Evaluation of protective efficacy, serological responses, and cytokine modulation induced by polyvalent Leptospira vaccines in hamsters.

Whole-cell inactivated vaccines (bacterins) are the only licensed vaccines available for leptospirosis prevention and control, especially in domestic and farm animals. However, despite their widespread use, inconsistencies in their efficacy have been reported. Because immunity induced by bacterins is mainly mediated by antibodies against leptospiral lipopolysaccharides, the involvement of cellular responses is not well-known. The aim of this study was to investigate the efficacy and characterize the humoral and cellular immune responses induced by whole-cell inactivated leptospirosis bacterin formulations containing serovars Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjoprajitno, and Pomona. For the potency test, hamsters were immunized with one dose of polyvalent bacterins (either commercial or experimental) and then challenged with a virulent Pomona strain. Serological (MAT and IgM and IgG-ELISA) and cellular (cytokine transcription in blood evaluated by RT-qPCR) analyses were performed. The results revealed that vaccination with either bacterin formulation was able to protect 90-100% of the hamsters infected with the Pomona serovar, although most of the surviving animals remained as renal carriers. Specific agglutinating antibodies and significant levels of IgM, IgG, and IgG2 (P < 0.05) that were able to react with the six serovars present in the vaccine formulations were produced, indicating that the vaccines can potentially provide immunity against all strains. The protective immunity of these vaccines was mainly mediated by balanced a Th1/Th2 response, characterized by increased IFN-γ, IL-10 and IL-α transcription. These data support the importance of characterizing immunological responses involved in bacterin efficacy and investing in the improvement of these vaccine formulations.

Characterization of cellular immune response in hamsters immunized with recombinant vaccines against leptospirosis based on LipL32:LemA:LigAni chimeric protein.

In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.

An indirect ELISA to detect antibodies to the gC of bovine alphaherpesvirus 1 (BoAHV1) displaying no crossreactivity with antibodies induced by bovine alphaherpesvirus 5 (BoAHV5).

Seroprevalence of bovine alphaherpesvirus type 1 (BoAHV1) infections may be contaminated by crossreactive antibodies to bovine alphaherpesvirus type 5 (BoAHV5). To avoid such crossreactivity, an indirect enzyme-linked immunosorbent assay prepared with a recombinant glycoprotein C (gC) antigen (ELISA-gC1) was developed, aiming the detection of antibodies to BoAHV1, with no crossreactivity with BoAHV5 antibodies. The antigen for the ELISA-gC1 was the product of the expression of 219 bp from the N-terminal portion of the BoAHV1 gC gene, which bears low homology between the two virus types. The test was validated on 131 bovine serum samples, including 26 sera from BoAHV1-experimentally immunized, 38 sera from BoAHV5-experimentally infected or immunized calves, and 67 sera from calves seronegative for both BoAHV1 and BoAHV5, as determined by serum neutralization (SN). When compared to SN for BoAHV1, the ELISA-gC1 presented 100% sensitivity, 95.5 % specificity, 100 % negative predictive value, 89.6 % positive predictive value, 98.8 % precision, and a kappa correlation coefficient (κ) 0.95. None of the 38 BoAHV5-seropositive calves was detected by the ELISA-gC1. The ELISA-gC1 proved highly effective for the identification of BoAHV1-positive sera, with no crossreactivity with anti-BoAHV5 antibodies, thus able to distinguish serological responses from BoAHV1- and BoAHV5-seropositive cattle. Its capacity to detect BoAHV1-specific antibodies should allow the determination of the actual BoAHV1 prevalence in herds, which cannot be serologically determined in countries where BoAHV5 is also prevalent due to antibody crossreactivity. Apart from recognizing exclusively BoAHV1-infected cattle, the ELISA-gC1 may also be used in support of BoAHV5 epidemiological studies by allowing the exclusion of BoAHV1-seropositive animals.

Challenges and Strategies for Developing Recombinant Vaccines against Leptospirosis: Role of Expression Platforms and Adjuvants in Achieving Protective Efficacy.

The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.

Pathogenesis and Genomic Analysis of a Virulent Leptospira Interrogans Serovar Copenhageni Isolated from a Dog with Lethal Infection.

Dogs are highly susceptible to leptospirosis and are a public health concern due to their important role as a source of spreading disease, particularly in urban settings. In this study, we present the pathogenesis, serological characterization, and complete genome sequencing of a virulent Brazilian strain (NEG7) of L. interrogans serovar Copenhageni isolated from the urine of a dog that died due to acute leptospirosis. Clinical investigation showed that the dog was presented with icteric mucous membranes, weakness, dehydration, anorexia, and kidney and liver failures. Necropsy followed by histopathological evaluation revealed lesions compatible with liver and kidney leptospirosis. The leptospires recovered from the urine were further characterized by genome analysis, which confirmed that the isolate belonged to L. interrogans serogroup icterohaemorrhagiae serovar Copenhageni. Multiple bioinformatics tools were used to characterize the genomic features, and comparisons with other available Copenhageni strains were performed. Characterization based on absence of an INDEL in the gene lic12008, associated with phylogenetic and ANI (99.99% identity) analyses, confirmed the genetic relatedness of the isolate with L. interrogans serovar Copenhageni. A better understanding of the diversity of the pathogenic Leptospira isolates could help in identifying genotypes responsible for severe infections. Moreover, it can be used to develop control and prevention strategies for Leptospira serovars associated with particular animal reservoirs.

Challenges for the development of a universal vaccine against leptospirosis revealed by the evaluation of 22 vaccine candidates.

Leptospirosis is a neglected disease of man and animals that affects nearly half a million people annually and causes considerable economic losses. Current human vaccines are inactivated whole-cell preparations (bacterins) of Leptospira spp. that provide strong homologous protection yet fail to induce a cross-protective immune response. Yearly boosters are required, and serious side-effects are frequently reported so the vaccine is licensed for use in humans in only a handful of countries. Novel universal vaccines require identification of conserved surface-exposed epitopes of leptospiral antigens. Outer membrane ß-barrel proteins (ßb-OMPs) meet these requirements and have been successfully used as vaccines for other diseases. We report the evaluation of 22 constructs containing protein fragments from 33 leptospiral ßb-OMPs, previously identified by reverse and structural vaccinology and cell-surface immunoprecipitation. Three-dimensional structures for each leptospiral ßb-OMP were predicted by I-TASSER. The surface-exposed epitopes were predicted using NetMHCII 2.2 and BepiPred 2.0. Recombinant constructs containing regions from one or more ßb-OMPs were cloned and expressed in Escherichia coli. IMAC-purified recombinant proteins were adsorbed to an aluminium hydroxide adjuvant to produce the vaccine formulations. Hamsters (4-6 weeks old) were vaccinated with 2 doses containing 50 - 125 µg of recombinant protein, with a 14-day interval between doses. Immunoprotection was evaluated in the hamster model of leptospirosis against a homologous challenge (10 - 20× ED50) with L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. Of the vaccine formulations, 20/22 were immunogenic and induced significant humoral immune responses (IgG) prior to challenge. Four constructs induced significant protection (100%, P < 0.001) and sterilizing immunity in two independent experiments, however, this was not reproducible in subsequent evaluations (0 - 33.3% protection, P > 0.05). The lack of reproducibility seen in these challenge experiments and in other reports in the literature, together with the lack of immune correlates and commercially available reagents to characterize the immune response, suggest that the hamster may not be the ideal model for evaluation of leptospirosis vaccines and highlight the need for evaluation of alternative models, such as the mouse.

Tp0684, Tp0750, and Tp0792 Recombinant Proteins as Antigens for the Serodiagnosis of Syphilis.

The incidence of syphilis has increased alarmingly over the years. Its diagnosis continues to be a challenge, leading to the search for new alternative and effective methods. The objective of this study was to select and evaluate three Treponema pallidum recombinant proteins for potential use in syphilis serodiagnosis. Bioinformatics analysis was performed with three T. pallidum antigens (Tp0684, Tp0750, and Tp0792) to assess their physical, antigenic, and structural characteristics. The antigens were chemically synthesized, recombinant plasmids were expressed in Escherichia coli BL21 Star™ (DE3), and the recombinant proteins were purified by nickel affinity chromatography. The antigenicity of the recombinant proteins was evaluated by western blotting and enzyme-linked immunosorbent assay (ELISA), using the sera from patients with primary and latent syphilis. In silico analysis indicated the antigenic potential once the exposed B cell epitopes were detected in the evaluated proteins. Sera from patients with primary and latent syphilis specifically recognized rTp0684, rTp0750, and rTp0792 recombinant antigens. Moreover, the rTp0684-ELISA receiver operating characteristic (ROC) analysis showed an area under the ROC curve of 0.99, indicating high diagnostic efficacy with 97.62% specificity and 95% sensitivity. In conclusion, rTp0684 showed better potential as an antigen for the development of syphilis serodiagnosis. Thus, bioinformatic analysis can be an important tool to guide the selection of antigens for serological diagnosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01017-w.

Viral isolation allows characterization of early samples of SARS-CoV-2 lineage B1.1.33 with unique mutations (S: H655Y and T63N) circulating in Southern Brazil in 2020.

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.

Auxotrophic Mycobacterium bovis BCG: Updates and Perspectives.

Mycobacterium bovis BCG has been used for a century as the only licensed vaccine against tuberculosis. Owing to its strong adjuvant properties, BCG has also been employed as an oncological immunotherapeutic as well as a live vaccine vector against other pathogens. However, BCG vaccination has limited efficacy in protecting against adult forms of tuberculosis (TB), raises concerns about its safety in immunocompromised populations, compromises the diagnosis of TB through the tuberculin test and lacks predictability for successful antigen expression and immune responses to heterologous antigens. Together, these factors propelled the construction and evaluation of auxotrophic BCG strains. Auxotrophs of BCG have been developed from mutations in the genes required for their growth using different approaches and have shown the potential to provide a model to study M. tuberculosis, a more stable, safe, and effective alternative to BCG and a vector for the development of recombinant live vaccines, especially against HIV infection. In this review, we provide an overview of the strategies for developing and using the auxotrophic BCG strains in different scenarios.

Polymerase chain reaction and loop-mediated isothermal amplification targeting lic13162, lic20239, and lipL32 genes for leptospirosis diagnosis.

Leptospirosis is a zoonotic disease caused by pathogenic species of Leptospira. Due to the similarity with clinical signs of other febrile diseases, early diagnosis remains challenging. Real-time PCR has been used for direct detection of Leptospira, but it requires thermocyclers and highly trained personnel. Loop-mediated isothermal amplification (LAMP) is a simple and rapid DNA-based assay. Therefore, here we have developed PCR and LAMP assays targeting two novel genes, lic13162 and lic20239, and also lipL32 gene to detect pathogenic Leptospira. Analytical and diagnostic performances were compared with bacterial isolates (including different Leptospira species and serovars) and clinical samples. The results demonstrated that PCR assays targeting lic13162 and lic20239 were successful to amplify Leptospira, but LAMP not. However, both PCR and LAMP targeting lipL32 could detect pathogenic Leptospira. LAMP lipL32 could be performed in 30 min with a detection limit of 156 cells/mL. Diagnostic performance of lipL32-LAMP presented 84.2% sensitivity and 93.2% specificity. In conclusion, lipL32 PCR and LAMP are effective methods to detect pathogenic Leptospira directly from clinical samples.

Development of ELISA Using Recombinant Proteins for the Diagnosis of Mycoplasma hyopneumoniae Infection.

In order to develop a more sensitive and reliable method for detection of serum antibodies against Mycoplasma hyopneumoniae infection in pigs, six recombinant proteins of M. hyopneumoniae (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of M. hyopneumoniae and 246 from farm with clinical signs of enzootic pneumonia and positive serology for M. hyopneumoniae) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against M. hyopneumoniae. Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.

Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis.

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection.

Development of Human Recombinant Leptospirosis Vaccines.

Leptospirosis is a bacterial zoonotic disease with significant impact on health all over the world. Currently, bacterins are the only vaccines available for prevention of this disease, despite several drawbacks. In an effort to develop a more effective vaccine against leptospirosis, reverse and structural vaccinology have been applied to design recombinant constructions composed of leptospiral surface-exposed antigens. Herein, we describe a protocol for design and development of Leptospirosis recombinant vaccines using immunoinformatic approaches.

TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis.

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires that are exposed on the bacterial surface and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/PpAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (p < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination. KEY POINTS: • Predicted surface-exposed epitopes were identified in three leptospiral TBDRs. • An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed. • Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.

Population-level seropositivity trend for SARS-Cov-2 in Rio Grande do Sul, Brazil.

OBJECTIVE: To describe the evolution of seropositivity in the State of Rio Grande do Sul, Brazil, through 10 consecutive surveys conducted between April 2020 and April 2021. METHODS: Nine cities covering all regions of the State were studied, 500 households in each city. One resident in each household was randomly selected for testing. In survey rounds 1-8 we used the rapid WONDFO SARS-CoV-2 Antibody Test (Wondfo Biotech Co., Guangzhou, China). In rounds 9-10, we used a direct ELISA test that identifies IgG to the viral S protein (S-UFRJ). In terms of social distancing, individuals were asked three questions, from which we generated an exposure score using principal components analysis. RESULTS: Antibody prevalence in early April 2020 was 0.07%, increasing to 10.0% in February 2021, and to 18.2% in April 2021. In round 10, self-reported whites showed the lowest seroprevalence (17.3%), while indigenous individuals presented the highest (44.4%). Seropositivity increased by 40% when comparing the most with the least exposed. CONCLUSIONS: The proportion of the population already infected by SARS-Cov-2 in the state is still far from any perspective of herd immunity and the infection affects population groups in very different levels.

Protective efficacy of whole-cell inactivated Leptospira vaccines made using virulent or avirulent strains in a hamster model.

Whole-cell inactivated vaccines remain the only licensed vaccines used to control human and animal leptospirosis worldwide. Although they are protective against lethal infections, the efficacy of these vaccines has been divergent. The manufacturing process often involves the use of standard bacterial strains subjected to serial in vitro passages, with a risk of loss of virulence, and may affect the immunogenicity and consequently decrease protection. Thus, the objective of this study was to perform a comparative analysis of the efficacy of in-house bacterins produced with standard (avirulent) and virulent strains. Hamsters were immunized with killed bacteria produced using avirulent and virulent strains of L. interrogans serovars Copenhageni and Canicola. Vaccine efficacy was determined in terms of protection against lethal homologous or heterologous challenges. The results showed that immunization with both avirulent and virulent Canicola strains resulted in 100% protection against homologous challenge. Conversely, Copenhageni bacterins produced using an avirulent strain conferred only 25-37.5% protection against homologous challenge (P > 0.05), while virulent Copenhageni bacterin conferred 100% protection (P < 0.001). A single vaccine dose was sufficient to induce protection, and administration of a prime boost significantly reduced the bacterial load in the kidneys and improved the humoral immune response to the virulent Copenhageni strain. These findings suggest that the maintenance of virulent strains in bacterin formulations is essential for improving the immunogenicity and efficacy of leptospirosis vaccines.

Time-dependent decay of detectable antibodies against SARS-CoV-2: A comparison of ELISA with two batches of a lateral-flow test.

BACKGROUND: Large-scale epidemiological studies of seroprevalence of antibodies against SARS-CoV-2 often rely on point-of-care tests that provide immediate results to participants. Yet, little is known on how long rapid tests remain positive after the COVID-19 episode, or how much variability exists across different brands and even among batches of the same test. METHODS: In November 2020, we assessed the sensitivity of three tests applied to 133 individuals with a previous positive PCR result between April and October. All subjects provided finger prick blood samples for two batches (A and B) of the Wondfo lateral-flow IgG/IgM test, and dried blood spot samples for the S-UFRJ ELISA test. RESULTS: Overall sensitivity levels were 92.5% (95% CI 86.6-96.3), 63.2% (95% CI 54.4-71.4) and 33.8% (95% CI 25.9-42.5) for the S-UFRJ test, Wondfo A and Wondfo B tests, respectively. There was no evidence of a decline in the positivity of S-UFRJ with time since the diagnosis, but the two Wondfo batches showed sharp reductions to as low as 41.9% and 19.4%, respectively, for subjects with a positive PCR in June or earlier. Positive results for batch B of the rapid test were 35% to 54% lower than for batch A at any given month of diagnosis. INTERPRETATION: Whereas the ELISA test showed high sensitivity and stability of results over the five months of the study, both batches of the rapid test showed substantial declines, with one of the batches consistently showing lower sensitivity levels than the other. ELISA tests based on dried-blood spots are an inexpensive alternative to rapid lateral-flow tests in large-scale epidemiological studies. FUNDING: The study was funded by the "Todos Pela Saúde" initiative, Instituto Serrapilheira, Brazilian Ministry of Health, Brazilian Collective Health Association (ABRASCO) and the JBS S.A. initiative 'Fazer o Bem Faz Bem'.

Slow Spread of SARS-CoV-2 in Southern Brazil Over a 6-Month Period: Report on 8 Sequential Statewide Serological Surveys Including 35 611 Participants.

Objectives. To evaluate the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) over 6 months in the Brazilian State of Rio Grande do Sul (population 11.3 million), based on 8 serological surveys. Methods. In each survey, 4151 participants in round 1 and 4460 participants in round 2 were randomly sampled from all state regions. We assessed presence of antibodies against SARS-CoV-2 using a validated lateral flow point-of-care test; we adjusted figures for the time-dependent decay of antibodies. Results. The SARS-CoV-2 antibody prevalence increased from 0.03% (95% confidence interval [CI] = 0.00%, 0.34%; 1 in every 3333 individuals) in mid-April to 1.89% (95% CI = 1.36%, 2.54%; 1 in every 53 individuals) in early September. Prevalence was similar across gender and skin color categories. Older adults were less likely to be infected than younger participants. The proportion of the population who reported leaving home daily increased from 21.4% (95% CI = 20.2%, 22.7%) to 33.2% (95% CI = 31.8%, 34.5%). Conclusions. SARS-CoV-2 infection increased slowly during the first 6 months in the state, differently from what was observed in other Brazilian regions. Future survey rounds will continue to document the spread of the pandemic.

High prevalence of symptoms among Brazilian subjects with antibodies against SARS-CoV-2.

Since the beginning of the pandemic of COVID-19, there has been a widespread assumption that most infected persons are asymptomatic. Using data from the recent wave of the EPICOVID19 study, a nationwide household-based survey including 133 cities from all states of Brazil, we estimated the proportion of people with and without antibodies for SARS-CoV-2 who were asymptomatic, which symptoms were most frequently reported, number of symptoms and the association with socio-demographic characteristics. We tested 33,205 subjects using a rapid antibody test previously validated. Information was collected before participants received the test result. Out of 849 (2.7%) participants positive for SARS-CoV-2 antibodies, only 12.1% (95% CI 10.1-14.5) reported no symptoms, compared to 42.2% (95% CI 41.7-42.8) among those negative. The largest difference between the two groups was observed for changes in smell/taste (56.5% versus 9.1%, a 6.2-fold difference). Changes in smell/taste, fever and body aches were most likely to predict positive tests as suggested by recursive partitioning tree analysis. Among individuals without any of these three symptoms, only 0.8% tested positive, compared to 18.3% of those with both fever and changes in smell or taste. Most subjects with antibodies against SARS-CoV-2 are symptomatic, even though most present only mild symptoms.