key: cord-0924203-n9m6u1gv authors: Freire-Paspuel, Byron; Vega-Mariño, Patricio; Velez, Alberto; Cruz, Marilyn; Perez, Franklin; Garcia-Bereguiain, Miguel Angel title: Analytical and clinical comparison of Viasure (CerTest Biotec) and 2019-nCoV CDC (IDT) RT-qPCR kits for SARS-CoV2 diagnosis. date: 2020-11-18 journal: Virology DOI: 10.1016/j.virol.2020.10.010 sha: 27eda3bba5f1bd268f7145f36dbb7fcdcc68581c doc_id: 924203 cord_uid: n9m6u1gv BACKGROUND: Several RT-qPCR kits are available for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA, but most of them lacking of proper evaluation studies due to covid19 emergency. OBJECTIVE: We evaluated Viasure RT-qPCR kit (CerTest Biotec, Spain) for SARS-CoV-2 diagnosis using FDA EUA 2019-nCoV CDC kit (IDT, USA) as gold standard. RESULTS: Although we found the lack of RNA quality control probe as the main limitation for the Viasure kit, the sensitivity was 91.9% and the specificity was 100%. The limit of detection (LOD) was 2000 copies/mL and 1000 copies/mL for Viasure and IDT kits, respectively. CONCLUSIONS: Viasure RT-qPCR kit is a relatively reliable tool for SARS-CoV-2 diagnosis but improvement of an alternative RT-qPCR reaction for RNA extraction quality control as RNaseP is recommended. The COVID19 pandemia has challenged public health systems worldwide, not only for patient care or surveillance and control, but also to guarantee the quality of SARS-CoV-2 related (1) (2) (3) (4) , and and RNase P as an RNA extraction quality control. Among the commercial kits available in the market, Viasure SARS-CoV-2 RT-qPCR kit (CerTest Biotec; Spain) includes "ORF1ab" and "N" probes for SARS-CoV-2 detection. However, no probe for RNA extraction quality control is included but simply an "internal positive control" to guarantee that PCR reaction performs well. Viasure SARS-CoV-2 kit is made in Spain, one of the countries leading COVID19 cases and deaths worldwide, where it has been used for SARS-CoV-2 diagnosis. Also, it was recently authorized for SARS-CoV2 diagnosis in Ecuador. However, it is not included on the list of FDA EUA kits (5) and only an evaluation study for Viasure SARS-CoV-2 RT-qPCR kit has been reported comparing to an automatized system like de Cobas 6800, besides the limited validation provided by manufacturer's manual (6, 7) . We herein present a comparison of the analytical and clinical performance of Viasure and 2019-nCoV CDC kits for SARS-CoV-2 RT-qPCR diagnosis from nasopharyngeal swab samples. Study design. 156 clinical specimens (nasopharyngeal swabs collected on 0.5mL TE pH 8 buffer) were included in this study, coming from individuals selected for SARS-CoV-2 surveillance at "LabGal" ("Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos") in Galapagos Islands, starting on April 8th 2020. Also, seven negative controls (TE pH 8 buffer) were included as control for carryover contamination, one for each set of RNA extractions. included on the study were tested following an adapted version of the CDC protocol: (1) using PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA) for RNA extraction method; (2) using CFX96 BioRad instrument (8-10). RT-qPCR for SARS-CoV-2 diagnosis using Viasure kit. Same RNA extractions from all the samples included on the study were tested using the Viasure SARS-CoV2, following manufacturer's manual (7). Analytical Sensitivity. Limit of detection (LoD) was performed using commercial positive controls provided by both manufacturers. For Viasure SARS-CoV2, a positive control is included on the kit; although the concentration is not detailed, the manufacturer reported that the positive control is provided 10.000 genome equivalents/mL upon request. For 2019-nCoV CDC kit, the 2019-nCoV N positive control (IDT, USA) was used, provided at 200.000 genome equivalents/mL. Ethics statement. All samples have been submitted for routine patient care and diagnostics. Ethical approval for this study was not required since all activities are according to legal J o u r n a l P r e -p r o o f provisions defined by the "Comité de Operaciones Especiales Regional de Galápagos" that is leading the Covid19 surveillance in Galapagos Islands. No extra specimens were specifically collected for this validation study. All data used in the current study was anonymized prior to being obtained by the authors. Clinical performance of "Viasure SARS-CoV-2 RT-qPCR kit" compared to the CDC gold standard protocol. 156 samples were tested for SARS-CoV-2 following both protocols described on the methods. For the 2019-nCoV CDC EUA kit, 86 samples tested positive and 70 samples tested negative (Table 1 and Supplementary Table 1 and 2). All the 70 samples tested negative for 2019-nCoV CDC kit were also SARS-CoV-2 negative for Viasure SARS-CoV-2 kit, so the specificity obtained in our study was 100%. (Tables 1 and Supplementary Table 1 and 2). Analytical sensitivity: calculation of the limit of detection (LoD) of "Viasure SARS-CoV-2 kit". The viral loads (copies/uL) detailed on Supplementary Table 1 were calculated running a calibration curve with 2019-nCoV N positive control (IDT, USA). The LoD for the CDC protocol was set at 1000 viral RNA copy per mL of sample (or 5 RNA copies/uL of RNA extraction solution) on previous studies (1, (8) (9) (10) . For Viasure SARS-CoV2 kit, the positive control included on the kit at a concentration of 10.000 genome equivalents/mL was used on serial dilutions to calculate de LoD for both viral probles. As the LoD is defined as the lowest viral load in which all replicates are detected (100% sensitivity), our data indicates that the LoD for "ORF1ab" probe was 1000 viral RNA copies/mL of sample (5 RNA copies/uL of RNA extraction solution) and for "N" probe 2000 viral RNA copies/mL of sample (10 RNA copies/uL of RNA extraction solution). As both "N" and "ORF1ab" gene targets must yield a positive result for a sample to be considered positive, the LoD for Viasure SARS-CoV-2 is set at 2000 viral RNA copies/mL of sample. Although the main limitation of our study is the sample size (156 specimens), our results support that Viasure SARS-CoV-2 RT-qPCR kit had a good performance in terms on sensitivity and specificity compared to 2019-nCoV CDC EUA, with values of 86,04% and 100%, respectively. Moreover, according to Viasure SARS-CoV-2 kit manufacturer's manual, when a sample only yields amplification for N gene target but Orf1ab gene target is negative, the result should be SARS-CoV-2 negative and a possible infection by other coronavirus must be considered (7). However, on our hands, all the Viasure SARS-CoV-2 N gene target positive but Orf1ab gene target negative were confirmed as SARS-CoV-2 positive by CDC protocol (1, 2) , and thus we J o u r n a l P r e -p r o o f referred to this samples as presumptive positive. So, if we calculate the sensitivity of Viasure SARS-CoV-2 kit including both true positives and presumptive positives samples, the value rise up to 91.9%. As we have described on the results, we could calculate de LoD of Viasure SARS-CoV-2 kit on 2000 viral RNA copies/mL of sample, that according to our experimental procedure is equivalent to 10 viral RNA copies/uL of RNA extraction solution, confirming the LoD indicated at manufacturer's manual (7). Actually, with the only exception of sample 448 (viral load of 4.300 copies/uL), all the presumptive positive and negative samples for Viasure SARS-CoV-2 kit yielded viral loads below 10 copies/uL (see Supplementary Table 1) . It came to our attention a recent study about the clinical performance of Viasure SARS-CoV-2 compared to Cobas 6800 system using a total number of 95 samples (6) . The authors reported a lost of sensitivity of 30,9% for Viasure SARS-CoV-2 kit, with only 47 out of 68 positive samples detected. However, all the samples that failed to be positive for Viasure SARS-CoV-2 kit had viral loads over 2000 copies/mL of sample (6) . Considering the viral loads frequency distribution for SARS-CoV-2 (11, 12), this study would have a bias toward ultra low viral loads below 2000 copies/mL as less than 10% of SARS-CoV-2 positive patients are expected to have those loads (11, 12). The lack of any probe for RNA extraction quality control like RNaseP is a limitation to be considered when using Viasure SARS-CoV-2 kit. Although in our experience, samples failing to yield any RNA after extraction are below 1%, an extra RT-qPCR reaction for a RNA extraction quality control probe would be recommended for laboratories starting SARS-CoV-2 diagnosis. On the other hand, Viasure SARS-CoV-2 kit is provided on precast format of 8 tubes strips containing a mix of enzymes, primers, buffer and nucleotides in stabilized format, so only rehydratation buffer and RNA samples have to be added. This format is a great advantage in terms of time saving and reduction on pippeting mistakes. Also, the manufacturer provided us upon request the concentration of the positive control included on Viasure SARS-CoV-2 kit (10.000 copies/uL). That means viral loads can be easily calculated without need of purchase any extra positive control, despite the manufacturer's manual does not include instruction for viral load calculation (7). Considering the worldwide high demand of reagents for SARS-CoV RT-qPCR diagnosis, supplies shortage is a fact, actually affecting harder to developing countries like Ecuador. Under this scenario, validation studies are helpful to guarantee the quality of the supplies in the market for every country in the world, as not necessary all the SARS-CoV-2 RT-qPCR kits show the performance indicated by manufacturer as it was the case for Viasure SARS-CoV-2 kit. This study was funded by Universidad de Las Americas (Quito, Ecuador). All authors contributed to study conceptualization, experimental procedures and revision and approval of final version of the manuscript. Byron Freire-Paspuel and Miguel Angel García Bereguiain analyzed the data and wrote the manuscript. All authors have no conflict of interest to declare. US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging Infectious Diseases Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19). Center for Diseases Control and Prevention, USA Comparison of Abbott ID Now, Diasorin Simplexa, and CDC FDA EUA methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from individuals diagnosed J o u r n a l P r e -p r o o f with COVID-19. Accepted Manuscript Posted Online 17 Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit. JCM Accepted Manuscript Posted Online 8 Lack of sensitivity of an IVD/CE-labelled kit targeting the S gene for detection of SARS-CoV-2. Clinical Microbiology and Infection Evaluation of nCoV-QS (MiCo BioMed) for RT-qPCR Detection of SARS-CoV-2 From Nasopharyngeal Samples Using CDC FDA EUA qPCR Kit as a Gold Standard: An Example of the Need of Validation Studies Cotton-Tipped Plastic Swabs for SARS-CoV-2 RT-qPCR Diagnosis to Prevent We thank the medical personnel from "Ministerio de Salud Pública" at Galapagos Islands and the staff from the "Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos" for their support. We also thank Dr. Ronald Cedeño from OPS/WHO for his work during Covid 19 surveillance in Galapagos Islands. We specially Oscar Espinosa and Dr Tannya Lozada from "Dirección General de Investigación de la Universidad de Las Américas", and also the authorities from Universidad de Las Américas, for logistic support to make SARS-CoV-2 diagnosis possible in Galapagos Islands.