Clinical failures associated with rpoB mutations in phenotypically occult multidrug-resistant Mycobacterium
Recently, Mycobacterium tuberculosis isolates have been described that test phenotypically susceptible to rifampicin (RMP) yet harbour genotypic rpoB mutations.
1) To investigate the impact of such mutations on clinical outcomes among RMP-susceptible isolates, and 2) to determine the prevalence of rpoB mutations among isoniazid (INH) monoresistant isolates at our laboratory and to describe the association between the presence of these mutations and clinical outcomes.
M. tuberculosis isolates were screened for mutations in the rpoB gene using the Cepheid Gene-Xpert® MTB/RIF assay. Clinical correlation was made by reviewing patient case notes.
Isolates from 94 patients were found to have INH-resistant, RMP-susceptible profiles. Clinical information was available for 52 patients, including three whose isolates had rpoB mutations. All three of these patients had treatment failures, compared to two of 49 patients whose isolates did not have rpoB mutations (P = 0.0005).
We demonstrate a significant association between the presence of rpoB gene mutations that are not detected at the current RMP critical concentration and treatment failure. We suggest that a review of the current RMP critical concentration is warranted to ensure that RMP is not used inappropriately for the treatment of phenotypically occult multidrug-resistant tuberculosis.
Available from: Padmapriya P Banada
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ABSTRACT: Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe
an asymmetric PCR of the M. tuberculosis RRDR by melting temperature (Tm) analysis. A three-point Tm code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated
on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted
from 589 geographically diverse clinical M. tuberculosis cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR
mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences
as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin
resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity,
99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using
the triple Tm code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations
when present in at least 40% of the total M. tuberculosis DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput
applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.
Journal of clinical microbiology 04/2012; 50(7):2194-202. DOI:10.1128/JCM.00143-12 · 3.99 Impact Factor
The International Journal of Tuberculosis and Lung Disease 05/2012; 16(5):706-7. DOI:10.5588/ijtld.11.0850 · 2.32 Impact Factor
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ABSTRACT: Recent reports suggest that false-positive rifampicin resistance may be assigned by the Xpert MTB/RIF assay. We analysed 169 specimens using the MTB/RIF assay. Using culture as the gold standard, we found that the assay had 100% sensitivity and specificity for detecting M. tuberculosis. However, we found that the assay incorrectly assigned rifampicin resistance in 4/13 (31%) of cases.
Diagnostic microbiology and infectious disease 07/2012; 74(2):207-9. DOI:10.1016/j.diagmicrobio.2012.06.013 · 2.46 Impact Factor
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