| Background and objectives: The emergence of drug-resistant tuberculosis(DRTB), especially extensively drug-resistant tuberculosis(XDRTB), is a serious impediment to global TB control. XDR TB patients require longer, more toxic treatment regimen, with higher mortality rates than those patients without resistance to second-line drugs because of the severe illness condition. Thus, reliable and fast diagnosis of XDRTB is essential for the appropriate treatment to interrupt transmission and lead to the successful treatment.At present, due to the slow-growing characteristic of mycobacterium(M.) tuberculosis, the conventional drug susceptibility test(DST) methods performed by culture on solid or liquid media require weeks to months to providing DST results, which is far beyond the needs of clinical treatment.Withthe development of molecular technology,using the correlation between gene mutation and drugresistance, we can detect the gene mutational regions associated with drugresistance, rapidly dilivering results of drug susceptibility test, and timely diagnose DRTB.The Geno Type MTBDRsl line probe assay(LPA) is based on the DNA?STRIP technology and permits the molecular genetic identification of the M. tuberculosis complex and its resistance to fluoroquinolones(e.g. ofloxacin and moxifloxacin) and/or aminoglycosides/cyclic peptides(injectable antibiotics as capreomycin, viomycin/kanamycin, amikacin) and/or ethambutol. The identification of resistance to fluoroquinolones is enabled by the detection of the most significant mutations of the gyr A gene(coding for DNA gyrase). For detection of resistance to aminoglycosides/cyclic peptides, the 16 S r RNA gene(rrs) and for detection of resistance to ethambutol the embB gene(which, together with the genes emb A and emb C, codes for arabinosyl transferase) are examined.This technique can detect multiple drug resistance gene locidirectly on clinical smear positive specimensat the same time, needing no expensive equipment, which is simple, fast, practical, inexpensive.In order to achieve the purpose of rapid diagnosis of extensively drug-resistant tuberculosis and ethambutol resistance and timely provide reliable dataonchemotherapy for clinical treatment, this subject adopts the the Geno Type MTBDRslline probe assay for detecting gene mutationsassociated with resistance to levofloxacin, amikacin, capreomycin and ethambutol, Therefore, There is important clinical significance for the application of Geno Type MTBDRsl line probe assayin rapid detection of resistance to second-line anti-TB drugs and ethambutol directly on sputum specimens. Methods:During the period of Jun 2013 to Jun 2014,there were 189 MDR-TB sputum specimens identified by the MTBDRplus line probe assay were consecutively analyzed using the Geno Type MTBDRsl line probe assay at the National Clinical Laboratory(NCL) on tuberculosis in Beijing Chest Hospital, China. The accuracy of the Geno Type MTBDRslline probe assay was assessedagainst culture-based phenotypicdrug susceptibility testing asa reference, the gene mutation patterns associated with drug resistance at Beijing Chest Hospital(BCH) were simulately evaluated, andsputum specimenswith discordant outcomes were assessed by gene sequencing. Results:1. The interpretability of the Geno Type MTBDRsl line probe assay was 96.8%(183/189). The sensitivity and specificity of the Geno Type MTBDRsl test were 81.6% and 91.5% for LFX, 52.6% and 99.2% for AMK, 58.1% and 97.7% for CAP, and 69.8% and 93.3% for EMB, 56.1% and 100% for extensively drugresistant tuberculosis.2. The turnaround time(TAT) was 3(range:1-6)days for Geno Type MTBDRsl line probe assay compared to 85(range:65-105) days for culturebased DST and was significantly shortened by 96%.3. Among the 80/98 phenotypic LFX-resistant specimens detected by the Geno Type MTBDRsl line probe assay, the most prevalent mutation was gyr A MUT 3C/D94G(36/80, 45%), followed by the gyr A-ΔWT3, MUT 3B/D94Y(11/80,13.75%) 、 gyr A-MUT1/A90V+3C/D94G(9/80,11.25%) 、gyr A-MUT3B/D94N+3C/D94G(8/80, 10.0%) 、 gyr A- MUT3C( 5/80,6.25%)、gyr A-ΔWT2,MUT1( 3/80, 3.75%)、 gyr A-ΔWT3,MUT3A( 2/80, 2.50%)、gyr A-ΔWT2,MUT2(1/80, 1.25%)and gyr A-ΔWT2,MUT1+3A(1/80,1.25%).The rest 4(5%)LFXresistance isolates showed absence of Geno Type MTBDRsl line probe assaywild-type bands without presence of mutant bands.Of the 20 sputum specimensresistant to AG/CP detected by the Geno Type MTBDRsl line probe assay, the most prevalent mutation occurred in rrs MUT 1/A1401G(15/20, 75.0%), 3(15.0%) specimensshowed mutations in rrs MUT 2/G1484 T and 1(4.8%) revealed mutations both in rrs MUT 1/A1401 G and MUT 2/G1484 T, while the rest1(4.8%) was diagnosed by the omission of Geno Type MTBDRsl line probe assay wild-type band. Of the 44/63 phenotypic EMBresistance sputum specimensdetected by Geno Type MTBDRsl line probe assay, 26/44(59.09%) harbored emb B MUT 1B/M306 V and 6/44(13.64%) showed mutation in emb B MUT 1A/M3061, while the rest12(27.27%) was confirmed by the absence of Geno Type MTBDRslline probe assaywild-type bands.4. DNA sequencing revealed the mutation patterns in some of discrepant isolates. The 11/23 discordant LFX isolates had mutations in gyr A gene(n=9) and gyr B gene(n=2), among which four were phenotypically susceptiblebut Genotype MTBDRsl resistant and seven were phenotypically resistantbut Genotype MTBDRsl susceptible;The 8/25 inconsistent EMB strains had mutations in emb B gene, among which five were phenotypically susceptiblebut Genotype MTBDRsl resistantand 3 were phenotypically resistantbut Genotype MTBDRsl susceptible The 3/19 discordant AMK isolates and 6/16 discordant CAP isolates had mutations in rrs gene, but none had mutation in tly A gene, among which one was AMK phenotypically susceptiblebut Genotype MTBDRsl resistant and two were AMK phenotypically resistantbut Genotype MTBDRsl susceptible, and three were CAP phenotypically susceptiblebut Genotype MTBDRsl resistant and other three were CAP phenotypically resistantbut Genotype MTBDRsl susceptible. Conclusion:5. Our findings provide critical information on the clinical performance of the Geno Type MTBDRslline probe assayin conjunction with the Geno Type MTBDRplusline probe assayin high drug resistant settings.The rapid diagnostics molecular tool is useful for timely screening of XDRTB using sputum samples. However, the Geno Type MTBDRslline probe assay has obvious limitations such as lower sensitivity compared to conventional culturebased DST, which largely restricts its wildlyclinical usage in hospital. To improve the ability of Geno Type MTBDRsl line probe assay in detection of XDRTB, more genetic mutations responsible for resistanceto second-line anti-tuberculosis drugs should be evaluatedandincluded.. |
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