aslan Alp

Rapid Determination of Rifampin Resistance in Clinical Isolates of Mycobacterium tuberculosis by Real-Time PCR

ABSTRACT 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.

MEMS biosensors for detection of methicillin resistant Staphylococcus aureus

This review presents the current state of the conventional methods, microfluidic based biosensors, and the commercial products used in the detection of methicillin resistant Staphylococcus aureus (MRSA), which is one of the most important threats of nosocomial infections in many parts of the world. The early detection of MRSA in the specimens of the patients is important to enable the appropriate treatment, to decrease morbidity and mortality rates, and to manage control actions in the healthcare units. Thus, rapid and inexpensive diagnostic systems with high sensitivity and specificity are essential to prevent MRSA to be an emerging public health threat. The design and fabrication of new diagnostic systems necessitates working in collaboration between different disciplines to make new challenges in the field of clinical diagnosis and to meet the demands of clinicians. It is certain that in the near future, MEMS and nanotechnology based detection methods will take the place of current methods in clinical diagnosis. The evaluation of new trends for specificity, sensitivity, cost effectiveness, disposability, low weight, ease of use, and facile access should be taken into consideration.

Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates by Heteroduplex Analysis and Determination of Rifamycin Cross-Resistance in Rifampin-Resistant Isolates

ABSTRACT Direct heteroduplex analysis and a universal heteroduplex generator assay were performed to detect rifampin resistance rapidly in Turkish Mycobacterium tuberculosis isolates. Cross-resistance to rifapentine, rifabutin, and rifalazil was investigated. A relationship between specific mutations and resistance patterns, which can guide the choice of an appropriate therapeutic regimen for tuberculosis patients, was identified.

Distribution of Spoligotyping Defined Genotypic Lineages among Drug-Resistant Mycobacterium tuberculosis Complex Clinical Isolates in Ankara, Turkey

Background Investigation 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 Findings A 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. Conclusions The 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.

Mycobacterial Strains That Stimulate the Immune System Most Efficiently as Candidates for the Treatment of Bladder Cancer

Background: Intravesical bacillus Calmette-Guérin (BCG) application is widely used in the treatment of superficial bladder carcinoma. Despite being an effective therapy, the pathogenicity and lethal side effects of BCG limits its usage. Intensive research has been carried out to find less toxic and more potent therapeutic agents for the treatment of bladder cancer. Researchers have focused on Mycobacterium phlei as an alternative. The cell wall extract of M. phlei is sufficient for antitumoral activity. Our preliminary experiments indicate that the fractions rich in cell wall proteins cause activation of tumor necrosis factor (TNF)-α and interleukin (IL)-12. This study aims to identify powerful and less harmful mycobacteria among 88 strains in terms of how they stimulate the immune system. Methods: Eighty-eight mycobacterial strains were grown in Middlebrook 7H9 medium. The bacterial cells were sonicated after heat treatment. The supernatants were incubated with the monocytic cell line THP-1, followed by measurement of TNF-α and IL-12 response. Results and Conclusion: In addition to M. phlei, the following 12 mycobacterial strains were selected as candidates for superficial bladder tumor treatment: M. agri, M. aichiense, M. aurum, M. brumae, M. chitae, M. chubuense, M. diernhoferi, M. gadium, M. murale, M. obuense, M. tokaiense and M. vaccae.

Utility of a commercial quantitative hepatitis C virus core antigen assay in a diagnostic laboratory setting.

In this study, the utility and impact of hepatitis C virus (HCV) core antigen (Cag) detection via a commercial assay have been evaluated in diagnostic laboratory conditions. In a total of 272 samples from 226 individuals, HCV RNA was detected in 81.3% and anti-HCV antibody prevalence was 86.4%. HCV Cag reactivity was identified in 59.9% of the samples and in 75.8% with detectable RNA. The sensitivity and specificity of HCV Cag assay have been calculated as 75.8% and 95.1%, respectively, and agreement between HCV RNA and HCV Cag was moderate (κ = 0.554). HCV Cag and RNA levels were highly correlated (r = 0.915 and 0.937). A viral load threshold of 10(3) IU/mL has been recognized, above which the correlation with RNA became statistically significant and sensitivity increased to 90.9%. Detection and quantification of HCV core antigen have been observed as a strong alternative to nucleic acid testing for HCV monitorization.

CYP17 and CYP2C19 gene polymorphisms in patients with endometriosis

Endometriosis seems to be the result of a complex interaction between environmental factors and various genes. In this regard, the cytochrome subfamily 17 (CYP17) may play an important role by altering the biosynthesis of sex steroids. CYP2C19 is also an important member of the cytochrome P450 (CYP) family, and related mutations may result in an inability to fully metabolize environmental chemicals and cytokines, leading to several diseases. This study sought to determine whether there is a relationship between endometriosis and CYP17 T>C, CYP2C19 *2 and CYP2C19 *3 polymorphisms. When samples from 46 patients with endometriosis and 39 healthy controls were analysed, A2A2 type mutation of the CYP17 gene was observed to be more frequent in patients with endometriosis (34.8 versus 7.7%, P = 0.003). No association was found between the severity of endometriosis and CYP2C19 *2 or CYP2C19 *3 polymorphisms of the CYP2C19 gene. These results suggest that mutations related with sex steroid metabolism seem to have an important role in endometriosis. However, the relation between detoxification ability and endometriosis should be examined in further studies with larger sample sizes.

Mycobacterium smegmatis pneumonia

Abstract:  Mycobacterium smegmatis is a non‐tuberculous mycobacterium that is usually associated with soft tissue or wound infections in humans. Pulmonary infections secondary to this pathogen are rarely seen and occur only in patients with an underlying condition, such as lipoid pneumonia. This report presents the first case of M. smegmatis pneumonia in an otherwise healthy individual who had no predisposing condition.

A Fully Microfabricated Electrochemical Sensor and its Implementation for Detection of Methicillin Resistance in Staphylococcus Aureus

On-chip detection of biological analytes can enable diagnosis at the point of care. Combining the advantages of microelectromechanical system (MEMS) technology and molecular methods, we present the design of an integrated microfluidic platform, a microelectrochemical sensor (μECS), and its implementation for the detection of methicillin resistance in Staphylococcus aureus. This platform is capable of electrochemically sensing the target analyte in a microfluidic reactor without the usage of bulky electrodes, rendering it useful for in vitro diagnostics. In our experiments, the functionality of the sensor was tested for detecting specific DNA sequences of mecA gene (an indicator of methicillin resistance) over a range of concentrations of DNA (down to 10 pM). Synthetic oligonucleotides and bacterial PCR product were used as a target analyte in Hoechst 33258 marker-based detection and horseradish peroxidase-based detection, respectively. The results revealed that this platform has high sensitivity and selectivity. Also, its compatibility to MEMS processes enables its use with different applications ranging from detecting various types of cancers to endemics. The designed μECS can enable the detection of biological analytes of interest at low cost and high throughput.

Comparison of 3 nucleic acid isolation methods for the quantification of HIV-1 RNA by Cobas Taqman real-time polymerase chain reaction system☆

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA viral load in blood is a clinically validated tool for the management and follow-up of patients infected with this virus. HIV-1 RNA quantification can be performed by several kinds of commercially available polymerase chain reaction (PCR) assays. The sensitivity of these assays is affected by the nucleic acid isolation procedures that are performed before PCR. Especially for the samples containing low numbers of HIV-1 RNA, an effective nucleic acid isolation method is very important to obtain a high nucleic acid output. The purpose of this study was to compare and evaluate the conventional manual extraction method, automated MagNA Pure system, and automated AmpliPrep system for the nucleic acid isolation before TaqMan real-time PCR assay for the quantification of HIV-1 RNA in plasma samples. Plasma samples of 40 patients in which anti-HIV antibodies were found as positive serologically were included in this study. Nucleic acid isolation from these samples was performed by manual High Pure Viral Nucleic Acid Isolation kit (Roche Diagnostics, Mannheim, Germany), automated MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics), and automated Cobas AmpliPrep kit (Roche Diagnostics). The nucleic acids obtained by these 3 methods were amplified and detected in the Cobas Taqman 48 (Roche Diagnostics) instrument. Viral RNA copies detected by AmpliPrep system, MagNA Pure system, and manual isolation method were found to be between 40 and 420, 000 (average, 100 135), 53 and 1 200 000 (average, 220 554), and 183 and 590 000 (average, 104 761) copies/mL, respectively. The results of isolation methods were evaluated for each sample, and it was found out that the number of HIV-1 RNA obtained by MagNA Pure system was higher than the other 2 methods. All of the 3 methods were found to be reliable in detecting very small amounts of HIV-1 RNA in samples. Manual isolation method is a cheaper alternative, but because the nucleic acid isolation can be performed in a shorter time by automated systems when compared with manual isolation method, these systems can be preferred especially in the laboratories that have high number of samples.