[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.

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.

Epidemiology of superficial mycosis (tinea pedis, onychomycosis) in elementary school children in Istanbul, Turkey.

The purpose of this study is to determine the prevalence of tinea pedis and onychomycosis in children of elementary school age and to examine the socio-demographic attributes that may be effective in correlation of both mycoses. 3,390 female and 3,768 male children between ages 6-14 have been examined in seven schools. Skin scrapings and nail samples were taken from 13 students who were suspected to have tinea pedis and from 49 students who were suspected to have onychomycosis. According to direct microscopy (10-15% KOH+calcofluor white) and culturel examination (Sabouraud dextrose agar and dermatophyte test medium) 11 students were diagnosed as tinea pedis and 24 were diagnosed as onychomycosis. Trichophyton rubrum was isolated in 3 students with tinea pedis whose culture was positive and five Candida albicans, five Candida glabrata and one Candida tropicalis cases were isolated from 11 samples with onychomycosis. Tinea pedis prevalence has been found to be 3.3%0. Differences between onychomycosis prevalence based on age have been found to be significant (p < 0.001). In conclusion, it has been determined that the prevalence of tinea pedis and onychomycosis among children is low. Candida spp. was isolated from all of the 14 samples diagnosed as onychomycosis. Our study shows similar results with previous studies done in Turkey and that Trichophyton rubrum continues to be the most isolated agent.

Synthesis of new triazolyl-N,N-dialkyldithiocarbamates as antifungal agents.

N,N-Dialkylditihiocarbamate derivatives have been well known as broad-range fungicides. In this study, the triazole derivatives of ten new N,N-disubstituted dithiocarbamates (3a-j) were synthesized and their structures were identified by spectral and elemental analysis. Results of the antifungal activity studies showed that some of the compounds tested were active against M. canis, M. gypseum, and T. rubrum at the concentration of 12.5 microg/mL when clotrimazol was used as a standard.

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.