How are viruses studied in vitro

Other important facets not addressed in these studies would be the matrix effects of the test material and the differences in behaviors of viral variants: not only the difference between wild-type viruses and culture-adapted strains, as discussed above, but also between different culture-adapted variants e. In the current era, new assays are required to be validated according to the principles outlined in the ICH guidance document[ 15 ].

However, compendial methods are required only to be verified for their intended use. Thus, these routine tests have not been subjected to the more stringent assay validation principles. Although these studies did not attempt to fully validate all relevant performance parameters, two aspects were studied — the sensitivity in terms of a limit of detection for each method cell line or in vivo test system and each virus and the specificity in terms of breadth of viruses detected by each method.

Also, even though the tests are intended to be broad spectrum, due to pragmatic limitations, only 16 model viruses could be studied. It should be noted that despite the long history of use of these routine tests, no international standards or reference standard materials exist for the purposes of standardizing the assays or for proficiency assessments.

An assumption that has been made to support the continued use of the in vivo methods has been that some viruses do not grow readily in cell culture and would be more sensitively detected by the in vivo test systems. These studies included viruses that were expected only to be detectable in vivo or to be more sensitively detected in vivo. These included the Coxsackie viruses and mumps virus, for which mice and eggs were considered most sensitive, respectively.

Coxsackie viruses could be adventitiously introduced from the operators, environment, or starting isolate for the vaccine viral seed, and mumps virus, for instance, could result from cross-contamination in viral vaccines in facilities that also make mumps vaccines. Rubella virus was detected, but with uniquely poor sensitivity.

Apart from these exceptions, the in vitro indicator cell cultures were reliably and highly sensitive. The cell lines may vary in this routine test, because the requirements are to include human diploid cells which may include primary cell cultures, although often a non-diploid cell line is used , monkey kidney cells could be primary cell cultures, but generally the Vero cell line is used , and a cell line of the same species and tissue type as the production cell line.

Since these studies were undertaken generally and not to support a specific product, a decision was made to include the 3 most commonly used indicator cell lines for vaccines that might be propagated in a human or simian cell line e. Although chick embryo fibroblasts CEF are usually included when propagating a vaccine virus in eggs or CEF, and Chinese Hamster Ovary CHO cells would be used when producing a biological from CHO cells, these cells were not included due to the pragmatic limitations of the study.

For human or non-human primate cell lines used in production of vaccines, the 3 cell lines used most frequently in this test are MRC-5, HeLa, and Vero, and therefore we choose these cell lines to include for study. Often amountsas low as 0. It is important to remember that the titer values were in most cases determined on the production cell line for the particular RVS and the in vitro test is designed to be more sensitive than typical titration assays in that there is ample opportunity for amplification of input virus and the test is of much longer duration.

In fact, for several viruses, substantially less than 1 infectious unit as defined by titration assays was defined as the LOD in 6-well plates Table 4.

Notably, this happened even on detection cell lines that were the same as used to determine the titers. This suggests that the format of the detection assay was more sensitive than the format of the titration assay, in which there were several differences including the length of incubation and observation.

Therefore, standardization of the format of the assay should be considered to control for potential differences in LOD due solely to assay format e. Development of international standards might also help to ensure reliable uniformity of the results from the in vitro methods. Viruses that were expected to be detected in the in vivo test systems surprisingly were not detected in the test systems used or were only detected at such high titers that the in vitro methods were orders of magnitude more sensitive.

Mumps virus was only detected in chicken eggs yolk sac route at an amount of 4. Mumps vaccine is prepared in chicken embryo fibroblast cultures, but this virus did not propagate readily in chicken eggs inoculated via the routes used in these studies. Historically, Coxsackie A viruses were expected only to be reliably detected in suckling mice, and both Coxsackie A and B viruses should be detectable in this test system.

Coxsackie B virus was reliably detected in all 3 indicator cell lines with Vero and HeLa cells being the most sensitive, each detecting 0. Thus, the indicator cells were 4 logs more sensitive for detecting the strain of Coxsackie A virus used and 2 logs more sensitive for detecting the strain of Coxsackie B virus used.

Interestingly, neither measles virus nor rubella virus were detected in eggs at any titer, even though measles vaccine is normally propagated in chicken embryo fibroblast cultures and the vaccine strain was used in these studies. However, we cannot exclude that attenuating mutations arose during passage to produce the RVS. Historically, rubella virus was propagated in duck embryos, rather than in chicken embryos, but only chicken eggs are used in the routine tests and thus, in this study.

Measles virus was detected in all threeindicator cell lines, with detection in Vero cells at a titer as low as 0. Rubella was not readily detected in the 3 indicator cell lines, including the MRC-5 cells in which only the undiluted stock was detected reliably, despite the vaccine strain being grown on WI cells, a similar human embryonic lung cell line.

Rubella virus was readily detected on the positive control RK cell line, though. However, this cell line is not routinely included in the standard test, but it can be used and sometimes is. Reflecting on the original hypothesis of the study, there were in fact only two viruses that were detected with more sensitivity in vivo than in vitro.

Both VSV and influenza were detected more sensitively in eggs, and VSV was detected more sensitively in post-weaning mice, than in cell culture. Whether inclusion of alternative cell cultures that might detect influenza virus or VSV more sensitively than the standard indicator cells was not studied.

However, it is feasible that inclusion of Madin-Darby canine kidney MDCK cells might have improved the ability of the in vitro test to detect influenza viruses. Consideration needs to be given to whether particular viruses like these two might be potential contaminants of concern for a given cell substrate or vaccine to determine whether it is warranted to continue including the test in eggs in order to not miss one of them.

It should be noted that some strains of influenza virus, particularly H3N2, do not propagate well in eggs until they have been adapted for egg growth, and so this enhanced sensitivity may be reflective of the particular strain chosen an H1N1 strain. Based on the results from these studies, several conclusions may be drawn. Firstly, the results support consideration of using a broader panel of cell lines for adventitious agent detection, especially if justifying replacement of the in vivo tests.

Currently, for vaccines made in human or simian cell lines e. One might question the value of including two human cell lines. For example, MRC-5 cells were more sensitive for detection of echovirus, HSV, and mumps virus,whereas, HeLa cells were more sensitive for detection of adenoviruses, Coxsackie B virus, and rhinovirus. Secondly, the longer duration 28 days of testing in vitro is supported by the data. In many cases, 14 days of incubation were adequate to detect a positive result at the LOD for the in vitro tests.

In some cases, only a higher titer was detected by 14 days, and the LOD was improved with the day culture two day cultures with a blind passage between them. These results suggest that while there should be continued use of the 28day culture period for the in vitro tests, decisions might be made on the basis of negative results at 14 days in situations where some level of risk is acceptable, e. However, lot release for clinical use should continue to rely on the day data.

Thirdly, the longer duration ofthe suckling mouse assay, which includes a blind sub-passage was not supported by the data. There were no examples where a test or a dilution that gave a positive result did not do so in the original inoculations and required the blind sub-passage into new suckling mice.

While this method should permit amplification of an undetectable contaminant that has amplified somewhat in the first passage to achieve detectable levels during the second, this benefit was not seen in these studies. Consideration should be given to refining this test method by reducing the number of animals used by eliminating the blind sub-passage. Fourthly, the in vivo tests may not be as sensitive or as broadly susceptible or otherwise superior to the in vitro test, as may have been previously thought.

Our results suggest that the in vivo tests, as routinely performed, are not capable of reliable and sensitive detection of most of the viruses tried, even those expected to be detected by these systems e.

Based on these public reports and the data reported herein, the value added by the in vivo methods can be questioned. While it is true that many viruses do not propagate in cell culture, and thus, the cell culture tests would be incapable of detecting them, we suggest that the adventitious agents of greatest concern are those that will propagate in the production cell substrate, because only those will be of high enough concentrations at harvest or cell banking to: 1 be detectable in the small volumes used in the in vivo tests, 2 challenge and perhaps overwhelm the ability of any purification or viral clearance steps in the production process to remain present in the final product, and 3 be a sufficient inoculum in the final product to infect human recipients.

An argument frequently given for continued reliance on the in vivo tests is that they can detect unknowns that might not propagate and be detectable in cell culture. This argument may be valid when primary cell cultures or eggs are used for vaccine production; however, if the potentially adventitious virus cannot propagate in cell culture, then the processes of cell expansion and perfusion or batch feeding of production cultures will dilute the potential contaminant.

This dilution effect will further reduce a low-level non-amplifying contaminant to such low levels that the probability of a sufficient inoculum being present in the small sample taken from a large bioreactor, wave bag, or other culture vessel and used in the in vivo tests will be miniscule.

Unlike cell cultures, where the inoculum can amplify from cell to cell and thus, increase the odds of detection, in vivo , the inoculum will not readily spread from animal to animal to amplify detection because of the manner in which animals are housed in groups of five in the case of post-weaning mice although sometimes they are housed in pairs; separately in the case of eggs, and by litter in the case of suckling mice.

Only in the case of suckling mice is there a real opportunity for animal-to-animal spread, but in that situation, the pups that die are often cannibalized by the dam and if there is excess death in one but not both litters, often it is concluded that the dam is a poor mother and little proof remains that an spreading adventitious agent would be the cause.

Such a result often leads to a retest, where again, the probability of having sufficient concentration in the inoculum into one animal drives the ability to detect the putative contaminant.

In suckling mice, these volumes are 0. If large bioreactors L or 10,L are used, the detection of a low-level contaminant would become highly unlikely considering the sample volumes used the in vivo tests. Thus, only those contaminants that can amplify in the production system are likely to be detectable by the adventitious agent tests, particularly the in vivo tests, and these should be detectable by the cell culture test that permits further amplification, if the proper endpoints are used for detection.

Based on our findings, it seems appropriate to suggest considering expanding the panel of cell linesin the in vitro test to potentiallyincrease assay sensitivity for some virusesif the in vivo methods are reduced or eliminated.

Consideration could be given to incubation of indicator cells at lower temperatures e. Furthermore, consideration could be given to optimizing the test for detection of cell-associated viruses. Cell debris may interfere with the test, but may contain the cell-associated viruses that one desires to detect.

It might be appropriate to add additional endpoints as well e. Decisions about which cell lines or which endpoints could be added would need to take into account the likely contaminants for the material under test. Thus, a thorough risk assessment should be undertaken to identify potential contaminants in order to rationally guide a proposed testing strategy. For example, if bovine serum or bovine or porcine trypsin were used in the legacy of the cell substrate or viral seed, inclusion of cell lines and endpoints capable of detecting bovine and porcine viruses could be considered in the panel of cell lines used in the in vitro testing.

In some cases, e. Although not tested in these studies, when avian cell substrates CEF or chicken eggs are used for production or development of the viral seed , CEF could be incorporated in the panel of cell lines.

Consideration must be given to the value added by the in vivo tests for a given situation. It may be the case that they are not of sufficient value to justify the use of animals for product safety testing when production is conducted in culture of established cell lines although use of primary cell cultures might warrant continued use of the test methods.

It would be important to acknowledge that changes in regulated tests would require the approval of the relevant regulatory agencies prior to implementation. To help developers of new assay methods compare the capabilities and performance parameters such as sensitivity and selectivity of their methods withthe routine methods employed in these studies, the viral stocks prepared for these studies will be made publicly available through the DAIDS Reagent Resource Support Program for AIDS Vaccine Development repository[ 16 ].

In addition, protocols may be provided on the methods used to propagate and characterize the viral stocks and to conduct the studies. It was a goal of the project to facilitate method developers and manufacturers wanting to implement the new methods in place of or to supplement the routine tests to have baseline data upon which to judge the relative capabilities and make decisions.

It should be noted that these materials are not international standards or reference materials, but are research reagents to be made available to researchers. Future directions that might be considered to expand the findings reported here would be: to increase the viral families represented, to include variants and other strains of the viruses tested, to include wild-type isolates, to test by spiking the viral stocks into various test sample matrices that might represent actual test samples e.

Also, completing the matrix orevery cell of the checkerboard for the in vivo tests might be considered. As discussed above, all of these potential factors were outside the scope of the project supporting the studies undertaken. Another useful future consideration would be to help new method developers outline how they might go about comparing their methods, which are mostly molecular genomics methods, to the routine tests that have infectivity and pathogenicity endpoints.

Molecular genomic or transcriptomic methods, like deep sequencing, mass spectrometry following broad-spectrum PCR, and microarrays, are capable of detecting both intact and fragmented viral nucleic acids NA. These NA may or may not represent a riskand may only be impurities of a fragmented residuum of an inactivated contaminant that may no longer be hazardous.

Depending on how they are applied, these new methods may not readily distinguish between inactive sequence copies and those resulting from amplification of infectious viruses. This same concern has been raised in considering using PCR to detect mycoplasma contamination and much consideration has been given to how best to do this. Whether similar approaches could be taken for viruses, which have a much greater breadth of characteristics than do mycoplasmas [e.

Consideration should be given to using standard viral stocks that have been characterized for both infectivity titer and genomic copy numbers as a basis for comparison or using the same stocks for each assay method and simply comparing the highest dilution at which each demonstrates detection.

In summary, the results of the studies reported here provide the first systematic data on comparing the breadth of detection and sensitivities of the in vivo adventitious virus tests and the in vitro tests. The data point to limitations with the in vivo tests and suggest ways that the in vitro tests might be refined or improved, such as the potential inclusion of additional cell lines.

Combining expanded in vitro testing with the new generation of molecular methods for hybrid assays might provide, in time, a pathway for implementing the 3 R's, i. Although the methods used for decades have been largely successful at keeping contaminated vaccines off the market, in the few cases that they have failed, the contaminants have not posed a risk to human health [ 17 ]. Nonetheless, in the spirit of Quality by Design, it is incumbent upon us to seek to continuouslyimprove our methods to assure safe and pure vaccines are available to protect and promote the public health.

The authors would like to acknowledge Kathryn Butler for initiation of this project, Sveta Sherbaty, Amy Bennett, and Marci Henkin for excellent technical assistance in the preparation and characterization of the research virus seeds, Cheryl Harper and Christine Nogier for critical data review, Michael Nicholson for thorough Quality review, and Michelle Walker for project coordination.

We also thank Dr. Barry Rosenblatt for many helpful discussions of the study design and for presentation of preliminary data at public meetings, and Christine Simko for excellent assistance during preparation of this manuscript. Further, we would like to acknowledge Jack Hill for his budget management and assistance in this project.

Keith Peden served on the expert panel that guided the project initially and has reviewed the manuscript and we thank him for his advice and guidance. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.

As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. National Center for Biotechnology Information , U. Author manuscript; available in PMC Aug 5.

Sheets h, i. Paul Duncan g Merck and Co. Rebecca L. Author information Copyright and License information Disclaimer. Box 4, West Point, PA Sheets, Ph. Copyright notice. The publisher's final edited version of this article is available at Vaccine.

See other articles in PMC that cite the published article. Associated Data Supplementary Materials 1. Abstract Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, which might contaminant them. Open in a separate window. RESULTS Detection of Adventitious Viral Agents by In Vitro Assays The purpose of this study was to systematically assess the sensitivity of commonly used cell lines to detect infection by a number of different viruses, representing those thatmight contaminate vaccines in production adventitiously from the production environment e.

Inoculation with Diluted Virus Stocks Based on the sensitivity protocol titration protocol , stocks that scored positive at the undiluted concentration, were serially diluted and each dilution tested to establish an LOD or amount at which a particular viral stock would score positive with that amount or more and negative with less than that amount.

Pathology noted in infected animals There was no pathology information collected on animals assigned to the breadth study. Comparison of sensitivity and breadth of in vitro and in vivo methods For almost every virus studied the in vitro assay was more sensitive than the in vivo assay Figure 1.

Supplementary Material 1 Click here to view. Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. Federal Register Doc. Sheets R, Petricciani J. Vaccine Cell Substrates , Expert Rev. European Pharmacopoeia Vers. International Conference on Harmonisation — Quality5D.

US Food and Drug Administration; Poliovirus Vaccine Live Oral Trivalent. Code of Federal Regulations. Additional Tests for Safety. Part Subpart B.

Additional Standards for Viral Vaccines. Measles Virus Vaccine Live. Test for Safety. Mumps Virus Vaccine Live. Rubella Virus Vaccine Live. The Vacuolating Virus, S. Proc Soc Exp Biol and Med. Update on Recommendations for the Use of Rotavirus Vaccines. Emerging Infectious Diseases. Nims RW. Detection of adventitious viruses in biological sea rare occurrence.

Dev Biol Basel ; — National Institute of Allergy and Infectious Diseases. Sheets RL. Opinion on adventitious agents testing for vaccines: Why do we worry so much about adventitious agents in vaccines? Support Center Support Center. External link. Please review our privacy policy. For questions regarding this document contact Tamara Feldblyum at or by email at tamara.

You may submit written comments and suggestions at any time for Agency consideration to the Division of Dockets Management, Food and Drug Administration, Fishers Lane, rm. Identify all comments with the docket number FDAD Comments may not be acted upon by the Agency until the document is next revised or updated.

Additional copies are available from the Internet. You may also send an e-mail request to dsmica fda. Please use the document number to identify the guidance you are requesting. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance.

If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance. FDA is issuing this guidance to provide industry and agency staff with recommendations for studies to establish the analytical and clinical performance of in vitro diagnostic devices IVDs intended for the detection, or detection and differentiation, of influenza viruses. These devices are used to aid in the diagnosis of influenza infection.

They include devices that detect one specific type or subtype, as well as devices that detect more than one type or subtype of influenza virus and further differentiate among them. This guidance provides detailed information on the types of data FDA recommends submitting in support of Class I and Class II premarket submissions for these devices. The guidance includes a list of influenza virus strains recommended for analytical sensitivity studies, a list of microorganisms recommended for analytical specificity studies, and an example of a suggested format for presenting data from cross-reactivity studies.

The scope of this document is limited to types of data intended to establish the performance characteristics of devices that detect either influenza viral antigen s or influenza viral gene segment s. It includes devices detecting influenza virus protein or nucleic acid targets, either single unit test formats or multi-test formats.

It does not address performance for assays detecting serological response of the host to the viral antigen, nor does it address establishing performance of non-influenza components of multi-analyte or multiplex devices. The use of the word should in Agency guidance documents means that something is suggested or recommended, but not required.

This document recommends studies for establishing the performance characteristics of in vitro diagnostic devices for the detection, or detection and differentiation, of influenza viruses directly from human specimens or from culture isolates. FDA believes that these recommended studies will be relevant for premarket submissions e. A manufacturer who intends to market an in vitro diagnostic device for detection, or detection and differentiation, of influenza viruses must conform to the general controls of the Federal Food, Drug, and Cosmetic Act the Act and, unless exempt, obtain premarket clearance or approval prior to marketing the device sections k , , of the Act; 21 U.

This document is intended to supplement 21 CFR It also discusses the FDA's thinking on premarket pathways for new or modified products intended to detect influenza A viruses, including a novel influenza A virus, or to detect and differentiate a specific influenza A virus. This special control guidance document includes recommendations for establishing device performance, as well as recommendations for labeling and postmarket measures.

Devices classified under 21 CFR This guidance is intended to complement the two preceding guidance documents by describing the types of studies FDA recommends for establishing the analytical and clinical performance of in vitro diagnostic devices IVDs intended for the detection, or detection and differentiation, of influenza viruses. As previously described, this document recommends studies for establishing the performance characteristics of in vitro diagnostic devices for the detection or detection and differentiation of influenza viruses, including those for the detection of novel influenza viruses in either human specimens or culture isolates.

This document is limited to studies intended to establish the performance characteristics of devices that either detect influenza viral antigens or influenza viral gene segments protein or nucleic acid. This guidance references serological reagents but does not address detection of serological response from the host to the viral antigen, nor does it address establishing performance of non-influenza components of multi-analyte or multiplex devices.

The scope of this document includes the devices described in existing classifications, as indicated below, and may also be applicable to future influenza diagnostic devices that may not fall within these existing classifications. Those future devices may include devices that will be subject to requests for initial classification under section f 2 of the act " de novo classification" , as well as subsequent devices that seek determinations of substantial equivalence to future de novo classified devices.

Influenza virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to influenza in serum. The identification aids in the diagnosis of influenza flu and provides epidemiological information on influenza. Influenza is an acute respiratory tract disease, which is often epidemic.

Class I general controls. Although devices within the classification described in 21 CFR Specifically, an IVD for detection of influenza is not exempt from submission of a k to the extent that it meets the limitations on exemption defined in 21 CFR Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a nucleic acid amplification test to directly detect specific virus RNA in human respiratory specimens or viral cultures.

Detection of specific virus RNA aids in the diagnosis of influenza caused by specific novel influenza A viruses in patients with clinical risk of infection with these viruses, and also aids in the presumptive laboratory identification of specific novel influenza A viruses to provide epidemiological information on influenza. These reagents include primers, probes, and specific influenza A virus controls. Human influenza is a highly contagious acute respiratory tract disease.

There are three genera of human influenza viruses: A, B and C. Infection with influenza A virus is the most severe, with several notable pandemics during the past century. Influenza A viruses are classified into subtypes according to the antigenic composition of their hemagglutinin HA and neuraminidase NA glycoproteins on the viral envelope. Illness caused by commonly circulating influenza viruses can cause high morbidity and mortality, particularly in special populations such as the elderly and the very young.

The development of acquired immunity to seasonal influenza viruses is limited because influenza viruses mutate in small but important ways from year to year a process known as antigenic drift. More dramatic changes or major antigenic shifts may result in the emergence of a new subtype of influenza A virus, or novel virus that has never circulated or has not circulated in humans for several decades.

This lack of immunity, as well as additional pathogenic factors that may also increase virulence, results in a greater likelihood of morbidity and mortality among those infected. In vitro diagnostic devices for the detection, or detection and differentiation, of influenza viruses are important for establishing the diagnosis of influenza, for differentiating seasonal from novel influenza virus strains, and for obtaining epidemiologic information on influenza outbreaks. Public health officials have emphasized the need for reliable influenza diagnostic devices that can differentiate seasonal from emerging viral strains and provide rapid test results.

Failure of devices for detection of influenza viruses to perform as expected, or failure to correctly interpret results, may lead to incorrect patient management decisions and inappropriate public health responses. In the context of individual patient management, a false negative report could lead to delays in providing or failure to provide definitive diagnosis and appropriate treatment and infection control and prevention measures.

A false positive report could lead to unnecessary or inappropriate treatment or unnecessary control and prevention actions. Therefore, establishing the performance of these devices and understanding the risks that might be associated with the use of these devices is critical to their safe and effective use.

You must identify a legally marketed predicate device in your k. You should also identify the regulation and the product code for your device.

We recommend including a table that outlines the similarities and differences between the predicate and the new device. You should include the following descriptive information to adequately characterize the new device that is intended to detect or detect and differentiate influenza viruses.

The intended use statement should specify the influenza virus types and subtypes the device detects and identifies, the nature of the analyte e. The intended use statement should state whether the test is qualitative, whether analyte detection is presumptive, and any specific conditions of use.

You should describe in detail the methodology used by your device. For example, the following elements, as applicable to the device, should be included:. Your k should include performance information supporting the conclusion that design control requirements for your device have been met as described in 21 CFR For instruments and systems that measure multiple signals, and for other complex laboratory instrumentation that has not been previously cleared, refer to the guidance document "Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems,"[1] for details on the types of instrument-related data you should provide to support clearance.

You should determine the level of concern prior to the mitigation of hazards. In vitro diagnostic devices of this type are typically considered a moderate level of concern; software flaws could indirectly affect the patient and potentially result in injury because inaccurate information may be given to the healthcare provider and the patient.

You should clearly describe how raw signals are converted into a result including adjustment to the background signal for normalization, if applicable. We also recommend that you include the following information for software development and implementation in the submission:.

Before beginning clinical studies, the configuration of the hardware and software components should be very similar or identical to the final version of the device. A risk assessment should be performed if any significant changes are made to the hardware or software after the completion of the clinical studies and before the clearance and distribution of the device.

Below are additional references to help you develop and maintain your device under good software life cycle practices consistent with FDA regulations. For example, if your device labeling specifies the use of Brand X DNA amplification enzyme, and use of any other DNA amplification enzyme may alter the performance characteristics of the device from that reported in the labeling, then Brand X DNA amplification enzyme is an ancillary reagent of concern. If the instructions for use of your device specify ancillary reagents of concern, you should address how you will ensure that the results of testing with your device and these ancillary reagents, in accordance with the instructions, will be consistent with the performance established in the premarket submission.

The plan may include an application of quality systems approaches, product labeling, and other measures. You should include the elements described below in your submission. In addition, you should provide testing data to establish that the quality controls supplied or recommended are adequate for detecting performance or stability problems with the ancillary reagents.

You must include a statement of limitations of the procedure in the labeling accompanying the product 21 CFR This should include potential issues that may affect the performance of your device and were not addressed in your analytical or clinical studies. You should include any potential risks associated with using the device in the Warning and Limitation sections of the device labeling. We recommend including statements such as those listed below as they pertain to your device:. When conducting the performance studies described below, we recommend that you run appropriate external controls every day of testing for the duration of the analytical and clinical studies.

Examples of appropriate external controls include vaccine or prototypic vaccine strains, low pathogenic viruses, and inactivated viruses. Specific information about controls for nucleic acid based devices is provided in Section 9. We recommend that you describe in your submission how positive, negative, equivocal if applicable , or invalid results are determined and how they should be interpreted.

If the assay has an equivocal zone, the cut-off values limits for the equivocal zone should also be defined. If your interpretation of the initial equivocal results requires re-testing, you should provide 1 a recommendation whether re-testing should be performed from the same nucleic acid preparation, a new extraction, or a new patient specimen and 2 an algorithm for defining a final result by combining the initial equivocal result and the results after re-testing.

Note that this algorithm should be developed before the pivotal clinical study that confirms the significance of the assay cut-offs, and the algorithm should be followed precisely e. If controls are part of the determination of invalid results, you should describe each possible combination of control results for defining the invalid result. You should provide a recommendation on how to follow up any invalid result, i.

If re-testing is recommended, provide the information similar to the one for re-testing of equivocal results whether re-testing should be performed from the same nucleic acid preparation, a new extraction, or a new patient specimen. You should include in the submission and the labeling the number of initial invalid results and the number of re-tests that were needed to determine a final result during your studies.

We recommend that you establish the following analytical performance characteristics for your assay:. You should determine the LoD for each specimen type and each analyte that will be tested with your device utilizing the entire test system from sample preparation to detection when evaluating assay LoD. This can be accomplished by limiting dilutions of propagated and titered viral stocks.

The study should include serial dilutions of at least two strains representative of types or subtypes for each claimed influenza virus please see Table 1 for suggested viral strains and replicates for each dilution. Since the nucleic acid based devices detect not only the infective viral particles but also the total viral RNA present in the specimen, an additional reference method, quantifying nucleic acids, e.

We recommend that you determine the LoD for each analyte in the most commonly used or most challenging matrix tested by the device. When selecting an appropriate matrix for your analytical studies you should choose one of the two alternatives outlined below:. We recommend that you demonstrate that the test can detect at least 5 strains for influenza B and 10 strains for each influenza A subtype detected by your device.

Influenza A detection should be tested across all subtypes that have infected humans and at viral levels at or near the LoD. Influenza B strains representing both lineages Victoria and Yamagata should be included.

Influenza strains selected should reflect temporal and geographical diversity with an emphasis on contemporary strains. For each claimed influenza subtype an additional selection of strains representing known lineages and clades should be included. For subtypes for which it is difficult to obtain sufficient number of strains to demonstrate reactivity, we recommend that you contact the Division of Microbiology Devices to discuss your study. All virus identities and titers should be confirmed.

Examples of recommended strains for the LoD and the analytical reactivity studies are shown in Table 1. Vaccine strains wild type from recent flu seasons can be included. Vaccine strains may vary from one influenza season to another.

Table 1. Examples of influenza strains for analytical sensitivity LoD studies. We recommend that you demonstrate analytical specificity of your assay with influenza types and subtypes not detected by your device.

An exclusivity panel could be comprised of well characterized seasonal or novel influenza strains not detected by your device as well as non-human influenza viruses that have been shown to infect humans.

Pertaining to nucleic acid-based devices, for large panels or for influenza strains that are difficult to culture, purified nucleic acids may be quantified, e. Nucleic acids can also be quantified in cases when highly purified influenza viruses are used in the study, e. We recommend that you test for potential cross-reactivity with non-influenza respiratory pathogens and other microorganisms with which the majority of the population may have been infected. The microorganisms recommended for cross-reactivity studies are listed in Table 2.

Pertaining to nucleic acid-based devices, for large panels or for organisms that are difficult to culture, purified nucleic acids may be quantified, e. Nucleic acids can also be quantified in cases when highly purified organisms are used in the study, e. Table 2. Microorganisms recommended for analytical specificity cross- reactivity studies. For devices detecting multiple analytes, e. We encourage sponsors to present cross-reactivity testing data for devices detecting multiple pathogens in the format shown in Table 3.