C-205. Comparison of culture with Auramine Rhodamine Stain, the Amplified Direct Test and a M. tuberculosis Real-Time PCR Assay for Respiratory Specimens

S. P. Buckwalter, L. Buschur, S. Clark, C. Ruth, G. Short, J. VanDorp, S. Wohlfiel, N. L. Wengenack;
Mayo Clin., Rochester, MN.

Background: Mycobacterium tuberculosis is considered a serious public health issue due to its high risk of person to person transmission and mortality. There are 8 million new cases of tuberculosis every year, and tuberculosis is responsible for nearly 2 million deaths a year. Early detection of M. tuberculosis from respiratory specimens is needed for the appropriate management of patients. Methods presently being used in our laboratory are either time consuming (culture), subjective (auramine rhodamine stain), or laborious (Amplified Direct Test ). We developed a LightCycler PCR assay, targeting the catalase-peroxidase gene (katG) to detect species within the M. tuberculosis complex from respiratory specimens in less than an hour. The assay was designed over an area of the katG gene so to simultaneously detect M. tuberculosis resistance to isoniazid, a first line antibiotic used for treatment of tuberculosis. In this study, our goal was to compare methods currently being used in our laboratory to our newly developed assay LightCycler assay (MTB) in order to determine if a more rapid method would deliver sensitive and specific results. Methods: Raw respiratory specimens (bronchoalveolar lavage fluid, bronchoscopy fluid, pleural fluid, tracheal secretions, and sputa) were collected from excess specimens in the clinical laboratory that were submitted for culture, smear and/or MTD testing. For the MTB real-time PCR assay, the respiratory specimens were liquefied, heat killed, lysed, and then extracted on the MagNA Pure Compact. Extracts from the MagNA Pure were then tested on the LightCycler instrument. Results from culture were compared to auramine rhodamine stain, the Amplified Direct Test (MTD) and our MTB real-time PCR assay. Results and Conclusions: To date, when compared to culture, the sensitivities of auramine rhodamine stain, MTD, and MTB real-time PCR assay were 23%, 40%, and 38% and specificities were 100%, 98%, and 100% respectively.