key: cord-272734-kawim93f
authors: Freire-Paspuel, Byron; Vega-Mariño, Patricio; Velez, Alberto; Castillo, Paulina; Cruz, Marilyn; Garcia-Bereguiain, Miguel Angel
title: 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
date: 2020-05-22
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104454
sha: 
doc_id: 272734
cord_uid: kawim93f

BACKGROUND: Several qPCR kits are available for SARS-CoV-2 diagnosis, mostly lacking of evaluation due to covid19 emergency. OBJECTIVE: We evaluated nCoV-QS (MiCo BioMed) kit using CDC kit as gold standard. RESULTS: We found limitations for nCoV-QS: 1) lower sensitivity 2) lack of RNA quality control probe. CONCLUSIONS: Validation studies should be implemented for any SARS-CoV-2 RT-qPCR commercial kit to prevent unreliable diagnosis.

Highlighted Corrections. 1 . Line 29 and Line 77. The RNA extraction control in the CDC assay is RNase P. Please correct.

Corrected on the text.

2. In the abstract, please remove or rephrase limitation #3. Since the assay is real time RT-PCR, a quantitated standard could be used to to generate viral load data. line 20 deleted 3) no capacity to quantify viral load.

Deleted from the text. We only mention on the discussion that viral load cannot be calculated with the control provided in the kit, but it is possible with other control as reviewer suggested.

Besides those suggested changes we made other typing corrections and small changes highlighted in red, among them, we want to detailed:

-In line 42 we eliminated any reference for 2 independent donors. That could be understood that one of the donations did not work, and we prefer to manage this information personally to donors.

-Line 32. We now point out the MicoBioMed SARS-CoV-2 RT-qPCR kit has not FDA EUA approval and has not been authorized in Korea neither (2 new referencees added endorsing that). Although the reference for Korean CDC is updated for March 17th 2020, personal communication from K-CDC to the corresponding aunthor confirms that MiCoBioMed do not have authorization to date.

Multiple in vitro RT-qPCR diagnosis kits are available on the market for the detection of SARS-CoV-2. Some of them have received emergency use authorization (EUA) from the U.S. Food & Drug Administration (FDA) while others only report validations made by manufacturers, and in general little is known about their performances using clinical specimens. The CDC designed 2019-nCoV CDC EUA kit (IDT, USA) is based on N1 and N2 probes to detect SARS-CoV-2 that have received positive evaluation on recent reports (1) (2) (3) , and and RNase P as an RNA extraction quality control. Other kit avalaible in the market is nCoV-QS (MiCo BioMed; South Corea) that include probes "ORF3a" and "N" probes for SARS-CoV-2 detection but no probe for RNA extraction quality control, with no EUA approval neither from FDA (USA) nor from Korean CDC (4,5,6).

This study compared the performance in terms of positive percent agreement (PPA) of nCoV-QS Fifty-four (54) clinical specimens (nasopharyngeal swabs collected on 0.5mL TE pH 8 buffer) from patients selected as suspicious for SARS-CoV-2 infection were included on this study during the surveillance in Galapagos Islands started on April 8th 2020. Also, six negative controls (TE pH 8 buffer) were included as control for carryover contamination. Both CoV-QS and 2019-nCoV CDC EUA kits were used at SARS-CoV-2 diagnosis laboratory "LabGal" at "Agencia de Regulación y Control de la Bioseguridad y Cuarentena para Galápagos" at Puerto Ayora in Galapagos Islands (Ecuador), where we considered this validation necessary to guarantee the sensibility of SARS-CoV-2 during the surveillance.

Twenty-five (25) samples were tested following an adapted version of the CDC protocol (1) Table 1 Table 1 and 2b. We used CFX96 BioRad to run qPCR but also results were confirmed using Veri-Q PCR316 instrument from MiCo BioMed (4). The assay sensitivity indicated on manufacturers manual (1.8 copies/uL for OFR3a and 4.24 copies/uL for N) could not be validated because positive control concentration was not provided.

In summary, overall PPA for nCoV-QS was 66.7% (22 out of 33 positives samples for 2019-nCoV CDC EUA; p<0.001), and 70.5% and 62.5% for MiCo BioMed and adapted CDC protocols, respectively. Additionally, considering the viral loads calculated following adapted CDC protocol with 2019-nCoV N positive control (IDT, USA), the limit of detection (viral copies/uL) for nCoV-QS kit is much higher than the one indicated at manufacturer's manual (6).

Although the main limitation of our study is the sample size (54 specimens), our results support that nCoV-QS kit had a significant lower performance in terms on PPA and sensitivity compared to 2019-nCoV CDC EUA. Also, the lack of any probe for RNA extraction quality control like RNase P and the unreported concentration of positive controls provided for the kit that does not allow viral load calculations, are limitations to be considered when using nCoV-QS kit.

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 J o u r n a l P r e -p r o o f 6 scenario, validation studies are helpful to guarantee the quality of the supplies in the market for every country in the world.

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 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.

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.

None.

All authors have no conflict of interest to declare.

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 thank Gabriel Iturralde, 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.

J o u r n a l P r e -p r o o f 

Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19)

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 with COVID-19. Accepted Manuscript Posted Online

Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit. Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit. JCM Accepted Manuscript Posted Online 8

COVID-19 Task Force and the Center for Laboratory Control of Infectious Diseases, the Korea Centers for Disease Control and Prevention. Guidelines for Laboratory Diagnosis of Coronavirus Disease 2019 (COVID-19) in Korea