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

Utility of In-House PCR for HLA-B27 Typing: Comparison of Concordance Rate between PCR Kit and In-House PCR / 대한진단검사의학회지

BACKGROUND: Commercial kits of PCR method are widely used in HLA-B27 typing; however, their cost is relatively high. In this study, we evaluated the utility of an in-house PCR method by comparing it with that of a commercial kit. METHODS: HLA-B27 typing was done in 188 patients by using two PCR methods, Absolute(TM) HLAB27 PCR kit (Biosewoom, Korea) and an in-house PCR method. The primers used in the in-house method were prepared by Bioneer (Korea). Both PCR tests were done by Gene Amp PCR System 9600 (Perkin-Elmer Centus Corp., USA). RESULTS: The commercial kit and in-house PCR showed 100% concordance rate with each other in HLA-B27 typing. Of 188 patients tested 72 (38.3%) were positive and 116 (61.7%) were negative by the both tests. Of 62 patients with ankylosing spondylitis, 50 were positive (80.7%). CONCLUSIONS: The in-house PCR is a reliable and cost-effective method and can replace or supplement commercial kits for HLA-B27 typing.

Detection of Mycobacterium tuberculosis in Sputum by using Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: The recently developed nucleic acid amplification methods may provide us with very sensitive, specific and rapid tests for the detection of M. tuberculosis. So the aim of this study was to compare the commercial Amplicor M. tuberculosis kit and our in-house polymerase chain reaction (PCR) with the conventional culture and direct AFB staining method. Materials and METHODS: Among the total of 2,340 clinical specimens, 1,314 sputum samples were tested for the presence of M. tuberculosis by Amplicor PCR and 1,026 sputum samples were tested by in-house PCR performing with resin matrix preparation and DNA extraction, synthesized primer pair, detection using agarose gel electrophoresis. RESULTS: One hundred-seventeen specimens were positive by Amplicor PCR, 105 were positive by in-house PCR, 185 were positive by culture. The sensitivity of the Amplicor PCR for all of the specimens and for smear-positive and smear-negative specimens was 92.9%, 97.9% and 88.2%, respectively after discrepant analysis. The sensitivity of the in-house PCR for all of the specimens and for smear-positive and smear-negative specimens was 80.0%, 93.6% and 65.5%, respectively after discrepant analysis. The specificity of the Amplicor PCR and in-house PCR for all of the specimens was 97.9% and 99.0%, respectively. CONCLUSIONS: Amplicor PCR was more sensitive than in-house PCR, but there was another problems such as high false positive rate and high cost. So PCR may certainly become very useful in microbiological laboratories if PCR method is selected according to the laboratory conditions.