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Resistant Cryptococcus Laurentii Received December 2000 Medical Mycology 2002, 40, 479–484 Accepted 17October 2001;Final revision received 24July 2002 Fungemia ina cancerpatient caused by ¯uconazole- resistant Cryptococcus laurentii D.AVERBUCH*, T. BOEKHOUT ,R.FALK*,D. ENGELHARD*, M. SHAPIRO , C. BLOCK ANDI. POLACHECK y z z z Departments of *Pediatrics, and Clinical Microbiologyand Infectious Diseases , The HebrewUniversity-Hadassah MedicalCenter, z Jerusalem,Israel; CBS Yeast Division, Utrecht, The Netherlands y Wereport the recentisolation of Cryptococcuslaurentii from the blood of apatient giventhe diagnosisof ganglioneuroblastoma.The organism was identied using physiologicaland molecular characteristics, including morphology, carbohydrate and nitrateassimilation, urease activity, inability to form melaninon appropriatemedia, positivestaining with diazoniumblue B andsequence analysis of the D1/D2domain of 26Sribosomal DNA. The isolate was resistant to uconazoleand 5- uorocytosine usingboth the Etest anda broth microdilutionassay. Repeated recovery of the organismfrom different blood cultures,and the patient’s good responseto treatment with amphotericinB support its etiologicalrole. C.laurentii hasrarely been implicatedas a causeof clinicallysigni cant infections. The identity of reported isolateshas not alwaysbeen adequately documented, and some appear to havebeen isolatedfrom lesionscaused by Cryptococcusneoformans ,emphasizingthe true rarityof diseasedue to this fungus. Keywords antifungal(s), Cryptococcuslaurentii ,cryptococcosis,fungemia For personal use only. Introduction Materialand methods Cryptococcosis was rare until the last decade. However, Case report its medical importance increased dramatically as a A16-year-old male given the diagnosis in 1998 of consequence of the AIDS epidemic [1–3]. The major ganglioneuroblastoma presented with alarge abdominal risk factors in non-AIDS patients are lymphoprolifera- tumor and metastases to the skull, right humerus, ribs, tive disorders, corticosteroid therapy, sarcoidosis, and pelvis and both femurs. He received ten courses of organ transplantation [2–4]. Infections caused by non- chemotherapy through aHickman catheter, with partial Cryptococcus neoformans cryptococci have been re- response. One month after the last course of chemother- ported rarely in humans in recent years, although reports apy he was admitted to hospital for elective resection of Med Mycol Downloaded from informahealthcare.com by University Library Utrecht on 07/16/12 of cases of infections due to Cryptococcus laurentii have the abdominal mass.On admission, the physical exam- been increasing [5–9]. ination was unremarkable except for the presence of a We report the isolation of C.laurentii fromthe blood large abdominal massand hepatomegaly. Laboratory of apatient with asolid tumor. In addition, we review the tests revealed aperipheral leukocyte (WBC)count of literature of infections caused by this rare pathogen and 3 6000 mm¡ with 70% granulocytes, hemoglobin 13.7 g%, discuss the problem of the emergence of fungi resistant 3 and platelet count 378 000 mm ¡ .One day after admis- to antimycotic agents. Finally, we discuss the salient sion he developed fever (38.2 oC),which lasted for characteristics of this yeast and the factors contributing several hours. Antibiotic treatment with cefuroxime to its appropriate classication. and gentamicin was initiated. Blood cultures fromtwo different blood samples, obtained at the onset of fever, yielded apure culture of yeast that was later identied as Correspondence:Itzhack Polacheck, Department of Clinical C.laurentii. Noother pathogens were isolated. The fever Microbiologyand Infectious Diseases, The Hebrew University- subsided and the antibiotic treatment was discontinued. HadassahMedical Center, PO Box12000, Jerusalem 91120, o Israel.Tel.: 9722 6776592; Fax: 9722 6769206; E-mail: One week later, pyrexia (39 C)recurred, with chills and ‡ ‡ [email protected] hypotensive episodes that lasted for several days, in spite ã 2002 ISHAM,ISHAM Medical Mycology , 40, 479–484 480 Averbuch et al. of reinstitution of the antibiotic therapy. In addition, used for the Etest were: RPMI-1640 medium (Sigma, St. 3 leukopenia of 2500 mm ¡ WBC,with 47% granulocytes, Louis, MO, USA)supplemented with 1.5% agar and 2% was observed. Therefore, the Hickman catheter was glucose (RPG)and buffered to pH7.0 with 0.165 M removed and treatment with amphotericin B(AmB) morpholinepropane sulfonic acid buffer (MOPS)for the 1 1 (0.5 mg kg¡ day¡ )was administered for three weeks. azoles; and modied Casitone agar for the amphotericin 1 The fever subsided and WBCcount gradually increased Band 5-uorocytosine [9gl ¡ Bacto Casitone (Difco), 1 1 to normal levels. After recovering fromthis infectious 5 g l¡ yeast extract (Difco),10 gl ¡ sodium citrate 1 episode, the patient underwent the planned laparotomy (Sigma) and 20 gl ¡ glucose]. For the Etest, 90-mm- for resection of his tumor. diameter plates containing agar at adepth of 4.0 mm Although C.laurentii was not isolated again from were used. The inoculum was prepared from48-h repeated blood cultures, it was suspected to be the most culture. Cell suspension was prepared in sterile 0.85% likely cause of the infectious episode as it was isolated NaCl adjusted to the turbidity of a1.0 McFarland fromdifferent blood samples at the onset, and because standard. The minimuminhibitory concentration (MIC) there was no response to antibacterial antibiotics but was dened as the lowest concentration of antifungal prompt response to amphotericin B. agent at which the border of the elliptical inhibition zone intercepted the readable scale on the strip. Candida Isolation and characterization ofthe organism krusei ATCC 6258 (American Type Culture Collection, Manassas, VA, USA) and Candida parapsilosis ATCC Blood was incubated at 37 oCin aerobic and anaerobic 22019 served as quality controls for all tests. BacTAlert1 blood culture bottles (Organon Teknika The in vitro susceptibility of the isolates was also Corp., Durham, NC,USA). After two days, broth from determined by the broth microdilution method according the positive BacTAlert 1 culture was streaked for to the recommendations of the National Committee for isolation over the surface of Emmons’modi ed Sabour- Clinical Laboratory Standards (NCCLSM27-A) [14] as aud’s glucose agar (SGA)supplemented with chloram- 1 1 described by Espinel-Ingroff et al. [15] for Cryptococcus phenicol (50 m g ml¡ )and gentamicin (5 m g ml¡ ). spp. These tests involved the use of 0.2 mlof RPMI-1640 Melanin formation was investigated on L-DOPA agar broth medium (Sigma) buffered to anal pHof 7.0 with [1], incubated at 30 oCand norepinephrine media 0.165 MMOPS and 1MNaOH, lter sterilized solu- incubated at 25 oC;colony color was examined after 2, tions, inoculum size of 10 4 cells per ml.The microtitra- For personal use only. 3and 5days. Selected colonies were transferred to and tion plate was incubated at 35 oCwith agitation for 48 h. maintained on SGA at 30 oC. Colonies from48-h SGA The MICwas dened as the lowest drug concentration cultures were evaluated for urease activity on Christen- that resulted in complete inhibition of visible growth. sen’s urea medium, and for carbohydrate and nitrogen assimilation patterns using the API ID32C and the API 20C AUXyeast assimilation systems (bioMe´ rieux, Results Marcy-l’Etoile, France). Fermentative capabilities and growth responses were further investigated using stan- Identi®cation ofthe clinical isolates dardized methods and results were compared with those White to cream-colored mucoid colonies with asmooth in recently published yeast monographs [10,11]. Conidial and glossy surface were recovered on SGA plates. Med Mycol Downloaded from informahealthcare.com by University Library Utrecht on 07/16/12 morphology and ontogeny were examined microscopi- Microscopic examination of cells fromthese colonies, cally after 7–10 days’incubation at 30 oCon cornmeal– as well as fromslide cultures, showed round to oval, Tween agar (DifcoLaboratories, Detroit, MI, USA) budding, encapsulated yeasts, without pseudo- or true slide cultures. Sequence analysis was performed of the hyphae. The overall micro-and macroscopicappearance D1/D2domains of the 26S ribosomal DNAaccording to was consistent with the genus Cryptococcus. The isolates Fell et al. [12]. The C.laurentii isolate (P-6723) is grew on SGA at 30 and 37 oCbut not at 45 oC, were maintained in the CBS culture collection (Centraalbur- sensitive to cycloheximide, hydrolyzed urea and lacked eau voor Schimmelcultures, Utrecht, The Netherlands) fermentative ability. However, the colonies did not as CBS 8833. produce any brown to black color on L-DOPA and norepinephrine agars and remained hyaline after 5days. Susceptibility to antifungal drugs Identication proles for C.laurentii were excellent with The in vitro antifungal susceptibility of the yeast isolate the ID32C yeast assimilation test system (biocode was determined using the Etest system (AB Biodisk, 5577766375), and good with the API 20C AUXsystem Solna, Sweden) according to the manufacturer’s instruc- (biocode 27455773). The growth pattern on awide tions and as described previously [13]. Agar formulations variety of carbon and nitrogen sources, growth at 37 oC, ã 2002 ISHAM, Medical Mycology , 40, 479–484 Cryptococcuslaurentii fungemia 481 growth in 0.1% cycloheximide, the production of starch- contaminant during the fermentation process of wine like compounds, urease activity and apositive staining and beer [11]. Itwas recently
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