PCR interna con ADN extraído directamente de portaobjetos positivos para confirmar o excluir el diagnóstico de tuberculosis: enfoque en la bioseguridad / In-house PCR with DNA extracted directly from positive slides to confirm or exclude the diagnosis of tuberculosis: focus on biosafety

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method

La posibilidad de obtener ADN a partir de frotis es una valiosa alternativa para remediar la falta de muestras cuando estas son totalmente utilizadas para la baciloscopia; esta opción soluciona, además, el problema de bioseguridad asociado a la posibilidad de accidente al transportar frascos que contienen muestras clínicas potencialmente infectivas. Por lo tanto, el propósito de este estudio fue utilizar para el diagnóstico de la tuberculosis la secuencia de inserción IS6110 para amplificación del ADN a partir de frotis que resultaron positivos por baciloscopia. Del análisis de 52 baciloscopias positivas surge que la sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de esta técnica fueron, respectivamente, del 52,3%, del 100%, del 100% y del 89,7%; y la precisión fue del 90,7%. La PCR IS6110 para el diagnóstico de tuberculosis, desarrollada con ADN extraído de frotis positivos, es un método rápido, simple, específico y seguro

Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques.

Introduction: Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. Materials and Methods: The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. Results: There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of Mycobacterium tuberculosis or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8% and 96%, of ZNS was 40.8% and 99%, of culture was 40.8% and 100% and of IS6110 gene PCR test was 100% and 92.1%. Conclusion: IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

Uso clínico de la reacción en cadena de la polimerasa e hibridización en el diagnóstico de tuberculosis / Clinical use of the polymerase chain reaction and hybridization in diagnosing tuberculosis

La tuberculosis (TBC) es una causa importante de morbimortalidad en nuestro país. Las herramientas de diagnóstico no son 100% eficientes y rápidas para ayudar al clínico en su decisión médica. Nuevas pruebas, como la reacción en cadena de la polimerasa (PCR), son necesarias. Evaluar la utilidad clínica de una PCR para IS6110, en muestras clínicas de pacientes con sospecha de tuberculosis pulmonar o extrapulmonar. Sesenta y una muestras de 46 pacientes del Hospital General del Este, Dr. Domingo Luciani de Caracas, Venezuela, fueron procesadas para la detección del M. Tuberculosis. Se practicó baciloscopia, cultivo en LJ y PCR e hibridación. Los pacientes se clasificaron como TBC positivos si tenían evidencia clínica o radiológica, y positivas algunas de estas pruebas: PPD, ZN, cultivo, biopsia positiva o respuesta favorable al tratamiento. Los TBC negativos con evidencia clínica o radiológica positiva y todas las pruebas negativas. La sensibilidad de la PCR sola fue del 75% y la especificidad del 61%. La PCR e hibridación juntas mostraron sensibilidad del 90% y especificidad del 57%. Los valores predictivos positivos y negativos fueron del 62% y 88%, respectivamente

Tuberculosis (TBC) remains a leading cause of morbidity and mortality in Venezuela. The diagnos tic tools used are not 100% specific, sensitive, efficient or fast enough to help clinicians take the right clinical decision. New tests, like polymerase chain reaction (PCR), are necessary. We evaluated the clinical usefulness of an IS6110 in-house PCR in clinical samples from patients suspected of having pulmonary or extrapulmonary TBC. Sixty one samples from 46 patients were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, and PCR and hybridization assay. The patients were classified as TBC positive when they had clinical or radiological evidence plus any positive of the following tests: PPD, ZN, LJ culture, biopsy or anti-TBC treatment response and TBC negative when they had clinical or radiological evidence but all the applied tests used were negative. PCR sensibility was 75% and specificity was 61%, using PCR alone, but when PCR and hybridization were evaluated together the sensibility was 90% and specificity was 57%. The predictive positive and negative values were 62% and 88% respectively

Clinical Utility of Bronchial Washing PCR for IS6110 and Amplicor for the Rapid Diagnosis of Active Pulmonary Tuberculosis in Smear Negative Patients / 결핵

BACKGROUND: There is a well recognized interlaboratory variation in the results using the polymerase chain reaction(PCR) to detect the IS6110 sequence. The clinical utility of a commercially developed PCR test(Amplicor) in bronchial washings for detecting pulmonary tuberculosis in smear negative patients was evaluated. The sensitivity and specificity of Amplicor was compared with that of an in-house PCR test used for detecting the IS6110 sequence of Mycobacterium tuberculosis(M.tbc) in the bronchial washing fluid. METHODS: 66 patients whose sputum smear for M.tbc were negative or who could not produce any sputum were recruited from January 1999 to July 1999. They all had a bronchoscopy performed to determine if there were signs of hemoptysis, patients who could not cough up sputum, lung lesion that exclude pulmonary tuberculosis. Pulmonary tuberculosis was diagnosed on the basis of a positive culture or a response to anti-tuberculosis therapy. RESULTS: 19 patients with tuberculosis were identified and samples from 16 patients were later confirmed by culture. Bronchial washing for Amplicor PCR revealed a sensitivity, specificity, positive and negative predictive values of 94.7%, 97.9%, 94.7%, 97.9%, respectively. Using IS6110 based PCR, the sensitivity, specificity, positive and negative predictive values were of 73.7%, 87.2%, 70%, 89.1% respectively. CONCLUSION: Bronchial washing for Amplicor PCR proved to be more useful than IS6110 based PCR in rapidly diagnosing smear negative pulmonary pulmoary tuberculosis in patients where tuberculosis was likely to be differential and rapid diagnosis was essential for optimal treatment.