Sabrina Esteves de Matos Almeida

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. J. Med. Virol. 83:1682–1688, 2011.

Metabolic changes associated with antiretroviral therapy in HIV-positive patients

OBJECTIVEnTo evaluate metabolic changes associated with highly active antiretroviral therapy (HAART) in HIV-positive patients, and to identify risk factors associated.nnnMETHODSnRetrospective study that included 110 HIV-positive patients who where on HAART in the city of Porto Alegre (Southern Brazil) between January 2003 and March 2004. Data on demographic variables, cigarette smoking, diabetes mellitus, cholesterol and triglyceride levels, stage of HIV infection, antiretroviral therapy and HCV coinfection were collected. General linear models procedure for repeated measures was used to test the interaction between HAART and HCV coinfection or protease inhibitor treatment.nnnRESULTSnTotal cholesterol, triglycerides, and glucose levels significantly increased after receiving HAART (p<0.001 for all variables), but no interaction with protease inhibitors was seen for total cholesterol, glucose and triglyceride levels (interaction treatment*protease inhibitors p=0.741, p=0.784, and p=0.081, respectively). An association between total cholesterol levels and HCV coinfection was found both at baseline and follow-up (effect of HCV coinfection, p=0.011). Glucose levels were increased by HAART (treatment effect, p=0.036), but the effect was associated to HCV coinfection (treatment*HCV effect, p=0.018). Gender, smoking habit, intravenous drug use and age were not significantly associated with cholesterol, triglyceride and glucose changes.nnnCONCLUSIONSnHCV-infected patients at baseline were significantly less likely to develop hypercholesterolemia. The results provide further evidence of the role of HAART for the development of metabolic disturbances.OBJETIVO: Evaluar las alteraciones metabolicas asociadas a la terapia anti-retroviral potente en pacientes HIV-positivos e identificar factores de riesgo asociados. METODOS: Estudio retrospectivo con 110 pacientes HIV-positivos que estaban en terapia anti-retroviral potente (HAART) en la ciudad de Porto Alegre (Sur de Brasil), entre enero de 2003 y marzo de 2004. Los datos colectados incluyen variables demograficas, tabaquismo, diabetes mellitas, niveles de colesterol y trigliceridos, fase de la infeccion viral, terapia anti-retroviral y co-infeccion con hepatitis C. El analisis multivariado para medidas repetidas (General Linear Model procedure for Repeated Measures) fue utilizada para analizar la interaccion entre el efecto de uso de HAART y el uso de inhibidores de proteasa o co-infeccion por hepatitis C. RESULTADOS: Fueron observados aumentos significativos en los niveles de colesterol total, trigliceridos y glucosa posterior al tratamiento con HAART (p<0.001, para todas las variables). Sin embargo, ninguna interaccion del tratamiento con inhibidores de proteasa fue observada para colesterol total, glucosa y trigliceridos (interaccion tratamiento *inhibidores de proteasa p=0.747, p= 0.784 y p= 0.081, respectivamente). Una asociacion entre los niveles de colesterol total y co-infeccion por HCV fue observada tanto antes como despues del tratamiento (efecto de la co-infeccion por hepatitis C, p= 0.0011). Lo niveles de glucosa fueron aumentados por el uso de la HAART (efecto del tratamiento, p= 0.036), siendo este dependiente de la co-infeccion por hepatitis C (efecto interaccion tratamiento *hepatitis C p= 0.018). Genero, tabaquismo, uso de drogas intravenosas y edad no influenciaron significativamente los niveles de colesterol total, trigliceridos y glucosa durante el tratamiento. CONCLUSIONES: Pacientes infectados por el virus de la hepatitis C en el inicio del tratamiento tuvo aumento menos significativo en los niveles de colesterol total. Los resultados refuerzan las evidencias del papel de la HAART en el desarrollo de desordenes metabolicos.

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 4.6u2009×u200910−5 to 2.3u2009×u200910−3, and the overall mutation rate estimate was 1.2u2009×u200910−3. The average of the paternal mutation rate (1.8u2009×u200910−3) was five times higher than the maternal rate (0.36u2009×u200910−3). 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.

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 500xa0ml 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.

Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus

Persistent infection with high-risk human papillomavirus (HR-HPV) has been associated with cervical cancer. Developing assays for the identification of these viral types is of great importance for monitoring patients and controlling strategies. The development of the MCHA (microplate colorimetric hybridization assay), a PCR-based method for identifying six of the most common HR-HPV types (HPV 16, 18, 31, 33, 39 and 45) is described. The MCHA combines the amplification with the GP5+/GP6+ consensus primers followed by PCR reverse hybridization with specific probes and detection through a colorimetric assay. The performance of the MCHA was evaluated using 108 DNA samples typed previously by the PapilloCheck(®). The agreement between both methods was 69.4% for HPV 16; 79.1% for HPV 45; 82.4% for HPV 18; 93.6% for HPV 31; 87.9% for HPV 33, and 17.6% for HPV 39. The assay had higher sensitivity than the Papillocheck(®), particularly for identifying HPV 16 and 18. The MCHA seemed to be sensitive and specific for the identification of the most prevalent HPV types in invasive cervical cancer, HPV 16, 18, 45, 33 and 31. It requires low-cost reagents and common laboratory apparatus.

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 Eagles 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.

QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES

SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

Use of FTA elute card impregnated with cervicovaginal sample directly into the amplification reaction increases the detection of human papillomavirus DNA.

This study aimed to evaluate the use of the FTA elute cardTM impregnated with cervicovaginal sample directly in the PCR amplification for detection of HPV-DNA. The results were compared to a reference technique. This method was more efficient than the protocol indicated by the manufacturer, identifying 91.7% against 54.2% of the positive samples.

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.

Comparison of urine and self-collected vaginal samples for detecting human papillomavirus DNA in pregnant women

To investigate the utility of urine sampling for detecting human papillomavirus (HPV) DNA among pregnant women and to compare HPV DNA detection in urine with detection in vaginal samples.