Dora Méndez del Castillo

Comparison of HPV genotyping by type-specific PCR and sequencing

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide and there is a strong link between certain high-risk viral types and cervical carcinogenesis. Although there are several typing methods, it is still unclear which test is the best. This study compared the effectiveness of type-specific PCR (TS-PCR) and sequencing, with a focus on their clinical application. A total of 260 cervical samples from HPV-positive patients were tested for types 6, 11, 16, 18, 31, 33 and 35 using TS-PCR and sequencing. The genotype was identified in 36% of cases by TS-PCR and in 75% by sequencing. Sequencing was four times more likely to identify the viral type in positive samples than TS-PCR (p = 0.00). Despite being more effective for virus genotyping, sequencing was unable to identify viral types in multiple infections. Combining both techniques resulted in highly sensitive detection (87% of cases), showing that they are complementary methods. HPV genotyping is an important step in HPV management, helping to identify patients with a higher risk of developing cervical cancer and contributing to the development of type-specific vaccines.

Alta prevalência do genótipo 1 em portadores de hepatite C crônica em Belo Horizonte, MG

The hepatitis C virus is characterized by significant genetic heterogeneity. It is currently classified into six main genotypes and several subtypes. Determining the genotype of the virus is important in clinical practice for guiding the treatment for individuals with chronic hepatitis C. The prevalence of different genotypes and subtypes of the hepatitis C virus has not been fully studied in some regions of Brazil. In this study, 788 samples from patients with chronic hepatitis C who were attended at the Viral Hepatitis Reference Centers in Belo Horizonte were analyzed between 2002 and 2006. The genotyping of the virus was performed by direct sequencing of the 5 UTR region. Additionally, phylogenetic analysis was performed, including all of the genotypic variants obtained. High prevalence of genotype 1 (78.4%; 1b [40.4%], 1a [37.5%] and 1a/b [0.5%]) was observed, followed by genotypes 3a (17.9%) and 2b (3.1%). Three samples were identified as genotype 2a/c (0.4%) and two as genotype 4 (0.2%). The phylogenetic analysis showed the expected segregation of the sequences obtained, with regard to the reference sequences for genotypes 1, 2, 3 and 4, except for two samples of genotype 1a. The high prevalence of genotype 1 (78.4%) found in this population from Belo Horizonte was similar to previous reports from other cities such as Rio de Janeiro, but it was higher than what has been described in São Paulo and in the south of the country. The presence of rare atypical sequences from the 5 UTR region suggests that variants in the hepatitis C virus exist in this population.

Associação entre a contagem de linfócitos T CD4+ e a gravidade da neoplasia intra-epitelial cervical diagnosticada pela histopatologia em mulheres infectadas pelo HIV

OBJETIVO: avaliar a associacao entre a contagem de linfocitos T CD4+ e a gravidade da neoplasia intra-epitelial cervical em pacientes HIV positivas. METODOS: estudo transversal no qual foram incluidas 87 pacientes infectadas pelo HIV, confirmado por testes sorologicos previos. Todas eram portadoras do HPV cervical, diagnosticado por meio da reacao em cadeia da polimerase. Foram realizados anamnese, exame fisico e colposcopia de todas em pacientes. A biopsia do colo uterino foi realizada quando indicada pelo exame colposcopico. Os resultados histopatologicos foram classificados com neoplasia intra-epitelial de baixo grau (NIC I) ou de alto grau (NIC II e II). A associacao entre a contagem de linfocitos T CD4+ e a gravidade da lesao foi verificada por meio da comparacao de medias utilizando a analise da variância (ANOVA). RESULTADOS: entre as 60 pacientes biopsiadas foram encontrados 24 casos (40,0%) com NIC I, oito (13,3%) NIC II, tres (5%) NIC III, 14 (23,3%) pacientes somente com cervicite cronica e 11 (18,3%) apresentando efeito citopatico produzido pelo HPV, mas sem perda da polaridade celular. Isso equivale a 35 mulheres com lesao intra-epitelial de baixo grau (NIC I + HPV) (58,3%) e 11 (18,3%) com lesao intra-epitelial de alto grau (NIC II + NIC III). A associacao entre a media da contagem de linfocitos T CD4+ e a gravidade da lesao intra-epitelial cervical nao foi significativa (p=0,901). CONCLUSOES: nao houve associacao entre a contagem de linfocitos T CD4+ e a gravidade da lesao intra-epitelial do colo uterino, diagnosticada pelo exame histopatologico.

Prevalence and risk factors for cervical intraepithelial neoplasia among HIV-infected women.

OBJECTIVES To evaluate the prevalence and the risk factors for cervical intraepithelial neoplasia (CIN) among HIV-infected women. METHODS Cross-sectional study of 494 HIV-infected women in Brazil, between 1998 and 2008. Gynecologic exam was performed, and samples were collected for cervical cytology and for HPV DNA detection. Cervical biopsy was carried out when indicated. HPV infection, CD4 T-lymphocyte count and HIV viral load were compared with cervical histopathology. Univariate and multivariate statistical analyses were performed to evaluate the statistical association of several risk factors. RESULTS CIN prevalence detected by histopathology was 23.4% (6% of CIN2/3 and 17.4% cases of CIN1). Multivariate analysis confirmed an independent association of CIN with CD4 T-lymphocyte count below 200 cells/mm³ (OR 5.0, 95% CI 2.5-10.1), with a positive detection of HPV DNA (OR 2.0, 95% CI 1.2-3.5), and with age < 34 years old (OR 1.5, 95% CI 1.0-2.4). HIV viral load and antiretroviral use were not independent risk factors for CIN. CONCLUSIONS Severity of immunosuppression, presence of HPV infection and younger age are strong predictors of CIN among HIV-infected women.

Prevalence and multiplicity of HPV in HIV women in Minas Gerais, Brazil

OBJECTIVE To detect the frequency and subtypes of HPV in the uterine cervix of HIV-infected women. METHODS Sample consisted of 288 HIV-infected women, recruited from the public health system of five cities of Minas Gerais, Brazil. Women were seen from August 2003 to August 2008. Cervical samples were collected for cytological analysis and for HPV DNA detection, using polymerase chain reaction (PCR). HPV DNA was classified according to its oncogenic potential in low risk (types 6, 11) and high risk (types 16, 18, 31, 33, 35). Colposcopy was performed, followed by cervical biopsy when necessary. Categorical variables were compared using the Chi-squared test, with a significance level established at the 5% level. RESULTS HPV prevalence was 78.8%. Most frequent genotypes were HPV-6 (63.9%) and HPV-16 (48.5%). High-risk HPV were observed in 70.5% of the women; low-risk in 71.4%; both high and low-risk HPV were detected in 55.1% of the patients. Multiple HPV genotypes were detected in 64.8% of the patients; two genotypes in 23.8%, and three in 18.9%. CONCLUSION HPV prevalence was high among HIV-infected women. Multiple HPV genotypes were common in samples from the uterine cervix of HIV-infected women.

Incidence of cervical intraepithelial neoplasia in a cohort of HIV-infected women

To assess cervical intraepithelial neoplasia (CIN) incidence in HIV‐positive women and the risk factors for these lesions.

Populational genetic data for 15 STR markers in the Brazilian population of Minas Gerais

Fifteen short tandem repeats (STR) markers were analyzed (TPOX, D2S1338, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D19S433, and D21S11) in unrelated individuals undergoing paternity studies from Minas Gerais state, Brazil. Allele frequencies and statistical parameters for the 15 loci were calculated.

High Incidence of Biotinidase Deficiency from a Pilot Newborn Screening Study in Minas Gerais, Brazil

OBJECTIVE To assess the incidence of biotinidase deficiency among newborns and their clinical outcome up to one year of age in a large pilot screening study in Minas Gerais, Brazil. METHODS A prospective cohort study was conducted from September 2007 to June 2008 with heel-prick blood samples collected on filter paper for the purpose of newborn screening. A qualitative colorimetric test was used as the primary screening method. Colorimetric-positive cases were further tested with a serum confirmatory assay. Gene sequencing was performed for eight children suspected with biotinidase deficiency and for some of their parents. Positive cases were daily supplemented with oral biotin and were followed up for approximately six years. RESULTS Out of 182,891 newborns screened, 129 were suspected of having biotinidase deficiency. Partial deficiency was confirmed in seven children (one was homozygous for p.D543E) and profound deficiency in one child (homozygous p.H485Q). Thus the incidence was one in 22,861 live births (95% confidence interval 1:13,503 to 1:74,454) for profound and partial biotinidase deficiency combined. Two novel mutations were detected: p.A281V and p.E177K. In silico analysis and estimation of the enzyme activity in the children and their parents showed that p.A281V is pathogenic and p.E177K behaves like p.D444H. CONCLUSION The incidence of biotinidase deficiency in newborn screening in Minas Gerais was higher than several international studies. The sample size should be larger for final conclusions. Oral daily biotin apparently precluded clinical symptoms, but it may have been unnecessary in some newborns.

Discordância de apresentação da doença celíaca em gêmeos monozigóticos

CONTEXTO: A doenca celiaca e uma enteropatia autoimune causada pela sensibilidade ao gluten em individuos geneticamente predispostos. Apesar da caracteristica genetica da doenca, estudos demonstram discordância de 30% na sua apresentacao em gemeos monozigoticos. OBJETIVO: Apresentar dois pares de gemeos monozigoticos, comprovados por estudos geneticos, discordantes para apresentacao da doenca celiaca. METODO: Os pacientes foram acompanhados no Servico de Gastroenterologia Pediatrica do Hospital das Clinicas da Universidade Federal de Minas Gerais desde 1990, sendo submetidos a exames clinicos periodicos, biopsias intestinais e sorologia para anticorpos IgG e IgA antigliadina, determinados pela tecnica de ELISA (ensaio imunoenzimatico), e anticorpos classe IgA antiendomisio, determinados pela tecnica de imunofluorescencia indireta. Estudos geneticos foram realizados atraves da tecnica de amplificacao por PCR e posterior tipagem de loci de microssatelites do tipo STR (short tandem repeats). RESULTADOS: Em cada par de gemeos, apenas um apresentou doenca celiaca ate o momento, mostrando que, apesar do genotipo identico, este nao foi o unico determinante para a expressao da doenca. CONCLUSAO: Outros fatores, ambientais e geneticos, parecem contribuir para determinacao da doenca.

Incidence of Cervical Human Papillomavirus and Cervical Intraepithelial Neoplasia in Women with Positive and Negative HIV Status

Objectives To evaluate the incidence and factors associated with cervical intraepithelial neoplasia (CIN) and cervical infection by human papillomavirus (HPV) among HIV-positive and HIV-negative women. Methods A cohort of 103 HIV positive and 113 HIV negative women were monitored between October 2008 and February 2012, for at least one year. Procedures included cervical cytology, DNA/HPV detection by polymerase chain reaction, colposcopy with biopsy if necessary, followed by an interview for exposure characteristics data. CIN was based on the histopathological results. Results The incidence of CIN was of 8.8 and 4.6 cases/100 women-years in HIV-positive and HIV-negative women, respectively. HIV-positive women presented a hazard ratio (HR) of 2.8 for CIN and developed lesions earlier (0.86 year) than HIV-negative women (2 years) (p = 0.01). The risk of developing CIN decreased with age (HR = 0.9) and marital status (HR = 0.4). HPV patients presented a higher incidence of CIN when compared HIV-positive and HIV-negative women (p = 0.01). The incidence of HPV cervical infection was 18.1 and 11.4 cases/100 women-years in HIV-positive and HIV-negative women, respectively. Those HIV-positive presented earlier HPV infection(p = 0.002). The risk of developing HPV infection decreased with age and was higher among HIV-positive women. HPV 16 was the most common type in HIV-positive women, and also the type most closely associated with CIN in HIV-negative women. Conclusions HIV-positive women had a greater incidence of HPV and CIN, and in a shorter time interval. More rigorous and timely clinical control is required for this group.