Validation of Serological Methods for COVID-19 and Retrospective Screening of Health Employees and Visitors to the São Paulo University Hospital, Brazil.

Reliable serological tests for the detection of SARS-CoV-2 antibodies among infected or vaccinated individuals are important for epidemiological and clinical studies. Low-cost approaches easily adaptable to high throughput screenings, such as Enzyme-Linked Immunosorbent Assays (ELISA) or electrochemiluminescence immunoassay (ECLIA), can be readily validated using different SARS-CoV-2 antigens. A total of 1,119 serum samples collected between March and July of 2020 from health employees and visitors to the University Hospital at the University of São Paulo were screened with the Elecsys® Anti-SARS-CoV-2 immunoassay (Elecsys) (Roche Diagnostics) and three in-house ELISAs that are based on different antigens: the Nucleoprotein (N-ELISA), the Receptor Binding Domain (RBD-ELISA), and a portion of the S1 protein (ΔS1-ELISA). Virus neutralization test (CPE-VNT) was used as the gold standard to validate the serological assays. We observed high sensitivity and specificity values with the Elecsys (96.92% and 98.78%, respectively) and N-ELISA (93.94% and 94.40%, respectively), compared with RBD-ELISA (90.91% sensitivity and 88.80% specificity) and the ΔS1-ELISA (77.27% sensitivity and 76% specificity). The Elecsys® proved to be a reliable SARS-CoV-2 serological test. Similarly, the recombinant SARS-CoV-2 N protein displayed good performance in the ELISA tests. The availability of reliable diagnostic tests is critical for the precise determination of infection rates, particularly in countries with high SARS-CoV-2 infection rates, such as Brazil. Collectively, our results indicate that the development and validation of new serological tests based on recombinant proteins may provide new alternatives for the SARS-CoV-2 diagnostic market.

Surrogate test performance for SARS-CoV-2 neutralizing antibodies (nAbs) for convalescent plasma (CCP): How useful could they be?

BACKGROUND: COVID-19 high-titer CCP selection is a concern, because neutralizing antibody (nAb) testing requires sophisticated labs and methods. Surrogate tests are an alternative for measuring nAb levels in plasma bags, including those that are pathogen-reduced. STUDY DESIGN/METHODS: We studied a panel consisting of 191 samples from convalescent donors tested by nAb (CPE-VNT), obtained from 180 CCP donations (collection: March 20-January 21) and 11 negative controls, with a total of 80 and 111 serum and plasma samples (71 amotosalen/UV treated), with nAb titers ranging from negative to 10,240. Samples were blindly tested for several surrogates: one anti-RBD, two anti-spike, and four anti-nucleocapsid tests, either isolated or combined to improve their positive predictive values as predictors of the presence of high-titer nAbs, defined as those with titers ≥160. RESULTS: Except for combined and anti-IgA/M tests, all isolated surrogate tests showed excellent performance for nAb detection: sensitivity (98.3%-100%), specificity (85.7%-100%), PPV (98.9%-100%), NPV (81.3%-100%), and AUC (0.93-0.96), with a variable decrease in sensitivity and considerably lower specificity when using FDA authorization and concomitant nAb titers ≥160. All surrogates had AUCs that were statistically different from CPE-VNT if nAb≥160, including when using combined, orthogonal approaches. CONCLUSIONS: Surrogate tests (isolated or in combination) have an indirect good performance in detecting the presence of nAb, with lower sensitivity and specificity when high nAb titer samples are used, possibly accepting a considerable number of donors whose nAb titers are actually low, which should be evaluated by each laboratory responsible for CCP collection.

Anti-A and SARS-CoV-2: an intriguing association.

BACKGROUND: Blood groups and anti-A isohemagglutinin may be involved in susceptibility to SARS-CoV-2 infection. MATERIALS AND METHODS: We retrospectively studied 268 COVID-19 convalescent plasma donors and 162 COVID-19 inpatients (total 430 subjects, confirmed by RT-PCR) and 2,212 healthy volunteer first-time blood donors as a control group. These were further divided into two groups: those with anti-A (blood types O and B) and those without it (types A and AB). Titres of nucleoproteins, and neutralizing SARS-CoV-2 antibody were measured in the convalescent plasma donors and inpatients. Multivariate logistic regression and non-parametric tests were applied. RESULTS: Persons having types O or B showed less infection prevalence than those of types A or AB (OR = 0·62, 95% CI 0·50-0·78; P < 0·001), but there was no difference when COVID-19 inpatients were analysed. Immunoglobulins M, G and A were lower in COVID-19 subjects of types O or B group than those of A or AB (0·16 vs. 0·19; P = 0·03, 2·11 vs. 2·55; P = 0·02, 0·23 vs. 0·32; P = 0·03, respectively). CONCLUSION: In this retrospective cohort, COVID-19 individuals were less likely to belong to blood types O and B, and also had lower SARS-CoV-2 antibody titres than A and AB individuals. COVID-19 severity did not associate with the blood groups.

In Nasal Mucosal Secretions, Distinct IFN and IgA Responses Are Found in Severe and Mild SARS-CoV-2 Infection.

Likely as in other viral respiratory diseases, SARS-CoV-2 elicit a local immune response, which includes production and releasing of both cytokines and secretory immunoglobulin (SIgA). Therefore, in this study, we investigated the levels of specific-SIgA for SARS-CoV-2 and cytokines in the airways mucosa 37 patients who were suspected of COVID-19. According to the RT-PCR results, the patients were separated into three groups: negative for COVID-19 and other viruses (NEGS, n = 5); negative for COVID-19 but positive for the presence of other viruses (OTHERS, n = 5); and the positive for COVID-19 (COVID-19, n = 27). Higher specific-SIgA for SARS-CoV-2, IFN-ß, and IFN-γ were found in the COVID-19 group than in the other groups. Increased IL-12p70 levels were observed in OTHERS group as compared to COVID-19 group. When the COVID-19 group was sub stratified according to the illness severity, significant differences and correlations were found for the same parameters described above comparing severe COVID-19 to the mild COVID-19 group and other non-COVID-19 groups. For the first time, significant differences are shown in the airway's mucosa immune responses in different groups of patients with or without respiratory SARS-CoV-2 infection.

Natural versus Recombinant Viral Antigens in SARS-CoV-2 Serology: Challenges in Optimizing Laboratory Diagnosis of COVID-19

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection;the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA;however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

Natural versus Recombinant Viral Antigens in SARS-CoV-2 Serology: Challenges in Optimizing Laboratory Diagnosis of COVID-19

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection;the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA;however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

High prevalence of SARS-CoV-2 infection among symptomatic healthcare workers in a large university tertiary hospital in São Paulo, Brazil.

BACKGROUND: Brazil became the epicenter of the COVID-19 pandemic in Latin America since May 2020, reporting the highest number of cases and deaths in the region. Healthcare workers (HCWs) are at increased risk of SARS-CoV-2 infection, experiencing a significant burden from COVID-19. Identifying and understanding the clinical characteristics and risk factors associated with infection are of paramount importance to inform screening strategies and infection control practices in this scenario. The aims of this study were to investigate the prevalence and clinical characteristics of HCWs with COVID-19 symptoms. METHODS: Between March 21st and May 22nd, 2020 a cross-sectional study was performed in a tertiary university hospital in São Paulo. Prevalence of SARS-CoV-2 infection among HCWs with COVID-19 symptoms was determined by RT-PCR testing on nasopharyngeal and oropharyngeal samples. Participants were asked to complete an electronic structured questionnaire including clinical and demographic data. RESULTS: Overall, 125 (42.37%) of 295 symptomatic HCWs tested positive for SARS-CoV-2. Over the 10-week study period, positivity rates varied from 22.2% (95% CI 15.9-60.3%) in the second week to 55.9% (95% CI 43.2-68.6%) in the sixth week, reaching a plateau (38-46%) thereafter. Median (SD) age was 34.2 (9.9) years and 205 (69.5%) were female. We did not find significant differences in the prevalence of the most commonly reported underlying medical condition among healthcare workers that tested positive or negative for SARS-CoV-2 infection. After multivariable analysis, using logistic regression, anosmia (adjusted OR 4.4 95% CI 2.21-8.74) and ocular pain (adjusted OR 1.95 95% CI 1.14-3.33) were the only symptoms independently associated with positivity for SARS-CoV-2 infection. Follow-up information on clinical outcomes showed that 9 (7.2%) HCWs were hospitalized (seven were male) and 2 (1.6%) died. CONCLUSIONS: The findings of this study confirmed the high burden of SARS-CoV-2 infection among healthcare workers in the hardest hit city by the pandemic in Latin America. Anosmia and ocular pain were symptoms independently associated with COVID-19 diagnosis. In low and middle-income countries, where limited availability of tests is frequent, these findings may contribute to optimize a targeted symptom-oriented screening strategy.

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES: The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION: We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.

Multisystem Inflammatory Syndrome Associated With COVID-19 With Neurologic Manifestations in a Child: A Brief Report.

Although first considered a benign infection, recent studies have disclosed severe and potentially lethal inflammatory manifestations of COVID-19 in children. We report the case of a 4-year-old child with a post-infectious multisystem inflammatory syndrome associated with COVID-19, with a Kawasaki-like shock and prominent neurologic features, for whom a cytokine storm and reduced brain-derived neurotrophic factor were well documented.

Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR.

In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. E gene primers previously determined for TaqMan assays by Colman et al. (2020) were adapted in SYBR Green assay and RT-PCR conventional. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. The conventional PCR detected until 107 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. Both of the proposed assays are less sensitive than the current gold standard; however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis.