Detection Of Rifampin Resistance Research Articles

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system.

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.

Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay.

An adaption of an RNA/RNA duplex, base pair-mismatch assay is capable of detecting rifampin resistance in Mycobacterium tuberculosis. The specificity and sensitivity of the mismatch assay in detecting rifampin resistance were 100% and 96%, respectively, when tested against 46 rifampin-resistant and rifampin-susceptible strains of M. tuberculosis. By use of a range of mycobacterial and nonmycobacterial prokaryote pathogens, the mismatch assay was shown to be specific for M. tuberculosis and Mycobacterium bovis. The assay is cost-effective compared with DNA sequencing and other molecular methods and is simple to perform and interpret. Furthermore, the assay can return a result within 24 h after receipt of an isolated organism and potentially can be used directly with smear-positive specimens.

Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.