[Comparison of the Results of Galactomannan and Beta Glucan Antigen Tests in the Suspicion of Invasive Pulmonary Aspergillosis]. / Invaziv Pulmoner Aspergilloz Süpheli Hastalarda Galaktomannan ve Beta Glukan Antijen Testlerinin Karsilastirilmasi.

Opportunistic fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Invasive aspergillosis (IA) has an important place among these infections with ~ 250.000 cases annually. Reducing the mortality rate due to invasive aspergillosis is possible with early diagnosis and treatment of the disease. Because of the low sensitivity in microscopic examination, the time consuming of culture growth, and the difficulties in distinguishing colonization/infection, serological methods are frequently used in the diagnosis of invasive aspergillosis. The aim of this study was to determine the diagnostic performance of galactomannan and beta glucan tests for the diagnosis of invasive pulmonary aspergillosis (IPA). Sixty patients, followed up with the suspicion of invasive pulmonary aspergillosis in Gazi University Hospital were included in the study. The clinical classification of the patients was made according to the revised European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) criteria. A total of 10 patients were classified as probable invasive aspergillosis and 20 patients were classified as possible invasive fungal disease. Demographic data of the patients and various risk factors were recorded. One hundred and thirty serum and nine bronchoalveolar lavage (BAL) fluid samples were studied with Plateliaᵀᴹ Aspergillus Ag (Bio-Rad, France), Dynamiker Aspergillus Galactomannan and Dynamiker Fungus (1-3)-beta-D-Glucan (Dynamiker, China) kits. Sensitivity and specificity values were calculated according to U.S. Food and Drug Administration (FDA) approved Plateliaᵀᴹ Aspergillus Ag test. According to this study, the most important risk factors in the development of IPA were the use of steroids and immunomodulatory drugs. The sensitivity of the galactomannan test in the probable group was 77.8%, the specificity was 96.7%, the sensitivity of the beta glucan test was 61.1%, and the specificity was 92.6%. When these two tests were evaluated together, it was observed that the sensitivity in the probable group increased to 83.3% and the specificity decreased to 89.3%. The combined use of galactomannan and beta glucan tests increases the diagnostic sensitivity. Although the presence of prolonged neutropenia is an important risk factor for IA, the use of steroids and immunomodulatory drugs should be kept in mind in non-neutropenic patients.

In vitro activity of eravacycline in combination with colistin against carbapenem-resistant A. baumannii isolates.

The synergistic activity of eravacycline in combination with colistin on carbapenem-resistant A. baumannii (CRAB) isolates was evaluated in this study. Minimum inhibitory concentrations (MICs) of eravacycline and colistin were determined by the broth microdilution method. MICs values ranged between 1 to 4 mg and 0.5 to 256 mg l-1 for eravacycline and colistin, respectively. In vitro synergy between eravacycline and colistin was evaluated by using the chequerboard methodology. Synergistic activity was found in 10% of the strains, and additive effect in 30%. No antagonism was detected. Similar activity was also observed in colistin-resistant CRAB isolates. The result of this study indicates that eravacycline and colistin combination may be a potential therapeutic option for the treatment of CRAB related infections.

[Characterization of Klebsiella pneumoniae strains exposed to subinhibitory concentrations of chlorhexidine]. / Düsük düzeyde Klorhekzidin ile karsilastirilmis Klebsiella pneumoniae izolatlarinin karakterizasyonu.

Chlorhexidine, a topical antiseptic, acts as a cationic biguanide altering the osmotic transport of the bacterial cell wall that has been used throughout the world to prevent healthcare-associated infections for decades. The routine application of chlorhexidine can result in decreased susceptibility of bacteria over time. The aim of this study was to develop Klebsiella pneumoniae strains after exposure to chlorhexidine and characterize these adapted strains in terms of their virulence ability both by in vivo and in vitro methods. Two clinical strains of K.pneumoniae were included in the study. One strain was completely susceptible and the other was resistant to certain antibiotics. Susceptible strain was subjected in the exposure assay as parent/wild strain. Exposure was performed by increasing chlorhexidine concentrations in agar plates. Chlorhexidine concentrations were gradually decreased reaching a final concentration of 0.12 mg/L after five weeks. Chlorhexidine-adapted viable colonies were selected and isolated. Minimal inhibitor concentrations of chlorhexidine, sodium hypochloride, benzalkonium chloride and triclosan for K.pneumoniae strains were determined using broth microdilution method. Reverse transcription-polymerase chain reaction analysis were performed for efflux pumps named cepA, kdeA and acrKp expressions. Fluorimetric efflux assay by using Rhodamine 6G was performed. Galleria mellonella killing assay and in vitro virulence determinants such as esculin hydrolysis, biofilm production, lecithinase, DNase activity, hemolytic activity, lipase production, mucoviscocity, casein hydrolysis and complement-mediated serum killing were evaluated. K.pneumoniae strains exposed to chlorhexidine did not show any antibiotic resistance. MICs for chlorhexidine, sodium hypochloride, and benzalkonium chloride were increased in the adapted strain. Efflux pumps of cepA and kdeA were over-expressed in the chlorhexidine adapted strain. Rhodamine 6G assay showed an increased efflux in the adapted strain. G.mellonella killing assay showed median virulence score. All strains, were esculin positive, while biofilm production, lecithinase, DNase, hemolytic activity, lipase production, mucoviscocity, casein hydrolysis were all negative. The susceptible parent/wild strain was susceptible to the complement-mediated serum killing, while the chlorhexidine adapted strain showed intermediate susceptibility. Chlorhexidine adapted strains of K.pneumoniae showed increased efflux pump expression, enhanced G.mellonella killing and raised resistance to serum killing. No difference was determined for other determinants. Minimal correlation was found between chlorhexidine resistance and virulence in K.pneumoniae.

[Detection of virulence factors and antifungal susceptibilities of Fusarium strains isolated from keratitis cases]. / Keratit olgularindan elde edilen Fusarium izolatlarinin virülans faktörlerinin ve antifungal duyarliliklarinin belirlenmesi.

Fusarium species have gained importance as a cause of keratitis. The pathogenicity and virulence factors of genus Fusarium remain largely unknown. Several putative virulence factors have been reported for fungal pathogens, including biofilm formation, production of proteinases and other hydrolytic enzymes. It has been emphasized that Fusarium species are generally resistant to antifungals but the resistance may vary depending on the species and even according to the isolate. For this reason, pathogenic features and antifungal susceptibility of the clinical isolates gained importance for the management of keratitis cases. The aim of this study was to identify clinical Fusarium isolates, to evaluate their virulence factors and to show antifungal susceptibility patterns. The identification of Fusarium was made on genus level isolated from 25 keratitis cases. Among them, 13 of the isolates were identified by ITS sequencing on species complex level. The production of hemolytic activity, caseinase, esterase, proteinase and phospholipase activity were investigated in 13 of the isolates. Biofilm production was searched among all 25 isolates. Galleria mellonella larvae was used as in vivo infection model. Antifungal susceptibility for amphotericin B, itraconazole, voriconazole and posaconazole was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 microdilution assay guidelines. As the subcommittee on antifungal susceptibility tests did not determine the clinical resistance breakpoints (CBP) specific to Fusarium species complex, the epidemiological cut off values (ECV) were used for the interpretation of the minimum inhibitory concentration (MIC) values of the antifungal drugs. Isolates were identified as six F.oxysporum, six F.solani species complex and one F.brachygibbosum. One F.solani, one F.oxysporum were positive for hemolytic activity; all isolates were caseinase positive; three F.oxysporum and two F.solani isolate were esterase positive; one F.solani isolate was proteinase positive; five F.oxysporum and two F.solani isolates were phospholipase positive; biofilm activity was positive in 52% of the 25 isolates. The larvae survived for seven days after Fusarium inoculation in the G.mellonella larvae model. MIC range was 0.5-8 µg/ml for amphotericin B, 2-32 µg/ml for itraconazole, 0.5-8 µg/ml for voriconazole, 0.5-16 µg/ml for posaconazole and according to the ECV values F.solani and F.oxysporum isolates were determined as wild type for four antifungal agents. As a result, it was shown that Fusarium isolates have some virulence factors, there was a concordance between in vitro virulence properties and in vivo virulence characteristics and some of the isolates were classified as antifungal susceptible wild type isolates.

[Emerging pathogen: Candida kefyr (Kluvyeromyces marxianus)]. / Önem kazanan patojen: Candida kefyr (Kluvyeromyces marxianus).

In the central microbiology laboratory of Gazi University Hospital Candida kefyr was isolated from different clinical samples as 5.3% in 2016 and in 2017 this rate increased to 9.3% which was nearly two-fold and this has drawn our attention. The aim of this study was to evaluate the special characteristics, antifungal susceptibility and virulence properties of C.keyfr species. Germ tube, corn meal-tween 80 agar morphology and carbohydrate assimilation profiles on ID32C yeast identification system were used for the diagnosis of Candida species. In this study, DNA sequencing was performed using ITS1 and ITS4 primers amplifying fungal gene between 5.8S and 18S regions of rRNA. Antifungal susceptibility was performed using M27A microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI). Minimum inhibitory concentration (MIC) values for amphotericin B, fluconazole, voriconazole and itraconazole were determined. MIC distribution, MIC50 and MIC90 values and geometric mean (GM) were detected. The existence of virulence factors caseinase, secreted aspartyl proteinase, esterase and phospholipase were investigated in vitro. A total of 865 Candida species were isolated from different clinical samples in the central microbiology laboratory of Gazi University Hospital in 2016. Among them, 46 (5.3%) were C.kefyr. In the first four months of 2017, 30 (9.3%) C.kefyr were identified among 320 Candida isolates. Ten isolates which have shown atypical morphology on corn meal agar were selected. Among these 10 isolates, nine of them were identified as C.kefyr by using ID32C system and DNA sequencing method. Amphotericin B MIC value was 2 µg/ml for one isolate, and fluconazole MIC value was 8 µg/ml for another isolate among 46 isolates. Among the 30 isolates of the year 2017, one of them presented MIC value for fluconazole as 8 µg/ml. No marked antifungal resistance was detected in our isolate group. Caseinase was positive in one C.kefyr isolate, and phospholipase were positive in eight of nine isolates. As a result, the reason of increase in the incidence of this Candida species, which does not show significant resistance and presents mostly phospholipase activity as a virulence factor, should be investigated in more detail.