Shirley Vasconcelos Komninakis

Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock.

Objectives:Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis. Design:Prospective, observational study. Settings:Two emergency rooms and two intensive care units in one university and one teaching hospital. Patients and Controls:A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled. Interventions:None. Measurements and Main Results:The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(−&Dgr;&Dgr;Ct)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in &Dgr;CT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease. Conclusions:TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.

Influence of Hepatitis B Virus (HBV) Genotype on the Clinical Course of Disease in Patients Coinfected with HBV and Hepatitis Delta Virus

OBJECTIVE We evaluated the influence of hepatitis B virus (HBV) genotype on the course of disease in patients coinfected with HBV and hepatitis delta virus (HDV). METHODS We evaluated HBV genotypes in 190 patients, 140 of whom had chronic HBV monoinfection and 50 of whom had chronic HBV-HDV coinfection. Real-time polymerase chain reactions for the amplification of HBV DNA and HDV RNA were developed, and we compared the patient groups with respect to HBV genotype, viral load, alanine aminotransferase (ALT) and bilirubin levels, and disease severity. RESULTS Coinfected patients had higher ALT and bilirubin levels as well as a higher prevalence of liver cirrhosis and liver carcinoma. ALT levels were higher among individuals coinfected with HDV and HBV genotype F than among individuals infected only with HBV genotype F. Among HDV-HBV-coinfected patients, HDV load was lower among those infected with HBV genotype A than among those infected with HBV genotype D or genotype F. CONCLUSION Liver inflammation and HDV load are influenced by HBV genotype in individuals coinfected with HBV and HDV.

Prevalence of transmitted HIV-1 antiretroviral resistance among patients initiating antiretroviral therapy in Brazil: a surveillance study using dried blood spots

In Brazil, the use of antiretrovirals is widespread: more than 260,000 individuals are currently undergoing treatment. We conducted a survey targeting antiretroviral‐naïve individuals who were initiating antiretroviral therapy (ART) according to local guidelines. This survey covered five Brazilian regions.

Spread of Chikungunya Virus East/Central/South African Genotype in Northeast Brazil

We investigated an outbreak of exanthematous illness in Maceió by using molecular surveillance; 76% of samples tested positive for chikungunya virus. Genetic analysis of 23 newly generated genomes identified the East/Central/South African genotype, suggesting that this lineage has persisted since mid-2014 in Brazil and may spread in the Americas and beyond.

Short Communication: High Polymorphism Rates in the HR1 and HR2 gp41 and Presence of Primary Resistance-Related Mutations in HIV Type 1 Circulating in Brazil: Possible Impact on Enfuvirtide Efficacy

We analyzed the gp41 sequences of 80 HIV-infected enfuvirtide-naive individuals who were eligible to receive this antiretroviral according to Brazilian guidelines. We analyzed the genetic diversity of pol and the heptad repeat 1 and 2 (HR1 and HR2) regions of gp41, and compared the genetic profile of HR1 and HR2 found in PBMCs with the profile found in plasma. The similarity between sequences obtained from DNA and RNA in the HR1 and HR2 regions was, on average, 98.6% and 98.9%, respectively. We detected mutations related to enfuvirtide resistance (L44M or N43K) in HR1 DNA samples from three individuals (3.8%) and RNA samples from three individuals (4.6%). Other polymorphisms frequently detected were E137K (10% and 13.8%), L130I (8.8% and 9.2%), S129N (6.3% and 10.8%), L44M (2.5% and 4.6%), S138A (2.5% and 1.5%), and N43K (1.3% and 0%) in DNA and RNA, respectively. Subtype B was identified in 68.8% of the samples [protease (PR) B, reverse transcriptase (RT) B, gp41 B], subtype F in 5.0%, subtype C in 1.3%, and the remaining sequences presented with a mosaic profile. These results suggest that genotyping the gp41 region prior to introducing an expensive and complex approach, such as enfuvirtide, may be cost effective. Moreover, assessment of proviral DNA may be less expensive than RNA, as well as being sufficient for this purpose.

HIV-1 Proviral DNA Loads (as Determined by Quantitative PCR) in Patients Subjected to Structured Treatment Interruption after Antiretroviral Therapy Failure

ABSTRACT The impact of Structured Treatment Interruption (STI) in peripheral blood mononuclear cell (PBMC) proviral reservoirs in 41 highly active antiretroviral therapy (HAART)-treated viremic individuals at baseline and 12 weeks after STI was determined using quantitative PCR (qPCR). Viral load increased 0.7 log10 and CD4 decreased 97.5 cells/mm3 after 12 weeks. A total of 28 of the 41 individuals showed an increased proviral load, 19 with a statistically significant increase above 10%. An increase in active viral replication is an important factor in the replenishment of the proviral reservoir even for short time periods.

The Detection of in Vivo and in Vitro HIV Type 1 B/F Profiles in Brazil Using a Real-Time PCR Assay for Five HIV Type 1 Genomic Regions

We sought to determine the frequency and profile of HIV-1 BF recombinants in vitro and in vivo. Laboratory HIV-1 strains from subtypes B and F were cocultured and evaluated. Clinical samples from the city of Santos, Brazil, where the first HIV-1 B/F circulating recombinant forms (CRF) were described, were also assessed. Five real-time PCR assays were developed to equally amplify subtypes B and F, and subtype-specific probes were developed and optimized. To validate the PCR systems, clinical samples from Santos were sequenced and phylogenetically analyzed. The real-time PCR assays were performed on these samples and on the supernatant of an in vitro competition assay to assess emergent recombinant strains. Out of 157 clinical samples, 62.1% were defined as subtype B, 3.0% were subtype F, 16.7% presented the CRF28_BF profile, and 13.6% of the samples presented the CRF29_BF profile. The specificity and sensitivity in the discrimination assay for this sample panel were 93% and 92%, respectively. The HIV that emerged from the coinfected cell culture closely resembled the CRF28_BF profile. The first-described CRFs are still fixed in this geographic region of Brazil, and the in vitro emerging strains detected by real-time PCR suggest that in addition to the shaping of recombinant strains by immune selection, viral structures may also play an important role in emerging CRFs.

Detection of lamivudine-resistant variants and mutations related to reduced antigenicity of HBsAg in individuals from the cities of Santos and São Paulo, Brazil

BackgroundContinuous long-term treatment is recommended to reduce the hepatitis B virus (HBV) viral load. However, as a consequence, resistance mutations can emerge and be transmitted to other individuals. The polymerase (POL) gene overlaps the surface (S) gene. Thus, during treatment, mutations in the POL gene may lead to changes in hepatitis B surface antigen (HBsAg). The purpose of this study was to evaluate the frequency of lamivudine and vaccine escape mutations in HBsAg-positive blood donors from the city of Santos and in untreated HBV mono-infected patients from the city of São Paulo, Brazil.MethodsHBV DNA was extracted from 80 serum samples, of which 61 were from volunteer blood donors and 19 were from untreated HBV patients. A fragment of the POL/S genes containing 593 base pairs was amplified using nested PCR. Thirty four were PCR-positive and sequencing was performed using an ABI Prism 3130 Genetic Analyzer. Alignments and mutation mapping were performed using BioEdit software.ResultsHBV DNA from 21 blood donors and 13 untreated patient samples were characterized using nucleotide sequencing PCR products from the POL/S genes. We were able to detect one sample with the resistance mutation to lamivudine rtM204V + rtL180M (2.94%), which was found in a volunteer blood donor that has never used antiviral drugs. The other samples showed only compensatory mutations, such as rtL80F (5.88%), rtL80V (2.94%), rtL82V + rtV207L (2.94%), rtT128P (5.88%), rtT128N/S (2.94%) and rtS219A (5.88%). We found modifications in the S gene in 14 of the 34 samples (41.16%). The mutations detected were as follows: sM133L + sI195T (2.94%), sI195M (2.94%), sP120T (2.94%), sY100S/F (2.94%), sY100C (17.64%), sI/T126P + sQ129P (2.94%), sM198I + sF183C (2.94%) and sS210R (5.88%).ConclusionsOur results suggest the transmission of lamivudine-resistant forms. Thus, the evaluation of HBV-infected subjects for lamivudine resistance would improve treatment regime. Moreover, the mutations in the S gene may impair HBsAg antigenicity and contribute to HBsAg failure detection and vaccine escape.

Is Hepatitis Delta infections important in Brazil

BackgroundThe Hepatitis Delta Virus (HDV) can increase the incidence of fulminant hepatitis. For this infection occurs, the host must also be infected with Hepatitis B Virus. Previous studies demonstrated the endemicity and near exclusivity of this infection in the Amazon region, and as a consequence of the difficulty in accessing this area we used dried blood spots (DBS) in sample collection. The aims of this study were to investigate the presence of recombination, to analyze the epidemiology, ancestry and evolutionary pressures on HDV in Brazil.MethodsBlood samples from 50 individuals were collected using dried-blood spots (DBS 903, Whatman), and sent via regular mail to Retrovirology Laboratory from Federal University of São Paulo, where the samples were processed. In the analysis the following software were used: PhyML, RDP, BEAST, jModelTest and CODEML.ResultsOur results confirm the prevalence of HDV-3 in the Amazon region of Brazil, with the absence of inter-genotypic recombination. It was identified a positive selection in probable epitopes of HDV on B lymphocytes that might indicate that the virus is changing to escape the humoral response of the host. The analysis of the time of the most common ancestor demonstrated the exponential growth of this virus in late 1970s that lasted until 1995, after which it remained constant. It was also observed a probable founder effect in two cities, which demonstrate the need to focus on prevention methods against HBV/HDV infection.ConclusionWe confirmed the prevalence of HDV-3 in the Amazon region of Brazil, without inter-genotypic recombination. The analysis of the time of the most common ancestor showed that this infection remain constant in the studied area. Taking into account the probable founder effect established in the cities of Rio Branco and Porto Velho, a focus on preventive methods is recommended against these infections.

Analysis of Transmitted Resistance to Raltegravir and Selective Pressure among HIV-1-Infected Patients on a Failing HAART in São Paulo, Brazil

ABSTRACT We studied the presence of primary resistance to raltegravir (RAL), natural polymorphisms, and selection pressure on HIV-1 integrase. We found a high frequency of integrase polymorphisms related to the resistance to RAL and sequence stability. Further studies are needed to determine the importance of these polymorphisms to RAL resistance.