The recent increase in new cases of Mycobacterium tuberculosis infection and rapid emergency of rifampin resistant and multidrug resistant strains have prompted new interest in tuberculosis worldwide. It has been reported that rifampin resistance
can
also be used as the surrogate marker for multidrug resistance. Therefore, rapid detection of rifampin resistant strain of M. tuberculosis is the key to control M. tuberculosis infection. Molecular biological methods based on rpoB gene sequencing
and
reverse hybridization line probe assay after polymerase chain reaction have a greater potential for early detection of rifampin resistance.
This study was conducted to evaluate the clinical effectiveness of new rapid assays to detect rifampin resistance on the basis of molecular biological methods. The mutation pattern of rpoB gene in rifampin resistant strains isolated from sputum
of
patients in Korea was also studied. The results of rpoB gene sequencing and INNO-LiPA(INNO-LiPA Rif. TB Kit: Line Probe Assay, Innogenetics, Belgium) tests after polymerase chain reaction from clinical specimens were compared with that of
conventional
antimicrobial susceptible strains of M. tuberculosis were collected ; and those were then subjected to rpoB gene sequencing and INNO-LiPA testing. The findings were as summarized below.
Twenty eight patients(93.3%) out of thirty who had resistant strain showed multidrug resistance while two(6.7%) showed single drug resistance to rifampin.
The rpoB gene sequencing revealed twenty eight(93.3%) sensitive strains agreed with results of conventional culture and susceptibility testing, but two(6.7%) strains disagreed with the conventional test results. Those two showed substitution of
526
histidine(CAC) by tyrosine(TAC) and asparagine(AAC). Twenty five(83.3%) of thirty resistant strains showed amino acid substitution within rpoB gene region. The patterns of this substitution by sequence were as following: ten(33.3%) strains of 531
serine(TCG) to leucine(TTG) ; seven(23.3%) of 516 aspartic acid(GAC) to valine(GTC), tyrosine and histidine ; seven(23.3%) of 526 histidine to arginine(CGC), tyrosine, aspartic acid, cystidine and serine ; and one(3.3%) of 533 leucine to
proline(CCG).
Of those the substitution of 526 histidine by serine has not been reported previously.
INNO-LiPA test showed twenty eight(93.3%) of thirty sensitive strains agreed with conventional test results. Twenty seven(90.0%) of thirty resistant strains were agreeable with the INNO-LiPA results. The profiles of INNO-LiPA of resistant strains
revealed absence of S5 band(43.4%), S4 band(23.3%), S2 band(13.3%), S1 band(10.0%) and S3 band(0%) in sequence. Of twenty seven strains showing absence of S bands, seventeen(63.3%) had confirmatory mutant R-probes.
The retrospective review of patients' record showed 73.3% of patients who had resistant strains also had cavitary lesions of lungs(p<0.01). Twenty two(73.3%) of thirty resistant strains were isolated from patients having cavitary lesions of
lungs.
The
most frequently observed mutation pattern of the resistant strains from the cavitary lesions was 531 substitution of serine by leucine(90.0% of 531 serine ->leucine). Twenty seven(90.0%) of thirty sensitive strains and twenty four(80.0%) of
thirty
resistant strains were agreeable with both methods.
Therefore, it is concluded that rpoB gene sequencing and INNO-LiPA test after polymerase chain reaction are effective methods for rapid detection of rifampin resistant. M. tuberculosis which is the surrogate marker for multidrug resistance. In
particular, rpoB gene sequencing is the choice of method to discover a new mutant strain.
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