Comparación de las técnicas Inmunofluorescencia directa y Biología molecular RT-PCR para detección de SARS-CoV-2 a partir de muestras de pacientes Covid-19

Abstract

SARS-CoV-2 causes COVID-19 disease, and early detection is important. In the presence of endemic SARS-CoV-2, a classical technique is necessary for initial screening in the differential diagnosis. We previously reported the development of a detection method based on the determination of viral antigen by immunofluorescence (IFD). In this work we compare results of this artisanal methodology for diagnostic purposes with those obtained by commercial molecular method (real-time RT-PCR).

A total of 174 nasopharyngeal secretions from swabs of adult patients with Covid-19 symptoms were analyzed. There were 115 positive by RT-PCR (DisCoVery) and 59 negative. From these samples, 224 respiratory epithelial cell imprints were made, with which the IFD technique was standardized, using polyclonal and monoclonal primary antibodies (post-infection human polyclonal serum/SP, polyclonal gamma globulin fraction/GP and anti-N and anti-RBD monoclonal/MAB antibodies, with corresponding fluorescein-labeled secondary/conjugated antibodies). Thirty-three imprints were tested per primary antibody tested. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the IFD technique were determined using the statistical program R-medic.

The fluorescence images resulting from the application of the technique with mono- and polyclonal antibodies on the RT-PCR-positive patient imprints were similar and showed the expected fluorescence pattern. For SP, sensitivity was 82%, specificity 64%, PPV 82% and NPV 64%. For GP, sensitivity 85%, specificity 67%, PPV 92% and NPV 50%. For MAB, both anti-N and anti-RBD: sensitivity 92%, specificity 78%, PPV 92% and NPV 78%. The data showed a ratio of 3 positives by RT-PCR to 1 or 2 by IFD.

In conclusion, the developed IFD technique could be successfully implemented for detection as an initial screening alternative. Furthermore, it demonstrates that this development of a classical direct diagnostic method of SARS-CoV-2 by DIF not only contributes to the detection in public health, but also, in the global ecological context of environmental care, it respects the sustainable development goals such as SDG 3 referred to ensure health and SDG 12 alluding to sustainable production and consumption (UN, SDG, 2022).