Avaliação de micrornas como biomarcadores moleculares no câncer de próstata

A incidência do câncer de próstata (CaP) vem aumentando na população brasileira. Os diagnósticos atuais de CaP são baseados na detecção do antígeno específico da próstata (PSA) no sangue, no exame digital retal (DRE) e na biópsia da próstata. No entanto, o uso do DRE e do exame de triagem de PSA têm valor diagnóstico limitado. MicroRNAs (miRNAs) são pequenas sequências de RNA não codificador que regulam genes específicos envolvidos no início e no desenvolvimento do CaP. MiRNAs estáveis foram encontrados em fluidos biológicos, como plasma e urina, surgindo como uma nova classe não invasiva de biomarcadores para detecção do CaP. O objetivo deste estudo foi investigar o perfil de expressão dos miRNAs em amostras de plasma, urina e tecido da próstata de pacientes com diagnóstico de CaP e indivíduos controles, visando utilizar estes marcadores como exame de triagem para CaP. O estudo foi realizado com três conjuntos de amostras, divididos nas fases de “descoberta”, “triagem” e “validação”. Na fase de descoberta foram selecionados 44 miRNAs a partir de um estudo piloto e dados da literatura para serem analisados na fase de triagem por RT-qPCR. A expressão dos 44 miRNAs foi investigada nos três espécimes clínicos de 40 pacientes (20 pacientes com CaP e 20 controles), usando cartões de miRNA TaqMan Custom Array (Thermo Fisher Scientific). Nove miRNAs foram diferencialmente expressos na fase de triagem entre casos e controles nas amostras de plasma, urina e tecido. Destes nove miRNAs, quatro foram selecionados para fase de validação em 22 pacientes com CaP e 28 indivíduos controles, utilizando a mesma metodologia de PCR. Na etapa de validação foi constatado que os miR-200b-3p, miR-21-5p e miR-375 foram significativamente mais expressos no tecido de CaP em relação ao controle, apresentando valores AUC de 0,640, 0,740 e 0,815, respectivamente. Apenas o miR-375 foi significativamente regulado positivamente (AUC = 0,677) em amostras de plasma de pacientes com CaP em comparação com os controles. O mir-200b-3p plasmático mostrou valor AUC semelhante (0,664) em pacientes com CaP, mas sem significância estatística (P> 0,05). Os miRNAs urinários não mostraram valores de AUC com relevância estatística. A associação de pares de miRNAs ou sua combinação com valores de PSA não resultou em melhoria adicional aos valores de AUC observados, quando comparado com os valores de um único miRNA. Nossos resultados sugerem que o miR-375 deve ser considerado um potencial biomarcador de triagem para o diagnóstico de CaP, uma vez que a diferença de expressão significativa no plasma e no tecido da próstata. Entretanto, estudos prospectivos de grande escala ainda são necessários para corroborar nossos achados como biomarcadores adjuntos aos exames de PSA e DRE, auxiliando no diagnóstico de câncer de próstata.Prostate cancer (PCa) incidence has been rising in Brazilian population. Current PCa diagnostics are based on the detection of prostate specific antigen (PSA) in blood, digital rectal examination (DRE) and prostate biopsy. However, DRE and PSA screening have diagnostic value limited. MicroRNAs (miRNAs) are small sequences of non-coding RNA regulating specific genes involved in the onset and development of PCa. Stable miRNAs have been found in biofluids, such as plasma and urine, emerging as a non-invasive new class of biomarkers for PCa detection. The aim of the study was to investigate microRNA expression profile in plasma, urine and prostate tissue samples from patients with diagnosis of PCa and subject controls, using these markers as a screening test for PCa. The study was performed with three sample sets, divided in “discovery”, “screening” and “validation” phases. In the discovery phase 44 miRNAs were selected from a pilot study and data from the literature, to be carried out in the screening phase by RT-qPCR. Expression of 44 miRNAs was screened in three different clinical specimens of 40 patients (20 PCa patients and 20 controls) using TaqMan Custom Array miRNA cards (Thermo Fisher Scientific). Nine miRNAs were differentially expressed in the screening phase between cases and controls among plasma, urine and tissue samples. Then, four of the dysregulated miRNAs were selected for validation in 22 PCa patients and 28 subject controls, using the same PCR methodology. After validation, miR-200b-3p, miR-21-5p and miR-375 were significantly overexpressed in PCa tissue with AUC of 0.640, 0.740 and 0.815, respectively. Only miR-375 was significantly upregulated (AUC = 0.677) in plasma samples of PCa patients in comparison to the controls. Plasmatic mir-200b-3p showed similar AUC value (0.664) in PCa patients, but without significance (P > 0.05). Urinary miRNAs showed no significant statistical AUC values. Testing pair of miRNA or their combination with PSA values resulted in no further improvement of AUC values observed for single miRNA. Our results suggest that miR-375 should be considered as potential screening biomarker for the diagnosis of PCa, since the difference in expression was significant in plasma and prostate tissue. However, large-scale prospective studies are still needed to validate our findings as adjunct biomarkers for PSA and DRE in diagnosis of prostate cancer

Enhancing tuberculosis diagnosis by polymerase chain reaction: An experience at a tertiary hospital

Introduction: Tuberculosis (TB) persists as a severe global public health issue. The aim of the present study was to evaluate the performance of an in-house TB PCR (polymerase chain reaction) in sputum.Methods: DNA from sputum specimens were submitted to a nested-PCR protocol for the IS6110 region detection. PCR results were compared to those of the traditional methods for TB diagnosis, i.e., acid-fast bacilli (AFB) smear microscopy and culture. We analyzed sputum samples obtained from 133 patients.Results: A total of 48 (36%) cultures yielded indeterminate results due to contamination. This high contamination rate may be explained by the fact that samples from fibrocystic patients were included in this study. Additionally, other five samples were positive for nontuberculous mycobacteria (NTM). Therefore, it was possible to compare 80 patients for M. tuberculosis detection. We found 14 positive samples: five presented positive results in the three methods (5/14; 35.7%), two were positive in culture and PCR (2/14; 14.3%), one was positive in AFB and PCR (1/14; 7.1%), five were positive only in PCR (5/14; 35.7%) and 1 was positive only in culture (1/14; 7.1%). Thus, positivity rates for each technique were: 7.5% for AFB (6/80), 10% for culture (8/80) and 16.25% for PCR (13/80). Among the 48 patients who had indeterminate results in sputum culture, two samples were positive in PCR.Conclusion: Considering the limitations of the traditional methods, the use of PCR as a molecular technique could be advantageous for TB diagnosis.