Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Marcelo Fruehwirth
* ORCID logo ,
Açucena Veleh Rivas
,
Andressa Faria Rahyn Fitz
,
Aline Cristiane Cechinel Assing Batista
,
Cleypson Vinicius Silveira
,
Robson Michael Delai
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)

A peer-reviewed article of this Preprint also exists.

Fruehwirth M, Rivas AV, Fitz AFR, Batista ACCA, Silveira CV, Delai RM. False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors. J. Bras. Patol. Med. Lab.2020;56(1):1-8 Fruehwirth M, Rivas AV, Fitz AFR, Batista ACCA, Silveira CV, Delai RM. False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors. J. Bras. Patol. Med. Lab.2020;56(1):1-8

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®) for extraction. 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 Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, 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 29.8% 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.

Keywords

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

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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