BackgroundInvestigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.Methods and FindingsA total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.ConclusionsThe results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.
Real-time PCR was used to determine rifampin resistance in clinical isolates of Mycobacterium tuberculosis.Ninety-six rifampin-resistant isolates and 23 rifampin-susceptible isolates were included in the study. A 305-bp region covering the 81-bp "rifampin resistance-determining region" of rpoB was amplified. Two hybridization probe pairs that covered the most frequent mutation sites in rpoB, codon regions 526 to 531 and 513 to 516, were used. The results obtained by real-time PCR were compared to those obtained by the proportion method. For detection of rifampin resistance, the real-time PCR assay yielded a sensitivity of 92.7% and a specificity of 100%. Real-time PCR is a very rapid method, and it can be especially helpful for the reporting of resistant clinical isolates in a very short period of time.Tuberculosis is still one of the infectious diseases that causes high fatality rates worldwide. In recent years, the emergence of drug-resistant strains has been an important problem and is threatening the control of the disease (4, 5). Therefore, the rapid detection of drug resistance is essential in order to begin effective therapies and protect the community from tuberculosis.Rifampin was introduced as an antituberculosis drug in 1972, and since then it has been a key component of shortcourse multidrug antituberculosis therapy. It is a bactericidal agent. It binds to the  subunit of the DNA-dependent RNA polymerase and inhibits the initiation of transcription (2, 15). The widespread use of rifampin and other rifamycin derivatives has led to the emergence of rifampin resistance (3, 8).Ninety-six percent of the cases of rifampin resistance are associated with mutations in the 81-bp "rifampin resistancedetermining region" of the rpoB gene coding for amino acids 507 through 533. The frequencies of frequent mutations were reported to be 41% in codon 531, 32 to 36% in codon 526, and 7 to 9% in codon 516 (15,22).Real-time PCR is a method that enables both the monitoring of amplification and the detection of mutations with the help of fluorescently labeled DNA probes (1,11,24). In previous studies, real-time PCR was used for the detection of rifampin resistance in isolates with known mutations (18,19,21). In this study, use of real-time PCR for the detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis was evaluated. The "resistance-determining region" of the rpoB gene was amplified, and the melting temperatures (T m s) of the probes were obtained on a LightCycler instrument (Roche Diagnostics). The change in the T m was considered an indicator of a mutation, and isolates for which the probe had a T m other than that for M. tuberculosis H37Ra were considered resistant to rifampin (1,11,24). The results obtained by realtime PCR were compared to the drug susceptibility results obtained by the conventional proportion method.We also performed DNA sequencing with certain strains, determined the rifamycin cross-resistance in rifampin-resistant strains, and showed the correlation between the anti...
The PP-g-PEG, especially Ag-PP-g-PEG polymer-coated ventricular catheters are more effective in preventing the catheter-related infection and created the least inflammatory reaction in the periventricular parenchyma.
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