False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors

ABSTRACT

Introduction:

Although reverse transcription-polymerase chain reaction (rRT-PCR) is the gold standard method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), some factors, such as the presence of amplification inhibitors, lead to false-negative results.

Objective:

Here we describe the differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to the presence of amplification inhibitors.

Material and method:

Viral ribonucleic acid (RNA) from samples of nasopharyngeal swabs from 20 patients previously detected as “Negative” and 21 patients detected as “Positive” for SARS-CoV-2 was performed with the EasyExtract DNA-RNA kit (Interprise^®). The rRT-PCR was performed with the OneStep/COVID-19 kit (IBMP), with normal and diluted (80 µl of H₂O RNAse free) samples, totaling 82 tests.

Results:

The results indicate that there is an average variation (a < 0.05) delaying the Cq between the results of amplification of the internal control (IC), N gene (NG), and ORF1ab (OF), 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively.

Discussion:

The extraction kit does not completely purify the inhibitor compounds; therefore, no amplified product result may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution; this process reduces the efficiency of rRT-PCR to 29.8% in detecting SARS-CoV-2.

Conclusion:

Knowing the rRT-PCR standards of diluted samples can assist in the identification of false-negative cases and, consequently, avoid incorrect diagnosis.

Key words:
COVID-19; rRT-PCR; dilution; viral diagnosis; RNA extraction