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FDA Issues New Guidance for SARS-CoV-2 Pooled Sample Testing

NEW YORK – The US Food and Drug Administration has issued new guidance for laboratories and commercial manufacturers with qPCR-based diagnostic tests to detect SARS-CoV-2 virus specifying pathways to authorization for pooled testing indications.

Pooling has been the subject of increased interest of late, with the White House Coronavirus Response Coordinator Deborah Birx recently endorsing the strategy in comments to an Association of Molecular Pathology virtual event.

The new pooling guidance came in the form of updates to the Emergency Use Authorization templates for laboratory developed tests and commercial molecular diagnostic tests. Guidance is provided for tests that already have EUA as well as those that are still in the EUA process.

The updated FDA template notes that the need for COVID-19 testing in the US remains greater than available resources. "Combining multiple patient samples to create one pooled sample for testing could be considered to enable broader access to testing," the template says.

Specifically, it guides labs and developers to use a simple pooling approach, otherwise known as a Dorfman approach, in which patient samples are combined into non-overlapping pools and each sample pool is tested.

"A negative result implies that all samples in the pool are negative. A positive result indicates that at least one sample in the pool is positive," the template notes. A positive pool is then split back out for individual testing, so the volume of sample initially collected must be sufficient for pooled testing as well as individual follow-up testing, if needed. Also, sample pools cannot combine different specimen types.

The utility of pooling as a way to scale up testing depends on prevalence, test sensitivity, and the number of low-positive samples. Pooling dilutes the samples, which can reduce sensitivity, which can be particularly detrimental for weakly-positive cases in which viral concentration may already be near the limit of detection.

The FDA said in the template it believes a pool of five samples is a reasonable starting point for validation of pooling for a high-sensitivity test in populations with a positivity rate of approximately 5 percent to 6 percent, although smaller pools may be needed in populations with higher prevalence rates.

When implementing a sample pooling strategy, the agency suggests labs and developers may wish to test a random sampling of patient samples without pooling to evaluate the positivity rate and percent of weak positive samples in the testing population and to identify differences in positivity rate between those tested individually and those tested through pooling.

To validate pooled testing, labs and developers should characterize the reduction in assay analytical sensitivity indicated by a shift in Ct score for RT-PCR assays with respect to the number of samples to be pooled to ensure the selected sample pooling strategy will maintain appropriate sensitivity.

The maximum number of samples acceptable to pool should be determined for each specimen type.

The FDA recommends conducting a clinical validation study in the intended use population that includes testing each sample individually and using the proposed pooling strategy.

To add a pooling strategy to a test that already has EUA, labs and developers should submit an EUA amendment request with the appropriate validation data, but they do not need to establish assay performance with a separate comparator test. 

For validation, a clinical study of a five-sample pool strategy should include at least 20 individual positive samples and 180 negative samples, either archived samples or freshly collected. The study should compare the performance of the EUA-authorized assay when testing single specimens to the performance of the assay when testing sample pools. 

In the template for developers, the FDA said it strongly encourages them to "work with their customers to gather existing data." For example, 100 Ct scores from individually tested positive patient samples can be used to evaluate the percentage of samples with Ct scores close to the assay LoD, or weak positives, the template said.

The FDA said that a Ct shift of Log2(n), where "n" is the number of samples in a pool, can be estimated, such that for a pool of five samples a Ct shift of 2.3 is expected, for example. "Therefore, if a large percentage of positive patient samples are close to your assay LoD, you may want to consider a smaller n, which will reduce the observed Ct shift and maintain higher sensitivity," the FDA said in the template. "We recommend that at least 25 percent of the validation samples be within 2-3 Ct of the cut off, and no more than within 2-4 Ct."

For a lab-developed or commercial test that is not previously authorized, pooling can be included in the EUA filing, but must also include performance characterization with a high-sensitivity comparator assay and a clinical study of pooling involving at least 30 individual positive samples and enough negative samples to generate 30 five-sample pools with one positive sample plus 30 five-sample pools with only negative samples.