Development and validation of a variant detection workflow for BRCA1 and BRCA2 genes and its clinical application based on the Ion Torrent technology.

BACKGROUND: Breast cancer is the most common among women worldwide, and ovarian cancer is the most difficult gynecological tumor to diagnose and with the lowest chance of cure. Mutations in BRCA1 and BRCA2 genes increase the risk of ovarian cancer by 60% and breast cancer by up to 80% in women. Molecular tests allow a better orientation for patients carrying these mutations, affecting prophylaxis, treatment, and genetic counseling. RESULTS: Here, we evaluated the performance of a panel for BRCA1 and BRCA2, using the Ion Torrent PGM (Life Technologies) platform in a customized workflow and multiplex ligation-dependent probe amplification for detection of mutations, insertions, and deletions in these genes. We validated the panel with 26 samples previously analyzed by Myriad Genetics Laboratory, and our workflow showed 95.6% sensitivity and 100% agreement with Myriad reports, with 85% sensitivity on the positive control sample from NIST. We also screened 68 clinical samples and found 22 distinct mutations. CONCLUSIONS: The selection of a robust methodology for sample preparation and sequencing, together with bioinformatics tools optimized for the data analysis, enabled the development of a very sensitive test with high reproducibility. We also highlight the need to explore the limitations of the NGS technique and the strategies to overcome them in a clinically confident manner.

Gravidade das coinfecções virais em lactentes hospitalizados com infecção por vírus sincicial respiratório / Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection

OBJETIVO: Comparar a gravidade de infecções causadas por um único vírus (VSR) com a gravidade de coinfecções. MÉTODOS: Este estudo avaliou uma coorte histórica de lactentes com infecção aguda por VSR. Secreção de nasofaringe foi coletada de todos os pacientes rotineiramente para pesquisa viral usando técnicas de biologia molecular. Os seguintes desfechos foram analisados: tempo total de internação, duração da oxigenioterapia, admissão em unidade de terapia intensiva e uso de ventilação mecânica. Os resultados foram ajustados para os fatores confundidores (prematuridade, idade e aleitamento materno). RESULTADOS: Foram incluídos no estudo 176 lactentes com idade média de 4,5 meses e diagnósticos de bronquiolite e/ou pneumonia. Cento e vinte e um tinham infecção única por VSR, e 55 tinham coinfecções (24 VSR + adenovírus, 16 VSR + metapneumovírus humano e 15 outras associações menos frequentes). Os quatro desfechos de gravidade avaliados foram semelhantes entre o grupo com infecção única por VSR e os grupos com coinfecções, independente do tipo de vírus associado com o VSR. CONCLUSÃO: As coinfecções virais não parecem alterar o prognóstico de lactentes hospitalizados com infecção aguda por VSR.

OBJECTIVE: To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS: A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS: A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION: Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection.

OBJECTIVE: To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS: A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS: A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION: Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

The prevalence of human papillomavirus in the oropharynx in healthy individuals in a Brazilian population.

Oncogenic human papillomavirus (HPV), a causative agent of uterine cervical cancer, has also been detected in head and neck squamous cell cancers, especially in squamous cell carcinomas of the tonsils. However, the true HPV prevalence in normal and neoplasic oropharyngeal mucosa remains uncertain. To determine the prevalence of HPV DNA in normal oropharyngeal mucosa of cancer-free individuals, a study was carried out on 50 Brazilian subjects. PCR was performed to identify HPV DNA in samples from four sites in the oropharynx (tonsils, soft palate, base of the tongue, and back wall of the pharynx). For amplification of the HPV DNA, MY09/11 consensus primers were used, and specific genotypes were identified by dot-blot hybridization or cloning and sequencing. HPV DNA was present in 14.0% of the individuals, and the identified genotypes were 16, 18, 52, and 61. All these types are considered high-risk (HR) HPV. The tonsils and the soft palate were the sites with the highest HPV prevalence. This study shows the prevalence of HR HPV in the oropharynx of normal individuals. However, the prevalence of HPV is still unclear, and if HPV infection in a healthy it is not known individual predisposes to HPV-associated disease such as oropharyngeal cancer. Thus, it is important to assess the prevalence of HPV in cancer-free individuals, in order to compare it with the HPV prevalence in oropharyngeal carcinomas and to attempt to determine the true role of HPV in the development of head and neck squamous cell cancers.