Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR.(Research article)

Authors: Divya Pathak [1,3]; Soumitesh Chakravorty [1,4]; Mahmud Hanif [2]; Jaya Sivaswami Tyagi (corresponding author) [1]

Background

TB is an enormous public health challenge worldwide, with an estimated global incidence of 0.136% in 2005 [1] which is likely to be aggravated by the HIV/AIDS pandemic. Therefore there is great pressure on clinical laboratories to rapidly and accurately detect and identify clinically important mycobacteria. TB is routinely diagnosed worldwide by smear microscopy and culture. In spite of its lack of sensitivity and inability to distinguish between tubercle bacilli and other mycobacteria, the former technique is widely used in TB control programmes on account of its speed, simplicity and low cost. Apart from lungs, TB also occurs in other body sites wherein the paucibacillary load often poses a significant diagnostic challenge [2, 3]. Nucleic acid amplification technologies are being intensively assessed in diagnosing extrapulmonary TB owing to their speed, specificity and sensitivity [4]. However their application is limited to sophisticated laboratories in developing countries including India and samples are often stored prior to analysis. Studies that have evaluated the effect of sample shipment and storage on the performance of conventional and nucleic acids-based tests concluded that accurate diagnosis rests on minimal transport time and lowest possible storage and shipping temperatures [4, 5, 6]. Furthermore, in case of paucibacillary specimens, diagnostic methods are no different from those employed for high load or fresh samples. The inherent delay in obtaining culture results is a serious limitation of the gold standard; therefore it was proposed that when smear microscopy and DNA amplification were both positive, the diagnosis of active TB could be considered established [7]. For these various reasons PCR is being increasingly considered as an adjunct test for diagnosing TB in resource-poor settings also.

We developed a sample processing procedure, USP methodology, that is compatible with microbiology and inhibition-free PCR in both pulmonary and extrapulmonary samples [8, 9, 10]. In the present study, we assessed the effect of low temperature storage on the efficacy of

M. tuberculosis detection in fresh and stored USP-processed sputum. The key finding was that a significant number of fresh specimens contained lysed mycobacteria and lysis increased during storage. As a consequence supernatant PCR positivity increased with sample storage. Thus supernatant is a novel and useful sample for enhancing PCR performance in clinical samples.

Results

In a preliminary study conducted to assess the impact of sputum storage on PCR, maximum bacterial autolysis in untreated samples was noted during room temperature vs. 4[degrees]C or -20[degrees]C storage (Dudeja M, unpublished observations). The present study was designed to assess the effect of storage at -20[degrees]C of NALC-treated sputum on bacterial lysis. We chose 1 and 2 months of storage as it exceeded the likely interval between sample collection and PCR analysis. PCR and smear microscopy were employed to assess the speed of storage-associated changes in bacterial load. Bacterial lysis was confirmed by simultaneous analysis of sputum supernatants that are conventionally discarded

Effect of sample storage on PCR

All the samples were processed as outlined in Fig. 1. A total of 522 IS

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