Blood agar for susceptibility testing of Mycobacterium tuberculosis against first-line drugs.

OBJECTIVE: To evaluate the performance of blood agar for the susceptibility testing of 50 Mycobacterium tuberculosis clinical isolates against isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB). DESIGN: The activity of the drugs was determined by the proportion method on blood agar instead of Middlebrook 7H10 agar according to Clinical Laboratory Standard Institute recommendations. The final concentrations of INH, RMP, SM and EMB were 0.2 microg/ml, 1 microg/ ml, 2 microg/ml and 5 microg/ml, respectively. RESULTS: The results were compared with the radiometric proportion method as the reference, and the agreements were determined as 100% for INH and RMP, 92% for SM and 96% for EMB. The specificity, sensitivity, positive predictive value and negative predictive value were 90.4% and 97.5%, 100% and 90%, 66.6% and 90% and 100% and 97.5% for SM and EMB, respectively, while these values were 100% for INH and RMP. The results of susceptibility testing were obtained on the 14th day of incubation. CONCLUSION: According to this preliminary study, our results suggest that blood agar can be used as an alternative medium for the susceptibility testing of M. tuberculosis strains against INH, RMP, SM and EMB in resource-limited countries. However, further studies are needed before implementating the method in diagnostic laboratories.

Rapid susceptibility test for Mycobacterium tuberculosis to isoniazid and rifampin with resazurin method in screw-cap tubes.

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.

The resazurin microplate method for rapid detection of vancomycin resistance in enterococci.

Vancomycin-resistant enterococci (VRE) are a serious challenge for physicians because of the limited treatment options for infections caused by this organism. Prevention of VRE transmission in hospitals requires early detection of infected or colonized patients. Therefore rapid and correct detection of vancomycin resistance is essential. In this study, we use the resazurin microplate method (RMM), which is a modification of the NCCLS and BSAC broth microdilution methods to rapidly determine the susceptibilities of clinical enterococci isolates to vancomycin. The alteration in the RMM was relevant to the final bacterial count. In this method, inoculum that was 10-fold higher than standard methods was used. A total of 80 enterococci, including 11 VRE isolates and 6 vancomycin intermediate isolates, were screened with this modified colorimetric broth microdilution method. After 4 h of incubation 30 microl of 0.01% resazurin solution were added to each well and the plates were reincubated for color change for 5-10 min. The MICs were obtained at the 4th h. The results were in exact agreement with the NCCLS and the BSAC microdilution methods. Absolute and essential agreements were 100% and there were no minor, major or very major errors. In conclusion, this modified colorimetric broth microdilution method can be used as a reliable, easy, cheap and rapid method for early detection of VRE. Moreover, this method has the potential of being used to test the susceptibilities of different bacteria to other antibiotics.

In vitro effects of cefotaxime and ceftriaxone on Salmonella typhi within human monocyte-derived macrophages.

The main objective of this in vitro study was to assess the effects of cefotaxime and ceftriaxone in killing Salmonella typhi in infected human macrophages. Human monocyte-derived macrophages isolated from peripheral blood of human volunteers were cultured in vitro for macrophage differentiation, and subsequently infected with S. typhi strains (a clinical isolate and a standard strain TA-42) at a cell ratio of 10 : 1. MICs of cefotaxime and ceftriaxone were determined by broth microdilution, and the antibiotics were included in the culture medium at one and five times their MIC values. Samples of cell culture medium taken at 0, 3, 6 and 24 h of incubation were cultured for growth of S. typhi on nutrient agar. Gentamicin (10 mg/L) was included in each well except for the control wells, in order to prevent growth of extracellular S. typhi. Both antibiotics showed good in vitro antibacterial effects against S. typhi strains. There were no statistically significant differences between the extracellular and intracellular effects of antibiotics with regard to elimination of the bacteria. Cefotaxime and ceftriaxone are highly effective against extracellular bacterial growth. The results of our in vitro experiments suggest that cefotaxime and ceftriaxone might also be used clinically against susceptible intracellular pathogens such as S. typhi.

Determination of minumum inhibitory concentrations of Candida species isolated from vaginal swab specimens by using broth macrodilution and E-test.

The purpose of the present study was to evaluate the utility of the E-test in determining the antifungal susceptibility of Candida species. A total of 50 Candida strains, including 34 Candida albicans and 16 non-albicans were isolated from vaginal swab specimens from women suffering from vaginitis. The minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole and ketoconazole were detected by using broth macrodilution and the E-test. When the results of the two tests were compared, the MIC values were considered acceptable if the difference between the two assays was no more than two-fold (+/-1dilution). The acceptable rates were: 84% for amphotericin B, 97% for fluconazole and 78% for ketoconazole. Finally, MICs of C. albicans against the tested antifungal agents were generally lower than for non-albicans strains. These results suggest that the E-test can be used for the determination of MIC values for Candida species isolates.

Effects of acetyl salicylate and ibuprofen on fluoroquinolone MICs on Salmonella enterica serovar typhimurium in vitro.

In this study, the effects of acetylsalicylate and ibuprofen at 2, 4 and 8 mM concentration were investigated on ofloxacin, ciprofloxacin, levofloxacin and pefloxacin minimum inhibitory concentrations (MICs) for 14 Salmonella enterica serovar typhimurium clinical isolates, one standard strain (SZH KUEN 557), SH7616 (acr mutant), SH5014 (parent strain of acr mutant) and PP120 (soxRS mutant) strains. All isolates were susceptible to the 4 fluoroquinolones. In the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen, 2- to 8-fold increases were observed in fluoroquinolone MICs. This rise was higher, especially in the presence of acetylsalicylate. In spite of this rise, none of the MICs were in the range of resistance limits in vitro. Except for a 2-fold increase in levofloxacin MICs, we did not observe any difference in MICs of ofloxacin, ciprofloxacin, and pefloxacin in the presence of 2, 4 and 8 mM acetylsalicylate and ibuprofen for SH7616 and PP120 strains. According to the in vitro results of this study, it can be suggested that use of acetylsalicylate or ibuprofen together with clinical treatment of bacteria, especially bacteria which show intermediate resistance, will cause resistance. However, since clinical data are insufficient, further studies are needed.

Diabetic retinopathy and HLA antigens in type 2 diabetes mellitus.

PURPOSE: Diabetic retinopathy is the most common complication of diabetes mellitus. No single predisposing factor has been identified, and genetic factors may play a role in the development of severe retinopathy. In this study, we investigated the association between diabetic retinopathy and HLA antigens in type 2 diabetes mellitus. METHODS: This study was conducted at the retina unit of the Department of Ophthalmology of Ondokuz Mayis University between October 1999 and March 2000, and included 46 diabetics with non-proliferative retinopathy and 30 with proliferative retinopathy, with 30 nondiabetic controls. HLA class I (A, B, C) antigens were studied by Terasaki's microlymphocytotoxicity test and HLA class II (DR, DQ) typing was carried out using a polymerase chain reaction-sequence specific primer. RESULTS: HLA-DR4 and DQ8 frequencies were higherin patients with non-proliferative retinopathy than those with proliferative retinopathy, and HLA-DR7 frequency was higher in patients with proliferative retinopathy than non-proliferative cases (p<0.05). No significant differences in HLA antigens were found between patient groups and controls. CONCLUSIONS: The differences in HLA antigen frequencies between patients with and without proliferative retinopathy suggest a genetic contribution to diabetic retinopathy.

Rapid identification of the Candida species from direct blood cultures by CHROMagar Candida.

We evaluated the ability of CHROMagar Candida to identify Candida species isolated directly from blood cultures. A total of 50 clinical isolates of Candida were incubated at 35 degrees C, and once growth was established, an aliquot of each was plated onto CHROMagar Candida medium. A control specimen was plated directly from Sabouraud's dextrose agar. Following incubation at 30 degrees C, all yeast isolates were identified by colony morphology and colour. We were able to identify all isolates of C. albicans (n = 20), C. tropicalis (n = 14), C. glabrata (n = 6), and C. krusei (n = 5), which were isolated from blood or from control cultures. This study demonstrated that CHROMagar Candida reliably isolated and identified yeast taken directly from blood cultures. We conclude that this rapid and easy method of identifying Candida species will enable clinicians to quickly choose the appropriate antifungal agent. This should decrease patient morbidity and mortality.

HLA tissue types in nonresponders to hepatitis B vaccine.

Genetic factors are implicated in the response of normal subjects to hepatitis B vaccine. In order to investigate the immunogenetic factors associated with nonresponsiveness to hepatitis B vaccine, 93 health care workers were vaccinated with hepatitis B vaccine. Initial nonresponders (antibody not detected or antibody detected but < 10 mlU/ml) were revaccinated. Only 12 (12.9%) of the 93 health care workers, who had anti-HBs levels of 10 mlU/ml or less after revaccination were defined as absolute nonresponders. HLA typing were performed in these 12 nonresponders, Anti-HBs levels were determined by ELISA method in mlU/ml units. HLA-A,B,C,DR, and DQ typing was performed using the microcytotoxicity test. The HLA-A10 (pc less than 0.01) and CW4 (pc less than 0.006) were decreased whereas DR7 (pc less than 0.09) was increased in nonresponders. Although our initial results suggest the importance of genetic modulation of responsiveness to hepatitis B vaccine, a formal demonstration of the mode of inheritance of unresponsiveness to hepatitis B vaccine and the explanation of the role of genes in this matter will require further studies of families.

[The effect of drugs on the immune response]. / Bazi ilaçlarin immün yanit üzerine etkileri.

In this study the effects of some drugs on the humoral and cellular immune responses were carried out. On the studying of laboratory animals, the humoral response by the antibody formation against S.typhi and the cellular response by PPD tuberculin of vaccinated animals with BCG, were examined. According to the experimental results, it was observed that Trimethoprim/Sulfamethoxazole (Baktrim) and Chloramphenicol did not effect humoral response but inhibited cellular response. It was determined that Rifampin had any effect on the humoral and cellular responses. On the other hand it was observed that Cimetidine inhibited the humoral response and increased the cellular response.

[Non-A, non-B/C hepatitis]. / Non-A, non-B/C hepatiti.

A recombinant immunoscreening approach was used to clone and identified the genome of the non-A, non-B hepatitis virus. This agent is a positive-stranded RNA virus that appears to be distantly related to the flaviviridae family. A recombinant viral antigen (C100-3) was used to develop a "capture" assay for circulating antibody. Data obtained using this assay indicate that this agent, termed the Hepatitis C virus (HCV), is the major cause of post-transfusion, community-acquired and cryptogenic, NANB. HCV is associated with the development of hepatocellular carcinoma and possibly, other liver diseases. In this article the characteristic of Hepatitis C virus were discussed.

[Antibiotic susceptibility of gram negative bacteria isolated from urinary tract infections]. / Idrar yolu enfeksiyonlarindan izole edilen gram negatif bakterilerin çesitli antibiyotiklere duyarliliklari.

In this study antibiotic susceptibilities of 334 gram negative bacteria to aminoglycosides, penicillins, cephalosporins, trimethoprim-sulfamethoxazole, and tetracyclines were evaluated by using Sceptor (BELR) microdilution system. These bacteria were isolated from urine samples at the microbiology laboratory. The sensitivity pattern of these gram negative bacteria have been found 70%, 26%, 41% and 25% respectively.

[Comparison of the IFA, ELISA IgG, ELISA IgM, IHA and the direct agglutination tests in Toxoplasma gondii serology]. / Toxoplasma gondii serolojisinde IFA, ELISA IgG, ELISA IgM IHA ve direkt aglütinasyon testlerinin karsilastirilmasi.

In this study, toxoplasma antibodies were evaluated by using IFA, ELISA IgG, ELISA IgM, IHA and direct agglutination test in serum samples obtained from reproductive women. The specificity, sensitivity, unit costing and timing of tests compared with IFA test. In this evaluation IFA test are used. ELISA IgG specificity 98%, sensitivity 97.5%; IHA specificity 90%, sensitivity 97.5%; direct agglutination specificity 92%, sensitivity 95% have been found.

[Gram-negative bacteria isolated from wound infections and their susceptibility to various antibiotics]. / Yara enfeksiyonlarinda izole edilen gram negatif bakteriler ve çesitli antibiyotiklere duyarliliklari.

In this study gram negative pathogens isolated from wound infections and antibiotic susceptibilities of these bacteria were evaluated by using Sceptor (BBLR) microdilution system. According to the our results, it was observed that Enterobacter sp. were found to be major causative agent with the ratio of 22.67%. The isolated gram negative bacteria have been found to be highly resistant to the most of the antibiotics except amikacin.

[In vitro activities of aztreonam and sulbactam/ampicillin for gram-negative bacteria]. / Aztreonam ve sulbaktam/ampisilin'in gram negatif bakterilere in-vitro aktiviteleri.

In this study antibiotic susceptibilities of gram negative bacilli to Aztreonam and Sulbactam/Ampicillin were evaluated by using microdilution technique. Aztreonam inhibited 42% of the strain and Sulbactam/Ampicillin compound to 29% of them.

[Detection of gram-positive bacteria isolated from wound infections and their susceptibility to various antibiotics]. / Yara enfeksiyonlarindan izole edilen gram pozitif bakteriler ve çesitli antibiyotiklere duyarliliklari.

In this study gram positive pathogens isolated from wound infections and antibiotic susceptibilities of these bacteria were evaluated by using sceptor (BBL) microdilution system. According to our experimental results, it was observed that the staphylococci found to be major causative agent with the ratio of 72%. The sensitivity of gram positive bacteria have been found aminoglycosides 88%, trimethoprim/sulphamethoxazole 67%. The sensitivity of these bacteria to the other antibiotics have been found 13-59%.

[Hepatitis b seroprevalence in hospital personnel]. / Hastane personelinde hepatit B seroprevalansi.

To investigate the epidemiology of viral hepatitis B (VHB) infection in Ondokuz Mayis University Hospital staff sera of 243 workers and 100 blood donors examined for HBsAg and Anti-HBs with ELISA. The prevalences of HBsAg and Anti-HBs among the workers were 8.6% and 33.7% and among the blood donors were 5 and 24%, respectively. The overall prevalence of donors was 29%. The risk of infection showed a correlation with age and working period but there was no difference according to the jobs and departments. According to these results hospital staff have a great risk for VHB and should be vaccinated for protection from the infection.

[Importance of the enzyme-linked immunosorbent assay (ELISA) for the serologic diagnosis of tuberculosis]. / Tüberkülozun serolojik tanisinda enzyme-linked immunosorbent assay (ELISA) testinin önemi.

Tuberculosis is still a major granulomatosis disease in developing countries. The diagnosis of tuberculosis infection frequently creates problems which is related to clinical, radiological and bacteriological investigations. Recently, immunoenzymatic assays provide hopeful results for the serodiagnosis of tuberculosis. In this study, ELISA was evaluated as a serodiagnostic test for tuberculosis. Sera were obtained from 40 pulmonary tuberculosis patients and 15 healthy volunteer donors. The results showed that ELISA could be used in the serologic diagnosis of pulmonary tuberculosis.

Human leucocyte antigens and cytokine gene polymorphisms and tuberculosis.

PURPOSE: Several genes encoding different cytokines and human leucocyte antigens (HLA) may play crucial roles in host susceptibility to tuberculosis (TB). Our objective was to investigate whether these genes might be associated with protection from or susceptibility to TB. MATERIALS AND METHODS: Genomic DNA from patients with TB (n = 30) and ethnically matched controls (n = 30) was genotyped by using sequence-specific primers-polymerase chain reaction and sequence-specific oligonucletid methods. RESULTS: Our results demonstrated that HLA-CwFNx0101 [P = 0.05, odds ration (OR) (95% confidence interval) = 2.269 (1.702-3.027)] allele frequency was significantly more common in TB patients than in healthy controls, and HLA-CwFNx0101 may be associated with susceptibility to TB. Analysis of cytokine allele frequencies showed that interleukin (IL)-10, -819 C and -592 C alleles was significantly more common in TB patients than in controls (pc: 0.038 and 0.017, respectively). From the IL-10 cluster, a positive significant difference was found at positions -1082 and -592 C/C (pc: 0.027 and 0.054, respectively) genotypes. Although these differences could be explained by the highest frequency of C/C and G/G homozygous patients with TB, in contrast to the control group, statistically significant differences for the C/C genotype however were lost after Bonferroni correction of the P-values. CONCLUSION: Altogether, our results suggest that the polymorphisms in HLA (class I) and cytokine (IL-10) genes may affect the susceptibility to TB and increase the risk of developing the disease.