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

1 2 Marcelo Fruehwirth1, Açucena Veleh Rivas1, Andressa Faria Rahyn Fitz1, Aline Cristiane Cechinel 3 Assing Batista1, Cleypson Vinicius Silveira1, Robson Michael Delai1 4 5 1 Centro de Medicina Tropical da Tríplice Fronteira, Fundação de Saúde Itaiguapy, Av. Araucária, 6 1734 Vila A, 85866-010, Foz do Iguaçu, Paraná, Brazil 7 8 *Corresponding author: Marcelo Fruehwirth. Centro de Medicina Tropical da Tríplice Fronteira, 9 Fundação de Saúde Itaiguapy, Av. Araucária, 1734 Vila A, 85866-010, Foz do Iguaçu, Paraná, Brazil 10 E-mail: marcelo.fruehwirth@hmcc.com.br / Phone: +55 (45) 3029-1733. 11 12 Abstract 13 14 Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as 15 amplification inhibitors presence, lead to false-negative results. Here we describe differences between 16 rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for 17 dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs 18 samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for 19 SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®) for extraction. rRT-PCR was 20 realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80μl of H2O RNAse free) 21 samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Ct 22 between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 23 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor 24 compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% 25 false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 26 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the 27 identification of false-negative cases, and consequently avoid a wrong diagnosis. 28 29 30