Kemal Bilgin

Two New Colorimetric Methods for Early Detection of Vancomycin and Oxacillin Resistance in Staphylococcus aureus

ABSTRACT We developed two colorimetric methods for the detection of vancomycin- and oxacillin-resistant Staphylococcus aureus in ≤6 h: (i) a nitrate reductase assay and (ii) a resazurin microplate method. MICs agreed with results obtained by CLSI methods for oxacillin. However, detection of vancomycin resistance required a larger inoculum. These methods may be recommended for the detection of vancomycin- and oxacillin-resistant S. aureus.

Rapid Susceptibility Test for Mycobacterium tuberculosis to Isoniazid and Rifampin with Resazurin Method in Screw-Cap Tubes

Abstract Multi-drug resistant (MDR) Mycobacterium tuberculosis is still a serious public health problem all over the world. MDR tuberculosis (MDR-TB) caused by these strains has emerged within the last decade and rapid detection is critical for the effective treatment of patients. Recently, a resazurin microtiter assay plate for detecting MDR strains was developed. In this study, it was adapted to screw-cap tubes and the activity of isoniazid (INH) and rifampin (RIF) to 50 M. tuberculosis clinical isolates was tested by this method for the first time. Results were compared with the radiometric reference method for the susceptibility testing of M. tuberculosis complex. The results of both methods were in 100% and 96% agreement for RIF and INH, respectively. Specificity, sensitivity, positive predictive value and negative predictive value were 91.7%, 100%, 92.8% and 100% for INH, respectively. All of these values were 100% for RIF. Susceptibility testing results were obtained on the 8th day of incubation for 42 isolates and on the 9th day for the other eight strains. Our results indicate that this method is suitable for the early determination of INH and RIF resistance in developing countries because it is inexpensive, rapid and easy to perform.

Comparative Study for Determination of Mycobacterium tuberculosis Susceptibility to First- and Second-Line Antituberculosis Drugs by the Etest Using 7H11, Blood, and Chocolate Agar

ABSTRACT We investigated the performance of blood and chocolate agar as alternatives to Middlebrook 7H11 agar for testing the susceptibility of Mycobacterium tuberculosis to first-and second-line drugs by the Etest method. A total of 39 strains of M. tuberculosis including 22 multidrug-resistant M. tuberculosis strains and 17 susceptible strains were tested. In conclusion, our results showed that chocolate agar gave insufficient growth, needing up to 21 days of incubation, while results on blood agar were comparable to those on Middlebrook 7H11 agar and can be further explored as an alternative for Etest-based susceptibility testing of M. tuberculosis.

Susceptibilities of Mycobacterium tuberculosis to Isoniazid and Rifampin on Blood Agar

ABSTRACT In this study, blood agar was used instead of 7H10 agar for the susceptibility testing of 34 clinical isolates of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) in accordance with the NCCLS. The BACTEC 460 TB system (Becton Dickinson, Sparks, Md.) was used as a “gold standard.” Results for both media were in agreement for RIF and INH at 100 and 94.1%, respectively. For INH, the specificity, sensitivity, positive predictive value, and negative predictive value were found to be 71.4, 100, 93.1, and 100%, respectively, while these values were 100% for RIF. In addition, the results of the susceptibility test performed with blood agar were obtained on day 14 of incubation. In conclusion, results were obtained much earlier with blood agar (2 weeks) than with 7H10 agar (3 weeks), and the results of this study suggest that blood agar may be used as an alternative medium for the susceptibility testing of M. tuberculosis to INH and RIF.

In Vivo Efficacy of Drugs against Toxoplasma gondii Combined with Immunomodulators

The aim of this study is to evaluate the effects of pyrimethamine (PYR) and sulfadiazine (SDZ) combined with levamisole and echinacea on the survival of mice infected with Toxoplasma gondii. For this, we used 99 specific pathogen-free BALB/c mice. All the mice were infected intraperitoneally with 10(5) T. gondii tachyzoites and were divided into 11 groups, each including 9 mice. Except for the control group, oral treatment was initiated in all groups 24 h post infection and was continued for 10 days. The treatment regimen included dual combinations of PYR (dose, 6.25 and 12.5 mg/kg/day) and SDZ (dose, 100 and 200 mg/kg/day), triple combinations of PYR + SDZ, and levamisole (dose, 2.5 mg/kg/day) or echinacea (dose, 130 and 260 mg/kg/day) and echinacea alone (dose, 130 and 260 mg/kg/day). We observed that an effective dose of the combination of PYR + SDZ and levamisole resulted in a statistically significant increase in the survival rate from 33.3% to 88.9%. Similarly, half the dose of this combination resulted an increase in the survival rate from 0% to 44.4% (p < 0.05). Survival rate also increased in the groups treated with the combinations including echinacea; however, the difference did not reach statistical significance. The triple combination of PYR-SDZ-levamisole could be an alternative treatment option in case of infections caused by T. gondii.

An evaluation of false-positive rifampicin resistance on the Xpert MTB/RIF

BACKGROUND Mycobacterium tuberculosis (MTB) is one of the most significant causes of mortality and morbidity. Early diagnose is important especially in multiple drug resistant tuberculosis to avoid transmission. Traditional techniques requires at least one to three weeks for diagnosis of tuberculosis. Diagnostic delays with multiple drug resistant tuberculosis are associated with worse clinical outcomes and increased transmission The Xpert MTB/RIF assay is one of the new diagnostic device for the diagnosis of tuberculosis and rapid detection of rifampicin resistance. OBJECTIVE We assessed the performance of Xpert MTB/RIF assay for detecting rifampicin resistance using phenotypic drug susceptibility tests as automated BD MGIT 960. METHODS Total of 2136 specimens were included in the study. Xpert MTB/RIF testing was performed on samples, using version 4 cartridges, according to the manufacturers recommendations. The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer. Agar proportion was used in the case of inconsistency for rifampicin resistance. FINDINGS Thirty-four samples (19 respiratory and 15 nonrespiratory samples) were determined as positive for M. tuberculosis complex by Xpert MTB/RIF (Cepheid GeneXpert® System, USA). Xpert MTB/RIF assay detected 4/34 (11.7%) specimens as rifampicin resistant. One of the rifampicin resistant isolates was determined susceptible in MGIT 960 automated system. This isolate was also tested with agar proportion method and found susceptible to rifampicin. MAIN CONCLUSION The Xpert MTB/RIF assay can be used as first-line assay for the detection of M. tuberculosis. However, microbiologists must be aware of the limitations of the assay.

Comparison of a real-time polymerase chain reaction-based system and Erlich-Ziehl-Neelsen method with culture in the identification of Mycobacterium tuberculosis

BASCKGROUND/AIM Mycobacterium tuberculosis is still a major health problem throughout the world, especially in developing countries. Disease control heavily depends on the establishment of early diagnosis. The aim of this study is to compare the efficacy of culture, GeneXpert MTB/RIF device, and Erlich-Ziehl-Neelsen direct microscopic method. MATERIALS AND METHODS A total of 927 samples (243 respiratory and 684 nonrespiratory), which were sent to Ondokuz Mayıs University Medical Faculty Tuberculosis Laboratory on suspicion of M. tuberculosis, were included in the study. RESULTS When compared to standard culture, sensitivity, specificity, and positive and negative predictive values of the GeneXpert system for respiratory samples were 100%, 98.7%, 87%, and 100%, respectively; these values for nonrespiratory samples were 71%, 98.6%, 71%, and 98.6%, respectively. CONCLUSION New, reliable, rapid, and easy-to-use methods that display high specificity and sensitivity are required for an effective struggle against tuberculosis. According to these results, we suggest that GeneXpert MTB/RIF is a rapid and reliable system, and when used in company with conventional tests, it would make significant contributions to the diagnosis of tuberculosis.

Comparison of Culture, Real-Time DNA Amplification Assay and Ehrlich-Ziehl-Neelsen for Detection of Mycobacterium tuberculosis

OBJECTIVE Mycobacterium tuberculosis is still a substantial health problem universally. Although culture is the gold standard method, reliable, rapid and new methods are required for effective struggle with disease. We retrospectively compared the results of Ehrlich-Ziehl-Neelsen (EZN) stain and real-time DNA amplification assay (BD ProbeTec ET system) with culture. STUDY DESIGN Retrospective study. MATERIAL AND METHODS A total of 703 samples, 182 pulmonary and 521 extra pulmonary, collected from 630 patients between May 2008 and February 2011 were evaluated. Culture was considered the gold standard. RESULTS For pulmonary specimens, sensitivity, specificity, positive predictive and negative predictive values of BD ProbeTec ET and EZN were calculated to be 100%, 98.8%, 87.5%, 100% and 71.4%, 98.8%, 83.3%, 97.6%, respectively. For extra pulmonary specimens, sensitivity, specificity, positive predictive and negative predictive values of BD ProbeTec ET and EZN were calculated to be 80%, 98.7%, 76.9%, 98.9% and 24%, 98.3%, 42.8%, 96.2%, respectively. CONCLUSION According to these results, we suggest that the BD ProbeTec ET system is more reliable than EZN. In addition, the BD ProbeTec ET system produces faster results. Based upon these results, we consider that the BD ProbeTec ET system may be employed in the diagnosis of M. tuberculosis.

Research of Acinetobacter Baumannii Isolation From Clinical Samples in Second Step Hospital

Aim: Due to existing multi drug resistance and subsequently acquired resistance Acinetobacter genus bacteria continuously actual. Other characteristics are increasing treatment costs, patient hospitalization period, mortality and morbidity. Risk factors like extended hospitalization period, background immune system disorders are increasing isolation frequency of this bacteria from patients. Extended spectrum antibiotic usage is known to be a major risk factor. Aim of our study is to investigate cause of growing A.baumanii isolation rate and cross contamination between this isolates in a state hospital. Material and Method: In this study analysed increasing isolation frequency by years and specimen occurrence in level 2 hospital. At the same time detected amount of used imipenem and meropenem in hospital during last three years. A.baumanii strains isolated from respiratory and sputum specimens of patients from intensive care unit and thoracal departament during last month of 2013 year’s were tested using PFGE method for genotypic similarity. Results: Acinetobacters isolation frequency in years and carbapenem usage are subsequently increased. Specimens are generally from respiratory tract. Genotypic similarity not detected on studied 6 A.baumanii strain’s PFGE image. This condition interpreted like this strains origins not from cross contamination.

[Isosporiasis in an elderly patient with chronic diseases: case report].

Isospora belli is a coccidian protozoon that can cause serious diarrhea especially in immunocompromised patients. The laboratory diagnosis depends primarily on the identification of oocysts in stool specimens by direct microscopic examination with iodine or special stains. This case is presented in order to draw attention to isosporiasis among the diarrheas that can be seen in elderly patients with several chronic diseases. A 81 year-old debilitated male, who had a history of hypertension, Alzheimers disease, previous cerebrovascular accident and right hemiplegia, was admitted to our hospital complaining of malaise, anorexia, chills, abdominal pain, dysuria, cough, sputum and diarrhea of ten days duration. I. belli oocysts were detected by microscopic examination of the sample with iodine after concentration by formalin-ethyl acetate sedimentation. Then, modified acid-fast and trichrome stains were performed and I. belli oocysts were detected with both methods. Similar to this case, infections caused by I. belli can occur in elderly immunocompromised patients with several chronic diseases and inadequate nutrition and care. Consequently, in individuals with persistent diarrhea, examinations and tests should be carried out by taking their immune status into consideration and stool examinations should be done at frequent intervals using the concentrations methods and special stains.