RESUMEN
AIMS: The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A-D targeting the 5' untranslated region (5' UTR) of enteroviruses genome. METHODS AND RESULTS: The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID50 assay-1 while the sensitivity for Enterovirus A was 3·00 CCID50 assay-1 . The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A-D and validated on 20 clinical isolates. CONCLUSIONS: This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5' UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A-D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.
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Regiones no Traducidas 5'/genética , Colorimetría , Enterovirus/aislamiento & purificación , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Enterovirus/genética , Infecciones por Enterovirus/virología , Genoma Viral/genética , Humanos , Virus ARN/genética , Virus ARN/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
HPV16 infection is found in more than 50 % of cervical cancer cases worldwide, triggering the development of numerous molecular techniques for viral diagnosis. The present study focuses on the development of a colorimetric IsoPCR for HPV16 DNA detection. The methodology combines the advantages of PCR and LAMP, while the most significant aspect of the new established methodology is the visual detection of amplification products through hydroxynapthol blue dye, thus minimizing the time and labor needed. An experimental cut-off value was tested through reconstitution experiments, while the specificity was evaluated by assessing clinical samples. The analytical sensitivity of the new colorimetric IsoPCR was found to be 0.1 viral DNA copy per reaction, while the specificity was 100 % for the detection of HPV16 DNA. The assay enabled the amplification of viral DNA in cases with viral load lower than 1 copy. In conclusion, the new established colorimetric IsoPCR can be regarded as an attractive molecular tool that facilitates the specific, rapid and highly sensitive visual detection of HPV16 DNA even at the very early stages of viral infection.
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Colorimetría , Papillomavirus Humano 16 , Técnicas de Amplificación de Ácido Nucleico , Papillomavirus Humano 16/genética , Humanos , Naftalenosulfonatos , Sensibilidad y EspecificidadRESUMEN
Several members of the human tissue kallikrein-related peptidase (KLK) family are emerging cancer biomarkers. The aim of this study was to analyse the expression of a panel of KLKs in colorectal cancer and to find out if the multiparametric combination of them can increase the accuracy of prediction of patients survival beyond the traditional clinical information. Nine KLKs (KLK5-8, KLK10, KLK11, KLK13-15) were measured using ELISA assays in cytosolic extracts of 122 colon cancer tissues and their nearby normal mucosa, obtained during surgery. The mean levels of almost all KLKs in tumour tissues were significantly different from their counterparts of normal tissue (P<0.0001). KLK 5, 6, 7, 13, 14 were significantly associated with overall survival in univariate analysis, but after adjusting for age, TNM and differentiation stage, only KLK5 (HR: 1.24 (95% CI: 1.05-1.47)), KLK7 (HR: 1.57 (95% CI: 1.04-2.37)) and KLK14 (HR: 1.43 (95% CI: 1.05-1.94)) remained significant. Addition of a panel of selected KLK markers to clinical parameters gave an increment in AUC of 0.86 beyond the clinical factors at year 1, showing that it can increase the accuracy of prediction of overall survival beyond the traditional clinical information, particularly the short-term (1 year) survival after surgery.
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Biomarcadores de Tumor/análisis , Carcinoma/diagnóstico , Neoplasias Colorrectales/diagnóstico , Calicreínas/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinoma/metabolismo , Carcinoma/mortalidad , Carcinoma/patología , Estudios de Casos y Controles , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Endopeptidasas/análisis , Endopeptidasas/metabolismo , Humanos , Calicreínas/metabolismo , Persona de Mediana Edad , Pronóstico , Sensibilidad y Especificidad , Análisis de Supervivencia , Distribución TisularRESUMEN
OBJECTIVE: BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients. PATIENTS AND METHODS: We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood. RESULTS: Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR. CONCLUSIONS: Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.
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Virus BK , ADN Viral/orina , Trasplante de Riñón , Orina/citología , Replicación Viral , Adulto , Anciano , Virus BK/genética , Virus BK/fisiología , Niño , ADN Viral/sangre , Humanos , Trasplante de Riñón/efectos adversos , Tamizaje Masivo/métodos , Persona de Mediana Edad , Infecciones por Polyomavirus/orina , Estudios Prospectivos , Urinálisis/métodos , Adulto JovenRESUMEN
PURPOSE: Topoisomerase II alpha (Topo IIa gene location 17q21) is a nucleic enzyme involved in the DNA replication, transcription and chromosome topological formation. Topo IIa inhibition strategies include specific chemotherapeutic agents such as anthracyclines. Our aim was to investigate potential protein alterations of the enzyme comparing them to ki 67 proliferation marker expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS: Using tissue microarray (TMA) technology, 50 specimens consisting of histologically confirmed PTCs (n=20), multi-nodular goiters (n=20) and also normal thyroid epithelia (n=10) were cored and re-embedded in the final paraffin block. Immunohistochemical analysis was performed using monoclonal anti-Topo IIa and anti-ki 67 (MIB-1) antibodies. Digital image analysis assay was also applied for the evaluation of the protein expression results (Nuclear Labeling Index-NLI). RESULTS: Topo IIa and ki 67 proteins were overexpressed in 4/20 (20%) and 14/20 (70%) cases, respectively. Concerning multi-nodular goiters, overexpression was observed in 2/20 and 4/20 specimens, respectively. Statistical association was assessed correlating ki 67 expression to pathology type, capsular invasion and also to vascular infiltration (p=0.001, p=0.008, and p=0.012, respectively). Topo IIa protein expression was strongly correlated only to capsular invasion (p=0.004). Overall expression of the examined markers demonstrated a medium concordance (kappa=0.27), but a strong association (p=0.001). CONCLUSION: Topo IIa and also ki 67 overexpression are correlated to an aggressive phenotype in PTC. Topo IIa overexpression maybe is a reliable marker for a rational application of targeted chemotherapeutic strategies in some subgroups of patients.
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Antígenos de Neoplasias/análisis , Carcinoma Papilar/patología , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/análisis , Procesamiento de Imagen Asistido por Computador , Antígeno Ki-67/análisis , Neoplasias de la Tiroides/patología , Análisis de Matrices Tisulares/métodos , Carcinoma Papilar/química , Proliferación Celular , Femenino , Humanos , Persona de Mediana Edad , Neoplasias de la Tiroides/químicaRESUMEN
Topoisomerase IIa is a nucleic enzyme that affects the topological structure of DNA and also is a target for chemotherapy (ie, anthracyclines). In this study, we coevaluated its protein expression with chromosome 17 and gene status. Using tissue microarrays, 40 cases of sporadic, primary endometrial adenocarcinomas, 5 cases of atypical hyperplasia, and 5 cases of benign hyperplasia were obtained and reembedded into two paraffin blocks with a core diameter of 1 mm. Immunohistochemistry combined with chromogenic in situ hybridization was performed in 2 and 5 microm sections, respectively. Finally using a semiautomated Image Analysis System, we evaluated the levels of Nuclear labeling index of topoisomerase IIa expression. Statistical analysis was performed by SPSS version 11.0 software. The results indicate that chromosome 17 instability (aneuploidy in 7/40 cases) and Topo IIa gene deregulation (amplification in 3/40 and deletion in 1/40 cases) are significant genetic events correlated with biologic behavior in endometrial adenocarcinoma. Because protein overexpression was observed in a significant proportion of the tumors (18/40), detection of the specific gene deregulation mechanism is a crucial process for application of targeted chemotherapies, which are characterized by different levels of cardiotoxicity and other serious effects.
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Adenocarcinoma/enzimología , Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/metabolismo , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Análisis de Matrices Tisulares/métodos , Anciano , Antígenos de Neoplasias/análisis , Compuestos Cromogénicos/farmacología , Inestabilidad Cromosómica , Cromosomas Humanos Par 17/metabolismo , ADN-Topoisomerasas de Tipo II/análisis , Proteínas de Unión al ADN/análisis , Hiperplasia Endometrial/metabolismo , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Análisis por Matrices de Proteínas/métodosRESUMEN
OBJECTIVE: To evaluate proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) expression in urine ThinPrep (TP) specimens, to compare these findings with clinical and histological features and to determine whether these immunomarkers are predictive of clinical stage. PATIENTS AND METHODS: The TP processed urine samples and the corresponding tissue sections from 42 patients with newly diagnosed bladder cancer (18 non-muscle invasive and 24 muscle invasive) were included in our study. Urine was collected for cytological evaluation before transurethral resection. Tumour grade and clinical stage were assessed from the transurethral resection specimens. The EGFR and PCNA expression was obtained by an automated immunostainer. RESULTS: There was a remarkable concordance in the expression of both antibodies in TP smears and tissue sections. No significant association was detected for any of the immunomarkers examined with regard to tumour grade. The EGFR expression as well as grade of malignancy were significantly associated with stage of disease (P = 0.0001). PCNA was not found to be a significant predictor of stage (P = 0.210). CONCLUSION: Our data suggest that the evaluation of grade of malignancy and EGFR immunopositivity can be considered as reliable predictors of disease stage in urine TP specimens.