Genetic diversity in the partial sequence of the HIV-1 gag gene among people living with multidrug-resistant HIV-1 infection.

The group-specific antigen (gag) plays a crucial role in the assembly, release, and maturation of HIV. This study aimed to analyze the partial sequence of the HIV gag gene to classify HIV subtypes, identify recombination sites, and detect protease inhibitor (PI) resistance-associated mutations (RAMs). The cohort included 100 people living with HIV (PLH) who had experienced antiretroviral treatment failure with reverse transcriptase/protease inhibitors. Proviral HIV-DNA was successfully sequenced in 96 out of 100 samples for gag regions, specifically matrix (p17) and capsid (p24). Moreover, from these 96 sequences, 82 (85.42%) were classified as subtype B, six (6.25%) as subtype F1, one (1.04%) as subtype C, and seven (7.29%) exhibited a mosaic pattern between subtypes B and F1 (B/F1), with breakpoints at p24 protein. Insertions and deletions of amino acid at p17 were observed in 51 samples (53.13%). The prevalence of PI RAM in the partial gag gene was observed in 78 out of 96 PLH (81.25%). Among these cases, the most common mutations were R76K (53.13%), Y79F (31.25%), and H219Q (14.58%) at non-cleavage sites, as well as V128I (10.42%) and Y132F (11.46%) at cleavage sites. While B/F1 recombination was identified in the p24, the p17 coding region showed higher diversity, where insertions, deletions, and PI RAM, were observed at high prevalence. In PLH with virological failure, the analysis of the partial gag gene could contribute to more accurate predictions in genotypic resistance to PIs. This can aid guide more effective HIV treatment strategies.

Genetic diversity in the partial sequence of the HIV-1 gag gene among people living with multidrug-resistant HIV-1 infection

ABSTRACT The group-specific antigen (gag) plays a crucial role in the assembly, release, and maturation of HIV. This study aimed to analyze the partial sequence of the HIV gag gene to classify HIV subtypes, identify recombination sites, and detect protease inhibitor (PI) resistance-associated mutations (RAMs). The cohort included 100 people living with HIV (PLH) who had experienced antiretroviral treatment failure with reverse transcriptase/protease inhibitors. Proviral HIV-DNA was successfully sequenced in 96 out of 100 samples for gag regions, specifically matrix (p17) and capsid (p24). Moreover, from these 96 sequences, 82 (85.42%) were classified as subtype B, six (6.25%) as subtype F1, one (1.04%) as subtype C, and seven (7.29%) exhibited a mosaic pattern between subtypes B and F1 (B/F1), with breakpoints at p24 protein. Insertions and deletions of amino acid at p17 were observed in 51 samples (53.13%). The prevalence of PI RAM in the partial gag gene was observed in 78 out of 96 PLH (81.25%). Among these cases, the most common mutations were R76K (53.13%), Y79F (31.25%), and H219Q (14.58%) at non-cleavage sites, as well as V128I (10.42%) and Y132F (11.46%) at cleavage sites. While B/F1 recombination was identified in the p24, the p17 coding region showed higher diversity, where insertions, deletions, and PI RAM, were observed at high prevalence. In PLH with virological failure, the analysis of the partial gag gene could contribute to more accurate predictions in genotypic resistance to PIs. This can aid guide more effective HIV treatment strategies.

Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil.

BACKGROUND: Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. METHODS: The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. RESULTS: A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. CONCLUSION: Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

Genome-wide association study of early ischaemic stroke risk in Brazilian individuals with sickle cell disease implicates ADAMTS2 and CDK18 and uncovers novel loci.

Ischaemic stroke is a common complication of sickle cell disease (SCD) and without intervention can affect 11% of children with SCD before the age of 20. Within the Trans-Omics for Precision Medicine (TOPMed), a genome-wide association study (GWAS) of ischaemic stroke was performed on 1333 individuals with SCD from Brazil (178 cases, 1155 controls). Via a novel Cox proportional-hazards analysis, we searched for variants associated with ischaemic stroke occurring at younger ages. Variants at genome-wide significance (p < 5 × 10-8 ) include two near genes previously linked to non-SCD early-onset stroke (<65 years): ADAMTS2 (rs147625068, p = 3.70 × 10-9 ) and CDK18 (rs12144136, p = 2.38 × 10-9 ). Meta-analysis, which included the independent SCD cohorts Walk-PHaSST and PUSH, exhibited consistent association for variants rs1209987 near gene TBC1D32 (p = 3.36 × 10-10 ), rs188599171 near CUX1 (p = 5.89 × 10-11 ), rs77900855 near BTG1 (p = 4.66 × 10-8 ), and rs141674494 near VPS13C (1.68 × 10-9 ). Findings from this study support a multivariant model of early ischaemic stroke risk and possibly a shared genetic architecture between SCD individuals and non-SCD individuals younger than 65 years.

Surveillance for Zika, Chikungunya, and Dengue Virus Incidence and RNAemia in Blood Donors at 4 Brazilian Blood Centers During 2016-2019.

BACKGROUND: Except for public health case reports, the incidence of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) infection are not available to assess the potential blood transfusion safety threat in Brazil. METHODS: Pools of 6 donation samples (MP6) left over from human immunodeficiency virus, hepatitis B virus, and hepatitis C virus nucleic acid testing were combined to create MP18 pools (3 MP6 pools). Samples were tested using the Grifols triplex ZIKV, CHIKV, and DENV real-time transcription mediated amplification assay to estimate prevalence of RNAemia and incidence, and to compare these results to case reports in São Paulo, Belo Horizonte, Recife, and Rio de Janeiro, from April 2016 through June 2019. RESULTS: ZIKV, CHIKV, and DENV RNAemia were found from donors who donated without overt symptoms of infection that would have led to deferral. The highest RNAemic donation prevalence was 1.2% (95% CI, .8%-1.9%) for DENV in Belo Horizonte in May 2019. Arbovirus infections varied by location and time of year, and were not always aligned with annual arbovirus outbreak seasons in different regions of the country. CONCLUSIONS: Testing donations for arboviruses in Brazil can contribute to public health. Transfusion recipients were likely exposed to ZIKV, CHIKV, and DENV viremic blood components during the study period.

Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil

Abstract Background Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. Methods The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. Results A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. Conclusion Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

HIV primary drug resistance and associated HIV risk factors among HIV positive blood donors in Brazil from 2007 to 2017.

BACKGROUND: Acquisition of HIV primary drug resistant (PDR) infection can lead to poor virologic and clinical outcomes in individuals and hampers public health efforts in epidemic control. Monitoring PDR in HIV-positive blood donors can be used to inform nationwide trends in the spread of drug-resistant HIV strains. METHODS: We conducted a cross-sectional study using genetic sequence analysis to assess HIV pol sequences, PDR, and risk factors for infection using audio computer-assisted structured interviews in four large blood centers in Brazil from 2007 to 2017. RESULTS: Of 716 HIV-positive blood donors, 504 (70.4%) were successfully sequenced. HIV clade B (73.2%) was the most prevalent subtype, followed by a mix of non-B (21.2%) sub-types. A twofold increase (from 4% to 8%) in recombinants prevalence was observed during the study period. Sixty-four (12.7%) presented PDR. Overall, HIV PDR prevalence remained stable during the study period. Drug resistance mutations for non-nucleoside reverse transcriptase inhibitors were found in 39 (7.7%) donors, while for nucleoside reverse transcriptase inhibitors were found in 26 (5.1%), and for protease inhibitors in 24 (4.8%) of HIV-infected donors. We did not find statistically significant differences in demographics, behavioural risk factors, or HIV genotypes when comparing volunteers with and without PDR. CONCLUSION: The HIV PDR rate among donors remained stable during the study period. HIV-positive blood donors can be an informative population to monitor primary HIV resistance and ultimately may help to increase the knowledge and awareness of HIV risk factors and PDR.

Genomic Analyses of Potential Novel Recombinant Human Adenovirus C in Brazil.

Human Adenovirus species C (HAdV-C) is the most common etiologic agent of respiratory disease. In the present study, we characterized the nearly full-length genome of one potential new HAdV-C recombinant strain constituted by Penton and Fiber proteins belonging to type 89 and a chimeric Hexon protein of types 1 and 89. By using viral metagenomics techniques, we screened out, in the states of Tocantins and Pará, Northern and North regions of Brazil, from 2010 to 2016, 251 fecal samples of children between 0.5 to 2.5 years old. These children were presenting acute diarrhea not associated with common pathogens (i.e., rotavirus, norovirus). We identified two HAdV-C strains in two distinct patients. Phylogenetic analysis performed using all complete genomes available at GenBank database indicated that one strain (HAdV-C BR-245) belonged to type 1. The phylogenetic analysis also indicated that the second strain (HAdV-C BR-211) was located at the base of the clade formed by the newly HAdV-C strains type 89. Recombination analysis revealed that strain HAdV-C BR-211 is a chimera in which the variable regions of Hexon gene combined HAdV-C1 and HAdV-C89 sequences. Therefore, HAdV-C BR-211 strain possesses a genomic backbone of type HAdV-C89 and a unique insertion of HAdV-C1 in the Hexon sequence. Recombination may play an important driving force in HAdV-C diversity and evolution. Studies employing complete genomic sequencing on circulating HAdV-C strains in Brazil are needed to understand the clinical significance of the presented data.

Effectiveness of strategies to screen for blood donors with RH variants in a mixed population.

INTRODUCTION: Patients with RH variants presenting antibodies directed to RH high frequency antigens or multiple RH antibodies might, in some occasions, be better served with RH genotype-matched units, requiring screening for RH variants among blood donors. To date, strategies to identify donors with RH variants were restricted to selecting individuals of African descent based on self-reported race, what can be inaccurate in racially mixed population. Our goal was to: 1) Screen for donors with RH variants in a mixed population using self-declared race and Rh phenotype as selection criteria; and 2) Verify if including the Duffy null genotype in the screening algorithm increases its effectiveness. METHODS: Brazilian donors were included if self-declared as black and phenotyped as R0r or R1r. All individuals were genotyped for RHCE exons 1, 5, 6 and 7 and for the FY*B c.-67 T > C polymorphism in order to determine the Duffy null genotype. RHD variants were searched for in cases of altered RHCE. RESULTS: Among 2500 blood donors, 217 fulfilled the inclusion criteria and were enrolled. Fifty-three (24.4 %) had a predicted clinically relevant Rh phenotype (partial antigens or lack of high frequency antigens). Twelve donors (5.5 %) had a predicted RhCE phenotype lacking either hrB or hrS. Most cases with predicted lack of high frequency antigens (66.7 %) occurred in donors with the Duffy null genotype. CONCLUSION: Selecting donors based on self-declared race, Rh phenotype and Duffy null genotype is feasible and effective in identifying RH variants lacking Rh high frequency antigens among racially mixed donors.

Use of an automated pyrosequencing technique for confirmation of sickle cell disease.

BACKGROUND: The diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sß0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary. OBJECTIVES: To develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene. METHODS: We developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene. RESULTS: We identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sß thalassemia genotype, 84 samples were classified as Sß0 thalassemia and 81 as Sß+ thalassemia. The most frequent beta thalassemia mutations of Sß0 and Sß+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively. DISCUSSION: The PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common ß+ and ß0 mutations in SCD patients with Sß-thalassemia in a large multi-institutional SCD cohort in Brazil.

Molecular characterization of viruses associated with encephalitis in São Paulo, Brazil.

The objective of this study was to characterize the prevalence of viral encephalitis due to arbovirus infection of the Togaviridae and Flaviviridae families in São Paulo, Brazil. A total of 500 cerebrospinal fluid (CSF) samples collected between August 2012 and January 2013, from patients with symptoms of acute encephalitis were analyzed. Findings suggestive of viral encephalitis-elevations in cell concentration, glucose and total protein-were observed in 234 (46.8%) samples, designated as Group 1. The remaining 266 samples comprised Group 2. All samples were tested for Flaviviruses (dengue virus 1, 2, 3 and 4, yellow fever virus and West Nile virus), Alphavirus (NS5 region) and enterovirus by RT- PCR and for herpesviruses and enteroviruses using CLART-Entherpex. A presumptive viral etiological agent was detected in 26 samples (5.2%), 18 (8.0%) in Group 1 and 8 (3.0%) in Group 2. In Group 1 human herpesviruses were detected in 9 cases, enteroviruses in 7 cases, dengue viruses (DENV) in 2 CSFs and St. Louis encephalitis virus (SLEV) in one case. In Group 2 there were 3 CSFs positive for human herpesviruses, 2 for enteroviruses, 2 for DENV and 1 for SLEV. Detection of arboviruses, even though present in a minority of infected patients, identifies these viruses as a probable etiological agent of encephalitis. This is of special concern in regions where this class of viruses is endemic and has been linked to other recent epidemics.

Molecular characterization of viruses associated with encephalitis in São Paulo, Brazil

The objective of this study was to characterize the prevalence of viral encephalitis due to arbovirus infection of the Togaviridae and Flaviviridae families in São Paulo, Brazil. A total of 500 cerebrospinal fluid (CSF) samples collected between August 2012 and January 2013, from patients with symptoms of acute encephalitis were analyzed. Findings suggestive of viral encephalitis­elevations in cell concentration, glucose and total protein­were observed in 234 (46.8%) samples, designated as Group 1. The remaining 266 samples comprised Group 2. All samples were tested for Flaviviruses (dengue virus 1, 2, 3 and 4, yellow fever virus and West Nile virus), Alphavirus (NS5 region) and enterovirus by RT- PCR and for herpesviruses and enteroviruses using CLARTEntherpex. A presumptive viral etiological agent was detected in 26 samples (5.2%), 18 (8.0%) in Group 1 and 8 (3.0%) in Group 2. In Group 1 human herpesviruses were detected in 9 cases, enteroviruses in 7 cases, dengue viruses (DENV) in 2 CSFs and St. Louis encephalitis virus (SLEV) in one case. In Group 2 there were 3 CSFs positive for human herpesviruses, 2 for enteroviruses, 2 for DENV and 1 for SLEV. Detection of arboviruses, even though present in a minority of infected patients, identifies these viruses as a probable etiological agent of encephalitis. This is of special concern in regions where this class of viruses is endemic and has been linked to other recent epidemics.

Clinical and genetic ancestry profile of a large multi-centre sickle cell disease cohort in Brazil.

Approximately 3500 children with sickle cell disease (SCD) are born in Brazil each year, but the burden of SCD morbidity is not fully characterised. A large, multi-centre cohort was established to characterise clinical outcomes in the Brazilian SCD population and create the infrastructure to perform genotype-phenotype association studies. Eligible patients were randomly selected from participating sites and recruited at routine visits. A biorepository of blood samples was created and comprehensive demographic and clinical outcome data were entered in a centralized electronic database. Peripheral blood genome-wide single nucleotide polymorphism (SNP) genotyping was performed using a customized Transfusion Medicine (TM) Array. A total of 2795 participants at six Brazilian sites were enrolled between 2013 and 2015. The cohort included slight predominance of children <18 years (55·9%) and females (53·0%). Haemoglobin (Hb) SS was the most common SCD genotype (70·7%), followed by HbSC (23%), Sß0 (3·0%) and Sß+ (2·9%). SNP data from the TM Array were analysed to evaluate the genetic ancestry of the cohort and revealed significant admixture among the population. Demographics and clinical complications, stratified by age and SCD genotype, are summarized and future studies in this cohort are discussed.

High frequency of variant RHD genotypes among donors and patients of mixed origin with serologic weak-D phenotype.

BACKGROUND: The current transfusion policy recommended for individuals with serologic weak-D phenotype is based on data derived from European-descent populations. Data referring to the distribution of RH alleles underlying weak-D phenotype among people of mixed origin are yet incomplete, and the applicability of European-based transfusion guidelines to this specific population is questionable. GOAL: To evaluate the distribution of RHD variant genotype among individuals with serologic weak-D phenotype of both African and European descent. METHODS: Donors and patients of mixed origin and with serologic weak-D phenotype were selected for the study. They were investigated using conventional RHD-PCR assays and RHD whole-coding region direct sequencing. RESULTS: One hundred and six donors and 58 patients were included. There were 47 donors and 29 patients with partial-D genotype (47/106, 44.3%, and 29/58, 50%, respectively). RHD*DAR and RHD*weak D type 38 represented the most common altered RHD alleles among donors (joint frequency of 39.6%), while weak D types 1-3 accounted for 10.4% of the total D variant samples. RHD*DAR was the most common allele identified in the patient group (frequency of 31%), and weak D types 1-3 represented 29.3% of the total. CONCLUSION: The frequency of partial D among mixed individuals with serologic weak-D phenotype is high. They should be managed as D-negative patients until molecular tests are complete.

Genomic and epidemiological characterisation of a dengue virus outbreak among blood donors in Brazil.

Outbreaks caused by Dengue, Zika and Chikungunya viruses can spread rapidly in immunologically naïve populations. By analysing 92 newly generated viral genome sequences from blood donors and recipients, we assess the dynamics of dengue virus serotype 4 during the 2012 outbreak in Rio de Janeiro. Phylogenetic analysis indicates that the outbreak was caused by genotype II, although two isolates of genotype I were also detected for the first time in Rio de Janeiro. Evolutionary analysis and modelling estimates are congruent, indicating a reproduction number above 1 between January and June, and at least two thirds of infections being unnoticed. Modelling analysis suggests that viral transmission started in early January, which is consistent with multiple introductions, most likely from the northern states of Brazil, and with an increase in within-country air travel to Rio de Janeiro. The combination of genetic and epidemiological data from blood donor banks may be useful to anticipate epidemic spread of arboviruses.

Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil

ABSTRACTBACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo.METHODS:A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program).RESULTS:Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r = 0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r = 0.32). An evident exponential growth (r = 0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s.CONCLUSIONS:After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil.

BACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo. METHODS: A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program). RESULTS: Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r=0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r=0.32). An evident exponential growth (r=0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s. CONCLUSIONS: After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

Enhanced detection of viral diversity using partial and near full-length genomes of human immunodeficiency virus Type 1 provirus deep sequencing data from recently infected donors at four blood centers in Brazil.

BACKGROUND: Here, we report application of high-throughput near full-length genome (NFLG) and partial human immunodeficiency virus Type 1 (HIV-1) proviral genome deep sequencing to characterize HIV in recently infected blood donors at four major blood centers in Brazil. STUDY DESIGN AND METHODS: From 2007 to 2011, a total of 341 HIV+ blood donors from four blood centers were recruited to participate in a case-control study to identify HIV risk factors and motivations to donate. Forty-seven (17 from São Paulo, eight from Minas Gerais, 11 from Pernambuco, and 11 from Rio de Janeiro) were classified as recently infected based on testing by less-sensitive enzyme immunoassays. Five overlapping amplicons spanning the HIV genome were polymerase chain reaction amplified from peripheral blood mononuclear cells. The amplicons were molecularly barcoded, pooled, and sequenced by a paired-end protocol (Illumina). RESULTS: Of the 47 recently infected donor samples studied, 39 (82.9%) NFLGs and six (12.7%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Subtype B was the only nonrecombinant virus identified in this study and accounted for 62.2% (28/45) of samples. The remaining 37.8% (17/45) of samples showed various patterns of subtype discordance in different regions of HIV-1 genomes, indicating two to four circulating recombinant subtypes derived from Clades B, F, and C. Fourteen samples (31.1%) from this study harbored drug resistance mutations, indicating higher rate of drug resistance among Brazilian blood donors. CONCLUSION: Our findings revealed a high proportion of HIV-1 recombinants among recently infected blood donors in Brazil, which has implications for future blood screening, diagnosis, therapy, and vaccine development.

A double-blinded, prospective study to define antigenemia and quantitative real-time polymerase chain reaction cutoffs to start preemptive therapy in low-risk, seropositive, renal transplanted recipients.

BACKGROUND: Cytomegalovirus (CMV) disease occurs in 16% to 20% of low-risk, CMV-positive renal transplant recipients. The cutoffs for quantitative real-time polymerase chain reaction (qPCR) or phosphoprotein (pp65) antigenemia (pp65emia) for starting preemptive therapy have not been well established. METHODS: We measured qPCR and pp65emia weekly from day 7 to day 120 after transplantation, in anti-CMV immunoglobulin G­positive donor and recipient pairs. Patients and physicians were blinded to the test results. Suspicion of CMV disease led to the order of new tests. In asymptomatic viremic patients, the highest pp65emia and qPCR values were used, whereas we considered the last value before diagnosis in those with CMV disease. RESULTS: We collected a total of 1,481 blood samples from 102 adult patients. Seventeen patients developed CMV disease, 54 presented at least one episode of viremia that cleared spontaneously, and 31 never presented viremia. Five patients developed CMV disease after the end of the study period. The median (95% confidence interval) pp65emia and qPCR values were higher before CMV disease than during asymptomatic viremia (6 [9­82] vs. 3 [1­14] cells/10(6) cells; P<0.001 and 3,080 [1,263­15,605] vs. 258 [258­1,679] copies/mL; P=0.008, respectively). The receiver operating characteristic curve showed that pp65emia 4 cells/10(6) cells or greater showed a sensitivity and specificity to predict CMV disease of 69% and 81%, respectively (area, 0.769; P=0.001), with a positive predictive value of 37% and a negative predictive value of 93%. For qPCR 2,000 copies/mL or higher, the positive predictive value and negative predictive value were 57% and 91%, respectively (receiver operating characteristic area, 0.782; P=0.000). CONCLUSION: With these cutoffs, both methods are appropriate for detecting CMV disease.

HCV genotypes, characterization of mutations conferring drug resistance to protease inhibitors, and risk factors among blood donors in São Paulo, Brazil.

BACKGROUND: Hepatitis C virus (HCV) infection is a global health problem estimated to affect almost 200 million people worldwide. The aim of this study is to analyze the subtypes and existence of variants resistant to protease inhibitors and their association with potential HCV risk factors among blood donors in Brazil. METHODS: Repeat anti-HCV reactive blood donors are systematically asked to return for retest, notification, and counseling in which they are interviewed for risk factors for transfusion-transmitted diseases. We analyzed 202 donors who returned for counseling from 2007 to 2010 and presented enzyme immunoassay- and immunoblot-reactive results. The HCV genotypes and resistance mutation analyses were determined by the direct sequencing of the NS5b and NS3 regions, respectively. The HCV viral load was determined using an in-house real-time PCR assay targeting the 5'-NCR. RESULTS: HCV subtypes 1b, 1a, and 3a were found in 45.5%, 32.0%, and 18.0% of the donors, respectively. The mean viral load of genotype 1 was significantly higher than that of the genotype 3 isolates. Subtype 1a was more frequent among young donors and 3a was more frequent among older donors. Protease inhibitor-resistant variants were detected in 12.8% of the sequenced samples belonging to genotype 1, and a higher frequency was observed among subtype 1a (20%) in comparison to 1b (8%). There was no difference in the prevalence of HCV risk factors among the genotypes or drug-resistant variants. CONCLUSIONS: We found a predominance of subtype 1b, with an increase in the frequency of subtype 1a, in young subjects. Mutations conferring resistance to NS3 inhibitors were frequent in treatment-naïve blood donors, particularly those infected with subtype 1a. These variants were detected in the major viral population of HCV quasispecies, have replicative capacities comparable to nonresistant strains, and could be important for predicting the response to antiviral triple therapy.