Potential in vitro anti-periodontopathogenic, anti-Chikungunya activities and in vivo toxicity of Brazilian red propolis.

Bacterial and viral infections are serious public health issue. Therefore, this study aimed to evaluate the antibacterial, antibiofilm and antiviral potential of the Brazilian Red Propolis (BRP) crude hydroalcoholic extract, fractions, and isolated compounds, as well as their in vivo toxicity. The antibacterial activity was evaluated by determining the Minimum Inhibitory Concentration and the antibiofilm activity by determining the Minimum Inhibitory Concentration of Biofilm (MICB50). The viable bacteria count (Log10 UFC/mL) was also obtained. The antiviral assays were performed by infecting BHK-21 cells with Chikungunya (CHIKV) nanoluc. The toxicity of the BRP was evaluated in the Caenorhabditis elegans animal model. The MIC values for the crude hydroalcoholic extract sample ranged from 3.12 to 100 µg/mL, while fractions and isolated compounds the MIC values ranged from 1.56 to 400 µg/mL.The BRP crude hydroalcoholic extract, oblongifolin B, and gutiferone E presented MICB50 values ranging from 1.56 to 100 µg/mL against monospecies and multispecies biofilms. Neovestitol and vestitol inhibited CHIKV infection by 93.5 and 96.7%, respectively. The tests to evaluate toxicity in C. elegans demonstrated that the BRP was not toxic below the concentrations 750 µg/mL. The results constitute an alternative approach for treating various infectious diseases.

Impact of SARS-CoV-2 Gamma lineage introduction and COVID-19 vaccination on the epidemiological landscape of a Brazilian city.

Background: The emergence of the Brazilian variant of concern, Gamma lineage (P.1), impacted the epidemiological profile of COVID-19 cases due to its higher transmissibility rate and immune evasion ability. Methods: We sequenced 305 SARS-CoV-2 whole-genomes and performed phylogenetic analyses to identify introduction events and the circulating lineages. Additionally, we use epidemiological data of COVID-19 cases, severe cases, and deaths to measure the impact of vaccination coverage and mortality risk. Results: Here we show that Gamma introduction in São José do Rio Preto, São Paulo, Brazil, was followed by the displacement of seven circulating SARS-CoV-2 variants and a rapid increase in prevalence two months after its first detection in January 2021. Moreover, Gamma variant is associated with increased mortality risk and severity of COVID-19 cases in younger age groups, which corresponds to the unvaccinated population at the time. Conclusions: Our findings highlight the beneficial effects of vaccination indicated by a pronounced reduction of severe cases and deaths in immunized individuals, reinforcing the need for rapid and massive vaccination.

Frequency of carriers for rare recessive Mendelian diseases in a Brazilian cohort of 320 patients.

Several Mendelian disorders follow an autosomal recessive inheritance pattern. Epidemiological information on many inherited disorders may be useful to guide health policies for rare diseases, but it is often inadequate, particularly in developing countries. We aimed to calculate the carrier frequencies of rare autosomal recessive Mendelian diseases in a cohort of Brazilian patients using whole exome sequencing (WES). We reviewed the molecular findings of WES from 320 symptomatic patients who had carrier status for recessive diseases. Using the Hardy-Weinberg equation, we estimated recessive disease frequencies (q2 ) considering the respective carrier frequencies (2pq) observed in our study. We calculated the sensitivity of carrier screening tests based on lists of genes from five different clinical laboratories that offer them in Brazil. A total of 425 occurrences of 351 rare variants were reported in 278 different genes from 230 patients (71.9%). Almost half (48.8%) were carriers of at least one heterozygous pathogenic/likely pathogenic variant for rare metabolic disorders, while 25.9% of epilepsy, 18.1% of intellectual disabilities, 15.6% of skeletal disorders, 10.9% immune disorders, and 9.1% of hearing loss. We estimated that an average of 67% of the variants would not have been detected by carrier screening panels. The combined frequencies of autosomal recessive diseases were estimated to be 26.39/10,000 (or ~0.26%). This study shows the potential research utility of WES to determine carrier status, which may be a possible strategy to evaluate the clinical and social burden of recessive diseases at the population level and guide the optimization of carrier screening panels.

Diagnostic power and clinical impact of exome sequencing in a cohort of 500 patients with rare diseases.

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care.

Seroprevalence for dengue virus in a hyperendemic area and associated socioeconomic and demographic factors using a cross-sectional design and a geostatistical approach, state of São Paulo, Brazil.

BACKGROUND: São José do Rio Preto is one of the cities of the state of São Paulo, Brazil, that is hyperendemic for dengue, with the presence of the four dengue serotypes. OBJECTIVES: to calculate dengue seroprevalence in a neighbourhood of São José do Rio Preto and identify if socioeconomic and demographic covariates are associated with dengue seropositivity. METHODS: A cohort study to evaluate dengue seroprevalence and incidence and associated factors on people aged 10 years or older, was assembled in Vila Toninho neighbourhood, São José do Rio Preto. The participant enrolment occurred from October 2015 to March 2016 (the first wave of the cohort study), when blood samples were collected for serological test (ELISA IgG anti-DENV) and questionnaires were administrated on socio-demographic variables. We evaluated the data collected in this first wave using a cross-sectional design. We considered seropositive the participants that were positive in the serological test (seronegative otherwise). We modelled the seroprevalence with a logistic regression in a geostatistical approach. The Bayesian inference was made using integrated nested Laplace approximations (INLA) coupled with the Stochastic Partial Differential Equation method (SPDE). RESULTS: We found 986 seropositive individuals for DENV in 1322 individuals surveyed in the study area in the first wave of the cohort study, corresponding to a seroprevalence of 74.6% (95%CI: 72.2-76.9). Between the population that said never had dengue fever, 68.4% (566/828) were dengue seropositive. Older people, non-white and living in a house (instead of in an apartment), were positively associated with dengue seropositivity. We adjusted for the other socioeconomic and demographic covariates, and accounted for residual spatial dependence between observations, which was found to present up to 800 m. CONCLUSIONS: Only one in four people aged 10 years or older did not have contact with any of the serotypes of dengue virus in Vila Toninho neighbourhood in São José do Rio Preto. Age, race and type of house were associated with the occurrence of the disease. The use of INLA in a geostatistical approach in a Bayesian context allowed us to take into account the spatial dependence between the observations and identify the associated covariates to dengue seroprevalence.

Evidence of natural Zika virus infection in neotropical non-human primates in Brazil.

In Africa, Old World Primates are involved in the maintenance of sylvatic circulation of ZIKV. However, in Brazil, the hosts for the sylvatic cycle remain unknown. We hypothesized that free-living NHPs might play a role in urban/periurban ZIKV dynamics, thus we undertook an NHP ZIKV investigation in two cities in Brazil. We identified ZIKV-positive NHPs and sequences obtained were phylogenetically related to the American lineage of ZIKV. Additionally, we inoculated four C. penicillata with ZIKV and our results demonstrated that marmosets had a sustained viremia. The natural and experimental infection of NHPs with ZIKV, support the hypothesis that NHPs may be a vertebrate host in the maintainance of ZIKV transmission/circulation in urban tropical settings. Further studies are needed to understand the role they may play in maintaining the urban cycle of the ZIKV and how they may be a conduit in establishing an enzootic transmission cycle in tropical Latin America.

Molecular characterization of hepatitis C virus in end-stage renal disease patients under hemodialysis

New direct-acting antiviral (DAA) agents are in development or already approved for the treatment of chronic hepatitis C virus (HCV) infection. The effectiveness of these drugs is related to the previous existence of resistant variants. Certain clinical conditions can allow changes in immunological characteristics of the host and even modify genetic features of viral populations. The aim of this study was to perform HCV molecular characterization from samples of end-stage renal disease patients on hemodialysis (ESRD-HD). Nested PCR and Sanger sequencing were used to obtain genetic information from the NS5B partial region of a cohort composed by 86 treatment-naive patients. Genomic sequences from the Los Alamos databank were employed for comparative analysis. Bioinformatics methodologies such as phylogenetic reconstructions, informational entropy, and mutation analysis were used to analyze datasets separated by geographical location, HCV genotype, and renal function status. ESRD-HD patients presented HCV genotypes 1a (n=18), 1b (n=16), 2a (n=2), 2b (n=2), and 3a (n=4). Control subjects were infected with genotypes 1a (n=11), 1b (n=21), 2b (n=4), and 3a (n=8). Dataset phylogenetic reconstruction separated HCV subtype 1a into two distinct clades. The entropy analysis from the ESRD-HD group revealed two amino acid positions related to an epitope for cytotoxic T lymphocytes and T helper cells. Genotype 1a was found to be more diverse than subtype 1b. Also, genotype 1a ERSD-HD patients had a higher mean of amino acids changes in comparison to control group patients. The identification of specific mutations on epitopes and high genetic diversity within the NS5B HCV partial protein in hemodialysis patients can relate to host immunological features and geographical distribution patterns. This genetic diversity can affect directly the new DAA's resistance mechanisms.

Viral Load and Cytokine Response Profile Does Not Support Antibody-Dependent Enhancement in Dengue-Primed Zika Virus-Infected Patients.

Background: The pathogenesis of severe dengue disease involves immune components as biomarkers. The mechanism by which some dengue virus (DENV)-infected individuals progress to severe disease is poorly understood. Most studies on the pathogenesis of severe dengue disease focus on the process of antibody-dependent enhancement (ADE) as a primary risk factor. With the circulation of Zika virus (ZIKV) in DENV-endemic areas, many people infected by ZIKV were likely exposed to DENV. The influence of such exposure on Zika disease outcomes remains unknown. Methods: We investigated whether patients previously exposed to DENV exhibited higher viremia when exposed to a subsequent, heterologous dengue or Zika infection than those patients not previously exposed to dengue. We measured viral loads and cytokine profile during patients' acute infections. Results: Neither dengue nor Zika viremia was higher in patients with prior DENV infection, although the power to detect such a difference was only adequate in the ZIKV analysis. Of the 10 cytokines measured, only 1 significant difference was detected: Levels of interleukin 1ß (IL-1ß) were lower in dengue-infected patients who had experienced a previous dengue infection than patients infected with dengue for the first time. However, power to detect differences between groups was low. In Zika-infected patients, levels of IL-1ß showed a significant, positive correlation with viral load. Conclusions: No signs of ADE were observed in vivo in patients with acute ZIKV infection who had prior exposure to DENV.

Evidence of bottleneck effect on hepatitis C virus transmission between a couple under interferon based therapy.

Issues on the correlation of viral genetic diversity and treatment response to the hepatitis C infection remain uncertain. The bottleneck effect dictates the characteristics of the viral population that will establish the infection in a new host and is related to how the immune system and treatment will be effective against the virus. Here we evaluated the phylogenetic characteristics of quasispecies population and the treatment response pattern of a HCV infected couple. We also analyzed whether the viral population of these patients indicated that they were exposed to the same source for primer infection. This study included two patients (P10 and P11) HCV genotype 1b infected. The couple presented horizontal transmission. Viral RNA was isolated from serum samples collected before, during and after treatment, at specific time points. The HCV NS5A gene sequence was amplified, cloned and sequenced. Genetic and evolutionary analyses were performed to compare the quasispecies population of these two patients and local control patients. Genetic distance and diversity were calculated. Phylogenetic analyses were performed by using maximum likelihood and Bayesian methodologies. The analysis of the baseline samples showed that the genetic distance of the viral populations of patients P10 and P11 was significantly lower than when these patients and the control group based on sequences from local patients were analyzed, supporting the horizontal transmission hypothesis. Phylogenetic analysis with sequences from all the time point samples also demonstrated two patterns of evolution depending on the treatment response. The Bayesian analysis showed that one isolate corresponding to the baseline sample of P10 was grouped into the P11 clade, suggesting a way of infection and a bottleneck effect. Our data suggests that the patient P11 viral population may be originated from variants from P10 patient and consequently showing that clinical differences between treatment responses can emerge from the bottleneck effect on viral populations.

Evidence of bottleneck effect on hepatitis C virus transmission between a couple under interferon based therapy

Issues on the correlation of viral genetic diversity and treatment response to the hepatitis C infection remain uncertain. The bottleneck effect dictates the characteristics of the viral population that will establish the infection in a new host and is related to how the immune system and treatment will be effective against the virus. Here we evaluated the phylogenetic characteristics of quasispecies population and the treatment response pattern of a HCV infected couple. We also analyzed whether the viral population of these patients indicated that they were exposed to the same source for primer infection. This study included two patients (P10 and P11) HCV genotype 1b infected. The couple presented horizontal transmission. Viral RNA was isolated from serum samples collected before, during and after treatment, at specific time points. The HCV NS5A gene sequence was amplified, cloned and sequenced. Genetic and evolutionary analyses were performed to compare the quasispecies population of these two patients and local control patients. Genetic distance and diversity were calculated. Phylogenetic analyses were performed by using maximum likelihood and Bayesian methodologies. The analysis of the baseline samples showed that the genetic distance of the viral populations of patients P10 and P11 was significantly lower than when these patients and the control group based on sequences from local patients were analyzed, supporting the horizontal transmission hypothesis. Phylogenetic analysis with sequences from all the time point samples also demonstrated two patterns of evolution depending on the treatment response. The Bayesian analysis showed that one isolate corresponding to the baseline sample of P10 was grouped into the P11 clade, suggesting a way of infection and a bottleneck effect. Our data suggests that the patient P11 viral population may be originated from variants from P10 patient and consequently showing that clinical differences between treatment responses can emerge from the bottleneck effect on viral populations.