RT-LAMP Multicenter Study for SARS-CoV-2 Genome Molecular Detection in Brazilian Swab and Saliva Samples.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid method that can replace RT-qPCR. A simple molecular assay for SARS-CoV-2 RNA detection in gold-standard diagnosis through swabs and alternative specimens such as saliva could be helpful in promoting genomic surveillance. A multicenter study was conducted to evaluate the RT-LAMP assay method as an alternative for the molecular detection of SARS-CoV-2 lineages in swab and saliva samples. A total of 350 swabs from individuals with (n = 276) or without (n = 74) COVID-19 tested by RT-qPCR were collected. Paired saliva was also collected from 90 individuals who had SARS-CoV-2 RNA that was detectable (n = 30) or undetectable (n = 60) via RT-qPCR. For the RT-LAMP methodology, six primers were used for ORF1 gene amplification. As for SARS-CoV-2 genotyping, 39 swabs had the whole genome sequenced by MinION. The sensitivity of RT-LAMP to the swab was 90.2%. For the swab samples with Ct ≤ 30, the sensitivity improved by 96%. Considering saliva with Ct ≤ 30 in RT-qPCR testing, the RT-LAMP sensitivity was 100%. The RT-LAMP specificity was 100% for both the swab and saliva samples. This RT-LAMP assay was capable of detecting all the SARS-CoV-2 lineages circulating in the Brazilian swab samples. The RT-LAMP method has significant potential for use in clinical routines since it was capable of detecting SARS-CoV-2 RNA in swab and saliva samples.

Characteristics of COVID-19 Infection in a Hospitalized Autoimmune Hepatitis Patient.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become a major public health worldwide. Hepatic dysfunction has been seen in patients with COVID-19 and could be related to a viral cytopathic effect, an exacerbated immune reaction, or drug-induced liver damage. Currently, routine modification of immunosuppressive therapy in patients with autoimmune hepatitis (AIH) before and after SARS-CoV-2 infection remains an important topic to be discussed. However, there is little evidence about this thematic to support any recommendation. Here, we described a case report in which the use of an immunosuppressive drug by a patient with diagnosed AIH might have influenced the COVID-19 clinical course with altered laboratory hematological and biochemical parameters during infection.

Clinical and laboratory characteristics of hepatitis C and COVID-19 coinfection: Prolonged RNA shedding in nonhospitalized case.

Nonhospitalized COVID-19 and hepatitis C-coinfected patient presented prolonged RNA shedding and mild course of infection. This finding demonstrated the importance of long follow-up of these patients.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Comparison of four methods of genotyping IL28B polymorphisms in chronic hepatitis C patients.

BACKGROUND: Single nucleotide polymorphisms (SNPs) of the interleukin 28B (IL28B) gene are associated with viral clearance and treatment response in hepatitis C virus (HCV) infection; however, most of the available SNP genotyping methods are expensive. AIMS: This study sought to evaluate the cost effectiveness of four methods used to genotype the rs12979860 and rs8099917 SNPs of the IL28B gene. METHODS: Tetra-primer amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR), restriction fragment length polymorphism (RFLP), quantitative (q) PCR and direct sequencing methods were evaluated in terms of specificity, cost and run time in 281 blood samples obtained from chronic HCV patients. RESULTS: In ARMS-PCR method, the primers designed to target both SNPs produced PCR fragments of specific sizes that distinguished the alleles of rs12979860 and rs8099917. In RFLP, the band profile allowed the distinction between genotypes. The qPCR was the faster and easier to perform. Validation by nucleotide sequencing showed 100% agreement among the three methods. The cost for a single reaction was lowest for ARMS-PCR, followed in turn by RFLP, qPCR and sequencing. CONCLUSIONS: The methodology described for the ARMS-PCR showed the most favorable cost-benefit ratio. Moreover, this approach is fast and simple, requiring only equipment that is commonly used in molecular diagnosis, which is an essential parameter for use in developing countries where laboratories have scarce financial resources.