FDA Guidance on New Coronavirus Mutation Testing

Jan 12, 2022

The FDA recently released revised enforcement policies regarding the approval of molecular influenza and RSV tests during the COVID-19 public health emergency. Since many influenza and RSV tests use the same ingredients as those used for molecular analysis of SARS CoV-2, this policy was created to help with shortages of reagent supplies during flu season. This policy will help expand access to FDA-approved molecular tests to detect and characterize influenza viruses, including those that identify RSV.


In addition, the FDA Virtual Town Hall Series - Effective Immediate Release of Guidance on Diagnostic Testing for Coronavirus (COVID-19) (Q&A format), CAIVD has translated and distilled key content for readers' enjoyment. The FDA has about 100 people involved in the direct review of EUA and Pre EUA, and there are a large number of staff behind them who help in various ways. This Q&A focuses on technical issues related to the development and validation of SARS CoV-2 tests during public health emergencies. A brief update was presented by Dr. Timothy Stenzel, Director, Office of In Vitro Diagnostics and Radiological Health, and Deputy Director, Office of In Vitro Diagnostics and Radiological Health, CDRH's Product Evaluation and Quality Division.


Timothy Stenzel: As of this week, we have approved a number of EUA authorizations, one of the more specific is that we authorized the first antigen dipstick test. It is a normal test strip that is put into the test tube and has been authorized for point-of-care testing. A lot of people are interested in this easy and inexpensive test, and this antigen strip test doesn't even require a Cartridge.


This is the Quidel QuickVue SARS test, and like Abbott's BinaxNow, it's a very simple antigen test that doesn't require any equipment. We think it's unique in that it can significantly reduce the cost of getting test results so it can be mass-produced like Abbott's BinaxNOW.


There are many other great antigen test products out there. We generally prioritize approvals for applications that have a significant public health impact, such as home testing, home collection, point-of-care testing, and high-volume central laboratory testing. We mainly consider diagnostic tests, but also focus on serological tests.


Another topic I want to discuss is the new coronavirus mutation. Variants of coronavirus in the UK have also become a concern in the US, where we have seen some confirmed cases. The South African variant has some overlapping mutations with the British variant, but there are also clear differences.


Both variants appear to be highly infectious due to the virus mutating and enhancing binding to the receptor. There is evidence for this, some of which may have higher viral loads and then bind more strongly to the receptors to have a greater impact. Therefore, it is important to know which variant strains are circulating. We are interested in sequencing-based assays that provide complete genome sequences. And we've licensed at least one of this type of reagents -- Illumina kits. It is worth mentioning that it was empowered before we understood the importance of variation.


Another thing the FDA has been doing is periodically requiring all applicants, especially those of molecular tests, to check the sequence of known variants at least twice during the authorization process, and to clarify their primers about those variants in the known database and any issues that may arise with probe design.


In addition, FDA has bioinformatics analysis capabilities. We have a list of all EUA authorized molecular primers and probes. We have been querying sequencing databases to see if there are any mutations that might affect test performance. We have analysed mutant strains appearing in the UK and South Africa. We've also done this before for any variants that appear in the database that are 5% or more of the sequence in the database.


At least one test of the British new coronavirus variant that we talked about today was publicly mentioned as the Thermofisher Attack Path Assay with the S gene deletion. Due to the S gene deletion, we investigated many variants and sequences in the US database. So far, most do not have the characteristics of the British variant. There is currently only one subset in the United States - Thermofisher's S gene deletion. We believe that approximately 5% of Thermofisher-positive patients have S gene deletions. However, we believe that to date the vast majority do not carry the British variant. Therefore, at present, we recommend at least sequence verification of S gene deletion samples, and Illumina's EUA-authorized sequencing analysis can meet this requirement.



Q1: When using a molecular test as a comparator for rapid antigen testing, which infectivity-related CT cutoff is appropriate for reporting purposes?

A: We didn't use CT cutoffs or some kind of infectious cutoff because in reality CTs vary widely. For rapid antigen testing, we found a CT as high as 40 in the first five days of symptom onset. The exact cause is not yet determined. But we saw very clearly that in the first five to seven days of symptom onset, there were some high CT results. Therefore, we think it is important that we know the true performance of the antigen test when symptoms appear.

Therefore, we would recommend direct antigen testing. Most tests are approved for the symptomatic period of five to seven days. We want to know what the detection rate is for patients who have undergone antigen testing and are diagnosed as positive for Covid-19.



Q2: If clinical validation is used as a screening test, what is the minimum recommended number of positive and negative PCR cases?

A: If you're doing asymptomatic screening, and this is your first and only study, then I think you need 30 positive cases. If you want to add it to authorized inspections, or to synchronize with the first EUA authorization period. We believe the minimum depends on having at least 10 positive cases of asymptomatic patients prior to marketing or prior to authorization.

If you're going for an antigen test, it's recommended that you do a study to get the most out of what people who are asymptomatic are really doing. So, as I said, antigen testing peaks during the first five to seven days of symptoms. Virus levels and occult spikes within them typically peak during this period. It may be a little earlier but usually not delayed. After a week of virus concealment, antigen detection performance declined. While a good molecular test may still be positive after 14 days, some cases develop symptoms, which are beyond the scope of symptomatic studies.

Therefore, an optimal study design is to link a series of serial assays that will give you the best chance of demonstrating the superior performance of antigen assays. Screen at least weekly with a molecular comparator that can be used as a documented comparator for asymptomatic studies. This way you can enrich your database while waiting until someone confirms a positive through screening. Contact people who have tested positive to see if they would like to be tested for the new test. It is desirable to use at least the same number of negatives to ensure reliability. Perhaps this is the best way to enrich asymptomatic virus levels, hopefully to see better antigen performance tests.



Q3: What is the priority of EUA approval for different tests?

A: POCT testing, home testing, home sampling, mainly focused on diagnostic testing, namely molecular and antigenic testing. But serological testing will also be prioritized. This is by high-throughput central laboratory testing, which helps reduce the turnaround time of test results, on automated platforms or automated systems. These are priorities.



Q4: As far as serology is concerned, with vaccinations currently being implemented, what does the FDA have to say about serology testing of vaccinated individuals?

A: Vaccines are reviewed by the Center for Biologics Evaluation and Research (CBER), and questions need to be sent to the center for responses. Serology can be predicted in a variety of ways based on known data, which is important for reviewing any vaccine-related submission. There is no prohibition on its evaluation in the various serological tests used by licensed clinicians. It must be prudent that no serological tests are currently authorized by the FDA for any purpose, whether to determine whether a vaccine is needed, or to determine that a vaccine is effective or that immunity has been established. Nor is serological testing authorized for immunization applications. The FDA has approved semi-quantitative serology tests and neutralizing antibody serology reagents.



Q5: How will serological testing of vaccinated people be performed and will there be false positives? If someone has been vaccinated, is it possible to tell that antibodies have been produced?

A: No such reagents are currently authorized. The development of vaccine antibodies may be very similar to the development of actual new crown infection antibodies. It is a very important test and requires specific requirements. The FDA will consider the design but still requires multiple considerations.



Q6: Regarding the use of quality control, what is the FDA's recommendation for internal standards for antigen testing?

A: There is an internal standard to assess the quality of the sample, but this is not a requirement for EUA authorization. The only requirement or recommendation for such an internal standard has so far been home collection of samples, and we do not know whether patients have collected good enough samples themselves for analysis without an internal standard control. The FDA has updated some authorizations with internal standards programs, evaluated performance in home tests, reviewed thousands of data, and in some cases no longer requires internal standards. But now with new R&D facilities and new household collections, we would like to see these data.



Q7: Can you comment on an acceptable enrichment strategy for rapid antigen testing, especially screening everyone who comes to a COVID testing center for symptoms of COVID-19 infection?

We are looking for positive cases, some with symptoms and some without symptoms. To try and get a symptom-positive rather than asymptomatic claim, we wanted to screen people who had symptoms within the past 8 days or who started showing symptoms 8 days ago.

A: I don't think this is a particularly good study design. Due to the current high positive rate, as long as symptomatic patients are seen, at least 30 positive results can be obtained. We have great flexibility in asymptomatic detection. To do a good job of antigen detection, it should be a group of continuous detection, which will provide the best opportunity for antigen detection and demonstrate its excellence in the first 5-7 days of virus concealment. Performance. If you want to follow up, you need to submit a Pre-EUA application for review, but this is not recommended.



Q8: The CDC has agreed to conduct some tests on UK mutants of the new coronavirus against existing EUA-authorized tests. Does this also hold true for developers who are developing antigen tests?

A: I think the right time may be after the analysis has been locked. You can also construct pseudoviruses yourself for evaluation, as UK-positive mutated live virus and/or irradiated or heme-activated virus may eventually be present in the BEI library. But I don't think it's necessary to overload CDC early in development.

It is important that we hope that all antigen tests will not be affected. I think we might get a good direction with the first test we did. Typically antigen assays target the N gene, which may be much more conserved than the spec protein in some respects.

But once you've got it locked and ready to commit, I'll start checking its progress. Because at this moment, it would also be of great help to us at the FDA to be able to immediately understand and respond to the reagents that evaluate the tests for the British variant. We want to follow the product development process with you.



Q9: If we have permission to do a clinical validation study design, does this also apply to a 510(k)?

A: After the first De Novo is approved in Molecular or any other relevant classification, each subsequent assay will be submitted to a 510(k). For those developers who want to understand their transformation requirements, we are drafting a guidance document, but the specific details about all EUA products may not be fully covered in the first draft.

So we asked the team to draft some information that could be shared with the developers. First, SARS-CoV-2 differs from standard antigens in other infectious diseases in serological and/or molecular detection of respiratory infectious antigens. So our feedback will focus on how Covid and our response are different. We urge you to review our summary of recent decisions for De Novos and 510(k)s, which includes all frequently asked questions about special controls related to testing for respiratory viruses.

I don't think there's any rush to convert EUA to 510(k). Because right now we have a lot of EUA applications to process and a lot of new applications every week. So our focus now will still be mainly on the authorization of the EUA. Even if this comes to an end, the guidelines we're drafting will provide a very reasonable transition time, so we don't think unreasonable things will happen in the transition transition.

But I know very well that we're going to be asking for a lot of transformations, so it's wise for all developers to start thinking about those transformations. If transitioning at an important transition point is important to you, it is possible to collect samples during the current COVID-19 pandemic for translational research in preparation for transitioning your EUA to a 510(k).



Q10: Do you have any indication or information that CDC primers may be affected by the mutation that is spreading?

A: Yes. Once we have identified the potential impact on either molecular, antigenic or serological testing, we will make that information public as soon as possible. The primer and probe sequences for CDC analysis have been published, and you can view and understand mutations and how they affect these primers and probes.



original source

FDA Virtual Town Hall Series Immediately in Effect Guidance on Coronavirus (COVID-19) Diagnostic Tests

Source: FDA official website

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