HIV-1 genetic diversity and transmitted drug resistance to integrase strand transfer inhibitors among recently diagnosed adults in Porto Alegre, South Brazil.

OBJECTIVES: The HIV-1 genetic diversity and the presence of transmitted drug resistance mutations (TDRMs) against integrase strand transfer inhibitors (INSTIs) were assessed sequencing samples of antiretroviral (ARV)-naive HIV-1-infected individuals from South Brazil. METHODS: Viral RNA from 42 ART-naive individuals was submitted to complete HIV-1 integrase gene amplification by RT-PCR and sequencing. RESULTS: Viral strains carrying TDRMs against INSTIs were not detected in the present study. However, the polymorphisms L74M and L74I were each observed in 4.8% of the individuals. These accessory mutations have been reported as putative causes of TDRMs in ART with raltegravir, but only when associated with additional major mutations. When submitted to HIV-1 subtyping, 50% were classified as subtype C, 21% as recombinant BC, 19% as subtype B, 4.8% as subtype F1 and 4.8% as recombinant CF1. CONCLUSIONS: All 42 ARV-naive individuals were apparently susceptible to INSTIs, included in the Brazilian therapeutic guideline since 2009. To the best of our knowledge, this is the first study to evaluate TDRMs against INSTIs in Brazil. The most prevalent HIV-1 subtypes were subtype C, followed by the recombinant BC and subtype B, which is in agreement with previous studies. However, the presence of subtype F1 and recombinant CF1 reported herein was not observed in previous studies.

Association between human papillomavirus (HPV) DNA and micronuclei in normal cervical cytology.

The aim of the present study was to investigate the association between HPV-DNA and micronucleus (MN) frequency in women with normal cervical cytology. A total of 158 normal cervical smears were analyzed cytologically. The HPV genome was amplified using the GP5+/bioGP6+ consensus primers. HPV-DNA of high-risk types 16, 18, 31, 33, 39, 45 and 59 were also investigated. Of the 158 samples, 20 (12.7%) and 47 (29.7%) were positive for HPV-DNA and MN, respectively. Evidence for MN was found in 11 out of 20 (55%) HPV-DNA positive samples and in 36 out of 138 (26.1%) HPV-DNA negative ones. MN presence was significantly higher in HPV-DNA positive samples (p = 0.016). On the other hand, the absence of MN observed in a considerable number of HPV-DNA negative samples (102) may be of great value in predicting the absence of HPV. The mean age of HPV-DNA positive women (34.2 ± 12.6) was significantly lower than the mean age of HPV-DNA negative women (43.9 ± 13.7) (p = 0.003). Infection by one or multiple HPV types was found in 11 out of 20 (55.0%) and 9 out of 20 (45.0%) samples, respectively. The evaluation of MN using cervical smears collected for cytology tests could, thus, be used as additional information to monitor a population's exposure to HPV.

Mutation rate estimates for 13 STR loci in a large population from Rio Grande do Sul, Southern Brazil.

Short tandem repeat (STR) polymorphisms have been extensively used in forensic genetics analysis. Knowledge about the locus-specific mutation rates of STRs improves forensic probability calculations and interpretations of diversity data. To incorporate single-locus diversity information into autosomal STR mutation rate estimations, 13 STR loci were studied during 2007-2009 in 10,959 paternity investigation cases from Rio Grande do Sul, the southernmost state of Brazil, covering an overall number of 284,934 allelic transfers. A total of 355 mutations were identified; 348 repeats were gains or losses of one step, three were gains or losses of two steps, and four were gains or losses of not stepwise mutation. The mutation rates ranged from 4.6 × 10(-5) to 2.3 × 10(-3), and the overall mutation rate estimate was 1.2 × 10(-3). The average of the paternal mutation rate (1.8 × 10(-3)) was five times higher than the maternal rate (0.36 × 10(-3)). The observed mutational features for STRs have important consequences for forensic applications, including the definition of criteria for exclusion in paternity testing and the interpretation of DNA profiles in identification analysis.

Co-circulation HIV-1 subtypes B, C, and CRF31_BC in a drug-naïve population from Southernmost Brazil: analysis of primary resistance mutations.

In Southernmost Brazil HIV-1 subtypes B, C, and CRF31_BC co-circulates and, since 1996 with the implementation of free access to highly active antiretroviral treatment (HAART), this epidemic is under a quite characteristic selective pressure. The profile of mutations and polymorphisms in the protease (PR) and reverse transcriptase (RT) genes of HIV-1 from untreated patients living in Porto Alegre, Southernmost Brazil were evaluated in order to identify the subtypes and circulating drug resistant genotypes. Blood samples from 99 HIV-1 positive drugs-naïve patients were collected from 2006 to 2007 in Porto Alegre, Brazil. HIV PR and RT genes were amplified, sequenced, and subtyped. The HIV-1 genotyping was performed by partial sequence analysis of the pol in the HIV Drug Resistance Database of Stanford University. Phylogenetic analyses allowed to classify the HIV samples according to their subtypes: B (26.2%), C (39.4%), F (1.1%), CRF31_CB (19.2%), and URF (14.1%). Eight (8.1%) samples showed primary resistance mutations according to the Calibrated Population Resistance tool based in the 2009 Surveillance Drug Resistance Mutation list. Two samples presented resistance mutations to PI, three NRTI and three NNRTI. There was no significant association between presence of resistant genotypes and subtypes, but resistance mutations seem to be less frequent in the subtype C. In addition, this study describes for the first time the mutational profile of CRF31_BC to PI, NRTI, and NNRTI. Genetic analyses of HIV-1 from naïve patients are a promising and important method for surveillance of HIV infection.

Role of SEP15 Gene Polymorphisms in the Time of Progression to AIDS.

AIMS: HIV infection is a chronic disease that requires intensive treatment in its later phases, including dietary supplementation. Several studies have suggested clinical improvements in patients with high levels of selenium, linking these levels with a longer progression to AIDS. The objective of this study was to verify the association of two polymorphisms in the SEP15 gene, which encodes a selenoprotein that is responsible for the transport of selenium in cells, with the time of progression to AIDS in HIV-1-infected patients. METHODS: Blood samples were obtained from 139 HIV-1-positive individuals after they provided informed consent. DNA was isolated and genotyped using real-time polymerase chain reaction for the presence of SEP15 single nucleotide polymorphisms (rs5859 and rs561104). Questionnaires on sociodemographic features and behavior were answered, and the time of progression to AIDS was estimated based on a medical chart analysis. RESULTS: The allelic and genotypic frequencies did not differ between rapid and nonrapid progressors; however, the presence of the AA genotype of the rs5859 polymorphism was associated with a shorter time of progression to AIDS compared with GG homozygotes (hazard ratio = 3.62, 95% CI = 1.55-8.43, p = 0.003). CONCLUSION: These findings show the importance of genetic analysis of the SEP15 gene in individual patients with regard to predicting time of progression to AIDS.

Molecular detection of human adenovirus in sediment using a direct detection method compared to the classical polyethylene glycol precipitation.

Various effective methods have been developed to measure the concentration of viruses in sediment samples. However, there is need to standardize less laborious and simpler techniques. The objective of the present study was to compare two different methods to measure the concentration of viruses in soil samples. The use of polyethylene glycol (PEG) was compared with a direct extraction of viral nucleic acids from the samples diluted in modified Eagle's minimal essential medium (E-MEM). The presence of adenovirus in the samples was detected by real-time quantitative polymerase chain reaction (qPCR). Only six samples (30%) were positive for adenovirus when PEG technique was used. The direct method showed 16 (80%) samples positive for adenovirus. Therefore, direct detection (i.e. without previous concentration) demonstrated a higher rate of detection, better effectiveness, and shorter execution time. Furthermore, direct detection uses reagents that are often readily available in virology laboratories. Thus, it is an attractive alternative to other methods of detection of virus particles in sediments.

Surface water quality in the Sinos River basin, in Southern Brazil: tracking microbiological contamination and correlation with physicochemical parameters.

Around the world, enteric viruses are often found in surface waters. This study set out to evaluate the occurrence of adenoviruses (AdVs) in water samples, and its relation to different physical, chemical, and bacteriological parameters [total coliform (TC) and fecal coliform (FC), represented by Escherichia coli]. Monthly samples of 500 ml of raw water were collected from May 2011 to June 2013 in eight abstraction points water treatment stations along three stretches of the Sinos River Basin (SRB), in Southern Brazil and, subsequently, were analyzed using real-time polymerase chain reaction (qPCR). AdVs from different species, from human (HAdV), and from other animals (CAV1-2, BAdV, PAdV, and AvAdV) were detected along the three stretches of the basin, indicating fecal contamination from different sources and proving the inefficiency of the wastewater treatment in the waters of the SRB and intensifying the strong influence of human activities that can contribute to the presence of inhibitory substances such as organic acids in surface of these waters. Statistical analyses revealed no significant correlations between the concentrations of TC and FC and the concentrations of AdVs. We observed a small, nonconstant, and unstable correlation between viruses and physicochemical parameters. These correlations were not sufficiently consistent to establish a reliable association; therefore, this study corroborates that only the viral assay itself is reliable for the diagnosis of fecal contamination by viruses in environmental samples.

Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more extensive analysis and in vitro experiments.