Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis.
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.
Van Der Zanden
A G M
AG
Medical Microbiology and Infectious Diseases, Gelre Hospitals, Location Lukas, Apeldoorn, The Netherlands. agm.vd.zanden@gelre.nl
Te Koppele-Vije
E M
EM
Vijaya Bhanu
N
N
Van Soolingen
D
D
Schouls
L M
LM
D000595:Amino Acid Sequence; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial / Q000235:genetics; D006652:Histological Techniques; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000187:drug effects; D010940:Plant Proteins / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology*; D013183:Sputum / Q000382:microbiology
D000904:Antibiotics, Antitubercular; D004269:DNA, Bacterial; D010940:Plant Proteins; C101305:RpoB protein, Oryza sativa; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin