Dados do Trabalho

Título

Clinical assessment of SARS-CoV-2 infectivity in nasopharyngeal swab and saliva samples in Covid-19 patients by pre-qPCR treatments-based molecular test

Introdução

The pandemic caused by SARS-CoV-2, the causative agent of Covid-19, is a major public health problem and an unprecedented challenge for governments around the world. Currently, there is a variety of Covid-19 vaccines, that avoid severe cases and mortality, but they do not necessarily prevent the occurrence of infection. Therefore, avoiding its transmissibility is a matter of paramount importance in controlling the pandemic. However, the correlation between detectable viral RNA and transmissibility is still unclear for SARS-CoV-2 infection.

Objetivo (s)

Develop a molecular test based on the pre-treatment of clinical samples with endonuclease, followed by real-time PCR (RT-qPCR), to assess the infectivity of SARS-CoV-2 in patients with Covid-19.

Material e Métodos

The assay was optimized using 30 nasopharyngeal swabs (NPS) (28 RT-qPCR positive and 2 negative) and 18 saliva samples (16 RT-qPCR positive and 2 negative). All positive NPS and saliva samples were characterized as P2 variant of interest. For discrimination of infectious particles (encapsidated) from non-infectious (“naked” RNA), samples were treated with the Benzonase® enzyme. The Benzonase® performance was evaluated by comparing it with cell culture isolation (CPE).

Resultados e Conclusão

Most of the NPS (6/8) with low viral load (Cq ≥33) became negative after Benzonase® pre-treatment (BePt), and were also negative in CPE, indicating that these samples were non-infectious. Most of the NPS showing Cq from 28 to 32, remained positive after BePt, demonstrating the presence of infectious particles, however, they became negative in CPE, probably due to the lower sensitivity on CPE assay. While, NPS with higher viral load (Cq ≤27), showed a slight reduction in viral load after BePt and were strongly associated with CPE positivity, indicating a higher proportion of infectious virus. Regarding the saliva, from 16 positive samples, those with Cq ranging from 15 to 32, remained RT-qPCR positive after BePt (n=12), showing a slight viral load reduction, suggesting the co-existence of infectious and non-infectious particles. Whereas samples with Cq >32 (n=4) became qPCR negative, indicating an absence of SARS-CoV-2 virions. This simple and low-cost molecular method showed an efficient way to confirm the infectivity of the virus in positive patients and could be especially applicable for SARS-CoV-2 where isolation requires a biosafety level 3, which makes its application in routine laboratory diagnosis unfeasible.

Palavras-chave

SARS-CoV-2;Benzonase;Infectivity

Agradecimentos

IOC,CAPES,CNPq,FAPERJ