A Simplified Sanger Sequencing Method for Detection of Relevant SARS-CoV-2 Variants.

Molecular surveillance of the new coronavirus through new genomic sequencing technologies revealed the circulation of important variants of SARS-CoV-2. Sanger sequencing has been useful in identifying important variants of SARS-CoV-2 without the need for whole-genome sequencing. A sequencing protocol was constructed to cover a region of 1000 base pairs, from a 1120 bp product generated after a two-step RT-PCR assay in samples positive for SARS-CoV-2. Consensus sequence construction and mutation identification were performed. Of all 103 samples sequenced, 69 contained relevant variants represented by 20 BA.1, 13 delta, 22 gamma, and 14 zeta, identified between June 2020 and February 2022. All sequences found were aligned with representative sequences of the variants. Using the Sanger sequencing methodology, we were able to develop a more accessible protocol to assist viral surveillance with a more accessible platform.

SARS-CoV-2 Incidence, signs and symptoms and main risk factors for COVID-19 infection in Health Care Workers: A hospital-wide survey in Salvador, Brazil.

BACKGROUND: Brazil is the third country most affected by Coronavirus Disease 2019 (COVID-19) in the world. Health care workers (HCWs) are at higher risk of infection. Despite the increasing numbers of studies on the topic, There are gaps in the knowledge of characteristics and risk factors for infection of HCWS. This information is important to design preventive strategies and to mitigate the disease impact. The objective of this study was to estimate the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, to identify factors associated, and to describe symptoms reported by healthcare workers at a tertiary hospital in Salvador, Brazil. METHODS: All HCWs were evaluated in a cross-sectional study conducted between May and September 2020, using self-administered questionnaires, and screening all participants for SARS-COV-2 IgG and IgM antibodies by rapid tests. Reactive IgG samples were retested by ELISA and IgM-positive test had a saliva sample retest by RT-PCR. Univariate associations were estimated by a non-adjusted incidence proportion ratio. Variables associated with COVID-19 incidence at p < 0.20 were selected for inclusion in a binary logistic regression model. RESULTS: A total of 2083 HCWs were included, mean age 41±10 years, 71.8% women, and 77.8% non-white. Of these, 271 (13.0%) and 25 (1.2%) HCWs tested positive for IgG and IgM SARS-CoV-2 antibodies, respectively, and three had a positive RT-PCR. Ancillary work [Odds Ratio (OR): 4.96], elementary education (OR: 2.91), high school education (OR: 2.89), and catholic religion (OR: 2.16) were associated with an increased likelihood of a positive IgG antibodies against SARS-CoV-2. Anosmia [Incidence Proportion Ratio (IPR): 7.41] and ageusia (IPR:8.51) were the most frequent associated symptoms. CONCLUSION: HCWs with low mean family income, lower level of schooling, ancillary workor being black had a significantly higher likelihood of testing positive for SARS-CoV-2 antibodies. Social vulnerability was an important risk factor for COVID-19 infection.

Frequent Infection of Cats With SARS-CoV-2 Irrespective of Pre-Existing Enzootic Coronavirus Immunity, Brazil 2020.

Carnivores such as cats and minks are highly susceptible to SARS-CoV-2. Brazil is a global COVID-19 hot spot and several cases of human-to-cat transmission have been documented. We investigated the spread of SARS-CoV-2 by testing 547 domestic cats sampled between July-November 2020 from seven states in southern, southeastern, and northeastern Brazil. Moreover, we investigated whether immune responses elicited by enzootic coronaviruses affect SARS-CoV-2 infection in cats. We found infection with significantly higher neutralizing antibody titers against the Gamma variant of concern, endemic in Brazil during 2020, than against an early SARS-CoV-2 B.1 isolate (p<0.0001), validating the use of Gamma for further testing. The overall SARS-CoV-2 seroprevalence in Brazilian cats during late 2020 validated by plaque reduction neutralization test (PRNT90) was 7.3% (95% CI, 5.3-9.8). There was no significant difference in SARS-CoV-2 seroprevalence in cats between Brazilian states, suggesting homogeneous infection levels ranging from 4.6% (95% CI, 2.2-8.4) to 11.4% (95% CI, 6.7-17.4; p=0.4438). Seroprevalence of the prototypic cat coronavirus Feline coronavirus (FCoV) in a PRNT90 was high at 33.3% (95% CI, 24.9-42.5) and seroprevalence of Bovine coronavirus (BCoV) was low at 1.7% (95% CI, 0.2-5.9) in a PRNT90. Neutralizing antibody titers were significantly lower for FCoV than for SARS-CoV-2 (p=0.0001), consistent with relatively more recent infection of cats with SARS-CoV-2. Neither the magnitude of SARS-CoV-2 antibody titers (p=0.6390), nor SARS-CoV-2 infection status were affected by FCoV serostatus (p=0.8863). Our data suggest that pre-existing immunity against enzootic coronaviruses neither prevents, nor enhances SARS-CoV-2 infection in cats. High SARS-CoV-2 seroprevalence already during the first year of the pandemic substantiates frequent infection of domestic cats and raises concerns on potential SARS-CoV-2 mutations escaping human immunity upon spillback.

Self-prescribed Ivermectin use is associated with a lower rate of seroconversion in health care workers diagnosed with COVID, in a dose-dependent response.

BACKGROUND: Over-the-counter use of ivermectin amongst other drugs as SARS-CoV-2 treatment has been increasingly common, despite the lack of evidence on its clinical efficacy. OBJECTIVE: To evaluate the effect of ivermectin use on production of antibodies against SARS-CoV-2 in health care workers (HCW) diagnosed with COVID-19 and of Th1/Th2 cytokines by stimulated peripheral blood mononuclear cells of the same cohort (PBMCs). METHODS: This cross-sectional study evaluated seroconversion and neutralizing antibodies production in HCW at Complexo Hospitalar Universitário Professor Edgard Santos (Salvador, Brazil), diagnosed with COVID-19 from May to July, 2020, as well as in vitro production of antibody against SARS-CoV-2 and Th1/Th2 cytokines. Analyses were performed between December 2020 and February 2021. Participants were stratified according to the use of ivermectin (≤ 1 dose vs. multiple doses) for treatment of COVID-19. RESULTS: 45 HCW were included (62% women). Mean age was 39 years, and disease severity was similar across groups. Neutralizing antibodies were detected less frequently in multiple doses (70%) vs. ≤ 1 dose (97%) groups, p = 0.02). PBMCs of patients in multiple doses group also were less likely to produce antibodies against SARS-CoV-2 following in vitro stimulation with purified spike protein in comparison with patients in ≤ 1 dose group (p < 0.001). PBMC´s production of Th1/Th2 cytokines levels was similar across groups. Abdominal pain (15% vs 46%, p = 0.04), diarrhea (21% vs. 55%, p = 0.05) and taste perversion (0% vs. 18%, p = 0.05) were more frequently reported by participants that used multiple doses of ivermectin. CONCLUSIONS: Although there was no evidence for differential disease severity upon ivermectin use for treatment of COVID-19 it was associated with more gastro-intestinal side-effects and impairment of anti-SARS-CoV2 antibodies production, in a dose dependent manner. This potentially impacts the effectiveness of immune response and the risk of reinfection and warrants additional studies for clarifying the mechanisms and consequences of such immunomodulatory effects.

Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection

Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Although Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of respiratory specimens is the gold standard test for detection of SARS-CoV-2 infection, collecting nasopharyngeal swabs causes discomfort to patients and may represent considerable risk for healthcare workers. The use of saliva as a diagnostic sample has several advantages. Objectives The aim of this study was to validate the use of saliva as a biological sample for diagnosis of COVID-19. Methods This study was conducted at Infectious Diseases Research Laboratory (LAPI), in Salvador, Brazil. Participants presenting with signs/symptoms suggesting SARS-CoV-2 infection underwent a nasopharyngeal swab (NPS) and/or oropharyngeal swab (OPS), and saliva collection. Saliva samples were diluted in PBS, followed by RNA isolation and RT-Real Time PCR for SARS-CoV-2. Results of conventional vs saliva samples testing were compared. Statistical analyses were performed using Statistical Package for the Social Sciences software (SPSS) version 18.0. Results One hundred fifty-five participants were recruited and samples pairs of NPS/OPS and saliva were collected. The sensitivity and specificity of RT-PCR using saliva samples were 94.4% (95% CI 86.4-97.8) and 97.62% (95% CI 91.7-99.3), respectively. There was an overall high agreement (96.1%) between the two tests. Conclusions Use of self-collected saliva samples is an easy, convenient, and low-cost alternative to conventional NP swab-based molecular tests. These results may allow a broader use of molecular tests for management of COVID19 pandemic, especially in resources-limited settings.

Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Although Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of respiratory specimens is the gold standard test for detection of SARS-CoV-2 infection, collecting nasopharyngeal swabs causes discomfort to patients and may represent considerable risk for healthcare workers. The use of saliva as a diagnostic sample has several advantages. OBJECTIVES: The aim of this study was to validate the use of saliva as a biological sample for diagnosis of COVID-19. METHODS: This study was conducted at Infectious Diseases Research Laboratory (LAPI), in Salvador, Brazil. Participants presenting with signs/symptoms suggesting SARS-CoV-2 infection underwent a nasopharyngeal swab (NPS) and/or oropharyngeal swab (OPS), and saliva collection. Saliva samples were diluted in PBS, followed by RNA isolation and RT-Real Time PCR for SARS-CoV-2. Results of conventional vs saliva samples testing were compared. Statistical analyses were performed using Statistical Package for the Social Sciences software (SPSS) version 18.0. RESULTS: One hundred fifty-five participants were recruited and samples pairs of NPS/OPS and saliva were collected. The sensitivity and specificity of RT-PCR using saliva samples were 94.4% (95% CI 86.4-97.8) and 97.62% (95% CI 91.7-99.3), respectively. There was an overall high agreement (96.1%) between the two tests. CONCLUSIONS: Use of self-collected saliva samples is an easy, convenient, and low-cost alternative to conventional NP swab-based molecular tests. These results may allow a broader use of molecular tests for management of COVID19 pandemic, especially in resources-limited settings.