Mutation Site Analysis Of Rifampin-resistant Gene And Development Of A Single Multiplex Amplification Refractory Mutation System Pcr For The Detection Of Rifampin-resistant Mycobacterium Tuberculosis

BackgroundTuberculosis meningitis (TBM) caused by Mycobacterium tuberculosis (Mtb) is themost severe extrapulmonary tuberculosis (TB). Although studies have shown thatprevalence of Mtb drug-resistance in patients with TBM was lower than that in patientswith pulmonary infection, but the outcome of TBM patients with drug-resistant TB ismore severe or even to death. The failure to detect drug resistance results in theprescription of inappropriate regimens, treatment failure, increased mortality, and furthertransmission of drug-resistant tuberculosis, and even extensive drug resistance. Thesesituations make stop TB strategy more challenges. In STOP TB Strategy, WHOrecommends that according to the effective diagnosis of resistance, reasonable, sufficientand combined treatment in early stage of infection can improve prognosis and decrease theincidence of drug-resistant TB. However, the treatments for TBM patients are lack of strictguidelines, often relying on those of the pulmonary tuberculosis. Furthermore, in the process of the TBM treatments, since drugs cannot cross the blood-brain barrier to reachthe bacterium easier, large-dose and long-term usage of RIF can cause drug resistanceeasier. But for some reasons, drug resistance in TBM cases study were insufficient andthere is impossible to directly observed treatment. So the diagnosis of TBM more andmore research is needed.ObjectiveAnalyzing differences in tuberculosis meningitis and pulmonary tuberculosis caseswith rpoB mutation characteristics of rifampin resistance and establishing method ofM-ARMS-PCR, which is a rapid and simple method for the detection of drug-resistantMycobacterium tuberculosis, are critical for the efficient treatment and control of thispathogen in developing country.MethodThe PCR products containing81bp rifampin resistance-determining region (RRDR)of102Mycobacterium tuberculosis isolates, including63strains from pulmonarytuberculosis patients and39strains from tubercular meningitis patients, were analyzed bysequencing. Then we developed a single multiplex amplification refractory mutationsystem (M-ARMS) PCR. And using this method, we detected rifampinresistance-associated mutations at codons511,516,526and531in the rifampinresistance-determining region of rpoB gene.ResultsAmong102rifampin-resistant strains, mutations were found in the81bp RRDR of100strains. The most frequent mutation rates of rpoB gene of63pulmonary tuberculosisassociated strains occurred at codons:531(53.97%),526(20.63%), and516(6.45%). Themost common mutation rates of the39meningitis tuberculosis associated strains occurredat codons:531(61.54%),526(20.51%), and533(7.69%). The performance ofM-ARMS-PCR assay was evaluated with135cultured isolates of Mycobacteriumtuberculosis. The sensitivity and specificity were94.2%and100%, respectively comparedwith direct DNA sequencing and86.67%and89.71%, respectively compared withculture-based phenotypic drug susceptibility testing.ConclusionThe rifampicin resistant Mycobacterium tuberculosis, resulting in the tuberculosismeningitis and pulmonary tuberculosis, showed no significant differences in the mostcommon mutation rates of the rpoB gene at two codons:531and526. The Serâ†'Leu mutation at codon531was predominant. Here we developed a single multiplexamplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer andtemperature switch PCR (TSP) strategy were included. Using this method, we detectedrifampin resistance-associated mutations at codons511,516,526and531in the rifampinresistance-determining region of RpoB gene. The performance of M-ARMS-PCR assaywas evaluated with135cultured isolates of Mycobacterium tuberculosis. The sensitivityand specificity were94.2%and100%, respectively, compared with direct DNAsequencing, and86.67%and89.71%, respectively, compared with culture-basedphenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCRmethod is useful and efficient for rapid detection of rifampin resistance-associatedmutations.