Clinical Outcomes Of The Individualized Treatment For Patients With Drug Resistant Spinal Tuberculosis And Amplification Of DNA Microarrays For The Detection Of Drug Resistant M.Tuberculosis

Objective: Drug-resistant tuberculosis is a major public health concern globally. Asthe most common extrapulmonary form of tuberculosis, spinal tuberculosis has anaggressive behaviour of profound vertebral destruction and severe complications. Theinitial optimism with tuberculosis spondylitis lessens as we see the increased number ofrelapses and initial treatment failures owing to the emergence of drug-resistant tuberculosis.However, clinical characteristics, drug resistant profile and treatment of drug-resistantspinal tuberculosis have been received little attention in the literature. Optimal managementof drug-resistant tuberculosis of the spine relies on the early diagnosis. The methods ofDST using the BACTEC MGIT960system can provide definitive results, but they usuallyrequire at least1-2months to produce the strain-susceptibility profile, leading to thediagnosis delay. DNA microarrays can detect tuberculosis and its multi-drug resistant formin M. tuberculosis isolates and sputum specimens with high sensitivity and specificity.However, no performance data currently exists for its use in spinal tuberculosis specimens.Otherwise, because of the dissemination of other first-line and second-line drug-resistant M.tuberculosis strains, gene mutations that relate to other first-line and second-line drugresistance should be considered for incorporation into the further DNA microarray system.Therefore, the aim of this study was to investigate the clinical characteristics, drugsusceptibility patterns and the outcomes of management with a combination of surgery andindividualized chemotherapy for drug-resistant spinal tuberculosis. Moreover, to access thefeasibility and accuracy of the DNA microarrays for parallel species identification anddetection of mutations that confer INH and RMP resistance among consecutive patientswith spinal tuberculosis. In the end, to develop a novel DNA microarray system for the detection of the SM/EMB/LVFX/AMK/CPM resistant M. tuberculosis, and to ascess itsaccuracy and feasibility.Materials and Methods:1. We retrospectively analysed thirty-five patients with drug-resistant tuberculousspondylitis in Southwest Hospital between September2005and January2010. After surgery,individualized chemotherapy was tailored for each patient according to the drug-resistanceprofile and previous history of chemotherapy. The patients were followed up clinically andradiologically for an average period of35.8months.2. From March2009to December2011,153consecutive patients from SouthwestHospital with clinically and pathologically diagnosed spinal tuberculosis were enrolled intothis study. Specimens collected during surgery from the tuberculosis patients weresubjected to M. tuberculosis species identification and RMP/INH-resistance detection bythe DNA microarray, and results were compared with those obtained from conventionalDST.3. A set of111drug-resistant M. tuberculosis strains and26drug-susceptible M.tuberculosis stains were collected from Chongqing Infecious Disease Center. DNAmicroarrays were designed and prepared for the detection of rrs/rpsL/gyrA/embB genemutations, and the results were compared to the phenotypic DST. The feasibility andaccuracy of the DNA microarrays for the diagnosis of SM/EMB/LVFX/AMK/CPMresistant M. tuberculosis strains were evaluated.Results:1. Among35drug-resistant spinal tuberculosis cases,13were retreatment cases.Twelve were MDR-TB, and23were non-MDR-TB. The patients with MDR-TB andnon-MDR-TB had undergone previous chemotherapy for an average of14.50±2.00(0–60)months and4.56±1.54(0–74) months, respectively. A total of32cases underwent openoperations, and the other3had percutaneous drainage and local chemotherapy. Patientsreceived individualized chemotherapy for an average of23.6months postoperatively. Localrecurrence was observed in6patients. Thirty-three patients had been cured at the finalfollow-up, and the other2were still receiving chemotherapy.2. The DNA microarray achieved93.55%sensitivity for the correct M. tuberculosisspecies identification of the93specimens that tested positive for spinal tuberculosis through culture. In addition,27additional patients (45.0%) were detected by the DNAmicroarray to be positive for M. tuberculosis among60spinal tuberculosis patients whowere culture negative. Moreover, the DNA microarray had a sensitivity of88.9%and aspecificity of90.7%for RMP resistance, and had a sensitivity of80.0%and a specificity of91.0%for INH resistance. The mean turn-around time of M. tuberculosis speciesidentification and drug resistance detection using the DNA microarray was5.8(range,4-9)hours, which has been significantly shorten compared to that of conventional culture andDST.3. Among the137M. tuberculosis stains, involving83SM-resistant strains,54SM-susceptible strains,63EMB-resistant strains,74EMB-susceptible strains,37LVFX-resistant strains,100LVFX-susceptible strains,50AMK-resistant strains,87AMK-susceptible strains,29CPM-resistant strains and108CPM-susceptible strains. Thesensitivity and specificity for the detection of drug resistance using DNA microarrays were90.36%and70.37%for SM,61.90%and58.11%for EMB,94.59%and58.00%for LVFX,60.34%and93.67%for AMK/CPM.Conclusions:1. Drug-resistant tuberculous spondylitis is mainly acquired through previous irregularchemotherapy and the spreading of drug-resistant M. tuberculosis strains. Management witha combination of surgery and DST-guided individualized chemotherapy is feasible in thetreatment of severe complications and the prevention of acquired drug resistance. Moreover,the traditional DST usually requires at least1to2months to produce the strainsusceptibility profile, leading to inadequate treatment and local relapse during this period.Therefore, developing new, rapid and accurate molecular DST methods is essential for theearly diagnosis of drug-resistant tuberculosis.2. The DNA microarray is a simple and accurate tool for M. tuberculosis identificationand for the diagnosis of RMP and INH resistance, demonstrating that the system is likely tobe applicable to spinal tuberculosis specimens. The automated readout and short turnaroundtime make this assay suitable for testing and decreases the diagnosis delay, without the needto build large numbers of advanced biosafety facilities. Otherwise, the gene mutations thatrelate to other first-line and second-line drug resistance should be considered forincorporation into the further DNA microarray system because of the dissemination of other first-line and second-line drug-resistant M. tuberculosis strains. Additionally, it will benecessary to explore whether early and individualized treatment based on the DNAmicroarray can influence the clinical outcome of drug-resistant spinal tuberculosis patients.3. The DNA microarrays can detect SM-resistant strains with high sensitivity andspecificity, having a great prospect of clinical application. For the detection of EMBresistance, the sensitivity and specificity were rather low. For the detection of LVFXresistance, the sensitivity was as high as the previous molecular DST technologies, but thespecificity was rather low. Moreover, for the detection of AMK and CPM resistance, thesensitivity was much lower, but has a high sensitivity. Thus, there's a need to improve thedesign of the DNA chip and methods of the investigation. Otherwise, the limitation of thisstudy was the small scale samples, the selection of gold standard and samples.