SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains (preprint)

Introduction- The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods- We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested by RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results- A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

Online symptoms screening and testing of Covid-19 through RT-LAMP saliva of students and asymptomatic employees in a public school in Brazil. (preprint)

Objective To evaluate a respiratory symptoms screening system and test asymptomatic individuals (high school students and employees) at a public school in Brazil.Methods SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) of saliva samples was performed weekly.Results A total of 969 samples were tested (mean of 108 tests per week). No asymptomatic participants tested positive for COVID-19, and no cases of COVID-19 transmission occurred at school despite the high transmissibility of the Delta variant, the local predominant variant at the time of the study.Conclusion Implementation of an online system of respiratory symptom screening and testing of COVID-19 through saliva in asymptomatic individuals is a feasible, low-cost and practical option to be used, especially in low-income countries.

Clinical Characteristics of COVID-19 by SARS-CoV-2 Gamma Variant in Vaccinated and Non-Vaccinated Healthcare Workers (preprint)

Background: The Gamma variant has been considered the predominant SARS-CoV-2 lineage in Brazil during the first half of 2021. We aimed to characterise the clinical presentation of COVID-19 caused by the Gamma variant in comparison with strains that are not variants of concern (non-VoC).Method: We performed a prospective cohort study including symptomatic COVID-19 cases among healthcare workers from January 22 to May 15, 2021. Positive samples for SARS-CoV-2 RT-PCR underwent whole genome sequencing. COVID-19 symptoms, caused by the Gamma variant or non-VoC, and risk factors for Gamma variant infection were evaluated using multiple logistic regression analyses.Findings: We included 423 COVID-19 cases, of which 415 (98%) with mild disease. One hundred and seventy-five (41%) patients had been fully immunised, of which 173/175 (99%) had received CoronaVac. There were 313 (74%) Gamma variant cases and 110 (26%) non-VoC cases. Hyposmia/anosmia and dysgeusia were present in 129 (30%) and 108 (26%) of cases, respectively. Lower frequencies of hyposmia/anosmia (OR=0.304, p <0.001) and dysgeusia (OR=0.385, p =0.011) were the only symptoms significantly associated with Gamma variant infection. COVID-19 immunisation, previous COVID-19 and age were not associated with Gamma variant infection.Interpretation: The increase in Gamma variant cases should raise the awareness that COVID-19 may present more often with cold-like symptoms because of a decreased frequency of hyposmia/anosmia and dysgeusia.Funding: Supported by the Itau Unibanco “Todos pela saúde” program”.Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Hospital’s Ethics Committee (CAAE: 42708721.0.0000.0068).

A novel RT-LAMP workflow for rapid salivary diagnostics of COVID-19 and effects of age, gender and time from symptom onset (preprint)

Objectives: Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP workflow for viral detection in saliva, and to provide more information regarding its potential in COVID-19 diagnostics. Methods: Clinical and contrived specimens were used to screen/optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate clinical performance (n = 90) and to characterize saliva based on age, gender and time from onset of symptoms (n = 49). Results: The devised workflow achieved 93.2% sensitivity, 97% specificity, and 0.895 Kappa for salivas containing >102 copies/L. Further analyses in saliva showed peak viral load in the first days of symptoms and lower viral loads in females, particularly among young individuals (<38 years). NOP RT-PCR data did not yield relevant associations. Conclusions: This novel saliva RT-LAMP workflow can be applied to point-of-care testing. This work reinforces that saliva better correlates with transmission dynamics than NOP specimens, and reveals gender differences that may reflect higher transmission by males. To maximize detection, testing should be done immediately after symptom onset, especially in females.

Skin imprints to provide noninvasive metabolic profiling of COVID-19 patients (preprint)

As the current COVID-19 pandemic progresses, more symptoms and signals related to how the disease manifests in the human body arise in the literature. Skin lesions and coagulopathies may be confounding factors on routine care and patient management. We analyzed the metabolic and lipidic profile of the skin from COVID-19 patients using imprints in silica plates as a non-invasive alternative, in order to better understand the biochemical disturbances caused by SARS-CoV-2 in the skin. One hundred and one patients (64 COVID-19 positive patients and 37 control patients) were enrolled in the study from April 2020 to June 2020 during the first wave of COVID-19 in Sao Paulo, Brazil. Fourteen biomarkers were identified related to COVID-19 infection (7 increased and 7 decreased in COVID-19 patients). Remarkably, oleamide has shown promising performance, providing 79.0% of sensitivity on a receiver operating characteristic curve model. Species related to coagulation and immune system maintenance such as phosphatidylserines were decreased in COVID-19 patients; on the other hand, cytokine storm and immunomodulation may be affected by molecules increased in the COVID-19 group, particularly primary fatty acid amides and N-acylethanolamines, which are part of the endocannabinoid system. Our results show that skin imprints may be a useful, noninvasive strategy for COVID-19 screening, by electing a pool of biomarkers with diagnostic potential.

Evaluating Burnout Among Health Workers Routinely Screened for SARS-CoV-2 (preprint)

Background: Healthcare workers (HW) are a vulnerable group to develop burnout during the COVID-19 pandemic. The aims of this study were to evaluate the perception of HW about the antibody test, and, secondarily, the prevalence of burnout and factors associated with burnout among HW who took the test.Methods: In this cross-sectional study, we evaluated burnout among HW in a 600-bed building entirely dedicated to COVID-19 inpatients care at Hospital das Clinicas (HC), located in São Paulo, Brazil. The HW answered an online questionnaire that included questions on burnout, a single-item scale based on the Maslach Burnout Inventory; demographic data, professional category, type of Protective Personal Equipment (PPE) used, distancing from social support; and emotional reactions to their serology result. Bivariate and multivariate analyses were done to evaluate the risk of burnout.Outcomes: Among 4,417 HW tested, 528 (12.0%) were positive for SARS-CoV-2 and 1,945 answered the questionnaire. Burnout was reported by 308 (15.8%); anxiety, tenseness, and depression associated with COVID-19 were reported by 344 (17.7%); 292 (15.1%); and 181(9.3%) of the participants, respectively. The risk factors for burnout were: being a physician [adjOR:1.604;(95%CI 1.604-1.080;p=0.019)]; a physiotherapist [adjOR:2.047;(95%CI:1.285–3.261;p=0.003)]; perceiving a decrease in public safety[adjOR:1.983;(95%CI:1.229–3.199;p=0.005)]; anxiety [adjOR:2.721;(95%CI:1.812–4.085;p=<0.001)], and depression associated with COVID-19[adjOR:2.071;(95%CI:1.308–3.279;p=0.002)]; and having negative feeling towards had a previously negative SARS-CoV-2 serology[adjOR:1,989;(95%CI:1.484-2.664; p<0.001)]. Interpretation: Routine serological testing was one of the strategies used in our hospital to promote the well-being of HW. We observed that those who had negative feeling regarding testing negative to COVID-19 in previous serologies were at higher risk of burnout, suggesting that the risk of contracting the disease is a major stressor for HW.Funding Statement: Internal funding from the Hospital das Clínicas of University of São Paulo, Brazil trough donation from Banco Pactual.Declaration of Interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: grant funding from the Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.Ethics Approval Statement: This study was approved by the Hospital das Clinicas da faculdade de Medicina da Universidade de Sao Paulo (protocol number 30701920200000068).

Prolonged presence of replication-competent SARS-CoV-2 in mildly symptomatic individuals: A Report of 2 Cases (preprint)

It has been estimated that individuals with COVID-19 can shed replication-competent virus up to a maximum of twenty days after initiation of symptoms. This report describes two patients with mild forms of the disease who shed replication-competent virus for 24 and 37 days, respectively, after symptom onset.

Evaluating burnout among health workers routinely screened for SARS-CoV-2. (preprint)

Background: Healthcare workers (HW) are a vulnerable group to develop burnout during the COVID-19 pandemic. The aims of this study were to evaluate the perception of HW about the antibody test, and, secondarily, the prevalence of burnout and factors associated with burnout among HW who took the test.Methods: In this cross-sectional study, we evaluated burnout among HW in a 600-bed building entirely dedicated to COVID-19 inpatients care at Hospital das Clinicas (HC), located in São Paulo, Brazil. The HW answered an online questionnaire that included questions on burnout, a single-item scale based on the Maslach Burnout Inventory; demographic data, professional category, type of Protective Personal Equipment (PPE) used, distancing from social support; and emotional reactions to their serology result. Bivariate and multivariate analyses were done to evaluate the risk of burnout.Outcomes: Among 4,417 HW tested, 528 (12.0%) were positive for SARS-CoV-2 and 1,945 answered the questionnaire. Burnout was reported by 308 (15.8%); anxiety, tenseness, and depression associated with COVID-19 were reported by 344 (17.7%); 292 (15.1%); and 181(9.3%) of the participants, respectively. The risk factors for burnout were: being a physician [adjOR:1.604;(95%CI 1.604-1.080;p=0.019)]; a physiotherapist [adjOR:2.047;(95%CI:1.285–3.261;p=0.003)]; perceiving a decrease in public safety[adjOR:1.983;(95%CI:1.229–3.199;p=0.005)]; anxiety [adjOR:2.721;(95%CI:1.812–4.085;p=<0.001)], and depression associated with COVID-19[adjOR:2.071;(95%CI:1.308–3.279;p=0.002)]; and having negative feeling towards had a previously negative SARS-CoV-2 serology[adjOR:1,989;(95%CI:1.484-2.664; p<0.001)].Interpretation: Routine serological testing was one of the strategies used in our hospital to promote the well-being of HW. We observed that those who had negative feeling regarding testing negative to COVID-19 in previous serologies were at higher risk of burnout, suggesting that the risk of contracting the disease is a major stressor for HW.

EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION (preprint)

Background: COVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population. Methods: Here we evaluate the sensitivity and specificity of eleven different immunochromatografic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests. Results: Our results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%. Conclusions: We found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.

Covid-19 automated diagnosis and risk assessment through Metabolomics and Machine-Learning (preprint)

COVID-19 is still placing a heavy health and financial burden worldwide. Impairments in patient screening and risk management play a fundamental role on how governments and authorities are directing resources, planning reopening, as well as sanitary countermeasures, especially in regions where poverty is a major component in the equation. An efficient diagnostic method must be highly accurate, while having a cost-effective profile. We combined a machine learning-based algorithm with instrumental analysis using mass spectrometry to create an expeditious platform that discriminate COVID-19 in plasma samples within minutes, while also providing tools for risk assessment, to assist healthcare professionals in patient management and decision-making. A cross-sectional study with 728 patients (369 confirmed COVID-19 and 359 controls) was enrolled from three Brazilian epicentres (Sao Paulo capital, Sao Paulo countryside and Manaus) in the months of April, May, June and July 2020. We were able to elect and identify 21 molecules that are related to the diseases pathophysiology and 26 features to patients health-related outcomes. With specificity >97% and sensitivity >83% from blinded data, this screening approach is understood as a tool with great potential for real-world application.

Evolution and epidemic spread of SARS-CoV-2 in Brazil (preprint)

Brazil currently has one of the fastest growing SARS-CoV-2 epidemics in the world. Due to limited available data, assessments of the impact of non-pharmaceutical interventions (NPIs) on virus transmission and epidemic spread remain challenging. We investigate the impact of NPIs in Brazil using epidemiological, mobility and genomic data. Mobility-driven transmission models for Sao Paulo and Rio de Janeiro cities show that the reproduction number (Rt) reached below 1 following NPIs but slowly increased to values between 1 to 1.3 (1.0 - -1.6). Genome sequencing of 427 new genomes and analysis of a geographically representative genomic dataset from 21 of the 27 Brazilian states identified >100 international introductions of SARS-CoV-2 in Brazil. We estimate that three clades introduced from Europe emerged between 22 and 27 February 2020, and were already well-established before the implementation of NPIs and travel bans. During this first phase of the epidemic establishment of SARS-CoV-2 in Brazil, we find that the virus spread mostly locally and within-state borders. Despite sharp decreases in national air travel during this period, we detected a 25% increase in the average distance travelled by air passengers during this time period. This coincided with the spread of SARS-CoV-2 from large urban centers to the rest of the country. In conclusion, our results shed light on the role of large and highly connected populated centres in the rapid ignition and establishment of SARS-CoV-2, and provide evidence that current interventions remain insufficient to keep virus transmission under control in Brazil.

Serial Interval Distribution of SARS-CoV-2 Infection in Brazil (preprint)

Using 65 transmission pairs of SARS-CoV-2 reported to the Brazilian Ministry of Health we estimate the mean and standard deviation for the serial interval to be 2.97 and 3.29 days respectively. We also present a model for the serial interval probability distribution using only two parameters.

First Case Of SAR-Coronavirus-2 Sequencing In The Cerebrospinal Fluid Of A Patient With Suspected CNS Demyelinating Disease (preprint)

The association between coronaviruses and central nervous system (CNS) demyelinating lesions has been previously shown. However, no case has been described of an association between the novel coronavirus (SARS-COV-2) and CNS demyelinating disease so far. SARS-COV-2 was previously detected in cerebrospinal fluid (CSF) sample of a patient with encephalitis. However, the virus identity was not confirmed by deep sequencing of SARS-COV-2 detected in the CSF. Here, we report a case of a patient with mild respiratory symptoms and neurological manifestations compatible with Clinically Isolated Syndrome. The viral genome of SARS-COV-2 was detected and sequenced in CSF with 99.74 to 100% similarity between the patient virus and worldwide sequences. This report suggests a possible association of SARS COV-2 infection with neurological symptoms of demyelinating disease, even in the absence of relevant upper respiratory tract infection signs.

Epidemiological and clinical characteristics of the early phase of the COVID-19 epidemic in Brazil (preprint)

BackgroundThe first case of COVID-19 was detected in Brazil on February 25, 2020. We report the epidemiological, demographic, and clinical findings for confirmed COVID-19 cases during the first month of the epidemic in Brazil. MethodsIndividual-level and aggregated COVID-19 data were analysed to investigate demographic profiles, socioeconomic drivers and age-sex structure of COVID-19 tested cases. Basic reproduction numbers (R0) were investigated for Sao Paulo and Rio de Janeiro. Multivariate logistic regression analyses were used to identify symptoms associated with confirmed cases and risk factors associated with hospitalization. Laboratory diagnosis for eight respiratory viruses were obtained for 2,429 cases. FindingsBy March 25, 1,468 confirmed cases were notified in Brazil, of whom 10% (147 of 1,468) were hospitalised. Of the cases acquired locally (77{middle dot}8%), two thirds (66{middle dot}9% of 5,746) were confirmed in private laboratories. Overall, positive association between higher per capita income and COVID-19 diagnosis was identified. The median age of detected cases was 39 years (IQR 30-53). The median R0 was 2{middle dot}9 for Sao Paulo and Rio de Janeiro. Cardiovascular disease/hypertension were associated with hospitalization. Co-circulation of six respiratory viruses, including influenza A and B and human rhinovirus was detected in low levels. InterpretationSocioeconomic disparity determines access to SARS-CoV-2 testing in Brazil. The lower median age of infection and hospitalization compared to other countries is expected due to a younger population structure. Enhanced surveillance of respiratory pathogens across socioeconomic statuses is essential to better understand and halt SARS-CoV-2 transmission. FundingSao Paulo Research Foundation, Medical Research Council, Wellcome Trust and Royal Society.