This section is primarily intended for monkeypox test developers.
This section provides answers to frequently asked questions relating to the development and performance of tests for monkeypox. These questions and answers provide additional clarity on existing policies and do not introduce any new policies or modify any existing policies.
This section includes questions and answers regarding the policies described in the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency.
Tests being offered prior to or without an EUA as described in the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency have not been reviewed or authorized by the FDA. As stated in the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency, all such tests should be validated by the developer prior to being offered for clinical use.
Note: Throughout this section and the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency, references to laboratories that are "certified to perform high complexity testing under CLIA" or to "high-complexity CLIA-certified laboratories" are referring to single site laboratories that are certified under CLIA that meet the requirements to perform tests of high complexity.
A: As discussed in the guidance, at this stage in the outbreak, for monkeypox diagnostic tests, the FDA intends to prioritize review of EUA requests for high-throughput diagnostic tests, tests with home specimen collection, or rapid diagnostic tests, all from experienced developers with high manufacturing capacity, that inform the FDA of their intent to submit an EUA request within 30 days after publication of the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency.
The FDA recommends that test developers provide preliminary information to the FDA to indicate their intent to submit an EUA request for a monkeypox diagnostic test in an email to MPXDx@fda.hhs.gov according to section IV.A.1 of the guidance.
After an EUA request has been submitted, the FDA intends to notify test developers by email if the FDA authorizes a test or declines to review, declines to issue, or otherwise decides not to authorize a test for any reason, including lack of response from the developer or a determination that there is a lack of adequate data to support authorization.
A: The FDA understands that some laboratories certified to perform high complexity testing under the Clinical Laboratory Improvement Amendments (CLIA) are developing diagnostic PCR tests to detect the monkeypox virus. The FDA does not intend to object to the use of these tests for testing lesion swabs after the laboratory has validated the test and given notification of validation within five business days of offering the test to the FDA as described in section IV.A.4. of the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency. The FDA intends to accept notifications for 30 days after publication of the notice of availability of this guidance in the Federal Register, but the FDA will monitor the situation and may adjust, including shortening or lengthening this time period, as appropriate.
This policy does not apply to tests with home specimen collection or at-home tests or to tests using specimen types other than lesion swabs or technologies other than PCR.
In section IV.A.5 of the guidance, the FDA provides recommendations regarding test reports. Specifically, the FDA recommends that test reports should prominently disclose that the test has not been reviewed by the FDA and is being offered under section IV.A.2. of the guidance. The patient reports should not expressly state or imply that the test has been reviewed or authorized by the FDA, as such statements would be false. Similarly, any statements that state or imply that the FDA will authorize a test could be misleading.
Laboratories should report all results (positive, negative, equivocal) immediately to the appropriate Federal, State, and/or local public health agencies in accordance with applicable laws.
If the FDA identifies a significant problem or concern with a test, the FDA intends to notify the laboratory by email and provide the laboratory an opportunity to address the questions or concerns. If the concerns cannot be adequately addressed in a timely manner, the FDA generally would expect the laboratory to take appropriate steps, which could include that they stop offering the test, conduct a recall of the test whether modified or not, and/or notify end users concerning corrected test reports indicating prior test results may not be accurate.
A: No. The FDA does not expect any notification from laboratories that are performing testing using FDA-cleared or EUA-authorized tests. Laboratories performing monkeypox testing must be certified under the Clinical Laboratory Improvement Amendments, or CLIA, which is administered by CMS.
Laboratories offering EUA-authorized tests are subject to various conditions that can be found in the EUA Letter of Authorization. Each EUA letter also specifies the settings in which the test is authorized. For ease of reference, the settings authorized in the EUAs are also noted in the EUA table on the Monkeypox Emergency Use Authorizations for Medical Devices. Tests that are noted with an "H" in the Authorized Settings are limited to use in laboratories certified under CLIA that meet requirements to perform high-complexity tests. Tests that are noted with an "H" and "M" in the Authorized Settings may be performed in laboratories certified under CLIA that meet requirements to perform high complexity and/or moderate complexity tests. Tests that are noted with a "W" in the Authorized Settings are deemed to be CLIA-waived for use in patient care settings operating under a CLIA Certificate of Waiver. Tests noted with an "H," "M," and "W" may be used in laboratories certified under CLIA that meet requirements to perform high complexity and/or moderate complexity tests and in patient care settings operating under a CLIA Certificate of Waiver.
A: This CDC assay is FDA-cleared for use in CDC-designated CLIA-certified laboratories that meet the CLIA regulatory requirements to perform high complexity testing. At this time, the CDC has designated laboratories in the CDC Laboratory Response Network (LRN) as well as the following commercial laboratories:
- Quest Diagnostics
- Mayo Clinic Laboratories
- Sonic Healthcare
- Aegis Science
A: The policies regarding the modification of an FDA-cleared or EUA-authorized monkeypox molecular diagnostic test, where the modifications do not change the indication for use set forth in the 510(k) or EUA and do not change the analyte specific reagents, are discussed in section IV.A.3 of the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency. These policies address modifications made by a commercial manufacturer to its own FDA-cleared or EUA-authorized test, and modifications made by a high-complexity CLIA-certified laboratory to an FDA-cleared or EUA-authorized monkeypox molecular diagnostic test, including one for which such laboratory is not the developer of the original cleared or authorized test.
When a high-complexity CLIA-certified laboratory is modifying a cleared or authorized monkeypox molecular diagnostic test, the FDA does not intend to object to implementation of the modification to the diagnostic test without a new or amended EUA or a new premarket submission (e.g., 510(k)) where the test has been validated, the validation demonstrates that the modifications do not adversely affect test performance, and the laboratory submits notification of validation to the FDA, as described in section IV.A.4 of the guidance.
When a commercial manufacturer is modifying its own cleared or authorized monkeypox molecular diagnostic test, the FDA does not intend to object to implementation of the modification to the diagnostic test while the FDA conducts its review of the supplemental EUA request or premarket submission.
Unless and until an EUA or marketing authorization is issued that authorizes additional testing environments for a specific test, under the Clinical Laboratory Improvement Amendments (CLIA), section 353 of the Public Health Service Act (42 USC 263a), use of that test is limited to laboratories that are certified under CLIA, and meet the requirements to perform tests of high complexity, and at the point-of-care (POC) when the site is covered by the laboratory's CLIA certificate for high-complexity testing.
In order to provide transparency, when a developer is distributing or offering a test that is a modification of a cleared or EUA-authorized diagnostic test as discussed in section IV.A.3 of the guidance, the recommendations in Section IV.A.5 of the guidance apply. The FDA further recommends that the developer post data about the modified test's performance characteristics on the developer's website, and that the instructions for use or test protocol and the test reports accurately reflect the modification and prominently disclose that the test has been modified since clearance/authorization by the FDA and that the modified test has not been reviewed by the FDA.
If the FDA identifies a significant problem or concern with a modified test, the FDA generally expects the developer to take appropriate steps to address such problems, which could include conducting a recall of the test and/or notification concerning corrected test reports indicating prior test results may not be accurate.
A: As discussed in the guidance, in an emerging outbreak, use of serology tests can provide information that may further our understanding of the disease process. While monkeypox serology tests can be helpful, there is great potential for their misuse. At least at this time, monkeypox serology tests cannot be used to diagnose, or aid in the diagnosis of, an active infection and are not tests of immunity. Therefore, it is important that results from monkeypox serology tests be used for appropriate purposes and the result be properly communicated.
At this time, the FDA does not intend to object to the use of monkeypox serology tests that are developed and performed by a high-complexity, CLIA-certified laboratory that is part of an entity that conducts research on diseases and is integrated into the direct medical care of the patient (often referred to as academic medical center laboratories), where the laboratory gives notification of validation as described in section IV.C.2 and certain information is included in the test reports as described in section IV.C.1 of the FDA's Policy for Monkeypox Tests to Address the Public Health Emergency.
The FDA's recommendations in section IV.C.1 of the guidance, that laboratories include certain clarifying information in the reports for serology tests to help patients understand the test results, are intended to mitigate the potential misuse of monkeypox serology test results while also fostering research from availability of data from serology testing of patients.
The FDA encourages the laboratories to share their validation data with FDA as well as what they learn about the utility of such tests. Should clinical utility be demonstrated for monkeypox serology tests in the future, the FDA may decide it is in the best interest of public health to review EUA requests for serology tests.
If the FDA becomes aware of questions or concerns about a notified serology test, such as poor performance or misleading statements about the test, the FDA will communicate those concerns to the laboratory and provide the laboratory an opportunity to address the questions or concerns. If the concerns cannot be, or have not been, addressed in a timely manner, the FDA would expect the laboratory to stop offering their test, and the FDA may take additional actions as appropriate.
A: On the Monkeypox Emergency Use Authorizations for Medical Devices (EUA) webpage, the FDA is providing recommendations regarding validation testing for diagnostic monkeypox tests in two voluntary EUA templates that are part of the guidance. These recommendations include validation testing that should be performed to provide information on the analytical and clinical validity of the test and additional details, including study design considerations. Depending on the characteristics of your test, additional validation studies may be recommended. However, developers do not need to use either template when submitting an EUA request.
The FDA intends to update its recommendations regarding validation testing as the outbreak evolves. At this time, the FDA's initial validation recommendations are for clinical validation with contrived specimens, but if clinical samples become more widely available, the FDA may revise this recommendation. Note that the FDA recommends that each contrived clinical specimen should consist of a unique individual natural clinical matrix sample (i.e., each contrived specimen should be made by spiking quantified material into a human skin lesion material specimen that is negative for monkeypox virus).
Developers can use alternative approaches. The FDA encourages developers to discuss any alternative technological approaches to validating their test with the FDA through submission of a pre-EUA to MPXDx@fda.hhs.gov.
A: Below is information regarding various test materials for diagnostic assay validation that FDA is aware of. Links provided are for information purposes only and are not a recommendation by the FDA to use that product. The FDA encourages other suppliers of test materials to email MPXDx@fda.hhs.gov to discuss whether materials they have available may also be appropriate for use.
As noted in the molecular diagnostic templates, at this time, the FDA's initial validation recommendations are for clinical validation with contrived specimens, but if clinical samples become more widely available, the FDA may revise this recommendation. If no natural clinical specimens are available at the time of your EUA request for a monkeypox virus molecular diagnostic test, the FDA recommends that fully contrived specimens used for clinical evaluation to support initial authorization be prepared using a unique natural clinical specimen matrix (e.g., human skin lesion material specimens are spiked with quantified material to create the contrived specimen). Please refer to those EUA templates for information regarding clinical study design recommendations for molecular diagnostic tests.
When preparing contrived specimens, we recommend spiking quantified virus (e.g., live virus or inactivated via heat treatment, chemically modified, or irradiated, or genomic DNA or synthetic DNA until viral isolates of the currently circulating strain become publicly available) into natural clinical matrix (e.g., human skin lesion material specimens). If using contrived specimens for your initial assay validation, you may request appropriate test material for assay validation directly from:
- ATCC: Order through their ATCC Resources website
- Product # VR-3270SD: Quantitative Synthetic Monkeypox virus DNA; Quantitative Synthetic Monkeypox virus DNA - Preparation includes fragments from J2L, D14L, F3L, F8L, A27L, A29L, B6R, B7R, and N3R regions.
- BEI Resources: Order through BEI Resources website
- NR-2500 Monkeypox virus USA-2003
- NR-27 Monkeypox virus WRAIR 7-61
- NR-2324 Monkeypox virus Zaire 79 (V79-I-005)
- NR-21738.1 Monkeypox virus Zaire 79 (V79-I-005) CDC, Animal Challenge Pool
- NR-4928 Genomic DNA from Monkeypox Virus, USA-2003 –monkeypox virus
- Twist Bioscience: Order through Twist Resources website
- Product # 106056: Twist Synthetic hMPXV Control 1 (CB)
- Product # 106059: Twist Synthetic hMPXV Control 2 (WA)
- NIST: Order through NIST Resources website
- Product # RGTM 10223: Monkeypox Research Grade Test Material
A: The presence of viral mutations in the monkeypox virus in a patient sample can potentially impact test performance. The impact of viral mutations on a test's performance is influenced by several factors, including the sequence of the variant, the design of the test, and the prevalence of the variant in the population.
Tests with single targets are more susceptible to changes in performance due to viral mutations, meaning they are more likely to fail to detect new variants than tests with multiple targets. Multiple targets means that a molecular test is designed to detect more than one section of monkeypox genome or, for antigen tests, more than one section of the proteins that make up the monkeypox virus. Test developers should consider including multiple targets as they develop new monkeypox diagnostic tests.
As discussed in the monkeypox EUA templates, the FDA encourages developers to include a highly conserved monkeypox virus target (i.e., a target in a portion of the genetic code not restricted to a specific monkeypox virus variant) or non-variola Orthopoxvirus target as part of a multiple target test which may improve performance with new genetic variants; however, the number of targets in the test should be appropriate to provide resilience (i.e., a reduction of the risk that viral mutation will impact test performance) and most efficiently leverage developer and laboratory resources.
A: The FDA collaborates with stakeholders to better understand the public health impact of new virus variants and their impact on test performance. The FDA is monitoring publicly available sequence databases and is coordinating efforts to evaluate the impact of new virus variants on tests being used clinically, including those that are FDA cleared or authorized or under FDA review. The FDA will continue this monitoring on an ongoing basis throughout the pandemic.
One aspect of the FDA's monitoring program involves analyzing available sequence data from publicly available genomic databases, such as the GISAID database. GISAID is a global science initiative and primary source that provides open-access to genomic data of influenza viruses and the novel coronavirus responsible for COVID-19 (https://www.gisaid.org/). Additionally, any variants with multiple credible reports (e.g., peer-reviewed literature or, in the more immediate term, reports from the public health community, such as state public health laboratories) indicating the potential to impact patient care practices, increased virulence, or increased transmission risk are more closely monitored by the FDA because of the possible increased public health risk.
In order for the FDA to monitor the potential impact of viral mutations on all monkeypox tests being used for clinical testing in the U.S., we encourage laboratories offering monkeypox PCR tests as described in section IV.A.2. of FDA's Policy for Monkeypox Tests to Address the Public Health Emergency to provide the test gene target and primer and probe sequences to the FDA for reference. Providing these sequences to the FDA will enable the FDA to include these tests in our regular mutation (variant) monitoring. In such cases, the FDA will notify the laboratory if we detect a potential issue.
A: Some monkeypox virus specific sequences are more likely to mutate than others. Monkeypox virus specific primers and probes target gene sequences that are specific to monkeypox virus and are not present in other non-variola orthopoxviruses. Highly conserved monkeypox virus or non-variola Orthopoxvirus targets are targets in a portion of the genetic code not restricted to a specific monkeypox virus variant and, therefore, are less likely to mutate.
The FDA is aware of signals that there are mutations in the monkeypox virus genetic code impacting the performance of some tests. In particular, the FDA is aware of a report of three monkeypox virus cases in which preliminary data show a significant deletion in the tumor necrosis factor (TNF) receptor gene. This gene is the target for the CDC West African MPXV and Generic MPXV real-time PCR tests. At this point, the TNF receptor gene deletion appears to be rare. Molecular laboratory developed tests (LDTs) designed using the CDC published primers and probes that specifically target the TNF receptor gene monkeypox virus will not detect the virus because of the TNF receptor gene deletion in these specimens. This was discovered because the test that was being used included a second set of primers and probes that target sequences that are present in all non-variola orthopoxviruses, not just monkeypox virus. This additional set of primers and probes detected the virus, meaning there was no clinical impact as the patients still received the correct diagnosis. Since the test returned an unexpected result indicating that the non-variola Orthopoxvirus target was positive and the monkeypox specific target was negative, the laboratorians were able to investigate further to find the mutation.
The primers and probes in the FDA-cleared CDC Non-Variola OrthopoxvirusTest are able to detect monkeypox virus, including when this reported mutation is present in the monkeypox virus genetic code.
If you have additional questions, email MPXDx@fda.hhs.gov.