Development and implementation of multiplex RT-PCR for respiratory virus detection and quality control: insights from Brazil's national laboratory network

Introduction:During the pandemic efforts, several molecular diagnostic kits were developed in partnership with the Respiratory, Exanthematous, Enterovirus, and Viral Emergencies Laboratory (LVRE)/IOC and Laboratory of Molecular Kits (LAMOL/Bio-manguinhos), facilitating the molecular diagnostics of influenza A and B and SARS-CoV-2 (SC2).A pilot study was conducted in 2021 to demonstrate the utility of these kits, leading to the development of an expanded multiplex RT-PCR panel to identify respiratory viruses of public health importance.Further steps led to developing an expanded multiplex RT-PCR panel to identify respiratory viruses of public health importance, allowing the resolution of respiratory syndromes captured in the Brazilian publicly funded health care system (SUS).Objectives: To evaluate the results obtained by the Lacen network using the extended respiratory viruses RT-PCR reagent array, developed in partnership with LVRE and LAMOL and distributed by Biomanguinhos.Methodology: During 2022, Bio-manguinhos produced and distributed 100,000 reactions of this multiplex RT-PCR reagents to simultaneously detect the seven historically more prevalent respiratory viruses (influenza A, influenza B, SC2, human metapneumovirus, respiratory syncytial virus, adenovirus and rhinovirus) and distribute them to Central Public Health Laboratory (Lacen).During online meetings, the Lacens were trained and encouraged to share the RT-PCR runs, which allowed for the analysis of the results and refinement of the protocol.Data was extracted from 1,382 raw data files using Helper software (ACME Lab) and submitted for statistical analysis.Results: Almost 30,000 reactions (excluding positive and negative controls) were received from 12 Lacens nationwide.The results indicate that most Lacens performed well.However, we identified a considerable cycle threshold (Ct) variation, highlighting heterogeneity and non-adherence to standardised conditions.The rhinovirus was the most prevalent virus identified in the analysis, but all viruses were detected in variable proportions throughout the year. Conclusion:The use of Helper software allowed for the automation of data collection and result normalisation, improving efficiency and accuracy.Implementing this RT-PCR multiplex approach across the network signifies an important advancement in monitoring virus circulation while ensuring compliance with international quality standards and cost-effectiveness.