COVID-19 PCR: frequency of internal control inhibition in clinical practice
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Introduction. Diagnosis of COVID-19 (coronavirus disease 2019) is best performed with real-time (quantitative) PCR (qPCR), the most sensitive method for detection and quantification of viral RNA. Using the Centers for Disease Control and Prevention (CDC) protocol, for each sample tested for the virus, three qPCR tests are performed, targeting the viral genes N1 and N2, in addition to the internal control gene RNase P. Samples in which internal control fails to amplify should be labelled ‘invalid’.
Methods. This study aims to determine the frequency of inhibition of the RNase P gene used as an internal control in qPCR tests for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in a reference hospital in Southern Brazil during the COVID-19 pandemic (1 February 2021 to 31 March 2021).
Results. A total 10, 311 samples were available for analysis. The mean cycle threshold (Ct) value for the RNAse P gene was 26.65 and the standard deviation was 3.18. A total of 252 samples were inhibited (2.4%) during the study period: amongst these, 77 (30.5%) showed late amplifications (beyond 2 standard deviations from the mean Ct value), and 175 (69.4%) revealed no fluorescence at all for the RNase P gene.
Conclusions. This study showed a low percentage of inhibition using RNase P as an internal control in COVID-19 PCRs using the CDC protocol, thus proving the effectiveness of this protocol for identification of SARS-CoV-2 in clinical samples. Re-extraction was efficacious for samples that showed little or no fluorescence for the RNase P gene.
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This is a study that would be of interest to the field and community.
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
Abstract P1, Methods: The methods section should not start with 'thus', in fact the statement describes the aim not the methods used. Introduction P2L1: I would recommend rethinking the first sentence, the definition of a pandemic is that it is disseminated around the world. P2L5: Understandably, you are trying to reference qPCR being the most sensitive method, but since you are talking about Covid-19, could you provide a more recent reference (published after the pandemic). P2L8: Ref3 is not a CDC study, please refer to the original resource. Methods P2L18: Please provide the type of samples (e.g. nasopharyngeal swabs, sputum…etc), "mostly respiratory samples" is not clear. Also, sensitivities vary depending on whether the sample is acquired from the upper or lower RT. What is the status of these …
Comments to Author
Abstract P1, Methods: The methods section should not start with 'thus', in fact the statement describes the aim not the methods used. Introduction P2L1: I would recommend rethinking the first sentence, the definition of a pandemic is that it is disseminated around the world. P2L5: Understandably, you are trying to reference qPCR being the most sensitive method, but since you are talking about Covid-19, could you provide a more recent reference (published after the pandemic). P2L8: Ref3 is not a CDC study, please refer to the original resource. Methods P2L18: Please provide the type of samples (e.g. nasopharyngeal swabs, sputum…etc), "mostly respiratory samples" is not clear. Also, sensitivities vary depending on whether the sample is acquired from the upper or lower RT. What is the status of these patients? Are these samples from screening efforts, or diagnosis after symptoms? What were the symptoms of the participants? Were there an informed or implied consent provided for the participants? P2L21: Please use direct reference Was The qPCR done in duplicates or triplicates? P3L26: What was the reason behind using three extraction kits? With different samples? With varying number of samples? If it was for comparison reasons, shouldn't you extract the same samples with the three kits? While using three extraction kits, each kit would provide different rate of sensitivity and specificity; how would you overcome the potential discrepancy of results? P3L33: Could you provide the name of software used for the statistical analysis? Results P3L38: How much of the 10,311 samples were positive or negative? P3L40: The standard deviation (3.18) reported is high, maybe you could adjust the parameters (e.g. excluding outliers). P4L48: Please mention the corrective action used P4L49-52: On what basis the decision was made to re-extract or re-amplify the inhibited samples? 81 inhibited samples were not re-extracted, why? What is the definition of inhibited and inconclusive result? "20 were released without any corrective action" does that mean they are positive or negative? Why are they still considered inhibited if no corrective action was conducted? The provided numbers don't add up to 252 Doesn't it make more sense that a mistake happened during the first extraction or the amplification that resulted in the 'inhibited' status? How can you prove that the samples were actually inhibited? Duplicates? Discussion P4L56-58: Maybe at the time of writing the manuscript this statement could be true, however, there are many in the literature nowadays. P4L61-66: what is the purpose of this paragraph? P5L81: 'that that' duplication error P5L83-84: Instead, one can argue that it has lower sensitivity than other kits. Also, it is scientifically incorrect to compare extraction kits without using the same samples on all of them, that is a massive compromise to the study design rigor and reproducibility. P7Table1: Why NCA was chosen for some samples, yet others were chosen for re-extraction or re-amplification? Have you tried similar measures with negative samples? What is the difference between invalid and inconclusive? From the numbers provided, Invitrogen kit is the only one showing 100% accuracy, wouldn't the conclusion be that this kit is the superior one rather than Maxwell? Any statistical analysis to scientifically translate these numbers?
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Through this writing, I inform you of the following observations, hoping they will be useful for the work that is being evaluated. The objectives of the methodology are established at the beginning and are met at the end of the study. Correct reference is made to the published literature. Although the methodology used is not described in detail, reference is made to it. The methods used have been correct according to the search needs in this study. They also establish the use of descriptive statistics for data management and it is reflected in the reported results. According to what has been reported, the ethical guidelines are followed as the research is approved by the hospital's ethics committee. The study is interesting, since it addresses a specific point of the CDC protocol, measuring the …
Comments to Author
Through this writing, I inform you of the following observations, hoping they will be useful for the work that is being evaluated. The objectives of the methodology are established at the beginning and are met at the end of the study. Correct reference is made to the published literature. Although the methodology used is not described in detail, reference is made to it. The methods used have been correct according to the search needs in this study. They also establish the use of descriptive statistics for data management and it is reflected in the reported results. According to what has been reported, the ethical guidelines are followed as the research is approved by the hospital's ethics committee. The study is interesting, since it addresses a specific point of the CDC protocol, measuring the percentage of RNase P inhibition as an internal control in COVID-19 PCR reactions. Although it is known that the methodologies prior to their use were evaluated by parameters such as precision, reproducibility, detection limit, among other parameters for their validation. The importance of the study lies in the finding of the incidence in the inhibition, showing interesting data that indicate a low rate of inhibition using this methodology, and that in part was due to the re-extractions and re-amplifications carried out, which becomes great contribution to the improvement of the technique by being able to recover those results that were believed to be inhibited and therefore a greater certainty that the result obtained is highly reliable. Although only one table is reported, the information resulting from the study carried out is explained in a summarized and clear manner. For my part, this study has my approval. I appreciate the confidence in requesting this evaluation. M. Sc EM Durán-Manuel ORCID 0000-0001-8985-486X
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes