BACKGROUND The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) has changed unevenly over time around the world. Although whole genome sequencing is the gold standard for virus characterisation, the discovery of alpha VOC causing spike gene target failure (SGTF) result, when tested using an reverse transcription real-time polymerase chain reaction (RT-qPCR) assay, has provided a simple tool for tracking the frequencies of variants. OBJECTIVES The aim of this study was to investigate if a multiplex RT-qPCR assay (BioM 4Plex VOC) could be used to detect SARS-CoV-2 and to perform a VOC screening test in a single reaction tube. Here, we present the multicentre study evaluating this assay. METHODS Twelve laboratories have participated in the multicentre study. The BioM 4Plex VOC was distributed to them with detailed instructions of how to perform the test. They were asked to test the BioM 4Plex VOC in parallel with their routine Commercial SARS-CoV-2 diagnostic assay. Additionally, they were requested to select SARS-CoV-2-positive samples with genome sequenced and lineage definition according to PANGO lineage classification. FINDINGS The BioM 4Plex VOC and commercial RT-PCR assay are equally effective in detecting SARS-CoV-2. Results revealed a specificity of 96.5-100% [95% confidence interval (CI)], a sensitivity of 99.8-100% (95% CI), and an accuracy of 99.8-100% (95% CI). A 99% concordance rate was found between results from the BioM 4Plex VOC and that from available genome sequencing data. MAIN CONCLUSIONS The BioM 4Plex VOC provides an effective solution to detect SARS-CoV-2 infections and screening for VOCs in a single reaction. It is a straightforward method to help us monitor the frequency and distribution of VOCs and develop strategies to better cope with the pandemics.

ABSTRACT In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses.

Abstract Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays, direct-acting antiviral agents (DAAs) are the main treatment for HCV; however, the high level of virus variability leads to the development of resistance-associated variants (RAVs). Thus, assessing RAVs in infected patients is important for monitoring treatment efficacy. The aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serum samples from blood donors at the Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) were collected retrospectively in 2013 and evaluated in this study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. The frequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencing reveled 1 sample with L320F mutation and 4 samples with the C316N/R polymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%), S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%) presented the V170 I/V non-synonymous mutation. In conclusion, we have shown that mutations in NS3 and NS5b genes are present in Brazilian isolates from therapy-naïve HCV patients.

ABSTRACT The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p= 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values.