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

Abstract

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High prevalence of SARS-CoV-2 infection among food delivery riders. A case study from Quito, Ecuador

“…Nasopharyngeal swabs were collected on 0.5 mL TE pH 8 buffer for SARS-CoV-2 diagnosis by RT-qPCR following an adapted version of the CDC protocol by using PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA) as an alternate RNA extraction method and CFX96 BioRad instrument ( Interim Guidelines for Collecting, n.d. ; Freire-Paspuel et al, 2020a ; Freire-Paspuel et al, 2020b ; Freire-Paspuel et al, 2020c ; Freire-Paspuel et al, 2020d ; Freire-Paspuel and Garcia-Bereguiain, 2020 ). Briefly, the CDC designed RT-qPCR FDA EUA 2019-nCoV CDC kit (IDT, USA) is based on N1 and N2 probes to detect SARS-CoV-2 and RNase P as an RNA extraction quality control ( Interim Guidelines for Collecting, n.d. ; Freire-Paspuel et al, 2020a ; Freire-Paspuel et al, 2020b ; Freire-Paspuel et al, 2020c ; Freire-Paspuel et al, 2020d ; Freire-Paspuel and Garcia-Bereguiain, 2020 ). Also, negative controls (TE pH 8 buffer) were included as control for carryover contamination, one for each set of RNA extractions, to guarantee that only true positives were reported.…”

Considerations for diagnostic COVID-19 tests

“…Unfortunately, and despite this EUA registration, as described in a news article, there were problems with one of the reagents described in the CDC protocol, partially blocking rapid implementation of the test or leading to retraction of test results. On 29 February 2020, new guid ance was issued for laboratories to be able to develop and implement COVID19 molecular diagnostic tests before obtaining EUA [58][59][60][61] (see also Supplementary Table 1). The relative technical ease with which such diagnostic tests could be designed and the stable nature of the target genetic material of the pathogen were contributing factors to test quality and reproducibility, which resulted in var ious tests being developed within a few months (Box 1).…”

Low clinical performance of the Isopollo COVID-19 detection kit (M Monitor, South Korea) for RT-LAMP SARS-CoV-2 diagnosis: A call for action against low quality products for developing countries

“…The viral loads detailed in Supplementary Material Tables S1 and S 2 were calculated running a calibration curve with the 2019-nCoV N positive control (IDT, USA). The LoD for the CDC protocol was set at 1000 viral RNA copies per milliliter of sample (or 5 RNA copies/μl of RNA extraction solution) in previous studies ( Lu et al, 2020 , Freire-Paspuel et al, 2020a , Freire-Paspuel et al, 2020b , Freire-Paspuel et al, 2020c , Freire-Paspuel and Garcia-Bereguiain, 2020a , Freire-Paspuel et al, 2020 , Freire-Paspuel and Garcia-Bereguiain, 2020b ). Although the LoD could not be calculated for the Isopollo COVID-19 detection kit, as described in the Methods section, even for samples with viral loads above 100 000 RNA copies/ml (500 RNA copies/μl of RNA extraction solution), only 81 out of 88 (92.04%) samples were also positive with the Isopollo COVID-19 detection kit.…”

“…Among the kits available on the market, the US Centers for Disease Control and Prevention (CDC) designed 2019-nCoV CDC EUA kit (IDT, USA), which is based on N1 and N2 gene targets to detect SARS-CoV-2, has received positive evaluations in recent reports. This kit uses the RNaseP gene target as an RNA extraction quality control and is considered a gold standard for clinical evaluation ( Lu et al, 2020 , Center for Diseases Control and Prevention, 2021 , Rhoads et al, 2020 , Nallaa et al, 2020 , Freire-Paspuel et al, 2020a ). The Isopollo COVID-19 detection kit (M Monitor, South Korea) is a fluorescence-based RT-LAMP kit that includes two gene targets for SARS-CoV-2 detection, ‘RdRp’ and ‘N’, but has no target for RNA extraction quality control.…”