Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

An outbreak of Fusarium solani endophthalmitis after cataract surgery in an eye training and research hospital in Istanbul.

To report an outbreak of Fusarium solani endophthalmitis after uneventful cataract surgeries performed on the same day in the same operating room. Nine patients underwent phacoemulsification at 4th Clinic of Beyoglu Eye Training and Research Hospital in Istanbul. Cefuroxime axetyl was injected intracamerally from the same vial to all patients at the end of surgery. All patients developed acute postoperative endophthalmitis. Presentation, cultural studies, treatment, clinical responses and risk factors were evaluated. Cultural and DNA sequence findings revealed F. solani. Antifungal therapy was begun and pars plana vitrectomy, intraocular lens and capsule extraction were performed. Corneal involvement was correlated with old age and systemic disease. Fusarium solani should be considered in acute postoperative endophthalmitis. This infection can be controlled with early and aggressive combined antifungal and surgical treatment. The patients with corneal involvement had poor prognosis. It is important to use solutions prepared separately for each patient.

[Evaluation of the significance of molecular methods in the diagnosis of invasive fungal infections: comparison with conventional methods]. / Invazif Mantar Enfeksiyonlarinin Tanimlanmasinda Moleküler Yöntemlerin Öneminin Konvansiyonel Yöntemler ile Karsilastirilarak Degerlendirilmesi.

Direct microscopy and culture methods are still valuable standard conventional methods for the diagnosis of infections caused by true or opportunistic fungal pathogens, especially in high risk patients. However, some of the problems concerning the application and interpretation of those methods, indicate a need for more rapid, practical and reliable tests with high sensitivity and specificity. This study was conducted to compare the results obtained by molecular methods with the results of conventional methods performed simultaneously for the detection and identification of causative fungi in clinical samples. Clinical samples [24 bronchoalveolar lavage (BAL); 14 blood; 5 peritoneal, 4 pleural and 1 pericardial fluids; 1 cerebrospinal fluid (CSF), 1 urine] from 50 immunosuppressed patients were included in the study. All of the samples were cultivated on Sabouraud dextrose and brain-heart infusion agar media and incubated at 30°C and 37°C for 30 days. Samples other than blood were stained with 10-15% KOH + calcofluor white and examined by direct microscopy. Conventional identification of the isolates were performed by using basic morphological and biochemical characteristics. The isolation of fungal DNAs for polymerase chain reaction (PCR) was achieved by classical phenol-chloroform-isoamylalcohol procedure (9-10 hours) and commercial DNA extraction kit (6-7 hours) and general and species-specific primers (multiplex) from ITS1, ITS2, ITS3, ITS4, 5.8S rDNA and 28S rDNA regions were chosen for amplification. In PCR results, 550 base-paired (bp) bands obtained with universal primers were evaluated as fungal DNA positivity, and 273 bp, 320 bp, 423 bp, 357 bp, 136 bp and 385 bp bands with species-specific primers were evaluated as Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Cryptococcus neoformans and Aspergillus fumigatus positivities, respectively. Seventeen (34%) of the 50 samples yielded fungal growth on culture (C.albicans in 12 BAL, 3 blood, 1 urine sample, and C.parapsilosis in 1 urine), while seven BAL out of 36 (19.4%) non-blood samples gave positive result by direct microscopy. Of the samples 27 (54%) were found positive by PCR. All of the 17 culture positive samples were also found PCR positive, and all of the 23 culture negative samples were also found PCR negative. However, fungal DNAs were detected by PCR in 10 of the samples (5 BAL, 4 peritoneal fluids, 1 CSF) which were negative by direct microscopy and culture methods. These fungi were identified as C.albicans (n= 8), C.parapsilosis (n= 1, from peritonal fluid) and C.neoformans (n= 1, from CSF) by multiplex PCR. No samples yielded PCR negative, culture positive result. All of those 10 PCR positive, culture negative samples belonged to patients who were under antifungal treatment. The detection of C.neoformans DNA from CSF sample of a patient with suspected cryptococcosis only with PCR provided the chance for rapid therapy. In statistical evaluation, the concordance between culture and PCR methods were found significantly high (k= 0.61; p< 0.001), whereas it was minimal (k= 0.24; p< 0.001) between direct microscopy and PCR. When considering culture as the reference method, the sensitivity and specificity of PCR were estimated as 100% and 69.7%, respectively. In addition, multiplex PCR was as successful as culture and conventional identification methods in the identification of all fungal species. As a result, without disregarding conventional methods, use of PCR might be recommended for the identification of fungal species on the basis of clinical status of the patient and conditions of the laboratory.

Prevalence, susceptibility profile and proteinase production of yeasts causing vulvovaginitis in Turkish women.

In this study the prevalence of vulvovaginal candidiasis (VVC), antifungal susceptibility and proteinase production of isolated Candida species were investigated. Vaginal swabs were collected from symptomatic women with vulvovaginitis attending the Obstetrics and Gynecology Clinic of Kocaeli University, Turkey. The relation between risk factors, such as pregnancy, diabetes mellitus, antibiotic and corticosteroid use, history of sexually transmitted diseases and contraceptive methods, was recorded. Candida spp. were identified by conventional methods, then evaluated for proteinase secretion in a medium containing casein. Antifungal susceptibility was determined according to the NCCLS microdilution method. The prevalence of women with vulvovaginitis was 35.7% (170/6080) and 16% (28/170) of them were diagnosed as VVC. Candida albicans was the dominant species: 21 (75%), followed by 4 C. glabrata (14%), 2 C. tropicalis (7%), and one C. krusei (3.5%). All isolates were susceptible to fluconazole, itraconazole and amphotericin B, except one C. krusei, one C. glabrata and one C. albicans that were resistant to fluconazole. Proteinase production was determined in 19 (90.5%) C. albicans and in all C. tropicalis isolates. Proteinase activity was not associated with antifungal resistance. No association was found between risk factors and VVC.

In vitro susceptibility of yeasts isolated from patients in intensive care units to fluconazole and amphotericin B during a 3-year period.

Fungal infections have increased dramatically in recent years and candidemia is a major risk factor for morbidity and mortality in intensive care units (ICUs). Candidemia has been considered to be a nosocomial infection that is strongly associated with neutropenia, recent surgery or presence of intravascular lines, and previous colonization is an independent risk factor. We evaluated the in vitro efficacy of fluconazole and amphotericin B against yeasts isolated from various clinical specimens of colonized or infected patients treated in the ICUs of the Institute of Cardiology, Istanbul University. A total of 1397 ICU patients were treated at the Institute of Cardiology between January 2000 and December 2002. A total of 117 yeasts isolated from 97 patients were included in this study. These ICU patients were hospitalized for a mean of 29 days. All yeasts were identified by conventional methods and using the API (20C AUX, ID 32C) system (Bio Meriéux, France). Susceptibility to fluconazole and amphotericin B was evaluated using the E-test (AB Biodisk, Solna, Sweden). The most commonly isolated yeast was Candida albicans (72.6%), followed by Candida tropicalis (16.2%), Candida kefyr, Candida krusei, Candida parapsilosis, Trichosporon mucoides and Geotrichum spp. Fluconazole and amphotericin B MIC90 values were 0.75 microg/ml; 0.19 microg/ml and 1 microg/ml; 0.38 microg/ml for C. albicans and C. tropicalis, respectively. All Geotrichum spp. were found to be susceptible-dose dependent (SDD) (MIC=16-32 microg/ml) to fluconazole. Two C. albicans, two C. tropicalis, one C. krusei and one Geotrichum spp. had a MIC value of > or = 0.38 microg/ml for amphotericin B. The rate of colonization was 3.36% (47/1397). Only 10 (0.71%) patients out of a total of 1397 developed candidemia during the period of the investigation. Of these, 7 (70%) were caused by non-albicans Candida spp.