The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.
O Pará está entre os estados brasileiros com as mais elevadas taxas de prevalências da infecção pelo vírus T-linfotrópico humano (HTLV) nas populações onde este vírus já foi investigado. A infecção por HTLV geralmente apresenta prevalência mais elevada em mulheres com mais de 40 anos e com relacionamento estável. Além disso, as mulheres são mais susceptíveis à aquisição do vírus por via sexual do que os homens. Neste contexto, este estudo teve como objetivo detectar o genoma do provírus de HTLV em secreção cérvico-vaginal, com posterior confirmação em amostras de sangue, visando assim, propor uma nova metodologia de rastreamento desta infecção. Foram investigadas 400 mulheres de novembro de 2015 a dezembro de 2019, em Belém, Pará, Brasil. A coleta de secreção cérvico-vaginal se deu durante a realização do exame de Papanicolaou, e a de sangue periférico, durante contato posterior. O DNA das amostras foi extraído e realizada a análise molecular por Nested-PCR, seguida de digestão enzimática por Taq I, para pesquisa da infecção pelos tipos HTLV-1 e HTLV-2. Cinco (1,25%) das 400 mulheres tiveram resultado positivo para HTLV em secreção, sendo três para HTLV-1 e duas para HTLV-2. Por dificuldade de contato, só foi possível a confirmação da infecção no sangue em uma destas cinco mulheres. Nossos achados sugerem ser possível utilizar amostras de secreção cérvico-vaginal como mais uma forma de rastreamento da infecção por HTLV em mulheres.
São Paulo state has been the epicenter of the Coronavirus Disease 2019 (COVID-19) in Brazil, ranking first by state with over six million reported cases. In February 2021, the P.4 lineage was reported in 21 cities across the state by public health authorities due to the L452R mutation. Here, by analyzing 17,304 genome sequences of SARS-CoV-2 sampled between February and August of 2021 in 476 distinct cities in São Paulo, we assess the transmission dynamics of the P.4 lineage and other SARS-CoV-2 variants that were, at the time of the study, co-circulating in the state. Additionally, clinical parameters from the city of Araras, São Paulo (N = 251) were considered to estimate the potential risk and mortality rate associated with the P.4 lineage since its higher prevalence was observed in that city. Our data suggest a low frequency (0.55%) of the P.4 lineage across the state, with the gamma variant being the dominant form in all regions (90%) at that time. Furthermore, no evidence of increased transmissibility and disease severity related to the P.4 lineage was observed. The displacement through the time of different lineages in São Paulo highlights how challenging genomic surveillance appears to track the emergence of new SARS-CoV-2 lineages, which could better guide the implementation of control measures.
Our effort in SARS-CoV-2 genomic surveillance in Brazil has detected the Alpha Variant of Concern with a predominance higher than 75% in the population of Ilhabela island (São Paulo State) at a time when the Gamma VOC was already predominating the mainland raised concerns for closer surveillance on this island. Therefore, we intensified the surveillance for 24 weeks by generating data from 34% of local positive cases. Our data show that the patterns of VOC predominance dynamics and infection rates were in general distinct from the mainland. We report here the first known case of Alpha predominance in a Brazilian population, a delay greater than 3 months for the Gamma to dominate the previous variants compared to the mainland, and a faster dispersion rate of Gamma and Delta VOCs compared to the mainland. Phylogenetic analysis revealed the SARS-CoV-2 transmission dynamics in Ilhabela were characterized by multiple independent introduction events of Gamma and Delta, with a few events of Alpha introduction, two of them followed by community transmission. This study evidenced the peculiar behavior of SARS-CoV-2 variants in an isolated population and brought to light the importance of specific programs for SARS-CoV-2 genomic surveillance in isolated populations.
Objetivos: Analisar os níveis da carga proviral (CPV) em portadores de HTLV-1 com diferentes condições de comprometimento neurológico. Metodologia: Estudo transversal, realizado de março a outubro de 2017, com 43 pacientes com HTLV-1, divididos em três grupos por ordem decrescente de envolvimento neurológico, segundo proposta atualizada dos critérios de diagnóstico clínico para HAM/TSP: Definidos para HAM/TSP (Grupo 1, n=7); Prováveis/Possíveis para HAM/TSP (Grupo 2, n=9); Sem HAM/TSP (Grupo 3, n=27). O teste ANOVA (um critério) foi utilizado para verificar a diferença entre as médias de CPV dos grupos (p-valor≤0,05). Resultados: Os grupos apresentaram os seguintes valores médios de CPV: G1=9.00±11.18; G2=1.88±3.92; G3=2.81±4.03 cópias/106 PBMC. Na comparação intergrupo, da diferença entre as médias de CPV, foi observada diferença significativa entre os grupos 1 e 2 e entre os grupos 1 e 3 (p<0,05); não foi observada uma média significativamente maior de CPV nos grupos de infectados pelo HTLV-1 com comprometimento neurológico, frente aos infectados sem comprometimento neurológico (p=0,7063). Conclusão: A CPV em portadores de HTLV-1 parece auxiliar a avaliação clínica do comprometimento neurológico na classificação dos casos clássicos de HAM/TSP, mas não se mostra um indicador importante de condições clínicas iniciais da HAM/TSP.
The authors hereby request the inclusion of two authors (Olivia Teixeira and Maria Cristina Nonato) in the recently published article in Viruses entitled “Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results” [...]
Human T cell lymphotropic virus (HTLV) is the caustive agent of two main conditions i. e., the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the adult T-cell leukemia/lymphoma (ATLL). HTLV diagnosis is based on serological and molecular approaches; however, an accurate and validated method is still needed. The objective of this study was to establish a rapid and sensitive molecular test to confirm and discriminate HTLV 1/2 types. The test validation was performed as a multicentric study involving HTLV confirmation centers throughout Brazil. Proviral DNA was extracted from whole blood and the amplification was performed using in-house designed primer and probe sets targeting the pol genomic region. An internal control to validate the extraction and amplification was also included. The limit of detection (LoD) of the assay was four copies/reaction for HTLV-1 and 10.9 copies/reaction for HTLV-2. The diagnostic sensitivity of the platform was 94.6% for HTLV-1, 78.6% for HTLV-2, and the specificity was 100% for both viruses. Cross-reactions of the test with human viruses including HAV, HBV, HCV, HIV-1/2, and parvovirus B19 were not observed. During the multicentric validation, the test was used to screen a total of 692 blood samples obtained from previously confirmed HTLV-positive individuals. From these, 91.1% tested positive being concordant with the previously obtained results. In conclusion, our duoplex-RT-PCR-HTLV1 /2 presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Therefore, it can be a suitable tool for confirmation of suspected and inconclusive HTLV cases, prenatal and pre-transplant diagnosis, in Brazil and in other countries HTLV-endemic countries.
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