Molecular karyotyping of multiple yeast species isolated from nine patients with AIDS during prolonged fluconazole therapy

Journal of Medical & Veterinary Mycology 1996, 34, I I I-I 16 Accepted 5 September 1995 Molecular karyotyping of multiple yeast species isolated from nine patients with AIDS during prolonged fluconazole therapy A. ESPINEL-INGROFF,* A. QUART, t L. STEELE-MOORE,~ I. METCHEVA,* G. A. BUCK,* V. L. BRUZZESE* & D. REICH t *Medical College of Virginia/Virginia Commonwealth University, Richmond, VA; tNorth Central Bronx Hospital, Bronx, NY; and ~.Medical Center of Delaware, Wilmington, DE, USA Introduction In immunocompromised patients there is an increase in the oral carriage of yeasts, the most important of which is Candida albicans [1]. Indeed, C. albicans is the main cause of oropharyngeal candidiasis, which occurs at some time in the course of their illness in about 80-95% of patients with AIDS [2]. Other species, such as C. tropicalis, Torulopsis glabrata, C. krusei and C. parapsilosis, are occasionally isolated from the oral cavities of HIVinfected patients, but appear to be colonizers rather than pathogens in most cases [1]. Symptoms of oral candidiasis caused by C. albicans can be relieved and lesions cleared with topical or systemic use of an azole antifungal agent, but complete eradication of yeasts is seldom achieved and relapse generally occurs within several months [3-7]. Relapses of oropharyngeal candidiasis may be related to the sustained immunosuppression seen in HIV-infected patients, which predisposes Correspondence: Dr A. Espinel-Ingroff, MCV/VCU, Box 980049, Richmond, VA 23298-0049, USA. Tel.: (804) 828-9711; Fax: (804) 828-3097. © 1996 ISHAM them to opportunistic infections. Moreover, in those patients experiencing recurrences while receiving prolonged or repeated courses of antifungal therapy, in vitro studies using D N A subtyping techniques have found either persistence throughout of the same subtype of C. albicans with a gradual increase of MICs of fluconazole for sequential strains [4,8-12] or the introduction or selection of a different, generally more resistant, DNA subtype [4,8,9,11]. The present study was undertaken to monitor variations in molecular karyotype and fluconazole susceptibility of serial isolates of the species of Candida and T. glabrata obtained from the oral cavities of nine patients with AIDS treated with fluconazole for single or multiple episodes of oropharyngeal candidiasis. Methods Source of isolates A total of 266 yeasts were isolated from nine patients (identified as patients 1 9) aged 2 5 4 8 years who met the criteria for a diagnosis of AIDS and had CD4 cell counts Variations in molecular karyotype and fluconazole susceptibility of serial yeast isolates from the oral cavities of nine patients with A I D S receiving fluconazole for single or multiple episodes of oropharyngeal candidiasis were monitored. Multiple yeast species were isolated from the initial oral specimens in six patients. Molecular karyotyping identified at least eight different D N A subtypes of C. albicans, at least eight of T. glabrata and only one D N A subtype each of C. krusei, C. tropicalis and C. parapsilosis. A m o n g isolates of T. glabrata, fluconazole MICs in each patient were consistently within one or two dilutions, regardless of strain variations. Similarly, among five patients monitored during one course of therapy, the MICs of fluconazole of C. albicans isolates of either the same or different D N A subtypes remained within two dilutions. However, increases in M I C s of fluconazole of C. albicans were observed in four patients who received two or more courses of fluconazole, three of whom had the same D N A subtype and one of whom changed from one D N A subtype to another. Espinel-lngroff et aL Molecular kaqotyping CHEF electrophoresis The 266 isolates were characterized by molecular karyotype analysis using pulsed-field gel electrophoresis following modifications of established techniques [13-17]. Each isolate was coded with a number to permit typing without knowing the origin of the isolate. Isolates were grown first on Sabouraud glucose agar (Difco Laboratories) at 35 °C from 48 h to 5 days to obtain single colonies and to verify the purity of the culture. Two to three colonies ( > I mm) of each yeast were grown in the same 10 ml of Sabouraud glucose broth in an incubator shaker (170 rev min -1) for 18 h at 30 °C. The cells were harvested by centrifugation (1000g) for 10 rain and washed twice with 1 ml of 50 mM sodium EDTA (pH 8"0). Spheroplasts were prepared by suspending the washed cells in 1 ml of 50 mM of sodium EDTA supplemented with 1% mercaptoethanol and incubated at 30°C for 45 min. The cells were washed twice as described before and resuspended in 1 ml of SNE buffer (1 M sorbitol and 50mM EDTA pH 8-0 and 50mM Na2PO4 pH 7"4) containing 10 mg m1-1 of cell walldegrading enzyme suspension (Zymolyase-20T, ICN Biomedicals, Costa Mesa, CA) in 10mM Na2HPO4 pH 7.5 and incubated overnight at 37 °C in a water bath. Microscopic observation confirmed that there was a large percentage (approximately 90%) of spheroplast formation. The spheroplasts were washed carefully twice with 1 ml of SE buffer (1 M sorbitol, 250 mM EDTA pH 8-0), resuspended in 0-5 ml of SE buffer and incubated at 37 °C until plugs were prepared. Agarose plugs were prepared by adding 1 ml of 1% low melting temperature chromosome grade agarose (Bio-Rad Laboratories, Hercules, CA) at 70 °C to each 0"5 ml suspension of spheroplasts. These mixtures were mixed well, dispensed in individual moulds and stored at 4 °C for 1 h. Individual agarose plugs were removed from the moulds and lysed at 50 °C in a water bath shaker (120 rev min -~) for 48 h in 3 ml of 0.5 M EDTA buffer (pH 8-0) containing 10 mg ml- ~ of N-lauroylsarcosine (1%; Sigma Chemical, St Louis, MO) and 1 mg ml ~ of Proteinase K (Sigma). Agarose plugs were washed four times (1 h each) with 10ml of ET buffer (1 M Tris and 50ram EDTA, pH 8.0) in a shaker water bath at 50 °C and stored at 4 °C. The liberated chromosomes in the plugs were loaded into the wells of low melting pulsed field certified agarose (Biorad) gels (0"9%) and electrophoresed by modifications of the protocols of Lasker and co-workers [14] and Magee and co-workers [16]. Saccharomyces cerevisiae chromosome/DNA size markers (Bio-Rad) and C. albicans chromosomes were included in the gels as standards. Each gel was electrophoresed in a CHEF-DR III (Bio-Rad contour-clamped homogeneous electric field) system. Although other testing conditions and switch times were evaluated for each of the species, the best electrophoretic conditions for C. albicans were 12°C and 4.5 volts cm - l with pulse intervals of 120 s for 14 h followed by 240 s for 19 h and 420 s for 14 h. The conditions for T. glabrata were 6 volts c m - l with pulse intervals of 90s for 15h followed by 4.5 volts cm- 1 with pulse intervals of 200 s for 12 h and 400 s for 8 h. Completed gels were stained for 15 min in 0.5/tg ml-~ ethidium bromide to reveal the chromosomes and photograph (...truncated)