Review and application of pooled testing strategies

1. Review and application of pooled testing strategies Jim Kennedy Colorado State University Veterinary Diagnostic Laboratory Rocky Ford Branch

2. Why pooled testing? • Pooled testing offers advantages over individual testing • Allows the diagnostician to take advantage of highly sensitive and specific tests while minimizing cost • Diminishes cumulative testing error over individual tests

3. Where testing error happens • Pre-analytical error sources, wrong sample, mishandled sample, improper sample collection, etc. Starts from collection and goes until analysis begins. • Analytical error, analytic variation such as mechanical wear and tear or inherent error such as that seen with a set of spring type scales. • Biological variation, an average means some are higher and some are lower. • Post-analytical error, reporting errors misread values or misreported values transposition of figures, etc.

4. Why not pooled testing? • Potential impact of dilution diminishing Se • Logistical requirements for pooling samples (pooling of individual samples can be labor intensive) • Loss of samples for follow-up testing on positive pools

5. Assumptions associated with pooled testing • Pooled test Se must be approximately the same as individual test Se • Samples must be easily obtainable • Pools must represent a homogenous mixture of samples • The outcome is binomially distributed, i.e. a discrete probability distribution of the number of successes in a sequence of independent yes/no events each yielding success with a probability p

6. Our human counterparts institute pooled testing strategies • During WW II the military implemented pooled testing strategies for inductees • Pooled testing for HIV is common today. • The commonality between pooled testing in human medicine and veterinary medicine lies in herd diagnostics, more can be accomplished at less expense.

7. Veterinarians and screening tests • Limited applications of screening test strategies • Salmonella contamination of eggs • Johne’s fecal pools • BVD • T. foetus

8. Application of pooled testing for BVD and T. foetus • Push for BVD control/eradication stimulated a search for a method to screen herds for persistently infected animals. • The prevalence of PI’s was/is quite low • Individual tests are expensive • The use of a pooled strategy would lower the cost of testing and encourage more to adopt BVD control

9. In the past four years what have we learned about pooled BVD testing • A total of 4039 pools have been tested those pools represent 265,388 animals with an average pool size of 66. • 205 pools have been found positive (5.1%) • From the 205 positive pools 513 positive individuals have been identified for an overall prevalence of .19% • The cost of testing had pooling not been done would have been $1,061,552 while with pooling the cost was $319,508, 70% less.

10. Pooled T. foetus testing • Several states have mandatory T. foetus testing programs. • Programs were based on culture with PCR confirmation of positive culture. • Published Se for a single culture range from .7 to .9 while PCR Se is published to exceed .98. • Diagnostic labs charge $5 for culture and $25 for individual PCR. • By pooling the charge per PCR could be spread out over several animals, provided Se was not severely compromised.

11. Applying pooled testing for T. foetus • Maximum pool size of 5 samples was established. • 2370 pools have been tested with 103 pools found positive (from April 07 to July 08) • The 2370 pools represent 8567 bulls, 143 bulls were identified as T. foetus infected.

12. No laboratory cost advantage to pool T. foetus samples • Using culture the lab charge for testing the 8,567 bulls would have been $42,835, and assuming the 143 positives were found an additional charge for PCR confirmation at $25 each would be $3,575 for a total of $45,960. • Laboratory charges for pooling for the 2,370 pools would have been $59,250 plus the additional charge of $10,300 for identifying the positive member/s of the pool, for a total of $69,550

13. Then why pool for T. foetus • During the same period 4,693 requests for culture with a PCR confirmation of culture positives were received with 69 positives and 39 confirmed as T. foetus by PCR yielding an apparent prevalence of .8%. • This compares to 1.7% by pooled PCR for the 8567 samples. Assuming that both the culture group and the PCR group are representative of the same population it would appear that 86 bulls would have been misclassified as not infected. • A look from a different angle is that the cost per positive bull by culture was $667 versus $486 for pooled PCR $181 less, minor to the cost of missing 86 infected bulls.