Evaluation of the BDProbeTecTM ET System for the Direct Detection of Mycobacterium tuberculosis Complex from Smear Negative Respiratory Samples

In 1998, nearly 3 million people died of tuberculosis, more than any other year in history. The estimated 8.8 million new cases every year correspond to 52,000 deaths per week or more than 7,000 each day, which translates into more than 1,000 new cases every hour, every day. Between 1993 and 1996 there was a 13% increase in TB cases worldwide. One third of the increase in the incidence of TB in the last five years can be attributed to the HIV pandemic. Another factor contributing to the worldwide increase in TB infections is the emergence of multidrug resistant strains. Tuberculosis continues to be a global public health problem. Conventional methods for identification of M.tb from culture first require growth, which can take 2 to 3 weeks. Nonamplified probe tests can then identify M.tb within hours. At the very least, the time needed from sample collection to M.tb identification can take several weeks. The introduction of direct nucleic acid amplification tests has reduced this M.tb identification time to within hours of sample collection. In theory, these tests can detect a single organism in respiratory samples. Despite this, accurate detection of M.tb from AFB smear negative, M.tb culture positive samples with nucleic acid amplification tests continues to be problematic. Sensitivity obtained by commercial systems testing these sample types range between 29-85%. There are several reasons contributing to the poor sensitivity of M.tb amplification tests on smear negative samples. First, incomplete removal of amplification inhibitors from the respiratory sample can be a source of false negative results. Second, there are few organisms present in an AFB smear negative sample. The threshold for detection by AFB smear is approximately 1x10 cfu/ml. Actual numbers of organisms present in a smear negative, culture positive sample can range from an upper limit of 1x10 cfu/ml to a lower limit of 10 viable M.tb per ml for culture. Third, M.tb grows in clumping, cablelike arrangements. This uneven distribution of M.tb compounds the sampling errors caused by the few organisms present to start with. Finally, nucleic acid extraction from M.tb cells is difficult. Target nucleic acid is entrapped in a thick waxy cell wall. Inefficient extraction can contribute to decreased sensitivity. Here we report the development of a simple sample processing procedure designed to specifically overcome the challenges that AFB smear negative, culture positive samples present (Figure 1). The procedure has been coupled with the homogeneous SDA system for direct detection of both specific M.tb complex target sequence and an internal amplification target sequence (IAC) on the BDProbeTec ET system.