Review 1: "Blood Transcriptomics Reveal Persistent SARS-CoV-2 RNA and Candidate Biomarkers in Long COVID Patients"

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: Menezes SM et al.'s work suggests that there are non-invasive markers able to discriminate long-term COVID participants from participants without long-term sequelae of COVID-19. They looked for transcriptomic markers distinguishing 48 long COVID participants from 12 matched participants without long-term sequelae of COVID-19. They identified two genes, FYN and antisense SARS-CoV-2 (Orf3a?) RNA, both induced in long COVID participants. One of those two genes, SARS-CoV-2 antisense, suggests that the persistence of viral replication two years following infection is a biomarker of long-term COVID and that antiviral therapy could be considered for the treatment of long-term COVID.

The method section of the paper is telegraphic and requires an additional explanation of how the ROC analysis was performed. My main concern is the lack of a test set or a cross-validation procedure to minimize overfitting. Also, detecting SARS-CoV-2 transcripts in blood two years following acute infection is not in line with other studies describing low or undetectable SARS-CoV-2 viral load during the convalescent phase of COVID-19 disease. The lack of longitudinal sampling prevents us from knowing that the two genes identified (FYN and SARS-CoV-2 antisense) can identify long COVID-19 before the two-year time point used by the study's author. No data lets us know if those two genes are only surrogate markers of long COVID or if they are mechanistically implicated in symptoms persistence following acute infection with SARS-CoV-2.