Open AccessUse of DNA Extracts from Ziehl-Neelsen-Stained Slides for Molecular Detection of Rifampin Resistance and Spoligotyping of Mycobacterium tuberculosis
Author(s) -
Adri G. M. van der Zanden,
E. M. Te Koppele-Vije,
Natarajan V. Bhanu,
Dick van Soolingen,
Leo M. Schouls
Publication year - 2003
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.41.3.1101-1108.2003
Subject(s) - rpob , mycobacterium tuberculosis , ziehl–neelsen stain , tuberculosis , biology , rifampicin , microbiology and biotechnology , dna sequencing , polymerase chain reaction , virology , gene , sputum , genetics , medicine , antibiotics , pathology , acid fast
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.