Preprint Article Version 2 Preserved in Portico This version is not peer-reviewed

False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

Version 1 : Received: 18 September 2020 / Approved: 19 September 2020 / Online: 19 September 2020 (08:27:13 CEST)
Version 2 : Received: 16 October 2020 / Approved: 16 October 2020 / Online: 16 October 2020 (11:48:36 CEST)

How to cite: Fruehwirth, M.; Rivas, A.V.; Fitz, A.F.R.; Batista, A.C.C.A.; Silveira, C.V.; Delai, R.M. False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors. Preprints 2020, 2020090450 (doi: 10.20944/preprints202009.0450.v2). Fruehwirth, M.; Rivas, A.V.; Fitz, A.F.R.; Batista, A.C.C.A.; Silveira, C.V.; Delai, R.M. False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors. Preprints 2020, 2020090450 (doi: 10.20944/preprints202009.0450.v2).

Abstract

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

Subject Areas

COVID-19; rRT-PCR; dilution; viral diagnosis; RNA extraction

Comments (1)

Comment 1
Received: 16 October 2020
Commenter: Marcelo Fruehwirth
Commenter's Conflict of Interests: Author
Comment: Some corrections in the writing were made, and some figures were repositioned/removed.

The article is in the new format for publication in the journal in which it was accepted.

As soon as published, we'll communicate. Att. Marcelo Fruehwirth
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