Downloaded from www.microbiologyresearch.org by IP: 54.224.121.223 On: Wed, 20 Apr 2016 06:18:23 In vitro antifungal susceptibilities and molecular typing of sequentially isolated clinical Cryptococcus neoformans strains from Croatia Emilija Mlinaric ´ -Missoni, 1 3 Ferry Hagen, 2,3 3 William H. M. Chew, 4 Verica Vaz ˇic ´ -Babic ´, 1 Teun Boekhout 2,3 and Josip Begovac 4,5 Correspondence Emilija Mlinaric ´ -Missoni emilija.mlinaric-missoni@hzjz.hr Received 13 February 2011 Accepted 26 May 2011 1 Croatian National Institute of Public Health, Reference Centre for Diagnostics of Systemic Mycoses, Zagreb, Croatia 2 CBS Fungal Biodiversity Centre, Department of Yeast and Basidiomycete Research, Utrecht, The Netherlands 3 Department of Internal Medicine and Infectious Diseases, University Medical Center, Utrecht, The Netherlands 4 University Hospital for Infectious Diseases, Zagreb, Croatia 5 Department of Infectious Diseases, University of Zagreb School of Medicine, Zagreb, Croatia A collection of 48 clinical Cryptococcus neoformans isolates from Croatia was investigated retrospectively using in vitro antifungal susceptibility testing and molecular biological techniques to determine mating type and serotype by PCR and amplified fragment length polymorphism (AFLP) genotyping. These isolates were obtained from 15 patients: ten were human immunodeficiency virus (HIV)-negative (66.7 %) and five were HIV-positive (33.3 %). From five patients, only one isolate was available, whilst from the other ten patients, two to 11 isolates were isolated sequentially. Antifungal susceptibility was tested by a broth microdilution method. Serotype A (genotype AFLP1) and serotype D (genotype AFLP2) were both found in six patients (40 % each), and serotype AD (genotype AFLP3) in three (20.0 %) patients. Mating type a (n512; 80.0 %) predominated and a/a hybrids were identified in 20.0 % of patients diagnosed with cryptococcosis. Two AFLP genotypes of C. neoformans were isolated during a single episode from one patient. The in vitro antifungal MIC 90 and susceptibility ranges for C. neoformans isolates were 0.5 mg ml ”1 (range 0.031–0.5 mg ml ”1 ) for amphotericin B, 4 mg ml ”1 (range 1–4 mg ml ”1 ) for flucytosine and fluconazole, 0.25 mg ml ”1 (range 0.031–0.5 mg ml ”1 ) for itraconazole and 0.062 mg ml ”1 (range 0.031–0.25 mg ml ”1 ) for voriconazole. INTRODUCTION Cryptococcosis is a life-threatening systemic fungal infection caused by the opportunistic pathogenic yeast Cryptococcus neoformans or its primary pathogenic sibling Cryptococcus gattii. Worldwide, C. neoformans is the major cause of cryptococcosis in individuals with an underlying immuno- logical disorder, such as patients that are infected with human immunodeficiency virus (HIV), who have undergone solid organ transplantation (Bovers et al., 2008; Warkentien & Crum-Cianflone, 2010) or who have haematological malignancies (Bassetti et al., 2009; Vigouroux et al., 2000). It has been estimated recently that the global burden of HIV- associated cryptococcosis approximates to 1 million cases annually (Park et al., 2009). In Croatia, from 1985, when HIV was diagnosed for the first time, up until December 2009, nearly 800 HIV-infected persons were registered (http:/www. hzjz.hr; data accessed 24 December 2010). Croatia has a centralized system of healthcare for HIV/acquired immuno- deficiency syndrome patients who are all treated at the University Hospital of Infectious Diseases (UHID) in Zagreb. Since 1998, HIV-positive patients at this hospital have been treated with highly active antiretroviral therapy (HAART). Extrapulmonary cryptococcosis is an important oppor- tunistic infection and was found in 29 (8.7%) of the 332 HIV-positive patients treated at the UHID in the period 1986–2010. The most frequent and severe clinical presentation of cryptococcosis is a disseminated meningoencephalitis, 3These authors contributed equally to this work. Abbreviations: AFLP, amplified fragment length polymorphisms; CSF, cerebrospinal fluid; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; i.v., intravenous; RCDSM, Reference Centre for Diagnostics of Systemic Mycoses; UHID, University Hospital of Infectious Diseases. Journal of Medical Microbiology (2011), 60, 1487–1495 DOI 10.1099/jmm.0.031344-0 031344 G 2011 SGM Printed in Great Britain 1487