The United States announces the opening of monkeypox reagent EUA!

On September 7, local time in the United States (5 a.

On September 7, local time in the United States (5:00 a.

First of all, let's talk about one of the most important things: the sampling method of monkeypox nucleic acid reagents in the United States is mainly based on skin lesion samples.

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At present, the template is only for molecular (molecular diagnosis), and has not yet been introduced for antigen (antigen diagnosis), but the manufacturers of monkeypox antigen reagents, if you are interested in the UUA in the United States, welcome to contact Puri Pure Evidence, analysis, clinical protocol design, and submit pre-EUA to discuss the relevant clinical and analytical protocols

Dear manufacturers of monkeypox antigen reagents, if you are interested in the U.

Regarding chemical cracking and solid phase extraction, this is also required in the new crown EUA, and it is estimated that everyone is also familiar with it

At present, the only reagent authorized by the FDA EUA is the QUEST monkeypox detection reagent

In fact, in addition to the EUA-authorized RT-PCR reagent, there is also a non-smallpox positive pox virus detection kit developed by the CDC and approved by the FDA (note that it is cleared rather than EUA-authorized), which can be used for the detection of positive pox virus and can also be used for monkey pox virus detection (with the corresponding primer probe combination):

The kit is intended for use in CDC-authorized laboratories, and Preh Pure has now received the CDC Monkeypox Detection Primer Probe Kit

The kit is intended for use in CDC-authorized laboratories, and Preh Pure has now received the CDC Monkeypox Detection Primer Probe Kit

About profiling performance validation

About profiling performance validation

The analysis performance verification is interpreted as follows:

Viral material should be prepared

Live viruses and inactivated viruses are the materials of choice for analytical performance evaluation, but natural genomic DNA or synthetic DNA can also be used, and if the latter is chosen, the FDA recommends pre-EUA

If specimen collection involves the use of swabs, viral material should be incorporated into the swab and then tested

1.

1.

If the product description claims multiple clinical types, then each corresponding clinically negative matrix needs to be evaluated for LoD

If the product is a non-smallpox positive pox detection kit, at least two non-smallpox positive pox viruses need to be used for LoD verification, or synthetic DNA can be used instead

2.

2.
Inclusive research

Mainly complete inclusive research in
bioinformatics analysis.

Inclusive analysis of monkeypox viruses mainly included: West African evolutionary branches (clade II), Congo Basin evolutionary branches (clade I), and Zaire strains
provided by BEI.

Inclusive analysis of non-smallpox virus includes: monkeypox virus, cowpox virus, ratpox virus, camel pox virus, pox vaccine, etc
.

Inclusive analysis of nucleic acid sequences
applicable to publicly available databases (e.
g.
, NCBI, GISAID, etc.
).

If the pairing ratio is less than 100%, a risk assessment
of the impact of the mismatch on performance needs to be provided.

There is also a need for continuous monitoring of new viral variants
that may affect molecular testing performance.

3.
Cross-reaction studies

3.
Cross-reaction studies

Cross-reaction testing
of the 33 pathogens enumerated is required.

For pathogens that have not been tested, a reason should be provided, and the ratio of these pathogens to the product primer probe needs to be bioinformatics analyzed and the pathogens with > 80% homology between the two need to be identified
.

4.
Microbial interference research

4.
Microbial interference research

From cross-reaction studies, it was analyzed that the homologous > 80% of pathogens paired with product primer probes were detected
for microbial interference.

If testing is not performed, it is necessary to explain why the test performance of the product is not affected
by clinically co-infected pathogens.

Or interpret computer simulation results that are not
clinical.

5.
Research on internal and external interfering substances

5.
Research on internal and external interfering substances

Provides a list
of 20 interfering substances from internal and external sources.

It is recommended to perform three repeated tests
on each substance with 2-3 times LoD.

6.
Sample stability

6.
Sample stability

Sample stability assessment
at room temperature for 2 h on freshly prepared viral material with 3x LoD.

If frozen clinical samples need to be tested, the difference
in performance assessment between fresh and frozen samples needs to be assessed in an equivalent study.

Reagent stability, only protocols
are available.

As can be seen from the above, unlike the domestic registration guidelines, the US EUA does not make relevant requirements for precision, and when submitting eucalyptus, on the stability, only need to submit the program, and the test data can be supplemented after obtaining EUA authorization, which makes the monkeypox reagent EUA analysis performance test complete faster
than the domestic registration.

As can be seen from the above, unlike the domestic registration guidelines, the US EUA does not make relevant requirements for precision, and when submitting eucalyptus, on the stability, only need to submit the program, and the test data can be supplemented after obtaining EUA authorization, which makes the monkeypox reagent EUA analysis performance test complete faster
than the domestic registration.

Back to the clinical trials themselves
that everyone is most concerned about.
Monkeypox nucleic acid reagent clinical trials can be divided into two protocols for manufacturers to choose from:

Monkeypox nucleic acid reagent clinical trials can be divided into two protocols for manufacturers to choose from:

7.
Forward-looking, double-blind, randomized clinical trials, which are the best options

7.
Forward-looking, double-blind, randomized clinical trials, which are the best options

(1) Clinical trial samples can choose fresh samples or cryopreserved samples, if it is a frozen clinical sample, it must be tested equivalently with fresh clinical samples to prove that cryopreserved clinical samples will not affect the accuracy of the test;

(2) Blinding is required during the trial, the end user should not know the status of the clinical sample to be tested, and the end user should not know the test results of the comparison reagent of the sample to be tested (Prijun understands the end user here as the terminal tester, not the patient);

(3) If the results of the reagent to be registered and the comparison reagent are inconsistent, the second EUA authorized reagent should be used for re-testing;

(4) Clinical trials require ethical and informed consent to the subjects;

(5) In the clinical sample, 20% of the positive clinical samples should be weakly positive clinical samples, and the LOD value of the compared reagent should be the viral concentration of the weakly positive clinical samples, and the DEVIATION of the CT value of the 20% positive clinical samples should not exceed ±3
of the average CT value.

8.
Artificial samples are not the optimal option, but they are currently the fastest option to log in to EUA

8.
Artificial samples are not the optimal option, but they are currently the fastest option to log in to EUA

(1) When either prospective or retrospective clinical samples are not available, the FDA also allows the use of artificial samples for clinical trials
.

(2) The concentration of the artificial positive sample, half of which is the LOD value, and the other half is not higher than 5 times the LOD value;

(3) The clinically negative matrix cannot use the sample pool method, and must correspond to a unique clinically negative matrix for each positive sample;

(4) When the initial authorization of eucalyptus is obtained for clinical trials using artificial samples, it is also necessary to submit a positive clinical sample test as an additional supplement
.

No matter which of the above is chosen, 30 cases of positivity and 30 cases of negative are required, and whether it is positive or negative consistency rate, it needs to be greater than or equal to 95%.

No matter which of the above is chosen, 30 cases of positivity and 30 cases of negative are required, and whether it is positive or negative consistency rate, it needs to be greater than or equal to 95%.

Usually nucleic acid reagents will have matching PCR equipment and extraction kits, extraction equipment, clinical and analytical requirements for these specifications of the matching test system products, as follows:

Every PCR instrument needs to be tested for LoD:

○ First the product should determine the initial LoD and final LoD of the different extraction methods in a PCR instrument
.

○ Select the extraction method with the lowest sensitivity above for LoD detection in other PCR instruments
.

○ If the sensitivity of different extraction methods in the first PCR instrument is similar, then the extraction method will be randomly selected
.

Interfering substances should be evaluated
using a combination of extraction methods and PCR instruments with the lowest analytical sensitivity.

Inclusive research should also be evaluated
using a combination of extraction methods and PCR instruments with the lowest analytical sensitivity.

Cross-reactions should be evaluated using a combination of all extraction methods and PCR instruments
.

Clinical studies should be evaluated
using an extraction method with the lowest analytical sensitivity.

In general, if from the most comprehensive point of view, of course, to carry out forward-looking, blinded, random clinical trials, is the most perfect, but the world's things are often fish and bear paws can not be combined, the optimal solution is often accompanied by a longer time- and more uncertain risk (subject recruitment, ethics and other issues), and EUA itself is a temporary policy, according to the experience of the new crown, the earlier the submission of approval, the more relaxed the approval, the more later, the more difficult
.

If from the most comprehensive point of view, of course, to carry out forward-looking, blinded, randomized clinical trials, is the most perfect, EUA itself is a temporary policy, according to the experience of the new crown, the earlier the submission of approval, the more relaxed the approval, the more later, the more difficult

Therefore, the suggestion of Prijun is to conduct clinical trials of artificial samples first, and simultaneously prepare clinical trials of real clinical samples, such as preparation of ethics, informed consent, etc.
, and at the same time carry out relevant analytical performance testing requirements
.
At present, Puri Pure Is a whole genome DNA extracted from real monkeypox virus, NIST (National Institute of Standards and Technology) samples and real monkeypox clinical samples, which can help domestic manufacturers land on EUA
as soon as possible.

Conduct artificial sample clinical trials first, and simultaneously prepare clinical trials of real clinical samples, such as preparation ethics, informed consent, etc.
, and carry out relevant analytical performance testing requirements
.
At present, Puri Pure Is a whole genome DNA extracted from real monkeypox virus, NIST samples and real monkeypox clinical samples, which can help domestic manufacturers land on EUA
as soon as possible.

The above is the interpretation of the current monkeypox EUA template clinical and analytical aspects, at present, Prijun has submitted a pre-EUA application to the FDA on the monkeypox nucleic acid reagent, and the final clinical plan and analysis plan will be subject
to FDA pre-EUA feedback.

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