Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Etiological agents of viral meningitis in children from a dengue-endemic area, Southeast region of Brazil.

Meningitis is a disease with a global distribution that constitutes a worldwide burden, with viruses as the primary etiologic agents. The range of viral meningitis severity depends mainly on age, immune status and etiological agent. The aim of this work was to investigate the suspected cases of viral meningitis using molecular techniques to confirm the viral infection. The diagnosed virus was correlated with clinical findings and cytochemical parameters in cerebrospinal liquid (CSF) of patients. CSF of 70 children with the presumptive diagnosis of viral meningitis was analyzed by real time PCR (qPCR). Viruses were identified by qPCR in 44 CSF samples (62.9%). Among them, 31 were identified as Enterovirus (ENTV) (70.4%), six as Human herpes virus 3 (HHV-3) (13.6%), five as Dengue virus (DENV) (11.7%), one as Human herpes virus 1-2 (2.3%) and one as Human herpes virus 5 (2.3%). Patients in the HHV-positive groups had increased percentage of polymorphonuclear neutrophils (PMN) (mean of 81%) while the groups of patients with DENV and ENTV had a mean of 30.9%. This study contributes to the knowledge of the epidemiological distribution of viral agents in CNS infections in children. In addition, it raises the relevance of DENV as an agent of CNS infection, and reinforces the importance for molecular in the cases of CNV infection.

Horizontal study of vaccinia virus infections in an endemic area: epidemiologic, phylogenetic and economic aspects.

Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7% of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.