Received December 2000 Medical Mycology 2002, 40, 479–484 Accepted 17October 2001;Final revision received 24July 2002

Fungemia ina cancerpatient caused by ¯uconazole- resistant 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 Division, Utrecht, The Netherlands y

Wereport the recentisolation of Cryptococcuslaurentii from the blood of apatient giventhe diagnosisof ganglioneuroblastoma.The organism was identiŽ ed 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 clinicallysigniŽ 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 . 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 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 classiŽcation. and gentamicin was initiated. Blood cultures fromtwo different blood samples, obtained at the onset of fever, yielded apure culture of yeast that was later identiŽed 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 modiŽed 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 deŽned 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’modiŽ 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 aŽnal pHof 7.0 with [1], incubated at 30 oCand norepinephrine media 0.165 MMOPS and 1MNaOH,Ž 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 deŽned 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 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 , 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 CBSculture 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 IdentiŽcation proŽles 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 isolated at ahigh frequency with diazonium blue Bwere all in agreement with known fromwild pigeons, suggesting that these birds are a characters of C.laurentii [10,11]. The D1/D2sequence of possible reservoir in nature [18]. The species is avery the 26S rDNAof the isolate was found to be identical rare pathogen in humans. Only 16 cases of infections with two other isolates of C.laurentii ,namely CBS2174 from which C.laurentii was isolated have been reported. (AB035040) originally isolated froma tumor, and Most were in cancer patients or immunocompromised CBS7140 isolated fromsoil. The sequence differed in hosts, including seven cases of fungemia (Table 2). one nucleotide fromthat of CBS 139 (AF075469), the As Krajden et al. have indicated [19], interpretation of type strain of C.laurentii ,and in two nucleotides from published reports of these rare clinical isolations is CBS7235 [16]. problematic. Two main categories of problems are encountered: the absence of adequate demonstration of Antifungal susceptibility the organism in tissues, and the uncertainty of its identiŽcation as C.laurentii .The two cases reported by 1 The MICvalues (mgl ¡ )of antifungal drugs were Krajden et al. were of particular interest, as C.laurentii detected according to the Etest method and the NCCLS was undoubtedly grown fromtissue in which C. neofor- microbroth dilution method (Table 1), and compared mans was shown to be present by uorescent antibody with these of C.laurentii type strain (CBS139). Itis clear techniques but fromwhich tissue it did not grow in that our isolate (CBS8833), contrary to the type strain, culture. Afurther example is the repeated isolation of was in vitro resistant to uconazole according to both C.laurentii fromperitoneal uid in apatient undergoing 1 1 methods (MICvalue >256 mg l ¡ –Etest, or 50 mgl ¡ peritoneal dialysis via aTenckhoff catheter [20]. – NCCLS). Whereas the peritoneum is usually regarded as asterile site, the presence of along-term catheter raises questions Discussion as to the etiological signiŽcance of the isolates in this case. In addition, without proof of tissue involvement, Molecular characterization, phylogeneticstatus and colonization of skin or catheter could not be excluded. ecology of C.laurentii Despite these caveats, and in view of the paucity of Morphological, physiological and molecular data point information regarding the species in general, we have For personal use only. to the identity of the isolate as C.laurentii. This species included all published reports of clinical isolations. We differs phylogenetically from C.neoformans , as its type believe that all the information should be available strain (CBS139) belongs to adifferent cluster in the (Table 2), especially for clinicians who are required to order (cluster Indecorata) [12]. C.laurentii, make real-time judgments based on laboratory results. as currently deŽned, is genetically heterogeneous However, we would emphasize the need to evaluate each [10,11,16,17]. Consequently it is difŽcult to identify it report critically. phenotypically, e.g. with the ID32C or API 20C AUX systems. Sequence analysis of the D1/D2 domain of the Clinical manifestations of C.laurentii 26S rDNAis required for the accurate identiŽcation of clinical isolates belonging to this complex. According to The clinical manifestations of C.laurentii infection vary Med Mycol Downloaded from by University Library Utrecht on 07/16/12 arecent molecular phylogenetic analysis of C.laurentii fromlocal skin lesions or asymptomatic lung infection based on the D1/D2 sequence, our isolate belongs to [5,21] to systemic symptomswith fever or hypothermia phylogenetic group I[17]. and hypotension, as probably occurred in our case [8], or C.laurentii isfound in the environment in diverse meningitis [7]. Fever was noted in all patients with habitats, e.g. in soil, certain vegetable skins, and as a fungemia, except in one who had hypothermia [9]. Other

Table 1 Susceptibilityof Cryptococcuslaurentii isolatesto antifungal drugs

1 AntifungalMIC (mgl ¡ )

Isolate AmB Flu Itra Keto 5FC CBS 8833(current case) 0.032(0.03)* 4 256 (50) 0.5 (ND) 0.016 (ND) 432 (4500) CBS 139(type strain) 0.006(0.03) 8 (6) 0.006 (ND) 0.008 (ND) 4 32 (4)

AmB,amphotericin B; Flu, uconazole;Itra, itraconazole; Keto, ketoconazole; 5-FC,  ucytosine.The MIC values are according to theEtest method. *Numbersin parenthesesare the MIC valuethat weredetermined according to theNCCLS microbrothdilution method. ND, not detected.

ã 2002 ISHAM, Medical Mycology , 40, 479–484 482 Averbuch et al. , C y y y . F s ] ] ] ] ] ] ] ] ] - f d i 5 ] ] ] 4 ] 5 4 ] 1 9 9 3 0 2 ] e u h t ; 6 8 8 2 9 2 2 7 2 1 1 2 2 2 5 R T s [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ s u o - n e e e v m v a o h o r t t r t c e e e e e e e e e e e e e e s t a n r r r r r r r r r r r r r r n p i n u e e u u u u u u u u u u u u u u a , m O C C C C D C C C C C C C C C C I m V m I r ; o n f R o o i C t e , a n t B u n s l R a u m l F C c p , A s c t n , o C C C a n R R R c c F F F R r i e t o C 5 C C 5 5 t R R C R M m , , , , , , p w t C C e , C y o B B B B B B B B B a , , r r n C e r u u u u m m m m m m m m m o R r C l l l l a F T A F A F A A A F A A A N C 5 F A m y b e ) ) n d 2 2 o ) e ( ( y b 2 s ) ) s ( , u d d 1 1 ) p i i T ( ( a y 1 o u u c s i ( ) M y y   p b s s 1 e B l l * ( o p p l m ) ) ) ) ) ) ) ) b i ; a a n l a u o o 1 1 1 1 2 2 2 2 s b i e e e i i a ) t o ( ( ( ( ( ( ( ( u n n v t h 1 b b u n m o ( c o d d d d d d d d o o i p o d g g t t e n c s o o o o o o o o i i i m r e F n n s e o r r t e o o o o o o o o ) i d l l l l l l l l S r r u u e e e p 1 u r S B B B B B B B B C B ( L L P P V L l e c t n e s i h o t t n c i i a c k r e m t s l , e i b t a , s m m s R a i a a a a a a a h u u m l t i i i i i i i C t i n i e e l ; s s h o g m m m m m m m r n n i e e r p n t e e e e l e e e e o o s i e t o c g g g t t g g g g g g g u a C i i e n e d n n n w n n n n n n n c r r d h f e V n t u u u o u u u u u u u e e o n g a I C F F F n F F F F M L L L P P E L c n i s d t u s u s o l e y e n c l r c h e i , n s u o c t v i n n t n d a a i i , , , a r o o a a a l t s n i m s e p p n n u s n n o i o e a c n i e l l t o o s l l t n r e n i i p a a i i p s a l c s s s s h l s r c n n e k s y e i r n n i i y n m i e s c u f , r r t e e g d y h i For personal use only. e o n , a o t t o m a m i s i l i p i t n r s t y e s i s i o o o o l c a i a a c r i o d l r p v t m l i p p d d i s i r Ž e r t . n t r b , u t i i a y y m y c b b s h p e o n e g e c i n a t p e i o n h h c a a f c f , t e e s e l h n n i g p c a t , , , , i , r t n C e p u m o n e n o s t r r r r r Ž r r r r I r m u e o i s i i t f n o o e e e e e e e e e r s s P a m p n n m u r u c a l v v v v v v v r l v v o d m e ; y s c e e s r f o h r e e e e e e e e e e r i u n o s o n e P F F F F F H a i F F A m N H d P p F v F C G e t u n e c c h s t , o , u s a s c c c c c c i o i t s t n o t s r u c o p o i l o i e i t o o i y t a t i t b n r t s b e i a p d e i t t . o o y l s s C h t v t e i i r , n t n c u l e n f s s s l , a a e s C u s o o a c a y y s o c z , i l l r u d c y m m t t s s a i i m s a a i b y i i t n l g o n , d d o e r i i i e d d r o a o o s u a a a d a c i r i i b i t c o o r e a e l l l i i n r r t , b o n n n s t c d i a a i a e t s e e e e e m u r t C r n r e e t t c s e l a , p p p e Med Mycol Downloaded from by University Library Utrecht on 07/16/12 u s n s a u V a V n n d c c v t a f I o a o o s i i i , , , , o o C c r n r r t e e e e o o t t i , , t t t . s C C C C C C i i M k e r b n n n p p s r r s u r u u e C C ; e i o o o e V V V V V V x o I I e e o e a e e t g e s t l g R C C C P C n P p C n C n E S N N P P T N e o y h u z r t s t a n r s s n i e y e n l o r a m d c o a g r i e u m p n s h o  o o e e e c s n , l r i i s s h o s t u t a a d l p s e , n s n u e e i s i F s e r o s s m s h d a g k v ; i i a i i y n o l s e t n n l d d B s s g i e n s i o i a l i u s l l g t u s s l n n p a i i i d o a a i i i o o n c m e l r r c y r y n n s r k i b e i g t v e r e o o e e e m t h g i v , t m r i n T e y r r u s r i p d t e o a c m m u o a i a u s t r y e o o t c c c l m a M i l u u t l i i i u h p t n t a v b r p u m a B H n n n e p c o e o - e e e o t g m d d d d o o o r t m c d i i m p n k r n n e u r r r e u V e s l l d e y a t n y r c I e v d o u o o h h h o r i o y o r , u t e l i U S P H h D N P A l S H D N N C C C M o B s c p o n m e p o F / A c f R e ; M r o / r e e r e t u n x i t 2 t e a s e l a a b s o e / n t e l m m s y e M M o F M M M M F F M F F F M b u / / / / e / / / / / / / / / / a c g e a r 6 7 7 0 7 6 4 5 4 7 4 3 1 0 u N C T A 1 1 N 2 5 P 5 2 3 5 5 3 1 1 6 4  y *

ã 2002 ISHAM, Medical Mycology , 40, 479–484 Cryptococcuslaurentii fungemia 483

1 clinical manifestations in the fungemic patients were resistant to both uconazole (50 mgl ¡ , NCCLS or 1 1 infrequent (Table 2). 4256 mg l¡ ,Etest) and ucytosine ( 4 32 mg l¡ ). We The fungus can be isolated fromthe site of infection, assume that this type of resistance isinnate and is such as the skin, respiratory tract, peritoneal uid, or unrelated to drug exposure, as our patient had never CSF [5,7,20–23], or, as in our case, fromblood cultures been treated with azoles or ucytosine. This case [6,8,24,25]. Itmay be argued that in our case the isolation resembled another, recently reported fromIsrael, in of C.laurentii represents colonization by asaprobe which C.neoformans that was resistant to antimycotic rather than true infection. However, no other infectious agents was obtained froma patient who had not received agent was associated with the recrudescence of fever and these drugs previously [28,29]. recurrent episodes of hypotension during leukopenia. Itis becoming clear that clinically relevant isolates of Moreover, there was no response to antibacterial C.laurentii show different degrees of susceptibility to antibiotics or to removal of the Hickman catheter, antifungal agents. Opportunistic fungal infections due to whereas there was aprompt response to amphotericin B. species tending tobe less susceptible to antifungal agents Thus it islikely that our case was indeed atrue infection in clinical use have been increasing in recent years. with C.laurentii . Resistance to azole antifungals among Candida and Cryptococcus spp. constitutes by far the mostsigniŽ cant Therapeutic approaches problem, and we might expect to see resistant C. laur- entii added to the emerging list of species causing Six of the seven reported patients with fungemia and the opportunistic disease in vulnerable patients exposed to case described here had acentral venous catheter in antifungal agents [17,29–31]. place [6,8,24,25] that was removed as part of the In conclusion, C.laurentii ,which isusually considered treatment. However, there is no deŽnite evidence that to be asaprobe, may cause clinically signiŽcant fungemia the central venous catheter was the source of the and other localized infections, especially in immuno- fungemia. Culturing of the tip of the Hickman catheter compromised hosts. Patients with malignant diseases in our case, as well as in three other patients [6,8], failed with acentral venous catheter seem to be at particular to reveal the pathogen. Removal of acentral line upon risk for fungemia with this organism. The clinical microbiologically documented fungemia iscommon manifestations of C.laurentii fungemia are usually not practice. severe, and there is usually afavorable response to

For personal use only. There are various therapeutic approaches to treating appropriate antifungal therapy and catheter removal. patients with infection caused by C.laurentii. Our Finally, special consideration should be paid to the patient, three others with fungemia and patients with emergence of C.laurentii strains resistant to antifungals. respiratory tract disease were treated successfully with AmB[21,24,25]. Others with similar clinical syndromes were successfully treated with uconazole [6,8,24], or a Acknowledgements combination of AmBand ucytosine [8]. The duration of We thank MrsIlana Sivan-Maltzov for her technical treatment for fungemia varied from10 days to 4weeks assistance in the mycological study. [6,8,24,25].

Med Mycol Downloaded from by University Library Utrecht on 07/16/12 Antifungal resistance References There are few reports on the MICsof antifungal drugs 1Kwon-ChungKJ, BennettJE. Cryptococcosis.In: Kwon-Chung KJ, BennettJE, eds. MedicalMycology .Philadelphia:Lea and C.laurentii et al. for . Ryder [26] reported that the MIC Febiger,1992: 397– 446. of seven isolates for uconazole ranged between 1and 2MitchellT, PerfectJ. Cryptococcosisin the era of AIDS— 100 1 1 4 mg l¡ ,compared with 50 mgl ¡ (NCCLSmethod) in yearsafter the discovery of Cryptococcusneoformans . Clin our case. The MICof AmBin three clinical isolates was MicrobiolRev 1995; 8: 515–548. 1 1 3CasadevallA, PerfectJ. Cryptococcusneoformans .Washington, 0.037–0.25 mg l ¡ (0.03 mg l¡ in our case) [7,8]. John- DC:ASMPress, 1998. son et al. [8] reported two blood-culture isolates that 4DiamondR, BennettJ. Prognosticfactors in cryptococcal were resistant to ucytosine, as in our case. They also meningitis:a studyof 111 cases. AnnIntern Med 1974; 80: 1 had relatively high MICsto uconazole (8–16 mgl ¡ ), 176–181. which, however, are signiŽcantly lower than our isolate, 5KamalamA, YesudianP, ThambiahAS. Cutaneous infection by and below the breakpoint for other known yeasts such as Cryptococcuslaurentii . Br JDermatol 1977; 97: 221–223. 6KrcmeryV, Jr., KunovaA, MardiakJ. Nosocomial Cryptococ- Candida albicans [27]. Breakpoints for Cryptococcus cus laurentii fungemiain a bonemarrow transplant patient after spp. are not available. To our knowledge, our strain is prophylaxiswith ketoconazole successfully treated with oral the only clinical isolate of C.laurentii that has proven uconazole. Infection 1997; 25: 130.

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