Investigation of mutations in Erg11 gene of fluconazole resistant Candida albicans isolates from Turkish hospitals.

Widespread use of fluconazole has resulted in resistance in strains of Candida. The aim of our study was to investigate Y132H and other mutations in the ERG11 gene in conferring fluconazole resistance to C. albicans isolates. Seven fluconazole-resistant (R)/susceptible dose-dependent (SDD)/trailing and 10 fluconazole-susceptible (S) isolates were included. Restriction enzyme analysis was performed on all isolates for Y132H mutation and sequence analysis was performed for other mutations in the ERG11 gene. None of our strains had Y132H mutation. One single mutation (D153E, E266D, D116E, V437I) was detected in isolates 348, 533, 644, 1453, 2157, while the others had more than one nucleotide change. D116E and E266D, which were two mutations found in fluconazole R/SDD/trailing isolates with the highest frequency, were also detected in azole S strains. K143R, G464S, G465S and V488I mutations were determined in three of the R/SDD isolates. S412T and R469K mutations were detected only in this group of strains by sequence analysis. Mutations such as K143R, G464S, G465S, V488I, S412T and R469K in the ERG11 gene were determined to be effective mechanisms in our fluconazole R/SDD C. albicans isolates. Other mechanisms of resistance, such as overexpression of ERG11 and efflux pumps and mutations in the ERG3 gene should also be investigated.

Molecular identification, genotyping, and drug susceptibility of the basidiomycetous yeast pathogen Trichosporon isolated from Turkish patients.

Deep-seated infections due to Trichosporon species are emerging mycoses that have a very poor prognosis in patients with persistent neutropenia. This study elucidated the mycological characteristics of Trichosporon strains obtained from deep-seated infections in Turkish patients and identified by DNA sequence analysis of intergenic spacer (IGS) region 1 of the rDNA locus. In addition, we genotyped the major causative agent, T. asahii, and evaluated the in vitro drug susceptibility of the isolates. While 87 (81.3%) of the 107 isolates were T. asahii, the remaining 20 were T. faecale (14.0%), T. asteroids (0.9%), T. coremiiforme (0.9%), T. japonicum, (0.9%), T. lactis (0.9%), and a new species (0.9%). In addition to the eight known T. asahii genotypes, one novel genotype was identified. The distribution of the T. asahii genotypes in this study were genotype 1 (79.3%), followed by 5 (8.0%), 3 (6.9%), 6 (3.4%), 4 (1.1%), and 9 (1.1%). Turkish isolates showed low susceptibility to amphotericin B, 5-flucytosine, and fluconazole. Although relatively low minimum inhibitory concentrations (MICs) were found with all drugs, voriconazole appeared to be the most active. The MICs of the non-Trichosporon asahiiTrichosporon species were similar to those of the T. asahii strains. Our findings suggest that Trichosporon species isolated from Turkish patients are more diverse than those reported from other countries.

A case of Acremonium strictum peritonitis.

During the past two decades opportunistic fungal infections have emerged as important causes of morbidity and mortality in patients with severe underlying illnesses. A few cases of Acremonium spp. infections have been described in immunocompromised patients, but they have on occasion been reported as the cause invasive disease in immunocompetent individuals. Peritonitis is a common clinical problem that occurs in patients with end-stage renal disease treated by continuous ambulatory peritoneal dialysis (CAPD). Yeasts, or rarely molds, may also cause peritonitis in patients on CAPD and we present here a case caused by Acremonium strictum.

Molecular epidemiology of Candida species isolated from clinical specimens of intensive care unit patients.

Epidemiological analysis of nosocomial Candida infections has gained importance due to an increase in these infections during the recent years. This study investigated the prevalence of clinical infections of Candida in anesthesiology intensive care unit patients, and ascertains the level of genetic diversity in the Candida species. A total of 70 Candida isolates, consisting of 42 Candida albicans, 16 Candida glabrata and 12 Candida tropicalis strains isolated from various clinical sites of infection of anesthesiology intensive care unit patients, were analysed. The susceptibility of the isolates against amphotericin B and fluconazole was determined by microdilution method according to Clinical and Laboratory Standards Institute M27-A2 standards. The strains were typed by random amplified polymorphic DNA (RAPD)-PCR using OPE-03, OPE-18, RP4-2 and AP50-1 primers. In the patients with Candida infections, most isolates exhibited different RAPD patterns. Only three C. albicans pairs isolated within a short time period had the same RAPD pattern. Most of the Candida infections in the anesthesiology intensive care unit of our hospital seem to be caused by endogenous strains. Exogenous spread of C. albicans infections occurred less frequently.

[Detection of Candida DNA in simulated blood samples by polymerase chain reaction]. / Simüle kan örneklerinde polimeraz zincir reaksiyonu ile Candida DNA'sinin saptanmasi.

Although blood culture method is accepted as gold standard in the laboratory diagnosis of invasive candidiasis seen in immunocompromised patients, cultivation of blood is considered as not a reliable and rapid method for the diagnosis of candidemia, since it may be negative in approximately half of the patients, slow growth rate of Candida in routine culture media and requirement of large amounts of blood for the isolation. The aim of this study was to detect Candida DNA in simulated blood samples by using polymerase chain reaction (PCR). Simulated samples were prepared by using blood samples of healthy volunteers. These samples were inoculated into tubes with EDTA and BACTEC 9240 blood culture bottles in which no growth was detected and with standard strains of C. albicans, C. tropicalis, C. parapsilosis, C. krusei, Escherichia coli and Staphylococcus aureus together with the clinical isolates of Aspergillus fumigatus, C. kefyr, C. glabrata, C. lusitaniae, C. guilliermondii and Rhodotorula sp. Additionally, blood culture samples of 23 cases whose blood culture bottles signaled as positive and revealed growth of Candida in agar plates were examined. DNA extraction of all samples were performed according to the standard procedure proposed by the MN Nucleospin Tissue Kit (Macherey-Nagel, Germany) for tissue samples; following the pre-treatment with erythrocyte, leukocyte and fungus cell wall lysis buffers. DNAs were amplified with PCR, using primers specific for the 5S rDNA region (PCon 1 and PCon 2 primers) and PCR products were obtained by electrophoresis in 2% agarose gel. Presence of a 105 base pair (bp) product was considered as positive. The lowest detection limit of PCR has been determined as 10(2)-10(3) cfu/ml Candida for our simulated samples. The presence of a 105 bp band has been observed in samples prepared with all Candida strains included in the study. Blood samples spiked with E. coli, S. aureus, A. fumigatus and Rhodotorula sp. and negative blood samples has been found negative in terms of Candida DNA. The same 105 bp product has been observed for blood culture samples with Candida growth. The PCR method applied in this study takes approximately seven hours and the cost was calculated approximately 6.00 U.S. dollars per patient. As a result, it has been determined that Candida DNA can be detected in a shorter time by PCR using specific primers for 5S rDNA gene from simulated samples prepared with either blood or BACTEC blood culture bottles.

[Comparison of minimum inhibitory and minimum effective concentration values for the detection of in vitro susceptibilities of Aspergillus species against caspofungin]. / Aspergillus türlerinin kaspofungine karsi in vitro duyarliliginin saptanmasinda minimum inhibitör ve minimum etkili konsantrasyon degerlerinin karsilastirilmasi.

Caspofungin is a promising echinocandin-group antifungal agent used especially in the treatment of resistant invasive aspergillosis. The guidelines for in vitro susceptibility testing of Aspergillus species against caspofungin are not described by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentration that showed a prominent reduction of growth (MIC-2) and minimum effective concentration (MEC) endpoints are frequently used for the susceptibility testing of caspofungin as MIC determination criteria. The aim of this study was to evaluate the in vitro activity of caspofungin against Aspergillus species and to compare MIC-2 and MEC endpoints in the determination of MICs. A total of 32 Aspergillus species (18 A. fumigatus, seven A. flavus, five A. niger, and two A. versicolor) isolated from different clinical samples were included to the study. In vitro susceptibilities of the strains against 0.03-16 microg/ml caspofungin concentrations were searched by broth microdilution method as recommended by CLSI M-38A document, with the use of glucose supplemented 2% RPMI 1640 media. The MIC-2 and MEC endpoints were determined both at 24 and 48 hours. The concordance between MIC-2 and MEC endpoints of the strains at 24 and 48 hours incubations was found as 53% and 100%, respectively, with the difference of +/- 1 dilution. MIC-2 and MEC measurements showed the same values at the end of 48 hours, whereas 7% showed differences in +/- 1 dilution. MEC endpoints were also found to be more stable than MIC-2 in both of the incubation periods. In conclusion, MEC value is a more objective and stable endpoint and easier to use than MIC-2 for testing in vitro caspofungin activity against Aspergillus species.

The role of calcium utilization of intestinal flora in urinary calcium excretion.

OBJECTIVE: We aimed to evaluate whether Ca(+2) utilization of intestinal flora (IF) has an effect on urinary excretion of Ca(+2) (UCaE) levels. MATERIALS AND METHODS: Fecal samples (0.1 g/mL) of children who underwent UCaE examination in the past year were implanted in broths. Labeled (45)Ca (5 muL) was added to the samples and incubated. From these samples, a 200-muL quantity was filtrated with a 0.45-micrometer membrane and was rinsed in 200 muL pure water. (45)Ca activity in the membrane was measured and defined as percent activity per bacteria ((45)Ca(act) %/CFU). Levels of aerobic and anaerobic (45)Ca(act) %/CFU and their correlations with UCaE were compared between hypercalciuric (Group I) and normocalciuric (Group II) patients. RESULTS: Levels of (45)Ca %/CFU were similar between groups (P > .05). Aerobic and anaerobic (45)Ca(act)%/CFU levels were not significantly correlated to UCaE, either in normocalciuric (P = .079, r = -0.503; P = .260, r = -0.420, stray mart respectively) or in hypercalciuric children (P = .509, r = 0.223; P = .623, r = -0.257, respectively). CONCLUSION: Similar (45)Ca(act)%/CFU levels in the 2 groups imply that calcium utilization of IF does not have a distinct effect on UCaE.

[Investigation of Aspergillus galactomannan levels in antimicrobial agents]. / Antimikrobiyal ilaçlarda Aspergillus galaktomannan düzeylerinin arastirilmasi.

The diagnosis of invasive aspergillosis which is a serious infection of immunocompromized patients, depends on the detection of Aspergillus galactomannan antigen in the serum by enzyme immunoassay (EIA) in routine laboratories. However, it has been previously reported that false positive results in Aspergillus galactomannan test may be obtained in the sera of patients sera receiving piperacillin-tazobactam (PIP-TAZ). The aim of this study was to investigate the presence and levels of Aspergillus galactomannan antigen in the content of PIP-TAZ and some other antimicrobial agents that are often used for the treatment of infections in immunocompromised patients. The level of galactomannan antigen was determined for PIP-TAZ, ampicillin-sulbactam, ampicillin, penicillin G, ceftriaxone, cefepime, imipenem, clarithromycin, ciprofloxacin, vancomycin, gentamicin, trimethoprim-sulfamethoxazole, ornidazole, fluconazole and amphotericin B, by a commercial EIA (Platelia Aspergillus EIA, Bio-Rad, France) kit. Galactomannan index (GI) was estimated with the ratio of absorbance values of antimicrobials to cut-off value and evaluated as positive when GI was found >0.5. Amongst the 15 antibiotics studied, the only positive result was detected for ampicillin with the highest index value (GI = 0.540), followed by PIP-TAZ with a relatively high value (GI = 0.235) even though it was not in the range of positivity. GI values have ranged from 0.011 to 0.188 for the other antibiotics. In conclusion, the use of especially ampicillin (and probably PIP-TAZ) therapy should be questioned in patients whose sera are being tested for Aspergillus galactomannan antigen by EIA in order to evaluate the positive results in terms of false positivities due to cross reactivity.

Fungi and indoor conditions in asthma patients.

This study was carried out with 127 asthmatic patients and 127 controls, which aimed to compare and evaluate the environmental conditions in the homes of asthmatic patients and the control group. Air samples were obtained by using an air sampler and the mean mould colony counts were established. Aspergillus and Penicillium were the most common isolated species. No significant difference was observed with regard to various house conditions and the mean mould colony counts between the houses of patients and controls. The mould colony counts were found to be lower in houses with wooden parquet flooring. The odds ratio for stone floors vs. wood floors was 2.3 (95% CI 1.08-4.98) for mould growth.

[Microbiological results of bronchoalveolar lavage that was performed for opportunistic pulmonary infections]. / Firsatçi pulmoner infeksiyon ön tanisiyla yapilan bronkoalveoler lavajin mikrobiyolojik sonuçlari.

Between 2001-2002; in 62 cases, 33 (53%) male, 29 (47%) female, mean age 51.4 +/- 18.1 years) bronchoalveolar lavage (BAL) was performed for diagnosis of opportunistic pulmonary infection and specimens were evaluated for results of microbiological examinations. There was hematological malignancy in 18 (29%) and solid organ malignancy in 13 (21%) cases. Thirty-one (50%) cases were immunocompromised for reasons other than malignancy. By endoscopic evaluation endobronchial lesion was seen in 2 (3%) cases, indirect tumor signs were seen in 2 (3%) cases and signs of infection were seen in 11 (18%) cases. Forty-even (76%) cases were endoscopically normal. Acid-fast bacilli (AFB) direct examination was positive in 3 (5%) cases. In 4 (6%) cases mycobacterial culture was positive, Mycobacterium tuberculosis-polymerase chain reaction (PCR) was also positive in these four cases. Examination of gram-stained smears for bacteria was associated with infection in 14 (23%) cases. Bacteriologic cultures were positive for single potential pathogen in 10 (16%) cases, and for mixed pathogens in 7 (11%) cases for a total number of 17 (27%). Fungal cultures were positive in 3 (5%) cases all of which had hematological malignancy. As a result in 24 (39%) cases microbiological agent of infection is determined: in four mycobacteria, in 17 bacteria other than mycobacteria and in three fungi.

Nosocomial bloodstream infections associated with Candida species in a Turkish University Hospital.

In recent years, a progressive increase in the frequency of nosocomial candidaemia has been observed, especially among the critically ill or immunocompromised patients. The aim of this study was to evaluate the trend in incidence of candidaemia together with potential risk factors in an 850-bed Turkish Tertiary Care Hospital in a 4-year period. A total of 104 candidaemia episodes were identified in 104 patients. The overall incidence was 0.56 per 1000 hospital admissions and the increase in incidence of candidaemia from 2000 to 2003 was found to be statistically significant (P = 0.010). Candida albicans was the most common species (57.7%) and non-albicans species accounted for 42.3% of all episodes. The most common non-albicans Candida sp. isolated was C. tropicalis (20.2%) followed by C. parapsilosis (12.5%). The most frequent risk factors possibly associated with the candidaemia were previous antibiotic treatment (76.9%), presence of central venous catheter (71.2%) and total parenteral nutrition (55.8%). Our results show the fact that the incidence of candidaemia caused by non-albicans species is frequent and increasing significantly, although the most common isolated Candida species were C. albicans and further investigations are necessary to evaluate the mechanisms of increasing incidence of candidaemia caused by non-albicans species.

[Evaluation of species distribution of yeasts isolated from intensive care units during the four years period]. / Yogun bakim unitelerinden dört yillik dönemde izole edilen mayalarin tür dagiliminin degerlendirilmesi.

The aim of this study was to evaluate the distributions of yeast species according to the years and to detect the emerging pathogens in intensive care units (ICU). For this purpose, yeast isolation rates were detected retrospectively, in the following time periods: Period I: April-December 2001; period II: January-December 2002; period III: January-December 2003; period IV: January-December 2004. A total of 490 yeast isolates recovered from 462 clinical specimens obtained from 360 different ICU patients were investigated during these periods. Urine (62.1%), blood (13.6%) and tracheal aspirate (8.7%) samples were detected as the most common specimens. Of these isolates, 53.3% were identified as Candida albicans, 14.5% as C. tropicalis, 12.2% as C. glabrata, 6.5% as C. parapsilosis, 4.5% as Trichosporon spp., 3.9% as C. kefyr, 1.6% as C. krusei, 1.4% as Geotrichum candidum and 2.1% as other Candida species. The isolation rates of C. albicans in the periods of I to IV were found as 47.7%, 55.5%, 41.7% and 62.4%, respectively. The decrease between the second and third periods, and increase between third and fourth periods were statistically significant (chi2 = 4.15, p = 0.04 and chi2 = 8.32, p = 0.004). C. glabrata was the second most common species in the first and second periods (14.8% and 15.5%, respectively), followed by C. tropicalis (12.5% and 10.0%, respectively), however this array has changed in the third and fourth periods (C. tropicalis was the second with the rates of 16.7% and 16.8%, while C. glabrata placed in the third line with the rates of 14.8% and 7.6%, respectively). It was concluded that C. albicans has still been the most frequent species among yeast isolates of ICU's in our hospital; however, the incidence of non-albicans species like C. glabrata and C. tropicalis has increased.

The effect of bee propolis on oral pathogens and human gingival fibroblasts.

Propolis is one of the few natural remedies that have maintained its popularity over a long period of time. The aim of this study is to investigate the antimicrobial properties of six propolis solutions and evaluate their cytotoxicity on gingival fibroblasts at different dilutions. Two different solutions of powder propolis (Sigma) and Turkish propolis were prepared and propylene glycol (PG) and alcohol were used as solvents for each propolis sample. In addition to the four propolis solutions, two other propolis samples of far geographic regions (USA and Australia) were included in the study. The antibacterial effects of six solutions on oral pathogen microorganisms were tested and their cytotoxic effects on human gingival fibroblasts were evaluated by MTT assay. The effective dilutions of the six propolis samples on periodontopathogen microorganisms were found to be cytotoxic to gingival fibroblasts. All solutions had strong antifungal activity and the effective dilutions were safe for gingival fibroblasts. Propolis could have a promising role in the future medicine, if appropriate solutions can be prepared being strongly antibacterial and non-cytotoxic as well.

Microbial growth in various perfluorocarbon liquids.

A prospective in vitro study was conducted to investigate the potential for various perfluorocarbon liquids to support the growth of microbes, which may be introduced into these liquids as contaminants during intraocular surgery. Perfluorodecaline, perfluoro-noctane, and perfluorophenanthrene were tested for the growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans by using tryptone soy broth, pancreatic digest of casein, and Sabouraud broth as culture media for up to 10 days. No microbial growth was observed in any of these perfluorocarbon liquids. Perfluorocarbon liquids do not promote microbial growth. Thus, they do not increase the risk of endophthalmitis in vitreoretinal surgery.

[Case report: a Fusarium fungaemia]. / Olgu raporu: bir Fusarium fungemisi.

In this report, a case of Fusarium fungaemia developed in an acute lymphoblastic leukemia (ALL) patient was presented. A seven year old girl who had weakness, loss of appetite, paleness and ecchymosis on legs applied to Pediatric Hematology Service and cytotoxic chemotherapy was started after she had been diagnosed as ALL-L1. Her chemotherapy was stopped because of increase in fever, leukopenia and neutropenia. Central venous catheter and peripheral blood cultures were obtained. Fusarium thapsinum was recovered from blood cultures, obtained in two consecutive days. Thereupon central venous catheter of the patient was removed and intravenous amphotericin B was added to the therapy. On the fifth day of febrile neutropenia attack, her fever was decreased after the onset of antifungal therapy. Radiological examinations were normal and no fungal growth was observed in the later blood cultures. On the 21st day of amphotericin B therapy, chemotherapy was started again and amphotericin B was changed to peroral itraconazole (200 mg/day) at the fifth week. The patient whose itraconazole therapy was stopped after three months, was still in remission and continued receiving her prolonged therapy. In conclusion, Fusarium infections which manifest with fungaemia and fever as the only symptoms, should be taken into consideration in neutropenic patients receiving immunosuppressive therapy.

[Biofilm production and antifungal susceptibility patterns of Candida species]. / Candida türlerinin biyofilm üretimi ve antifungal duyarlilik paternleri.

In this study, biofilm production and antifungal susceptibility of various Candida species were examined and compared. A total number of 156 Candida species (94 C. albicans, 21 C. tropicalis, 18 C. glabrata, 12 C. parapsilosis, 9 C. krusei, 1 C. guilliermondii and 1 C. kefyr) isolated from different clinical specimens were included in the study. The biofilm production of the strains was searched by modified tube adherence and microplate methods. Their antifungal susceptibilities against fluconazole and amphotericin B were determined by microdilution method, according to NCCLS M27-A2 standards. Forty three (27.6%) and 26 (16.7%) of the strains were found to be slime producing by tube adherence and microplate methods, respectively. The agreement between the two methods were detected as 65 percent. The rate of biofilm formation by different species ranged between 17% and 55% by tube adherence test and 0 and 48% by microplate method. No significant difference was found between the biofilm production of C. albicans and non-albicans species by tube adherence test (p=0.29). However; a statistically important difference was found when microplate method was considered (p=0.04). MIC50 and MIC90 values for fluconazole ranged between 4-64 microg/ml and 32-->64 microg/ml for different Candida species while these values changed between 0.25-1 microg/ml and 0.5-2 microg/ml for amphotericin B, respectively. Forty-five (28.8%) and 23 (14.7%) of the isolates were found to be dose dependent susceptible and resistant to fluconazole, respectively. Eleven (6.7%) of the strains had MIC values >1 microg/ml for amphotericin B. When the relation between the biofilm production and the susceptibility categories of the strains for amphotericin B were searched, no statistical differences were found by any of the two methods (p=0.12 and p=0.50). A statistically important difference (p=0.03) was determined by tube adherence test and no important difference (p=0.11) was detected by microplate method when fluconazole susceptibility categories were considered. As a conclusion, it has been determined that biofilm production which is a potential virulence factor for Candida species seems to be in agreement with the antifungal susceptibility categories of the strains especially for amphotericin B when the planktonic cells were used for the susceptibility testing.

Isolated nasopharyngeal aspergillosis caused by A flavus and associated with oxalosis.

We report a case of isolated nasopharyngeal aspergillosis in a 52-year-old woman with Hashimoto's thyroiditis. We found the nasopharyngeal lesion incidentally while evaluating bilateral cervical lymphadenopathy, which we had discovered during a routine follow-up examination pursuant to the patient's thyroid problem. Biopsy analysis of the nasopharyngeal lesion revealed the presence of a mycelium made up of septate hyphae and associated oxalosis. Mycologic examination confirmed that Aspergillus flavus was the responsible pathogen. No systemic involvement or involvement of other head and neck sites was found. The patient had been exposed to a considerable amount of dust during the construction of her house, and this may have been the precipitating factor in the development of her infection. We treated the patient with a 4-week course of itraconazole. At the end of therapy, she exhibited no evidence of A flavus on physical and mycologic examinations.

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species.

BACKGROUND: The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. METHODS: A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37 degrees C. RESULTS: The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. CONCLUSIONS: It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.