Molecular epidemiology, antifungal susceptibility and virulence factors of Candida glabrata complex strains in Kayseri/Turkey.

BACKGROUND: Candida nivariensis and Candida bracarensis are included in Candida glabrata complex, which are usually misidentified as C. glabrata based on phenotypic identification methods. It was aimed to identify C. glabrata complex isolated from various clinical samples in Kayseri/Turkey to the species level and to determine antifungal susceptibilities, virulence factors, and molecular epidemiology. METHODS: Eighty three C. glabrata complex strains were studied in this study. Strains were phenotypically and molecularly identified. Phylogenetic analysis was done by the neighbor-joining method. Proteinase, phospholipase, esterase enzyme activity, and biofilm formation of strains were determined phenotypically. Antifungal susceptibility of strains were determined according to M60-Ed2 recommendations. RESULTS: All the 83 strains identified as C. glabrata complex by phenotypic tests were confirmed as C. glabrata sensu stricto (C. glabrata) by PCR amplification and sequence analysis, but other complex members C. nivariensis and C. bracarensis were not detected. Phylogenetic analysis results revealed 19 different genotypes. No clonal relationship was detected among the strains. Biofilm formation in 75.9% of strains and esterase activity in 7.2% were found positive. Antifungal resistance rates of strains were determined as 9.2% for fluconazole and 45.8% for itraconazole; 43.4% of the strains for voriconazole were determined as non-wild type. CONCLUSION: It was determined that biofilm and esterase activity might play an active role in the virulence of C. glabrata. In addition, high resistance rates to azoles in C. glabrata strains isolated in our hospital at Kayseri/Turkey emphasized the significance of epidemiological studies.

Mucormycosis experience through the eyes of the laboratory.

Background: Mucormycosis is a rare, worldwide fungal infection with high mortality, which mostly affects immunocompromised patients. Compared to large parts of Asia, Europe, and the USA, information on clinical expression and fungal species distribution in mucormycosis in Turkey is limited. Objectives and methods: The main aim of this study was to evaluate the demographic features of mucormycosis cases, identify the isolates at the species level by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), compare culture results with histopathological examination and determine the antifungal susceptibility patterns of the pathogens. Results: Between 2016 and 2018, 10 mucormycosis cases (six female, four male; age range: 35-74 years) were evaluated retrospectively. The predominance of the cases were in late autumn and winter. Diabetes mellitus was the most common underlying condition. Seven patients had rhinoorbitocerebral, two had pulmonary and one had cutaneous mucormycosis. By mycological culture and direct microscopic examination nine strains were identified as Rhizopus spp. and one as Mucor spp. Seven of these strains were identified at the species level by MALDI-TOF. Histopathological examination of eight tissues were reported as compatible with mucormycosis. All isolates were resistant to azoles and echinocandins. Two isolates were resistant to Amphotericin B and one was resistant to posaconazole. Surgical debridement combined with antifungal therapy was the main treatment option. The mortality rate was 40% (n = 4) and the mean number of days between the onset of complaints and the initiation of treatment was 9.25. Conclusions: Early, rapid and accurate diagnosis of mucormycosis is of critical importance in the treatment of immunosuppressed patients.

[Determination of Candida colonization and Candida score in patients in anesthesia intensive care unit]. / Anestezi yogun bakim ünitesinde yatan hastalarda Candida kolonizasyonu ve Candida skorunun belirlenmesi.

The colonization rate of Candida spp. reaches up to 80% in patients who reside in intensive care units (ICUs) more than a week, and the mean rate of development of invasive disease is 10% in colonized patients. Since invasive candidiasis (IC) in ICU patients presents with septic shock and high mortality rate, rapid diagnosis and treatment are crucial. The aim of this study was to assess the relationship between invasive infection and the determination of Candida colonization index (CI) and Candida score (CS) in patients admitted to ICU who are at high risk for IC and likely to benefit from early antifungal therapy. A total of 80 patients (34 female, 46 male; age range: 12-92 years, mean age: 69.57 ± 16.30) who were in ICU over seven days or longer of Anesthesia Department of Kayseri Education and Research Hospital between April, 2014 and July, 2015 were included in the study. None of the patients were neutropenic. After admission, throat, nose, skin (axillary region), urine, rectal swab and blood cultures have been collected weekly beginning from day zero. Isolation and identification of Candida strains were performed by using conventional mycological methods. CI was calculated as the ratio of the number of culture-positive distinct body sites (except blood culture) to the total number of body sites cultured. CI> 0.2 was considered as fungal colonization, while CI≥ 0.5 as intensive colonization. CS value was calculated according to the components including total parenteral nutrition (TPN) (plus 0.908 points), surgery (plus 0.907 points), colonization in multiple areas (plus 1.112) and severe sepsis (plus 2.038 points), and cut-off value for CS was accepted as >2.5. In our study, overall 1009 cultures (mean: 13 cultures per patient) were taken from 80 patients, and yeast growth was detected in 365 (36.2%) of them. Accordingly, among 68 (85%) of 80 patients included, in at least one sample, yeast growth was determined. No yeast growth was observed in the blood cultures. Of 365 yeast-positive cultures, C.albicans was isolated from 184 (50.4%), C.glabrata from 66 (18%), C.parapsilosis from 42 (11.5%), C.tropicalis from 12 (3.3%), C.kefyr from three (0.8%), and C.krusei from one (0.3%) samples, whereas six (1.6%) samples yielded other yeasts (3 Saprochaete capitata, 3 Trichosporon spp.) and 51 (13.9%) samples yielded multiple yeast growth. The highest colonization rates were detected in rectal swabs (27.4%), urine (23.3%) and throat (22.5%) samples. CI value was found as >0.2 in 65% (52/80), and ≥0.5 in 25% (20/80) of the patients, whereas CS value was >2.5 in only 2.5% (2/80) of the patients. In the statistical evaluation, significant correlations were found between fungal colonization (CI> 0.2) and gender (p=0.032) and length of stay in ICU (p=0.004), and between intensive colonization (CI≥ 0.5) and gender (p=0.008) and age (p=0.012). However, the correlation between Candida colonization and the presence of underlying diseases, APACHE II score, Glasgow coma scale, invasive procedures, use of extended-spectrum antibiotics, presence of bacterial infections, haemodialysis, transfusion and history of previous hospitalization was not statistically significant. Our results have also indicated a statistically significant relationship between fungal colonization and the positivity of C.albicans, C.glabrata, C.parapsilosis ang C.albicans/C.glabrata (p=0.001, p=0.002, p=0.008 and p=0.028, respectively), emphasizing the importance of species-level identification of Candida isolates. The reason of lacking of IC development in our patients may be explained by their low CI and CS values. In conclusion, monitoring of ICU patients who are at high risk for IC in terms of CI and CS would be beneficial. However it is clear that our data need to be supported by multi-center and high-scale studies.

Antifungal Activity of Propolis Against Yeasts Isolated From Blood Culture: In Vitro Evaluation.

BACKGROUND: Due to the failure of available antifungal agents in the treatment of candidemia and the toxic activities of these drugs, a lot of researches are being conducted to develop new nontoxic and effective antifungal agents for optimal control of fungal pathogens. The aim of this study is to evaluate the in vitro antifungal activity of propolis against yeasts isolated from the blood cultures of intensive care unit patients. METHODS: Seventy-six strains were included in this study. The in vitro antifungal activity of propolis, fluconazole (FLU), and itraconazole (ITR) was investigated by the microdilution broth methods (CLSI guidelines M27-A3 for yeast). The propolis sample was collected from Kayseri, Turkey. RESULTS: Of the 76 isolates, 33 were identified as Candida albicans while 37 were C. parapsilosis, three were C. tropicalis, and three were identified as C. glabrata. The geometric mean range for MIC (µg/ml) with regard to all isolates was 0.077 to 3 µg/ml for FLU and ITR, and 0.375 to 0.70 µg/ml for propolis. It was shown that propolis had significant antifungal activity against all Candida strains and the MIC range of propolis was determined as 0185 to 3 µg/ml. CONCLUSION: This study demonstrated that propolis had significant antifungal activity against yeasts isolated from blood culture compared with FLU and ITR. The propolis MIC in azole-resistant strains such as C. glabrata was found lower than the FLU MIC.

Investigation of the relationship between virulence factors and genotype of Candida spp. isolated from blood cultures.

INTRODUCTION: The aim of study was to investigate the virulence factors of phospholipase, proteinase, esterase production and biofilm formation in Candida species isolated from patients with candidemia, and to assess their relationship with Candida genotypes derived after repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting. METHODOLOGY: Fifty-two strains were identified to species level according to conventional methods and sequencing. The DiversiLab system was used for the genotyping. Enzyme activities and biofilm formation were evaluated using microbiological methods. RESULTS: The 52 strains were identified as follows: 29 C. parapsilosis, 19 C. albicans, 2 C. glabrata, and 2 C. tropicalis. Phospholipase and proteinase activities were observed to have statistically significant differences between C. albicans and non-albicans Candida (NAC) strains (p < 0.05), with C. albicans strains showing higher virulence. Rep-PCR revealed eight major genotypes (A-H).The 19 C. albicans and the 33 non-albicans Candida isolates yielded seven (A-G) and four (A, B, C, H) genotypes, respectively. C. albicans strains were not shown to have a predominant genotype and showed higher phospholipase and proteinase activitiy than did NAC, regardless of genotype. Genotype H (52%) was the predominant genotype for the NAC including 27 C. parapsilosis strains, but the majority of strains showed low virulence. CONCLUSIONS: NAC species were the most common causative agent for candidemia. Genotyping showed low transmission of C. albicans strains, but transmission of C. parapsilosis was high. In candidemia, several Candida virulence factors may be responsible at the same time. However, different genotypes of Candida strains showed different virulence activity.

Effects of moxifloxacin exposure on the conjunctival flora and antibiotic resistance profile following repeated intravitreal injections.

AIM: To evaluate the effects of moxifloxacin exposure on the conjunctival flora and antibiotic resistance profile following repeated intravitreal injections. METHODS: Seventy-two eyes of 36 patients [36 eyes in control group, 36 eyes in intravitreal injection (IVI) group] were enrolled in the study. All the eyes had at least one IVI and had diabetic macular edema (DME) or age-related macular degeneration (ARMD). Moxifloxacin was prescribed to all the patients four times a day for five days following injection. Conjunctival cultures were obtained from the lower fornix via standardized technique with every possible effort made to minimize contamination from the lids, lashes, or skin. Before the application of any ophthalmic medication, conjunctival cultures were obtained from both eyes using sterile cotton culture. An automated microbiology system was used to identify the growing bacteria and determine antibiotic sensitivity. RESULTS: The bacterial cultures were isolated from 72 eyes of 36 patients, sixteen of whom patients (44.4%) were male and twenty (55.6%) were female. Average age was 68.4±9.0 (range 50-86). The average number of injections before taking cultures was 3.1+1.0. Forty-eight (66.7%) of 72 eyes had at least one significant organism. There was no bacterial growth in 8 (20.5%) of IVI eyes and in 16 (44.4%) of control eyes (P=0.03). Of the bacteria isolated from culture, 53.8% of coagulase negative staphylococci (CoNS) in IVI eyes and 47.2% CoNS in control eyes. This difference between IVI eyes and control eyes about bacteria isolated from culture was not statistically significant (P=0.2). Eleven of 25 bacteria (44.0%) isolated from IVI eyes and 11 (57.9%) of 19 bacteria isolated from control eyes were resistant to oxacillin. The difference in frequency of moxifloxacine resistance between two groups was not statistically significant (12.0% in IVI eyes and 21.1% in control eyes) (P=0.44). There were no cases of resistance to vancomycin, teicoplanin and linezolid. CONCLUSION: There was no difference in species of bacteria isolated from cultures, or in the frequency of resistance to antibiotics between eyes that had recurrent IVI followed by moxifloxacin exposure compared with control eyes. However, the number of eyes that had bacterial growth was higher in IVI group than in the control group.

[Investigation of cytomegalovirus positivity in the peripheral blood samples of risky patients by shell-vial cell culture, antigenemia test and real-time polymerase chain reaction]. / Riskli Hastalarin Periferal Kan Örneklerinde Sitomegalovirus Varliginin "Shell Vial" Hücre Kültürü, Antijenemi Testi ve Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Arastirilmasi

Cytomegalovirus (CMV) infections in immunocompromised patients and congenital infections in infants have high morbidity and mortality while it may lead to asymptomatic infections in immunocompetent subjects. Serological tests, culture methods, antigenemia tests and molecular methods are applied in the diagnosis of CMV infection. The aim of this study was to investigate the presence of CMV in peripheral blood samples of patients who were at risk for CMV disease by shell vial cell culture, antigenemia test and real-time polymerase chain reaction (RT-PCR) methods. A total of 141 blood specimens obtained from 91 patients (33 female, 58 male) with suspected CMV disease were included to the study. Five of the patients were newborns and the others aged between 17-79 years old were bone morrow (n= 81), kidney (n= 4) and liver (n= 1) transplantation patients. Shell vial (Vircell, Spain) cell culture method was applied for CMV isolation from the samples, while the detection of pp65 antigen in blood leukocytes was investigated by indirect immunofluorescence method (CINAkit Argene, Biosoft, France). The presence of CMV DNA in plasma samples was detected by RT-PCR (CMV QNP 2.0 kit; Fluorion, Iontek, Turkey) method. CMV was found positive in 72 (51%) of 141 samples by shell vial, 82 (58.2%) by antigenemia test and 49 (34.8%) by RT-PCR. Considering cell culture as the gold standard, the sensitivity and specificity of antigenemia test were calculated as 81.9% and 66.6%, respectively; and for PCR those rates were 43% and 73.9%, respectively. In addition DNA sequencing (ABI Prism 310 Genetic Analyzer; Perkin Elmer, USA) was performed for the samples of randomly selected three patients out of 15, who were yielded positive results with cell culture and antigenemia tests but negative CMV DNA by RT-PCR. In this analysis CMV DNA was found positive in three of the samples that were found negative by RT-PCR in spite of CMV isolation and positive antigenemia. DNA sequencing of those samples revealed multiple mutations in the probe binding region (gB) of CMV QNP 2.0 kit. It was concluded that for the detection of CMV viremia and viral load in patients under risk for CMV disease, antigenemia and PCR based methods could be applied, however, negative results obtained by PCR targeting CMV gB gene, should remind the possible presence of mutations in the related site and the results should be confirmed by sequence analysis.

Isolation of Acremonium strictum from pleural fluid of a patient with colon adenocarcinoma.

Although Acremonium strictum is environmentally widespread as opportunistic fungus, it may cause infection in patients who have immunodeficiency. In this study, A. strictum were isolated from the pleural fluid of a patient with colon adenocarcinoma. The patient did not receive antifungal therapy because the patient died on the same day after the isolation of the mould from the pleural fluid. Major risk factors for the fungal infection are surgery because of cancer, administration of parenteral hyperalimentation and broad-spectrum antibiotics, attaching chest tube and ventilation tube and hospitalization in intensive care unit. The minimal inhibitory concentrations (MICs) of amphotericin B, fluconazole, ketoconazole, itraconazole, voriconazole for the A. strictum strain isolated from pleural fluid were 0.125, 256, 2, 1.5 and 0.25 microg ml(-1), respectively. In conclusion, bacteria and fungi, especially opportunistic fungi should be taken into consideration in the developing pleuritis in the patients with predisposing risks for the fungal infection.

Pleuritis caused by Acremonium strictum in a patient with colon adenocarcinoma.

Although Acremonium strictum is environmentally widespread as opportunistic mold, it may cause infection in patients who have immunodeficiency problems. In this study, Staphylococcus aureus and A. strictum were isolated from the pleural fluid of a patient with colon adenocarcinoma. The patient did not receive antifungal therapy because the patient died after the isolation of mold. The minimal inhibitory concentrations of amphotericin B, fluconazole, ketoconazole, itraconazole, voriconazole for the A. strictum strain isolated from pleural fluid were 0.125, 256, 2 and 1.5, 0.25 mug ml(-1) respectively. In conclusion, bacteria and fungus, especially opportunistic mold, should be taken into consideration in developing pleuritis in the patients with immune-deficiency.