Evaluation of pooling in surveillance for white spot syndrome virus

Abstract

White spot syndrome virus (WSSV), initially identified in 1992, is recognized as a major global viral threat to shrimp farming. Testing pooled samples, which involve collectively assessing multiple individual animal specimens, is the most efficient method in surveillance programs. Currently, despite its advantages, pooling for WSSV surveillance is not typically recommended due to its insufficient evaluation. Therefore, this study focused on assessing the applicability of pooling for WSSV surveillance. By evaluating the analytical performance, a reduction was observed, evidenced by the increased C_T values in the mixed group (pleopod + hepatopancreas) compared to those in the pleopod group. From the evaluation of the influence on diagnostic performance, pools showed a detection rate greater than 82.50 %, regardless of pool size, for G2–G3 severity grades. For the G0–G1 grades, the detection rate increased from 58.33 % to 93.33 % as the number of positive samples increased from 1 to 3 in a pool size of 5, whereas for a pool size of 10, the rate increased from 31.67 % to 86.67 % as the number of positive samples increased from 1 to 5. Notably, the cutoff values for the applicability of pools were determined to be 10^3.39 and 10^4.61 copies/mg for a single positive sample with pool sizes of 5 and 10, respectively. Similarly, in a simulation with 2 % prevalence and 160 shrimp, the detection rate for a pool size of 5 increased from 42.85 % to 80.86 % as severity grades progressed from G0–G1 to G1–G2, whereas for a pool size of 10, the detection rate improved from 46.73 % to 80.40 % as severity grades advanced from G1–G2 to G2–G3. Furthermore, at G0–G1, although a pool size of 5 showed 71.39 % surveillance sensitivity with a sample size of 150, it increased to 95.20 % with 360 shrimp. To adequately apply the diagnostic method for WSSV surveillance, it is necessary to consider the seasonal outbreak characteristics of the disease and pathogen activation. Based on these results, to enhance the efficiency and sensitivity of surveillance during the low-risk seasons for white spot disease (WSD), employing a pool size of 5 along with a larger sample size is recommended. Moreover, pooling could aid in rapid screening for WSSV during the endemic period. This study anticipates that pooling strategies tailored to the disease status may significantly contribute to reducing the incidence and spread of WSD through efficient and rapid testing.