Isolated specific IgA against respiratory viruses, Influenza or SARS-CoV-2, present in the saliva of a fraction of healthy and asymptomatic volunteers.

OBJECTIVES: Defense against respiratory viruses depends on an immune response present in the mucosa, as saliva IgA secretes antibodies. During the pandemic, such as influenza or SARS-CoV-2, most infected patients are asymptomatic but retain specific antibodies post-infection. The authors evaluated IgG and IgA antibodies against SARS-CoV-2 and influenza in the saliva of asymptomatic volunteers, validated with controls or vaccinated individuals. METHODS: The authors detected specific antibodies by validated conventional ELISA using natural SARS-CoV-2 antigens from infected Vero cells or capture-ELISA for influenza using natural antigens of the influenza vaccine. RESULTS: Saliva from influenza-vaccinated individuals had more IgA than paired serum, contrary to the findings for specific IgG. In COVID-19-vaccinated samples, specific IgA in saliva increased after vaccination, but IgG levels were high after the first dose. In saliva from the asymptomatic population (226), anti-Influenza IgG was found in 57.5% (130) of samples, higher than IgA, found in 35% (79) of samples. IgA results were similar for SARS-CoV-2, with IgA present in 30% (68) of samples, while IgG was less present, in 44.2% (100) of samples. The proportion of influenza IgG responders was higher than that for SARS-CoV-2 IgG, but both populations presented similar proportions of IgA responders, possibly due to variable memory B cell survival. For both viruses, the authors found an important proportion (> 10%) of IgA+IgG- samples, suggesting the occurrence of humoral immunity directed to the mucosa. CONCLUSION: Specific antibodies for respiratory viruses in saliva are found in either infection or vaccination and are a convenient and sensitive diagnostic tool for host immune response.

Beneficial effects of a mouthwash containing an antiviral phthalocyanine derivative on the length of hospital stay for COVID-19: randomised trial.

The risk of contamination and dissemination by SARS-CoV-2 has a strong link with nasal, oral and pharyngeal cavities. Recently, our research group observed the promising performance of an anionic phthalocyanine derivative (APD) used in a mouthwash protocol without photoexcitation; this protocol improved the general clinical condition of patients infected with SARS-CoV-2. The present two-arm study evaluated in vitro the antiviral activity and cytotoxicity of APD. Additionally, a triple-blind randomized controlled trial was conducted with 41 hospitalized patients who tested positive for COVID-19. All the included patients received World Health Organization standard care hospital treatment (non-intensive care) plus active mouthwash (experimental group AM/n = 20) or nonactive mouthwash (control group NAM/n = 21). The adjunct mouthwash intervention protocol used in both groups consisted one-minute gargling/rinsing / 5 times/day until hospital discharge. Groups were compared considering age, number of comorbidities, duration of symptoms prior admission and length of hospital stay (LOS). The associations between group and sex, age range, presence of comorbidities, admission to Intensive care unit (ICU) and death were also evaluated. The in vitro evaluation demonstrated that APD compound was highly effective for reduction of SARS-CoV-2 viral load in the 1.0 mg/mL (99.96%) to 0.125 mg/mL (92.65%) range without causing cytotoxicity. Regarding the clinical trial, the median LOS of the AM group was significantly shortened (4 days) compared with that of the NAM group (7 days) (p = 0.0314). Additionally, gargling/rinsing with APD was very helpful in reducing the severity of symptoms (no ICU care was needed) compared to not gargling/rinsing with APD (28.6% of the patients in the NAM group needed ICU care, and 50% of this ICU subgroup passed way, p = 0.0207). This study indicated that the mechanical action of the protocol involving mouthwash containing a compound with antiviral effects against SARS-CoV-2 may reduce the symptoms of the patients and the spread of infection. The use of APD in a mouthwash as an adjuvant the hospital COVID-19 treatment presented no contraindication and reduced the hospital stay period.Trial registration: The clinical study was registered at REBEC-Brazilian Clinical Trial Register (RBR-58ftdj).

Lack of Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spillover in Free-Living Neotropical Non-Human Primates, Brazil.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of coronavirus disease 2019 (COVID-19), is responsible for the worst pandemic of the 21st century. Like all human coronaviruses, SARS-CoV-2 originated in a wildlife reservoir, most likely from bats. As SARS-CoV-2 has spread across the globe in humans, it has spilled over to infect a variety of non-human animal species in domestic, farm, and zoo settings. Additionally, a broad range of species, including one neotropical monkey, have proven to be susceptible to experimental infection with SARS-CoV-2. Together, these findings raise the specter of establishment of novel enzootic cycles of SARS-CoV-2. To assess the potential exposure of free-living non-human primates to SARS-CoV-2, we sampled 60 neotropical monkeys living in proximity to Manaus and São José do Rio Preto, two hotspots for COVID-19 in Brazil. Our molecular and serological tests detected no evidence of SAR-CoV-2 infection among these populations. While this result is reassuring, sustained surveillance efforts of wildlife living in close association with human populations is warranted, given the stochastic nature of spillover events and the enormous implications of SARS-CoV-2 spillover for human health.

COVID-19 convalescent plasma cohort study: Evaluation of the association between both donor and recipient neutralizing antibody titers and patient outcomes.

BACKGROUND: Current evidence regarding COVID-19 convalescent plasma (CCP) transfusion practices is limited and heterogeneous. We aimed to determine the impact of the use of CCP transfusion in patients with previous circulating neutralizing antibodies (nAbs) in COVID-19. METHODS: Prospective cohort including 102 patients with COVID-19 transfused with ABO compatible CCP on days 0-2 after enrollment. Clinical status of patients was assessed using the adapted World Health Organization (WHO) ordinal scale on days 0, 5, and 14. The nAbs titration was performed using the cytopathic effect-based virus neutralization test with SARS-CoV-2 (GenBank MT126808.1). The primary outcome was clinical improvement on day 14, defined as a reduction of at least two points on the adapted WHO ordinal scale. Secondary outcomes were the number of intensive care unit (ICU)-free days and the number of invasive mechanical ventilation-free days. RESULTS: Both nAbs of CCP units transfused (p < 0.001) and nAbs of patients before CCP transfusions (p = 0.028) were associated with clinical improvements by day 14. No significant associations between nAbs of patients or CCP units transfused were observed in the number of ICU or mechanical ventilation-free days. Administration of CCP units after 10 days of symptom onset resulted in a decrease in ICU-free days (p < 0.001) and mechanical ventilation-free days (p < 0.001). CONCLUSION: Transfusion of high titer nAbs CCP units may be a determinant in clinical strategies against COVID-19. We consider these data as useful parameters to guide future CCP transfusion practices.

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.

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.

Correlation between SARS-COV-2 antibody screening by immunoassay and neutralizing antibody testing.

BACKGROUND: The efficacy of convalescent plasma (CP), an alternative for the treatment of COVID-19, depends on high titers of neutralizing antibodies (nAbs), but assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clinical characteristics of CP donors. STUDY DESIGN AND METHODS: A total of 214 CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies (IgG) using two commercial immunoassays: EUROIMMUN (ELISA) and Abbott (Chemiluminescence). Quantification of nAbs was performed using the Cytopathic Effect-based Virus Neutralization test. Three criteria for identifying donors with nAbs ≥ 1:160 were tested: - C1: Curve ROC; - C2: Conditional decision tree considering only the IA results and - C3: Conditional decision tree including both the IA results and the clinical variables. RESULTS: The performance of the immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers ≥1:160. Regarding the studied criteria for identifying CP donors with high nAbs titers: (a) C1 showed 76.1% accuracy if S/CO = 4.65, (b) C2 presented 76.1% accuracy if S/CO ≥4.57 and (c) C3 had 71.6% accuracy if S/CO was ≥4.57 or if S/CO was between 2.68-4.57 and the last COVID-19-related symptoms were recent (within 19 days). CONCLUSION: SARS-CoV-2 IgG immunoassays (S/CO) can be used to predict high anti-SARS-CoV-2 nAbs titers. This study has proposed different criteria for identifying donors with ≥1:160 nAbs titers, all with high efficacy.

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.

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.