Measures of diagnostic precision (repeatability and reproducibility) for three test methods designed to detect spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a rhabdovirus of the Sprivivirus genus and the etiological agent of an internationally regulated aquatic animal disease in several fish species, including koi carp Cyprinus carpio L. The virus has a complex lifecycle with both acute and persistent stages of infection and can cause high mortality in affected populations. In this study, the diagnostic repeatability (within laboratory agreement) and reproducibility (between laboratory agreement) of 3 tests were investigated to assess their fitness as SVCV diagnostic tools. The tests, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays targeting either the SVCV glycoprotein (Q1G) or nucleoprotein (Q2N) genes and virus isolation by cell culture (VI), were performed in a blinded study with four Canadian laboratories. Test panels consisted of duplicate sets of 100 tissue samples collected from 3 SVCV prevalence populations of koi: a low-prevalence negative reference population (n = 20 fish) as well as moderate- (n = 50 fish) and high-prevalence (n = 30 fish) populations of koi experimentally infected with SVCV. The Q1G and Q2N tests were performed with kidney tissue in 3 laboratories and with brain tissue in 1 laboratory whereas pools of kidney, spleen and gill tissues were tested with the VI assay in 2 laboratories. Agreement of binary results was evaluated using the observed proportion of agreement, Cohen's kappa and Gwet's agreement coefficient (AC1) whereas the concordance correlation coefficient (ccc) and Bland Altman's limit of agreement were used to evaluate agreement of the RT-qPCR continuous data. Gwet's AC1 provided a more stable estimate of agreement than Cohen's kappa. Overall, high repeatability (AC1, 0.78-0.90) and reproducibility (AC1, 0.74-0.89) were observed for the Q1G and Q2N tests when kidney tissue was used. Lower agreement estimates of repeatability (AC1, 0.54-0.77) and reproducibility (AC1, 0.50-0.80) were obtained for the VI test. RT-qPCR reproducibility was low with kidney-brain tissue pairs (AC1, 0.09-0.46) and high with inter-test pairs of brain (AC1, 0.76-0.86) or kidney tissue (0.75-0.86). Tissue-specific differences in virus load affected test precision and informed final tissue selection. Repeatability (ccc, 0.94-0.97) and reproducibility (ccc, 0.91-0.97) estimates of agreement for paired continuous data from the RT-qPCR assays were similarly high with kidney tissue and lower with paired brain (ccc, 0.15-0.83) and kidney-brain tissues (ccc, 0.01-0.55). The high precision of Q1G and Q2N with kidney tissue suggests that the tests are performing similarly and are suitable candidates for assessment of their diagnostic accuracy.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.