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1.  Efficient Identification of HIV Serodiscordant Couples by Existing HIV Testing Programs in South Brazil 
PLoS ONE  2015;10(11):e0142638.
Objective
To examine the feasibility of identifying HIV negative at risk individuals in HIV serodiscordant couples, during voluntary HIV testing in South Brazil.
Methods
We surveyed HIV testers at 4 public testing sites in Rio Grande do Sul. We obtained information on risk behaviors and sexual partnerships. HIV testing and testing for recent infection were performed; HIV prevalence and risk behaviors were assessed among subjects who reported having a steady partner who was HIV positive (serodiscordant group) and compared with the general testing population.
Results
Among 3100 patients, 490 (15.8%) reported being in a steady relationship with an HIV positive partner. New HIV infections were diagnosed in 23% of the serodiscordant group (vs. 13% in the general population, p = 0.01); among newly positive subjects, recent HIV infections were more frequent (23/86, 26.7%) among testers with positive partners than among the general testing group (52/334; 15.6%; p = 0.016). Less than half of the serodiscordant testers reported having used a condom during the last sexual intercourse with their HIV-positive partner. Participants with inconsistent condom use with steady partner were four times more likely to test positive for HIV compared to those who reported always using condoms with the steady partner (OR: 4.2; 95% CI: 2.3 to 7.5).
Conclusion
It is highly feasible to identify large numbers of HIV susceptible individuals who are in HIV serodiscordant relationships in South Brazil testing sites. Condom use within HIV serodiscordant couples is low in this setting, suggesting urgent need for biomedical prevention strategies to reduce HIV transmission.
doi:10.1371/journal.pone.0142638
PMCID: PMC4642994  PMID: 26562436
2.  The Virological and Immunological Characteristics of the HIV-1-Infected Population in Brazil: From Initial Diagnosis to Impact of Antiretroviral Use 
PLoS ONE  2015;10(10):e0139677.
Background
Immunological and virological status of HIV-infected individuals entering the Brazilian public system over time was analyzed. We evaluated the impact of ART on virological, immunological and antiretroviral resistance over time.
Methods
CD4+ T cell counts, viral loads and genotypes from patients over 13 years old from 2001–2011 were analyzed according to demographic data. We compared groups using parametric t-tests and linear regression analysis in the R statistical software language.
Results
Mean baseline CD4+ T cell counts varied from 348 (2003) to 389 (2009) and was higher among women (p = 1.1 x 10−8), lower in older patients (p< 1 x 10−8) and lower in less developed regions (p = 1.864 x 10−5). Percentage of treated patients with undetectable viral loads increased linearly from 46% (2001) to 77% (2011), was lower among women (p = 2.851 x 10−6), younger ages (p = 1 x 10−3), and in less developed regions (p = 1.782 x 10−4). NRTI acquired resistance was 86% in 2001–3 and decreased over time. NNRTI resistance increased from 2001-3(50%) to 2006–9 (60%), PI resistance decreased from 2001–3 (60%) to 2009 (40%), and 3-class resistance was stable over time around 25%. Subtype prevalence comprised B (75.3%), B/F recombinants (12.2%), C (5.7%), F (5.3%) and B/C recombinants (1.5%), with regional variations. Three-class resistance was 26.5% among Bs, 22.4% among Fs and 17.2% among Cs.
Conclusions
HIV diagnosis occurs late, especially among elderly Brazilians. Younger individuals need special attention due to poor virological response to treatment. Antiretroviral Resistance profile is subtype related.
doi:10.1371/journal.pone.0139677
PMCID: PMC4624810  PMID: 26509441
3.  In Vivo HIV-1 Hypermutation and Viral Loads Among Antiretroviral-Naive Brazilian Patients 
Abstract
Hypermutation alludes to an excessive number of specific guanine-to-adenine (G- >A) substitutions in proviral DNA and this phenomenon is attributed to the catalytic activity of cellular APOBECs. Population studies relating hypermutation and the progression of infection by human immunodeficiency virus type 1 (HIV-1) have been performed to elucidate the effect of hypermutation on the natural course of HIV-1 infection. However, the many different approaches employed to assess hypermutation in nucleotide sequences render the comparison of results difficult. This study selected 157 treatment-naive patients and sought to correlate the hypermutation level of the proviral sequences in clinical samples with demographic variables, HIV-1 RNA viral load, and the level of CD4+ T cells. Nested touchdown polymerase chain reaction (PCR) was performed with specific primers to detect hypermutation in the region of HIV-1 integrase, and the amplified sequences were run in agarose gels with HA-Yellow. The analysis of gel migration patterns using the k-means clustering method was validated by its agreement with the results obtained with the software Hypermut. Hypermutation was found in 31.2% of the investigated samples, and a correlation was observed between higher hypermutation levels and higher viral load levels. These findings suggest a high frequency of hypermutation detection in a Brazilian cohort, which can reflect a particular characteristic of this population, but also can result from the method approach by aiming at hypermutation-sensitive sites. Furthermore, we found that hypermutation events are pervasive during HIV-1 infection as a consequence of high viral replication, reflecting its role during disease progression.
doi:10.1089/aid.2013.0241
PMCID: PMC4151062  PMID: 25065371
4.  Prevalence and factors associated with HSV-2 and hepatitis B infections among truck drivers crossing the southern Brazilian border 
Sexually transmitted infections  2011;87(7):553-559.
Objectives
The authors estimate the prevalence of HIV, syphilis, hepatitis B virus (HBV) and herpes simplex virus type-2 (HSV-2) infection and correlates of HBV and HSV-2 infection among truck drivers crossing the southern Brazilian border at Foz do Iguaçu.
Methods
Between October 2003 and March 2005, 1945 truck drivers were sampled while accessing voluntary counselling and testing services; 1833 (94.2%) were tested for HIV (ELISA and confirmatory immunofluorescence assay) and syphilis (non-treponemal (VDRL) and treponemal tests (FTA-ABS)). From these, 799 stored sera were tested for HSV-2 (type-specific ELISA test for detection of IgG) and HBV (core antibodies (anti-HBc) with positives tested for surface antigen (HBsAg)). The authors estimate HIV, syphilis, HSV-2 and HBV prevalence and determine socio-demographic and behavioural correlates of HSV-2 infection and HBV exposure.
Results
HIV prevalence was 0.3% (95% CI 0.1 to 0.6) and syphilis 4.5% (95% CI 3.6 to 5.4). Among those tested for HBV and HSV-2, 32.3% (95% CI 28.9 to 35.6) had serological evidence of exposure to HBV and 26.6% (95% CI 23.5 to 29.7) tested positive for HSV-2. Factors independently associated with HBV exposure included increasing age, Brazilian nationality and unprotected anal sex. Increasing age and reporting an unknown number of lifetime partners were associated with HSV-2 infection.
Conclusions
In this sample of truck drivers in southern Brazil, HIV prevalence was lower than national population estimates; exposure to HBV was higher than population estimates, while per cent positive for HSV-2 was similar to population estimates. The low prevalence of HIV in truck drivers indicates prevention successes; however, future HIV prevention programming should incorporate HBV vaccination and sexually transmitted infection prevention.
doi:10.1136/sextrans-2011-050186
PMCID: PMC4539241  PMID: 21968460
5.  Epigenetic Modulations in Activated Cells Early after HIV-1 Infection and Their Possible Functional Consequences 
PLoS ONE  2015;10(4):e0119234.
Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.
doi:10.1371/journal.pone.0119234
PMCID: PMC4395311  PMID: 25875202
6.  Data-intensive analysis of HIV mutations 
BMC Bioinformatics  2015;16(1):35.
Background
In this study, clustering was performed using a bitmap representation of HIV reverse transcriptase and protease sequences, to produce an unsupervised classification of HIV sequences. The classification will aid our understanding of the interactions between mutations and drug resistance. 10,229 HIV genomic sequences from the protease and reverse transcriptase regions of the pol gene and antiretroviral resistant related mutations represented in an 82-dimensional binary vector space were analyzed.
Results
A new cluster representation was proposed using an image inspired by microarray data, such that the rows in the image represented the protein sequences from the genotype data and the columns represented presence or absence of mutations in each protein position.The visualization of the clusters showed that some mutations frequently occur together and are probably related to an epistatic phenomenon.
Conclusion
We described a methodology based on the application of a pattern recognition algorithm using binary data to suggest clusters of mutations that can easily be discriminated by cluster viewing schemes.
doi:10.1186/s12859-015-0452-0
PMCID: PMC4344997  PMID: 25652056
HIV; Mutation; Cluster
8.  Prevalence of transmitted HIV-1 antiretroviral resistance among patients initiating antiretroviral therapy in Brazil: a surveillance study using dried blood spots 
Introduction
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.
Methods
The HIV Threshold Survey methodology (HIV-THS) of the World Health Organization was utilized, and subjects were selected from seven highly populated cities representative of all Brazilian macro-regions. Dried blood spots (DBS) were collected on SS903 collection cards and were transported by regular mail at room temperature to a single central laboratory for genotyping.
Results
We analysed samples from 329 individuals initiating highly active antiretroviral therapy (HAART), 39 (11.8%) of whom were harbouring transmitted drug resistance (TDR). The mean CD4+ T cell count was 253 cells/µL, and the mean viral load was 142,044 copies/mL. The regional prevalence of resistance was 17.0% in the Northeast, 12.8% in the Southeast, 10.6% in the Central region, 8.5% in the North and 8.5% in the South. The inhibitor-specific TDR prevalence was 6.9% for nucleoside reverse transcriptase inhibitors, 4.9% for non-nucleoside reverse transcriptase inhibitors and 3.9% for protease inhibitors; 3.6% of individuals presented resistance to more than one class of inhibitors. Overall, there were trends towards higher prevalences of subtype C towards the South and subtype F towards the North. Of the DBS samples collected, 9.3% failed to provide reliable results.
Discussion
We identified variable TDR prevalence, ranging from intermediate to high levels, among individuals in whom HIV disease progressed, thus implying that resistance testing before initiating ART could be effective in Brazil. Our results also indicate that the use of DBS might be especially valuable for providing access to testing in resource-limited and remote settings.
doi:10.7448/IAS.17.1.19042
PMCID: PMC4172689  PMID: 25249214
transmitted drug resistance; dried blood spots; Brazil; genotyping; antiretroviral therapy; HIV subtype
9.  Inhibitors of Urokinase Type Plasminogen Activator and Cytostatic Activity from Crude Plants Extracts 
Molecules (Basel, Switzerland)  2013;18(8):8945-8958.
In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 μg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study.
doi:10.3390/molecules18088945
PMCID: PMC4034058  PMID: 23896619
urokinase type plasminogen activator (uPA); uPA inhibitor; fibrin plate assay; Croton lucidus; metastasis; cytostatic activity
10.  Analysis of HIV-1 Protease Gene Reveals Frequent Multiple Infections Followed by Recombination among Drug Treated Individuals Living in São Paulo and Santos, Brazil 
PLoS ONE  2014;9(1):e84066.
The present study investigated the prevalence of HIV-1 multiple infections in a population composed by 47 patients under HAART failure and enrolled at the National DST/AIDS, Program, Ministry of Health, Brazil.Detection of multiple infections was done using a previously published RFLP assay for the HIV-1 protease gene, which is able of distinguishing between infections caused by a single or multiple HIV-1 subtypes. Samples with multiple infections were cloned, and sequence data submitted to phylogenetic analysis. We were able to identify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were mostly composed by a mixture of recombinant viruses (94%), with only one case in which protease gene pure subtypes B and F were recovered. This is the first study that reports the prevalence of multiple infections and intersubtype recombinants in a population undergoing HAART in Brazil. Based on the data there was a steep increase of multiple infections after the introduction of the combined antiretroviral therapy in Brazil. Cases of multiple infections may be associated with HIV-1 genetic diversity through recombination allowing for the generation of viruses showing a combination of resistance mutations.
doi:10.1371/journal.pone.0084066
PMCID: PMC3880281  PMID: 24404149
11.  T-Cell Response to Hepatitis B Core Antigen: Identification of Prior Exposure to and Confirmatory Testing for Screening for Anti-HBc 
Journal of biomarkers  2013;2013:812170.
Background. During routine donor screening in the blood bank, it is not uncommon to find isolated reactivity for anti-HBc in the absence of detectable HBV DNA in a first donation but absence of reactivity to anti-HBc in subsequent donations, suggesting a false-positive result for anti-HBc. Study Design and Methods. The blood donor population was screened between January 2010 and October 2011. We selected 2,126 donations positive only for anti-HBc from a total of 125,068 donations. During the process, OBI donors were identified, and their HBcAg-specific T-cell response was analyzed and compared to donors with chronic (HBsAg positive) and recovered (anti-HBc only) infection. We analyzed correlations between signal levels (Co/s) in the competitive assay for anti-HBc and HBV DNA detection. Results. In the 21-month study period, 21 blood donors with anti-HBc alone were identified as OBI (1 in each 5955 donors). The relevant finding was the observation that anti-HBc only subjects with Co/s ≥ 0.1 did not have either HBcAg-specific T-cells or detectable HBV DNA and OBI subjects presented with Co/s ≤ 0.1 and HBcAg T-cell response. In the subset of 21 OBI subjects, 9 donors remained positive for HBcAg T-cell response after four collections. In all 9 samples, we observed HBV DNA fluctuation. Conclusion. Our data suggest that HBcAg-specific T-cell response could be used to confirm anti-HBc serological status, distinguishing previous exposure to Hepatitis B virus from anti-HBc false-positive results.
doi:10.1155/2013/812170
PMCID: PMC4437383  PMID: 26317023
12.  Detection of lamivudine-resistant variants and mutations related to reduced antigenicity of HBsAg in individuals from the cities of Santos and São Paulo, Brazil 
Virology Journal  2013;10:320.
Background
Continuous 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.
Methods
HBV 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.
Results
HBV 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%).
Conclusions
Our 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.
doi:10.1186/1743-422X-10-320
PMCID: PMC3898369  PMID: 24165277
HBV; Genotypes; Transmitted drug resistance; Lamivudine; Vaccine escape mutations; Polymerase gene; Surface gene; Surface antigen
14.  Codon pairs of the HIV-1 vif gene correlate with CD4+ T cell count 
BMC Infectious Diseases  2013;13:173.
Background
The human APOBEC3G (A3G) protein activity is associated with innate immunity against HIV-1 by inducing high rates of guanosines to adenosines (G-to-A) mutations (viz., hypermutation) in the viral DNA. If hypermutation is not enough to disrupt the reading frames of viral genes, it may likely increase the HIV-1 diversity. To counteract host innate immunity HIV-1 encodes the Vif protein that binds A3G protein and form complexes to be degraded by cellular proteolysis.
Methods
Here we studied the pattern of substitutions in the vif gene and its association with clinical status of HIV-1 infected individuals. To perform the study, unique vif gene sequences were generated from 400 antiretroviral-naïve individuals.
Results
The codon pairs: 78–154, 85–154, 101–157, 105–157, and 105–176 of vif gene were associated with CD4+ T cell count lower than 500 cells per mm3. Some of these codons were located in the 81LGQGVSIEW89 region and within the BC-Box. We also identified codons under positive selection clustered in the N-terminal region of Vif protein, between 21WKSLVK26 and 40YRHHY44 regions (i.e., 31, 33, 37, 39), within the BC-Box (i.e., 155, 159) and the Cullin5-Box (i.e., 168) of vif gene. All these regions are involved in the Vif-induced degradation of A3G/F complexes and the N-terminal of Vif protein binds to viral and cellular RNA.
Conclusions
Adaptive evolution of vif gene was mostly to optimize viral RNA binding and A3G/F recognition. Additionally, since there is not a fully resolved structure of the Vif protein, codon pairs associated with CD4+ T cell count may elucidate key regions that interact with host cell factors. Here we identified and discriminated codons under positive selection and codons under functional constraint in the vif gene of HIV-1.
doi:10.1186/1471-2334-13-173
PMCID: PMC3637627  PMID: 23578255
HIV-1; Epistasis; APOBEC; Vif; Hypermutation; Positive selection; Co-evolution
15.  HIV-1 Proviral DNA Loads (as Determined by Quantitative PCR) in Patients Subjected to Structured Treatment Interruption after Antiretroviral Therapy Failure 
Journal of Clinical Microbiology  2012;50(6):2132-2133.
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.
doi:10.1128/JCM.00393-12
PMCID: PMC3372152  PMID: 22422851
16.  Incidence of Recent Human Immunodeficiency Virus Infection at Two Voluntary Counseling Testing Centers in Pernambuco, Brazil, from 2006 to 2009 
Journal of Clinical Microbiology  2012;50(6):2145-2146.
This study shows HIV-1 incidence in the northeastern region of Brazil, where the HIV epidemic has spread recently. Incidence was higher among men (1.34%; 95% confidence interval [CI], 1.00% to 1.69%) than among women (0.55%; 95% CI, 0.43% to 0.68%) (P < 0.0001), and there was an association between younger age and recent HIV infection (P < 0.004).
doi:10.1128/JCM.05107-11
PMCID: PMC3372162  PMID: 22403429
17.  Effect of Natural Polymorphisms in the HIV-1 CRF02_AG Protease on Protease Inhibitor Hypersusceptibility 
Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.
doi:10.1128/AAC.06079-11
PMCID: PMC3346603  PMID: 22330918
18.  Comparative Effectiveness of Dried-Plasma Hepatitis B Virus Viral Load (VL) Testing in Three Different VL Commercial Platforms Using ViveST for Sample Collection 
Journal of Clinical Microbiology  2012;50(1):145-147.
Ninety-six samples from hepatitis B virus (HBV)-infected individuals were used to compare ViveST samples to frozen samples in COBAS TaqMan, RealArt, and VERSANT. Correlation (r) between ViveST samples and frozen samples was 0.99 in all three platforms. Correlations among tests using frozen samples were 0.96 for COBAS and RealArt, 0.94 for COBAS and VERSANT, and 0.97 for VERSANT and RealArt. The results indicate that ViveST may be useful in clinical practice. Different HBV-VL platforms correlated well with one another.
doi:10.1128/JCM.01016-11
PMCID: PMC3256692  PMID: 22012021
19.  Relaxation of Adaptive Evolution during the HIV-1 Infection Owing to Reduction of CD4+ T Cell Counts 
PLoS ONE  2012;7(6):e39776.
Background
The first stages of HIV-1 infection are essential to establish the diversity of virus population within host. It has been suggested that adaptation to host cells and antibody evasion are the leading forces driving HIV evolution at the initial stages of AIDS infection. In order to gain more insights on adaptive HIV-1 evolution, the genetic diversity was evaluated during the infection time in individuals contaminated by the same viral source in an epidemic cluster. Multiple sequences of V3 loop region of the HIV-1 were serially sampled from four individuals: comprising a single blood donor, two blood recipients, and another sexually infected by one of the blood recipients. The diversity of the viral population within each host was analyzed independently in distinct time points during HIV-1 infection.
Results
Phylogenetic analysis identified multiple HIV-1 variants transmitted through blood transfusion but the establishing of new infections was initiated by a limited number of viruses. Positive selection (dN/dS>1) was detected in the viruses within each host in all time points. In the intra-host viruses of the blood donor and of one blood recipient, X4 variants appeared respectively in 1993 and 1989. In both patients X4 variants never reached high frequencies during infection time. The recipient, who X4 variants appeared, developed AIDS but kept narrow and constant immune response against HIV-1 during the infection time.
Conclusion
Slowing rates of adaptive evolution and increasing diversity in HIV-1 are consequences of the CD4+ T cells depletion. The dynamic of R5 to X4 shift is not associated with the initial amplitude of humoral immune response or intensity of positive selection.
doi:10.1371/journal.pone.0039776
PMCID: PMC3387245  PMID: 22768122
20.  Differential Persistence of Transmitted HIV-1 Drug Resistance Mutation Classes 
The Journal of Infectious Diseases  2011;203(8):1174-1181.
Background. Transmitted human immunodeficiency virus type 1 (HIV-1) drug resistance (TDR) mutations can become replaced over time by emerging wild-type viral variants with improved fitness. The impact of class-specific mutations on this rate of mutation replacement is uncertain.
Methods. We studied participants with acute and/or early HIV infection and TDR in 2 cohorts (San Francisco, California, and São Paulo, Brazil). We followed baseline mutations longitudinally and compared replacement rates between mutation classes with use of a parametric proportional hazards model.
Results. Among 75 individuals with 195 TDR mutations, M184V/I became undetectable markedly faster than did nonnucleoside reverse-transcriptase inhibitor (NNRTI) mutations (hazard ratio, 77.5; 95% confidence interval [CI], 14.7–408.2; P < .0001), while protease inhibitor and NNRTI replacement rates were similar. Higher plasma HIV-1 RNA level predicted faster mutation replacement, but this was not statistically significant (hazard ratio, 1.71 log10 copies/mL; 95% CI, .90–3.25 log10 copies/mL; P = .11). We found substantial person-to-person variability in mutation replacement rates not accounted for by viral load or mutation class (P < .0001).
Conclusions. The rapid replacement of M184V/I mutations is consistent with known fitness costs. The long-term persistence of NNRTI and protease inhibitor mutations suggests a risk for person-to-person propagation. Host and/or viral factors not accounted for by viral load or mutation class are likely influencing mutation replacement and warrant further study.
doi:10.1093/infdis/jiq167
PMCID: PMC3107558  PMID: 21451005
21.  Faster HIV-1 Disease Progression among Brazilian Individuals Recently Infected with CXCR4-Utilizing Strains 
PLoS ONE  2012;7(1):e30292.
Introduction
Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression.
Methods
A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/µL.
Results
Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4<350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4>350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4>350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype.
Conclusions
Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.
doi:10.1371/journal.pone.0030292
PMCID: PMC3266896  PMID: 22291931
22.  The Influence of HIV-1 Subtype in the Response to Therapeutic Dendritic Cell Vaccine 
The Open AIDS Journal  2012;6:289-292.
In the present study, we investigated the influence of HIV-1 subtype in the response to the dendritic cell (DC) therapeutic vaccine for HIV. HIV-1 viral load and TCD8+/TCD4+ cell counts for up to 48 weeks after vaccination. Out of 19 immunized subjects, 13 were infected by subtype B, 5 by subtype F, and 1 by subtype D. Overall, 42.1% (8/19) achieved a viral load decline of ≥ 1 log10 sustained up to 48 weeks after immunization. Such magnitude of viral load drop was seen in 80% (4/5) of subtype F infected patients, and in 23.0% (3/13) of the subtype B infected ones (p=0.08). Moreover, mean viral load decline was 1.32 log10, for subtype F infected individuals compared to 0.5 log10 among subtype B infected patients (p=0.01). The variation in TCD4+ cell count was not related to HIV-1 subtype. Larger studies are necessary to confirm the efficacy of this immunotherapy and the differential response according to the background genetic diversity of HIV-1.
doi:10.2174/1874613601206010289
PMCID: PMC3534477  PMID: 23289053
Dendritic cells; HIV-1 subtype; vaccine; immunotherapy.
23.  The Brazilian Network for HIV-1 Genotyping External Quality Control Assurance Programme 
The Brazilian network for genotyping is composed of 21 laboratories that perform and analyze genotyping tests for all HIV-infected patients within the public system, performing approximately 25,000 tests per year. We assessed the interlaboratory and intralaboratory reproducibility of genotyping systems by creating and implementing a local external quality control evaluation. Plasma samples from HIV-1-infected individuals (with low and intermediate viral loads) or RNA viral constructs with specific mutations were used. This evaluation included analyses of sensitivity and specificity of the tests based on qualitative and quantitative criteria, which scored laboratory performance on a 100-point system. Five evaluations were performed from 2003 to 2008, with 64% of laboratories scoring over 80 points in 2003, 81% doing so in 2005, 56% in 2006, 91% in 2007, and 90% in 2008 (Kruskal-Wallis, p = 0.003). Increased performance was aided by retraining laboratories that had specific deficiencies. The results emphasize the importance of investing in laboratory training and interpretation of DNA sequencing results, especially in developing countries where public (or scarce) resources are used to manage the AIDS epidemic.
doi:10.1186/1758-2652-14-45
PMCID: PMC3192700  PMID: 21936945
24.  Prevalence, Incidence Density, and Genotype Distribution of GB Virus C Infection in a Cohort of Recently HIV-1-Infected Subjects in Sao Paulo, Brazil 
PLoS ONE  2011;6(4):e18407.
Background
The results of previous studies elsewhere have indicated that GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus type 1 (HIV-1) due to similar transmission routes of both viruses. The aim of this study was to determine the prevalence, incidence density and genotypic characteristics of GBV-C in this population.
Methodology/Principal Findings
The study population included 233 patients from a cohort primarily comprised of homosexual men recently infected with HIV-1 in São Paulo, Brazil. The presence of GBV-C RNA was determined in plasma samples by reverse transcriptase-nested polymerase chain reaction and quantified by real-time PCR. GBV-C genotypes were determined by direct sequencing. HIV viral load, CD4+ T lymphocyte and CD8+ T lymphocyte count were also tested in all patients. The overall prevalence of GBV-C infection was 0.23 (95% CI: 0.18 to 0.29) in the study group. There was no significant difference between patients with and without GBV-C infection and Glycoprotein E2 antibody presence regarding age, sex, HIV-1 viral load, CD4+ and CD8+T cell counts and treatment with antiretroviral drugs. An inverse correlation was observed between GBV-C and HIV-1 loads at enrollment and after one year. Also, a positive but not significant correlation was observed between GBV-C load and CD4+ T lymphocyte. Phylogenetic analysis of the GBV-C isolates revealed the presence of genotype 1 and genotype 2, these sub classified into subtype 2a and 2b.
Conclusion/Significance
GBV-C infection is common in recently HIV -1 infected patients in Sao Paulo, Brazil and the predominant genotype is 2b. This study provides the first report of the GBV-C prevalence at the time of diagnosis of HIV-1 and the incidence density of GBV-C infection in one year.
doi:10.1371/journal.pone.0018407
PMCID: PMC3071701  PMID: 21483671
25.  Correction: Analysis of the Origin and Evolutionary History of HIV-1 CRF28_BF and CRF29_BF Reveals a Decreasing Prevalence in the AIDS Epidemic of Brazil 
PLoS ONE  2011;6(3):10.1371/annotation/25854e06-a961-4fec-8664-38667cc278b8.
doi:10.1371/annotation/25854e06-a961-4fec-8664-38667cc278b8
PMCID: PMC3061945

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