Dear All, 

I was just looking through the OIE manual again, based on Greg's suggestion, and was struck by the lack of validated PCR, especially qPCR assays in it. From my experience (and I believe that of others, though I won't speak for them), quantitative real time PCR is much more (analytically) sensitive than standard PCR (e.g., with the MCP4/5 primers in Mao et al. 1997) and more sensitive even than virus isolation in cell culture. We are consistently detecting the equivalent of <1 plaque forming unit in asymptomatic animals from experiments and from the wild. Jaramillo et al. have a validated assay for ranavirus in fish that seems to support the assertion that qPCR is more sensitive: 

"The assay had 100-fold greater analytical sensitivity compared to virus isolation in Bluegill fry (BF-2) cells, detected all viruses in a panel of 20 ranaviruses from Australia, America, Europe and the United Kingdom, did not detect two disputed members of the genus Ranavirus (doctorfish virus and guppy virus 6) and did not detect a megalocytivirus or two cyprinid herpesviruses."  (Note: I do understand that analytic sensitivity is not the same as diagnostic sensitivity, but if virus isolation is our gold standard then qPCR cannot, by definition, have greater diagnostic sensitivity!) 

So I guess my question is, if someone is interested in ensuring that their animals are ranavirus free, shouldn't we be pointing them to these more sensitive assays? What are your thoughts? Would it be worth "stressing out" some subset of the animals to see if animals carrying inapparent infections might recrudesce? The question of whether they can develop full blown, transmissible infections seems very relevant in these cases.

I would also like to point out that the protocols in the OIE manual state, in section 6: "Statistically valid sampling practices need to be used but these cannot presently be defined for amphibians." I am not sure why amphibians would present a special case in epidemiology, but I was not at these meetings, so perhaps someone could clear up my confusion. Barring that, I might suggest using the standard formulas for calculating the sample sizes required to be, say, 95% confidence that the prevalence of infection is at or below some level (e.g., 10%) given a population of N (e.g., 10^6)… here n = 30.  I'm sure there are other references for the formula, but this is the one I have:

I've attached a spreadsheet I created a while back to help me see the relationships between confidence-level, population sizes, the number of disease animals, and the sample size. It is rather busy, but some playing with the numbers in different colors should clear up which graph is associated with which equation. Or I'm happy to answer questions about it.  

In any case, if there is a better way of establishing statistically valid sample sizes, I would love to hear them. It would be great for the members of this list to be on the same page, more or less, about how best to sample.

Best wishes, 

Jesse