الفهرس 
? Mycobacterium TuberculosisOnly 14 pages are availabe for public view
 |  | from | 157 |  |  |
| | | from | 157 | | |
Abstract
Background: According to World Health Statistics, Total cases notified of TB in Egypt during 2015 was 8,155 new and relapse cases with an incidence rate of 15 per 100,000 population. Fortunately, Egypt is still not on the WHO’s list of 30 high-TB-burden countries or the 27 high MDR-TB burden countries. In Egypt, percentage of MDR among new TB cases was 14% while that among previously treated TB cases was 23% according to most recent year available data .Several methods have been developed for the rapid detection of drug resistance tuberculosis compared with conventional time consuming drug susceptibility testing (DST). These methods include genotypic assays which are easy to perform and shorten the turnout time for the diagnosis of MDR-TB.
Objective: The aim of this study was to evaluate real-time PCR as a rapid diagnostic method in detection of mutations associated with the resistance to second-line injectable agents (capreomycin, amikacin and kanamycin) and fluroquinolones in M. tuberculosis clinical isolates in comparison to proportional method.
Methods: This study was conducted on 600 M. tuberculosis clinical isolates obtained from sputum and bronchioalveolar lavage samples from the National Central Egypt, during the period from July 2015 to December 2016. Isolates were tested for first-line anti-tuberculous drugs, isoniazid (INH) 0.2 ug/, rifampicin (RMP) 40ug/ml, streptomycin (STM) 4ug/ml and ethambutol (EMB) 2ug/ml using PM. Nintey two (92) isolates found to be multi-drug resistant and they were further tested for susceptibility to four second-line anti-tuberculous drugs, amikacin (AMK) 20 ug/L capreomycin (CM) 40 ug/L kanamycin (KM) 30 ug/L and ofloxacin (OFX) 2 ug/L using PM. Out of 92 MDR-TB isolates forty eight (48) isolates were found to be resistant to at least one of the second line drugs. These 48 isolates were tested by real -time PCR using three dually labeled probes and three pairs of primers designed to detect mutations in gyrA, rrs and the promoter of eis.
Results: This study showed that 8 isolates were found to be resistant for gyrA, and 38 isolates for rrs, and 32 isolates for the promoter of eis using real time PCR. Comparing the results of gold standard PM to results of real time PCR, an agreement of 86.5%, 100%, and 69% were found to gyrA, rrs, and the promoter of eis respectively. In this study sensitivity of Rt-PCR for AMK and CM was 100%, specificity was 100%, PPV was 100% and NPV was 100%. Sensitivity of Rt-PCR for KM was 84.2%, specificity was 100%, PPV was 100% and NPV was 62.5%. Sensitivity of Rt-PCR for OFX was 80%, specificity was 100%, PPV was 100% and NPV was 95%.
Conclusions: The use of Real-time PCR for rapid detection of mutations associated with second-line anti-tuberculous drugs shows a high level of agreement with the proportional method. Real-time PCR is a simple, accurate, easy to perform and eliminates the need for electrophoresis after the cycling reaction. So it reduces the analysis time to three or four days, which is important in laborious and lengthy microbiological diagnoses, helping in early detection of MDR-TB and screening for XDR-TB strains.