In response to: "Diagnostic evaluation of nCoV-QS, nCoV-QM-N, and nCoV-OM detection kits based on rRT-PCR for detection of SARS-CoV-2 in Ecuador"

Abstract

Dear Editor, I have read with great interest the article “Diagnostic evaluation of nCoV-QS, nCoV-QM-N, and nCoV-OM detection kits based on rRT-PCR for detection of SARS-CoV-2 in Ecuador” by Salinas et al. [1]. In this study, the authors carried out an evaluation of commercial RT-qPCR kits for SARS-CoV-2 detection, available in Ecuador. This type of studies are very useful in the context of low and middle income countries where it has been described that low quality products for SARS-CoV-2 detection have been commercialized during COVID-19 pandemic [2]. However, I would like to express my concern about some of the information; also, some of the data provided in this study could question the main findings described by authors. First, in the introduction the authors said: “In fact in Ecuador, of the 35 kits with authorization for diagnosis of SARS-CoV-2 by the Ecuadorian regulatory entity—ARCSA, only 10 have been tested and they are included in the list of emergency authorization use by the FDA [16]". This affirmation is not accurate as the reader could easily understand that the Ecuadorian regulatory entity (ARCSA) carried out the experimental evaluation of 10 of the 35 kits available in Ecuador. This is not correct, as ARCSA did not carry any experimental evaluation of SARS-CoV-2 RT-qPCR commercial kits. Moreover, the reference 16 does not endorse that previous affirmation as it is only the list of SARS-CoV-2 RT-qPCR kits authorized in Ecuador, none of them experimentally evaluated by ARCSA. It is surprising that the authors were not aware of the several publications done in Ecuador related to the topic of this study by other universities and research centers, including my own laboratory. There is a recent publication that summarizes all the SARS-CoV-2 RT-qPCR kits evaluated in Ecuador [3]. Among those publications, there is one including a clinical performance evaluation of nCoV-QS Kits (MiCo BioMed, Korea), one of the kits evaluated by the authors [4]. Considering that the results of this study were different that the results obtained by the authors, it is striking that this is not discuss in their publication. Second, although the authors specifically referred to the lack of evaluation studies for authorization of SARS-CoV-2 RT-qPCR kits in Ecuador, I am missing some important information related to the kits evaluated in the present study: those kits were not authorized for “Korean Center for Disease Control” for clinical use in South Korea, their country of manufacture. I believe this information is important as the reader should be aware that those kits are commercialized in Ecuador but not in their country of production. This is a red flag for potential low quality products [2,5]. Third, related to the data presented by the authors; considering the LightMix E/RdRp kit based on the “Berlin Charite Hospital SARS-CoV-2 RT-qPCR protocol” as the gold standard in this study, the percentage of negative agreement would be equivalent to the specificity. That means that two of the evaluated kits have a strong reduction over 5% in specificity. This is usually not acceptable for a good quality SARS-CoV-2 detection by RT-qPCR, and the authors should have discussed this issue. This lack of specificity could be true and due to cross reactivity with other pathogens, or could be false and due to a lower sensitivity of the LightMix E/RdRp kit. Definitely, this is missing in the discussion, because the authors told about the importance of avoiding false positive results in the introduction. Fourth, the “Berlin Charite Hospital SARS-CoV-2 RT-qPCR protocol” based on the E gene is considered a high quality gold standard; but this is not totally equivalent to the LightMix E/RdRp kit used by the authors. In my own experience, when the “One-Step RT-PCR Polymerase Mix 5× Lyophilized (TIB Mol-biol, Germany)" is used, there is a reduction of sensitivity in the “Charite protocol” compared to other commercial RT-qPCR master mix like the ones recommended by the “Center for Diseases Control and Prevention” from the USA. Moreover, it is striking that the authors used the RdRp gene from the “Charite protocol” for SARS-CoV-2 detection, when it was reported in 2020 that the primers for RdRp included mismatching nucleotides [6]. So, RdRp primers used in this study should be definitely avoided. Fifth, based on the comment number four, it is hard to understand how the detection capacity for E gene (47%) was lower than for RdRp gene (49%). Moreover, the authors described: “the median Ct value of LightMix RdRp (Ct = 27.57) was significantly higher than (…) LightMix E (Ct = 23.85) assays”. Those results are totally controversial and make me question the validity of the data presented in this study. So far, such a switch toward higher Ct values for RdRp gene would explain a reduction in sensitivity for this gene as it has been described [6]; but I wonder how the authors could explain the opposite result. In summary, considering that the kits evaluated in the present study lack of clinical use authorization by the “Federal and Drug Administration” from USA or the “Korean Center for Disease Control” (regulatory agency at the country of production), and that some experimental mistakes could have happened, the main findings of this study (good quality of nCoV-QS, nCoV-QM-N, and nCoV-OM Detection Kits from MiCo BioMed, Korea) is questionable. More experiments are recommended, specially including a reliable gold standard method, to endorse the authors' conclusion. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: No funding was available for this work.