Neutralizing antibody response after immunization with a COVID-19 bivalent vaccine: Insights to the future.

The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.

Effectiveness of Heterologous Coronavirus Disease 2019 (COVID-19) Vaccine Booster Dosing in Brazilian Healthcare Workers, 2021.

BACKGROUND: Little is currently known about vaccine effectiveness (VE) for either 2 doses of Oxford-AstraZeneca (ChAdOx1) viral vector vaccine or CoronaVac (Instituto Butantan) inactivated viral vaccine followed by a third dose of mRNA vaccine (Pfizer/BioNTech) among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil from January to December 2021. VE was defined as 1 - incidence rate ratio (IRR), with IRR determined using Poisson models with the occurrence of laboratory-confirmed coronavirus disease 2019 (COVID-19) infection as the outcome, adjusting for age, sex, and job type. We compared those receiving viral vector or inactivated viral primary series (2 doses) with those who received an mRNA booster. RESULTS: A total of 11 427 HCWs met the inclusion criteria. COVID-19 was confirmed in 31.5% of HCWs receiving 2 doses of CoronaVac vaccine versus 0.9% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001) and 9.8% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 1% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). In the adjusted analyses, the estimated VE was 92.0% for 2 CoronaVac vaccines plus mRNA booster and 60.2% for 2 ChAdOx1 vaccines plus mRNA booster, when compared with those with no mRNA booster. Of 246 samples screened for mutations, 191 (77.6%) were Delta variants. CONCLUSIONS: While 2 doses of ChAdOx1 or CoronaVac vaccines prevent COVID-19, the addition of a Pfizer/BioNTech booster provided significantly more protection.

Nucleocapsid single point-mutation associated with drop-out on RT-PCR assay for SARS-CoV-2 detection.

BACKGROUND: Since its beginning, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been a challenge for clinical and molecular diagnostics, because it has been caused by a novel viral agent. Whole-genome sequencing assisted in the characterization and classification of SARS-CoV-2, and it is an essential tool to genomic surveillance aiming to identify potentials hot spots that could impact on vaccine immune response and on virus diagnosis. We describe two cases of failure at the N2 target of the RT-PCR test Xpert® Xpress SARS-CoV-2. METHODS: Total nucleic acid from the Nasopharyngeal (NP) and oropharyngeal (OP) swab samples and cell supernatant isolates were obtained. RNA samples were submitted to random amplification. Raw sequencing data were subjected to sequence quality controls, removal of human contaminants by aligning against the HG19 reference genome, taxonomic identification of other pathogens and genome recovery through assembly and manual curation. RT-PCR test Xpert® Xpress SARS-CoV-2 was used for molecular diagnosis of SARS-CoV-2 infection, samples were tested in duplicates. RESULTS: We identified 27 samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing of 2 of 27 samples revealed a single common mutation in the N gene C29197T, potentially involved in the failed detection of N target. CONCLUSIONS: This study highlights the importance of genomic data to update molecular tests and vaccines.

Sabiá Virus-Like Mammarenavirus in Patient with Fatal Hemorrhagic Fever, Brazil, 2020.

New World arenaviruses can cause chronic infection in rodents and hemorrhagic fever in humans. We identified a Sabiá virus-like mammarenavirus in a patient with fatal hemorrhagic fever from São Paulo, Brazil. The virus was detected through virome enrichment and metagenomic next-generation sequencing technology.

HCV inter-subtype 1a/1b recombinant detected by complete-genome next-generation sequencing.

Next-generation sequencing (NGS) provides a practical approach to HCV complete-genome sequencing, detecting low-frequency variants and allowing analysis of viral genetic diversity (quasispecies) in the sample, and so far, it is very useful for identifying preexisting drug-resistant mutants and emerging escape mutations, as well as detecting viral recombinants containing genomic regions from different genotypes and subtypes. The aim of this study was to analyze the complete coding region of hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) from patients with chronic infection who were direct-acting antiviral (DAA) naïve. Next-generation sequencing (Ion Torrent™ PGM) was used to determine the sequence of the complete coding region of 100 HCV-monoinfected DAA-naïve patients (51 and 49 subtypes 1a and 1b, respectively). We report the first description of nearly complete HCV genome sequences of subtype 1a and 1b isolates from a large population of Brazilian patients with chronic hepatitis C, and HCV-1a grouped in two different clades. Using this methodology, an inter-subtype 1a/1b recombinant was identified in this study.

Association of IFNL3 and IFNL4 polymorphisms with hepatitis C virus infection in a population from southeastern Brazil.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated complications such as liver cirrhosis and hepatocellular carcinoma (HCC). Viral and host factors are known to be predictors for antiviral therapy. Host factors that are predictors of sustained viral response (SVR) were discovered by genome-wide association studies (GWAS), including single-nucleotide polymorphisms (SNPs) in or near the interferon lambda gene (rs8099917, rs12979860 and rs368234815). The aim of the present study was to verify the genotype frequencies of SNPs rs8099917, rs12979860 and rs368234815 and to evaluate the association between SNPs and the outcome of HCV infection, taking into account the population ancestry. In this study, there was an association of the three polymorphisms with both clinical outcome and response to treatment with PEG-IFN and RBV. The polymorphisms rs12979860 and rs368234815 were associated with increased sensitivity (97.7 %, 95 % CI 87.2-100, and 93.3 %, 95 % CI 81.3-98.3; respectively) and with a greater predictive value of a positive response to treatment. In multivariable analysis adjusted by gender, age and ancestry, the haplotype G/T/ΔG was related to non-response to treatment (OR = 21.09, 95 % CI 5.33-83.51; p < 0.001) and to a higher chance of developing chronic infection (OR = 5.46, 95 % CI 2.06-14.46; p = 0.001) when compared to the haplotype T/C/TT. These findings may help to adjust our treatment policies for HCV infection based on greater certainty in studies with populations with such genetic characteristics, as well as allowing us to get to know the genetic profile of our population for these polymorphisms.

Metagenomic next-generation sequencing in patients with fever of unknown origin: A comprehensive systematic literature review and meta-analysis.

Metagenomic Next-Generation Sequencing (mNGS) holds promise in diagnosing fever of unknown origin (FUO) by detecting diverse pathogens. We systematically reviewed the literature to evaluate mNGS's accuracy, clinical efficacy, and limitations in FUO diagnosis. Nine studies revealed mNGS's positivity rate ranging from 66.7% to 93.5% for bacterial bloodstream infections and systemic infections. Meta-analysis of three studies involving 857 patients, including 354 with FUO, showed a sensitivity of 0.91 (95% CI: 0.87-0.93) and specificity of 0.64 (95% CI: 0.58-0.70). Despite lower specificity, mNGS demonstrated a higher Diagnostic Odds Ratio (DOR) of 17.0 (95% CI: 4.5-63.4) compared to conventional microbiological tests (CMTs) at 4.7 (95% CI: 2.9-7.6). While mNGS offers high sensitivity but low specificity in identifying causative pathogens for FUO, its superior DOR suggests potential for more accurate diagnoses and targeted interventions. Further research is warranted to optimize its clinical application in FUO management.

Effectiveness of two coronavirus disease 2019 (COVID-19) vaccines (viral vector and inactivated viral vaccine) against severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection in a cohort of healthcare workers.

OBJECTIVE: We investigated real-world vaccine effectiveness for Oxford-AstraZeneca (ChAdOx1) and CoronaVac against laboratory-confirmed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil between January 1, 2021 and August 3, 2021, to assess vaccine effectiveness. We calculated vaccine effectiveness as 1 - rate ratio (RR), with RR determined by adjusting Poisson models with the occurrence of SARS-CoV-2 infection as the outcome and the vaccination status as the main variable. We used the logarithmic link function and simple models adjusting for sex, age, and job types. RESULTS: In total, 13,813 HCWs met the inclusion criteria for this analysis. Among them, 6,385 (46.2%) received the CoronaVac vaccine, 5,916 (42.8%) received the ChAdOx1 vaccine, and 1,512 (11.0%) were not vaccinated. Overall, COVID-19 occurred in 6% of unvaccinated HCWs, 3% of HCWs who received 2 doses of CoronaVac vaccine, and 0.7% of HCWs who received 2 doses of ChAdOx1 vaccine (P < .001). In the adjusted analyses, the estimated vaccine effectiveness rates were 51.3% for CoronaVac, and 88.1% for ChAdOx1 vaccine. Both vaccines reduced the number of hospitalizations, the length of hospital stay, and the need for mechanical ventilation. In addition, 19 SARS-CoV-2 samples from 19 HCWs were screened for mutations of interest. Of 19 samples, 18 were the γ (gamma) variant. CONCLUSIONS: Although both COVID-19 vaccines (viral vector and inactivated virus) can significantly prevent COVID-19 among HCWs, CoronaVac was much less effective. The COVID-19 vaccines were also effective against the dominant γ variant.

NAFLD in Polycystic Ovary Syndrome: Association with PNPLA3 and Metabolic Features.

Background: The aim of this study was to determine the frequency of the rs738409 polymorphism in the patatin-like phospholipase domain containing 3 (PNPLA3) gene in patients with polycystic ovary syndrome (PCOS) and its impact on nonalcoholic fatty liver disease (NAFLD) risk and severity. We also evaluated other risk factors associated with NAFLD and advanced fibrosis. Methods: This was a cross-sectional study involving 163 patients with PCOS at a tertiary center. Genotyping for the PNPLA3 polymorphism was undertaken using a TaqMan assay. The degree of fibrosis was defined by transient elastography. Results: The prevalence of NAFLD was 72.4%, and the polymorphism was heterozygous in 41.7% and homozygous in 8% of patients. Homeostasis model assessment of insulin resistance ≥ 2.5 was the main factor associated with the risk of developing NAFLD (OR = 4.313, p = 0.022), and its effect was amplified by the polymorphism (OR = 12.198, p = 0.017). Age > 32 years also conferred a higher risk for NAFLD. HDL values ≥ 50 mg/dL conferred protection against the outcome. Metabolic syndrome (OR = 13.030, p = 0.020) and AST > 32 U/L (OR = 9.039, p = 0.009) were independent risk factors for advanced fibrosis. Conclusions: In women with PCOS, metabolic characteristics are more relevant than PNPLA3 polymorphism regarding the risk for NAFLD and its advanced forms, but these factors can act synergistically, increasing disease risk.

Updating the Phylodynamics of Yellow Fever Virus 2016-2019 Brazilian Outbreak With New 2018 and 2019 São Paulo Genomes.

The recent outbreak of yellow fever (YF) in São Paulo during 2016-2019 has been one of the most severe in the last decades, spreading to areas with low vaccine coverage. The aim of this study was to assess the genetic diversity of the yellow fever virus (YFV) from São Paulo 2016-2019 outbreak, integrating the available genomic data with new genomes from patients from the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP). Using phylodynamics, we proposed the existence of new IE subclades, described their sequence signatures, and determined their locations and time of origin. Plasma or urine samples from acute severe YF cases (n = 56) with polymerase chain reaction (PCR) positive to YFV were submitted to viral genome amplification using 12 sets of primers. Thirty-nine amplified genomes were subsequently sequenced using next-generation sequencing (NGS). These 39 sequences, together with all the complete genomes publicly available, were aligned and used to determine nucleotide/amino acids substitutions and perform phylogenetic and phylodynamic analysis. All YFV genomes generated in this study belonged to the genotype South American I subgroup E. Twenty-one non-synonymous substitutions were identified among the new generated genomes. We analyzed two major clades of the genotypes IE, IE1, and IE2 and proposed the existence of subclades based on their sequence signatures. Also, we described the location and time of origin of these subclades. Overall, our findings provide an overview of YFV genomic characterization and phylodynamics of the 2016-2019 outbreak contributing to future virological and epidemiological studies.

Prevalence and Pattern of Resistance in NS5A/NS5B in Hepatitis C Chronic Patients Genotype 3 Examined at a Public Health Laboratory in the State of São Paulo, Brazil.

PURPOSE: Globally, it is estimated that 71 million people are chronically infected with hepatitis C, and 10-20% of these will develop cirrhosis and hepatocellular carcinoma. The development of new direct-acting antiviral (DAA) drugs has contributed to sustained virological response (SVR), eliminating the infection and achieving cure of chronic hepatitis C. However, treated patients can develop HCV resistance to DAAs, which can contribute to the failure of treatment. Here, we aimed to evaluate the prevalence and specific pattern of NS5A and NS5B resistance-associated substitutions (RAS) in samples from patients chronically infected with HCV genotype 3a at a public health laboratory, Instituto Adolfo Lutz, São Paulo, Brazil. PATIENTS AND METHODS: Serum samples from the enrolled individuals were submitted to "in-house" polymerase chain reaction amplification of NS5A and NS5B non-structural protein genes, which were then sequenced by Sanger method. RESULTS: A total of 170 and 190 samples were amplified and analyzed for NS5A and NS5B, respectively. For NS5A, 20 (12.0%) samples showed some important RAS; 16 (9.0%) showed some type of substitution and 134 (79.0%) showed no polymorphism. No sample showed any RAS for NS5B. CONCLUSION: This study found important RAS in samples from naïve chronic HCV patients in some areas from São Paulo. The most prevalent were A62S, A30K, and Y93H, which could indicate an increase in resistance to some DAAs used in HCV treatment.

Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow.

Since the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient's samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results' display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.

Molecular evidence of horizontal transmission of hepatitis C virus within couples.

Hepatitis C virus (HCV) transmission has decreased with the adoption of universal blood donor screening and social policies to reduce the risk of infection in intravenous drug users, but remains a worldwide health problem. The objective of this study was to evaluate the phylogenetic relationships among sequences from different HCV genomic regions from sexual partners of infected patients. Nine couples with a stable relationship and without other risk factors for HCV infection and 42 control patients were selected, and the NS3 and NS5B regions were analysed. Phylogenetic analysis showed that viruses from five of the couples had a common origin, clustering in the same monophyletic group, with bootstrap values greater than 70. For the other couples, monophyletic groups were observed, but without bootstrap support. Thus, using two different viral genome regions, a common source of infection was observed in both members of five couples. These data strongly support HCV transmission within couples.

Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus.

BACKGROUND: Direct-acting antiviral agents (DAAs) permit the use of interferon (IFN)-free regimens to treat hepatitis C (HCV) in patients with chronic kidney disease (CKD) on hemo-dialysis (HD) or renal transplant (RTx) recipients, with excellent response rates and safety. However, the occurrence of basal or therapy-induced resistance-associated substitutions (RASs) to DAAs can result in treatment failure. The aim of this study was to estimate the prevalence of RASs to NS3A, NS5A and NS5B inhibitors, and particularly the Q80K polymorphism, in CKD patients on HD and RTx recipients infected with HCV. PATIENTS AND METHODS: HD and RTx patients infected with HCV-genotype 1 (GT1) were subjected to sequencing of the NS3, NS5A and NS5B regions. RESULTS: Direct sequencing of NS3 protease, NS5A and NS5B was performed in 76 patients (HD, n=37; RTx, n=39). The overall prevalence of RASs was 38.2%, but only 5.3% of the patients had mutations in more than one region. Substitutions were detected in NS3A (17.8%), NS5A (21.9%) and NS5B (8.4%). Q80K was detected in 1.5 % of the patients. Highly inhibitory RASs were uncommon (L31M, 2.6%; L159F+C316N, 2.6%). RASs were more prevalent in HCV-GT1a (42.9%) than in HCV-GT1b (32.4%), P=0.35. RASs were detected in 52.4% of treatment-naive patients and 27.8% of peg-IFN/ribavirin-experienced patients (P=0.12). The presence of RASs was associated with time of RTx (P=0.01). CONCLUSION: The Q80K polymorphism was uncommon in our sample of HD and RTx patients. Despite the high prevalence of naturally occurring RASs, most of the substitutions detected were associated with a low level of resistance to DAAs.

Resistance-associated variants in HCV subtypes 1a and 1b detected by Ion Torrent sequencing platform.

BACKGROUND: As a result of increased understanding of the HCV life cycle, a new generation of drugs known as direct-acting antivirals (DAAs) was developed and is constantly being improved. At baseline, HCV variants resistant to DAA therapy may pre-exist, increasing the likelihood of treatment failure. The aim of this study was to investigate the presence of resistance-associated variants (RAVs) in treatment-naive patients infected with HCV subtypes 1a and 1b. METHODS: Next-generation sequencing was used to assess the frequencies of NS3-4A, NS5A and NS5B RAVs in 100 HCV monoinfected DAA-naive patients (HCV-1a: n=51; HCV-1b: n=49). RESULTS: Complete HCV sequence information was obtained for most samples. RAVs were detected in the NS3-4A (T54S, V55A, Q80K and R155K), NS5A (Q30H/R, H58P and Y93C/H/N) and NS5B (A421V) regions in 10%, 22% and 8%, respectively, of patients infected with HCV subtype-1a. Among the patients infected with HCV subtype-1b, mutations in the NS3-4A (F43I, T54S, Q80H, D168E and M175L), NS5A (L28M, R30Q, L31M, Q54H, A92T and Y93H) and NS5B (L159F, C316N, A421V and S556G) regions were observed in 12%, 53% and 31% of patients, respectively. CONCLUSIONS: High-throughput DNA sequencing allows an easier and more complete analysis of DAA RAVs, including mutations that represent only a minor variant of the whole viral population. RAVs to the three different classes of DAAs were found in our population. The characterization of their profile in the circulating virus is relevant to determine the better treatment option for infected individuals or to guide the implementation of treatment policies.

Differences in viral kinetics between genotypes 1 and 3 of hepatitis C virus and between cirrhotic and non-cirrhotic patients during antiviral therapy.

AIM: To evaluate the impact of hepatitis C virus (HCV) infection with genotype 1 or 3 and the presence or absence of liver cirrhosis (LC) in the early viral kinetics response to treatment. METHODS: Naive patients (n = 46) treated with interferon-alpha (IFN-alpha) and ribavirin and followed up with frequent early HCV-RNA determinations were analysed. Patients were infected with genotype 1 (n = 28, 7 with LC) or 3 (n = 18, 5 with LC). RESULTS: The first phase decline was larger in genotype 3 patients than in genotype 1 patients (1.72 vs 0.95 log IU/mL, P < 0.001). The second phase slope decline was also larger in genotype 3 patients than in genotype 1 patients (0.87 vs 0.15 log/wk, P < 0.001). Differences were found in both cirrhotic and non-cirrhotic patients. Genotype 1 cirrhotic patients had a slower 2nd phase slope than non-cirrhotic patients (0.06 vs 0.18 log/wk, P < 0.02). None of genotype 1 cirrhotic patients had a 1st phase decline larger than 1 log (non-cirrhotic patients: 55%, P < 0.02). A similar trend toward a slower 2nd phase slope was observed in genotype 3 cirrhotic patients but the 1st phase slope decline was not different. Sustained viral response was higher in genotype 3 patients than in genotype 1 patients (72% vs 14%, P < 0.001) and in genotype 1 non-cirrhotic patients than in genotype 1 cirrhotic patients (19% vs 0%). A second phase decline slower than 0.3 log/wk was predictive of non-response in all groups. CONCLUSION: Genotype 3 has faster early viral decline than genotype 1. Cirrhosis correlates with a slower 2nd phase decline and possibly with a lower 1st phase slope decline in genotype 1 patients.