diagnostic process to control white spot disease. The analytical sensitivity of diagnostic assays (polymerase chain reactions; PCRs and rapid diagnostic kit) at different severity grades of WSSV infection were determined using the 95% limit of detection (LOD_95%). The LOD_95% of nested, real-time, and one-step PCRs and rapid diagnostic kit were 0.70, 4.67, 1.19, and 79,434.65 viral genome copies/reactions, respectively. From the intramuscular challenge tests conducted under different dose and temperature conditions, the WSSV severity grades of time-course collected whiteleg shrimp (Litopenaeus vannamei) and dead or live shrimps were determined based on the viral loads of the pleopod. By applying the WSSV severity grades, a total of 92 shrimps were classified as G0. Furthermore, 92, 66, 199, and 79 shrimps were classified as G1, G2, G3, and G4, respectively. Additionally, 222 shrimps were classified as negative as WSSV was not confirmed in the nested PCR assay. Diagnostic sensitivity (DSe) and specificity (DSp) values of molecular diagnostic assays were compared with those of nested PCR in artificial WSSV-infected shrimp to evaluate diagnostic performance. The real-time and one-step PCRs exhibited a DSe >92.4% for G0 grade (approximately 10¹-10² copies/mg), indicating WSSV detection at low copy numbers. The rapid diagnostic kit presented a DSe >92.4% for G2 grade (approximately 10⁴-10⁵ copies/mg), suggesting the detection of WSSV-infected shrimp with clinical signs during the endemic period. These results suggest that the strategy of presumptive diagnosis using one-step PCR and rapid diagnostic kit at different seasonal periods followed by confirmatory diagnosis using real-time PCR assay could aid in controlling WSD with rapidity, accuracy, and cost-effectiveness.