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Journal of Clinical Microbiology, March 2003, p. 1101-1108, Vol. 41, No. 3
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.3.1101-1108.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of Mycobacterium tuberculosis

Medical Microbiology and Infectious Diseases, Gelre Hospitals, Location Lukas, Apeldoorn,¹ Mycobacteria Reference Department, Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, Research Laboratory for Infectious Diseases,³ National Institute of Public Health and the Environment, Bilthoven, The Netherlands,⁴ Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland²

Received 29 July 2002/ Returned for modification 23 September 2002/ Accepted 28 November 2002

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the rpoB gene using reverse line blot hybridization and DNA sequencing. Analysis of the rpoB gene revealed that samples containing rifampin-resistant Mycobacterium tuberculosis carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the rpoB gene was more accurate in identifying mutations in the core region of the rpoB gene. Sequence analysis of the rpoB gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.

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