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Journal of Clinical Microbiology, November 2008, p. 3591-3594, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00856-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Identification of Mycobacteria from Smear-Positive Sputum Samples by Nested PCR-Restriction Fragment Length Polymorphism Analysis{triangledown}

Tsu-Lan Wu,1,2 Ju-Hsin Chia,1,2 An-Jing Kuo,1,2 Lin-Hui Su,1,2 Ting-Shu Wu,3,4 and Hsin-Chih Lai1*

Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan,1 Department of Clinical Pathology,2 Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan,3 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan4

Received 5 May 2008/ Returned for modification 24 June 2008/ Accepted 2 September 2008

The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and evaluated for its efficiency compared to that of traditional culture methods and 16S rRNA gene sequencing identification. A total of 204 smear-positive and culture-positive sputum specimens were prospectively collected for analysis between November 2005 and May 2006. The samples were classified according to an acid-fast bacillus (AFB) staining scale as rare/1+, 2+, or 3+. The results of the nested-PRA showed that the identification rate for AFB 3+, AFB 2+, and AFB rare/1+ samples was 100, 95, and 53%, respectively, and that the overall identification rate was 89%. All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. The nested-PRA appears to have clinical applicability when used for the direct identification of mycobacterial organisms (both MTBC and NTM) that are present in smear-positive sputum samples, especially for countries in which MTBC is endemic.

* Corresponding author. Mailing address: Department of Medical Biotechnology and Laboratory Science, Chang Gung University, No. 259, Wen-Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China. Phone: 886-3-2118800, ext. 3585. Fax: 886-3-2118700. E-mail: hclai{at}mail.cgu.edu.tw

{triangledown} Published ahead of print on 3 September 2008.

Journal of Clinical Microbiology, November 2008, p. 3591-3594, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00856-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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