| Tuberculosis is an ancient but very important disease which is still a critical threat to human health. With wide use of Directly Observed Treatment-Short course (DOTS), this disease has been effectively controlled while there has been a new challenge because of increase of resistance and dissemination of tuberculosis. Fluoroquinolones (FQ) are the major drugs for treatment of tuberculosis. But with abuse and inappropriate use of fluoroquinolones, the curative effect and clinical outcome has been affected by emerge of multidrug-resistant tuberculosis (MDR-TB). The characteristics and molecular mechanism of mycobacterium tuberculosis resistance to fluoroquinolones is still unclear now, so that there is lack of specific therapeutic strategies in clinic. This study was conducted to investigate the resistance profile, prognosis and molecular characteristics of fluoroquinolone-resistant mycobacterium tuberculosis, and to know characteristics, prognosis, phenotype and resistance related gene of MDR-TB. Finally to explore the early diagnosis method of fluoroquinolone-resistant mycobacterium tuberculosis to provide the basis for rational clinical use of anti-tuberculosis drugs.This study is divided into three parts. Firstly, the study on resistance characteristics of 213 MDR-TB patients was conducted. The TB isolates from the patients were divided into two groups:fluoroquinolone-resistant (FQr) and fluoroquinolone-sensitive (FQs). The difference in clinical manifestations and resistance phenotype were compared. And then the resistant genes of 206 isolates of fluoroquinolone-resistant mycobacterium tuberculosis were detected to find out molecular characteristics of fluoroquinolone-resistant mycobacterium tuberculosis in China for early clinical diagnosis.Finally, a retrospective study of prognosis in MDR-TB patients was conducted to compare the difference of prognosis of two groups:fluoroquinolone-resistant (FQr) and fluoroquinolone-sensitive (FQs).Part 1 Study of resistant profile in fluoroquinolone-resistant mycobacterium tuberculosis clinical isolatesObjective:To understand resistant characteristics of MDR-TB patients for guiding clinical therapy. Methods:A retrospective study was carried out in 213 MDR-TB patients diagnosed with sputum culture and drug susceptibility test in Shanghai Public Health Clinical Center from September 2007 to April 2010. The patients included 170 FQr MDR-TB patients and 43 FQS MDR-TB patients. Results: In FQr group, secondary pulmonary infection occurred in 60.6% of MDR-TB patients and 37.2% in FQs group. The incidence rate of secondary pulmonary infection in FQr group was obviously higher than that in FQs group (p<0.001). The results of susceptibility test showed that in fluoroquinolone-resistant mycobacterium tuberculosis,85.9% were resistant to streptomycin,74.1% were resistant to ethambutol,38.2% were resistant to amikacin,41.2% were resistant to capreomycin, 39.1% were resistant to amikacin and capreomycin simultaneously and 52.6% were sensitive to amikacin and capreomycin simultaneously. So that more than half of fluoroquinolone-resistant MDR-TB patients are still sensitive to amikacin and capreomycin which might be alternative treatment option in clinic. While because 39.1% of MDR-TB patients were resistant to both amikacin and capreomycin, the two drugs may have cross resistance. Conclusions:The incidence rate of secondary pulmonary infection in fluoroquinolone-resistant MDR-TB patients was obviously higher than that in fluoroquinolone-sensitive MDR-TB patients. More than half of fluoroquinolone-resistant MDR-TB patients were still sensitive to amikacin and capreomycin but the two drugs may have cross resistance, so when mycobacterium tuberculosis resistant to each of the two drugs, the other one is neither recommended.Part 2 Study of molecular mechanism of fluoroquinolone-resistant mycobacterium tuberculosisObjective:To investigate molecular characteristics of clinical isolates of fluoroquinolone-resistant mycobacterium tuberculosis in Shanghai. Methods: Proportion method was used to detect phenotype of clinical isolates of fluoroquinolone-resistant mycobacterium tuberculosis. Direct DNA sequencing was used to determine the mutations inside quinolone resistance-determining region (QRDR) of gyrA and gyrB genes in those isolates. The relations of genotype and phenotype of fluoroquinolone-resistant mycobacterium tuberculosis were assessed. Results:Total 206 clinical isolates of fluoroquinolone-resistant mycobacterium tuberculosis were screened for determination of fluoroquinolone resistance gene. The results showed that 44%(90/206) of strains were MDR,39.3%(81/206) of strains were extensively drug-resistant strains (XDR). Only 9%(19/206) were monoresistant to fluoroquinolones. In total,79.1%(163/206) of FQr isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7%(156/206) of FQr clinical isolates. Among those gyrA mutants, a majority (75.6%,118/156) harboured mutations at amino acid position 94, including 7 amino acid changes: D94G (the most frequent), D94A, D94C, D94Y, D94N, D94V and D94H. Mutation in gyrA QRDR showed 100% positive predictive value for FQr Mycobacterium tuberculosis in China. Mutation in gyrB were observed in 15.5%(32/206) of fluoroquinolone-resistant mycobacterium tuberculosis clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQr resistance in Mycobacterium tuberculosis.Conclusions:gyrA mutations were still main mutations in clinical isolates of FQr mycobacterium tuberculosis. gyrB mutations had limited effect in FQr mycobacterium tuberculosis.Mutations in gyrA were shown to be the major mechanism of FQr resistance in Mycobacterium tuberculosis isolates.The mutations in gyrA QRDR can be a good molecular surrogate marker for decting FQr Mycobacterium tuberculosis in China.Part 3 Analysis of prognosis in fluoroquinolone-resistant MDR-TB patientsObjective:To investigate prognosis in fluoroquinolone-resistant MDR-TB patients to provide basis for clinical treatment regimen establishment and monitoring. Methods:A retrospective study was carried out in 99 MDR-TB patients (71 were fluoroquinolone-resistant and 28 were fluoroquinolone-sensitive) to research clinical treatment effects. All the patients were treated with individualized regimens. The clinical effect, sputum smear, sputum culture and clinical outcomes of all patients were statistically analyzed following 24 months follow up. The logistic regression model was used to analysis the influencing factors. Results:The total cure rate of MDR-TB patients was 51.5%. Still 8.5% patients recrudesced after individualized therapy. Therapy on 93.5% of MDR-TB patients with positive sputum culture results was failed and only 6.5% were cured. While 90.6% of MDR-TB patients with negative sputum culture results were cured and only 9.4% failed in therapy. It is showed that in a single factor logistic regression analysis, compared with patients with failed treatment, less elder people (over 40 years old) (P=0.034,OR=2.4), lower fluoroquinolone resistance (P<0.001,OR=7.06), exist of fluoroquinolones in treatment (P=0.031,OR=0.40) in cured patients. It is indicated that fluoroquinolone resistance was lower in cured patients than in patients with treatment failed in a multiple factors logistic regression analysis for the risk factor of treatment failure when the confounding factors were eliminated (P=0.001,OR=7.06). More fluoroquinolones were used in cured patients than in patients with treatment failure (P=0.025,OR=0.33). Conclusions:The total cure rate of MDR-TB infection is not ideal. Cured patients who accept individualized treatment still have high risk of recrudesce. Sputum culture test have big value for prognosis in MDR-TB patients with 12 months individualized treatment and 12 months might be the important time for treatment regimen adjustment. Fluoroquinolone resistance is an independent risk factor for treatment failure of MDR-TB infection while fluoroquinolone included in regimen is a related factor for MDR-TB treatment success. |
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