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Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR

Soumitesh Chakravorty and Jaya Sivaswami Tyagi^*

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Received 12 February 2004/ Returned for modification 6 September 2004/ Accepted 13 February 2005

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.

This work is dedicated to the memory of T. A. Venkitasubramanian, who contributed greatly to the study of mycobacterial metabolism.

Present address: Division of Infectious Diseases, Department of Medicine, and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103.

This article has been cited by other articles:

• Haldar, S., Chakravorty, S., Bhalla, M., De Majumdar, S., Tyagi, J. S. (2007). Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J Med Microbiol 56: 1356-1362 [Abstract] [Full Text]  
• Chakravorty, S., Dudeja, M., Hanif, M., Tyagi, J. S. (2005). Utility of Universal Sample Processing Methodology, Combining Smear Microscopy, Culture, and PCR, for Diagnosis of Pulmonary Tuberculosis. J. Clin. Microbiol. 43: 2703-2708 [Abstract] [Full Text]