JOURNAL OF CLINICAL MICROBIOLOGY, May 1989, p. 927-9310095-1137/89/050927-05$02.00/0Copyright (O 1989, American Society for Microbiology

Clinical, Microbiological, and Experimental Animal Studies ofCandida lipolytica

THOMAS J. WALSH 1.2.3* IRA F. SALKIN,4 DENNIS M. DIXON,4 AND NANCY J. HURD4

Infectious Diseases Section, National Cancer Institute, Bethesda, Maryland 208921*; Baltimore Veterans AdministrationMedical Center, Baltimore, Maryland 212182; Department of Medicine, University ofMaryland School of Medicine,

Baltimore, Maryland 212013; and Wadsworth Center for Laboratories and Research, New York StateDepartment of Health, Albany, New York 122014

Received 28 July 1988/Accepted 28 December 1988

Candida lipolytica was recovered from six patients in three different clinical centers. The index isolate causeda persistent fungemia with catheter-associated Candida thrombophlebitis, the second isolate was from a

polymicrobial sinusitis, and the remaining four isolates were involved in tissue colonization. These and 20 otherisolates were consistent in their morphological and physiological characteristics. Ail formed true hyphae andblastoconidia on cornmeal-Tween 80 agar and all assimilated glucose, glycerol, and erythritol. In a murinemodel of disseminated candidiasis, the index isolate that caused clinical fungemia caused no mortality andproduced only two lesions on a kidney, as determined at necropsy. The nine isolates selected for in vitroantifungal susceptibility studies had interniediate susceptibilities to amphotericin B but were susceptible toketoconazole. We conclude that C. lipolytica is a weakly virulent pathogen which may require an intravascularforeign body to cause fungemia.

Candida lipolytica (Harrison) Diddens et Lodder hasseldom been identified as a cause of infection. Whenfungemia caused by C. lipolytica developed in one of our

patients, we found that there was no single study whichexamined the clinical manifestations, microbiological fea-tures, and in vivo characteristics of this fungus. C. lipolyticawas not included in several large case series as a cause offungemia (3, 7, 8, 13). Only one well-described case offungemia (18) and three cases of traumatic ocular infection(11) caused by C. lipolytica have been reported previously.While the morphological and physiological properties of C.lipolytica have been described in standard monographs (2,10), little is known about the characteristics of C. lipolyticain the new rapid diagnostic systems for yeasts. There arevirtually no studies of the in vitro susceptibility of C.lipolytica to antifungal agents. Finally, with the exception ofone study in which a strain from a nonclinical source wasused (6), there were no reported in vivo studies in which thevirulence of C. lipolytica has been evaluated.

Since relatively little was known about C. lipolytica, westudied the clinical features of six cases of infection orcolonization involving C. lipolytica, investigated the mor-

phological characteristics and biochemical properties of 26isolates, and evaluated the antifungal susceptibilities of 9isolates of this yeastlike fungus. We particularly examinedthe properties of these isolates of C. lipolytica in a new rapidyeast identification systems. We also studied the virulence ofthe isolate that caused clinical fungemia in experimentalanimals. Since clinical isolates of C. lipolytica were misiden-tified as Candida ingens van der Walt et van Kerken, we alsostudied the clinical microbiological features of this saprobicfungus in comparison with those of C. lipolytica.

MATERIALS AND METHODS

Cases. The six clinical cases of C. lipolytica infection wereidentified between 1980 and 1987 at one of the followinginstitutions: Baltimore Veterans Administration Medical

* Corresponding author.

Center, Baltimore, Md.; University of Maryland MedicalSystems, Baltimore; or the Clinical Center of the NationalInstitutes of Health, Bethesda, Md. Cases of infection were

classified as fungemia, localized infection, or colonization.(i) Fungemia. Fungemia and suppurative thrombophlebitis

developed in a 54-year-old male with a history of alcoholabuse who underwent cholecystectomy for cholelithiasis andcholecystitis. After cholecystectomy and placement of a Ttube in the common bile duct, he received six differentantibacterial agents in different combinations over 26 daysfor cholangitis. Blood cultures obtained on day 27 of hospi-talization for a fever of 39.2°C grew a pure culture of a germtube-negative Candida sp. in a bottle (6 A) of a radiometricblood culture system (BACTEC 460; Becton-Dickinson Di-agnostic Instrument Systems, Towson, Md.) after 24 h ofincubation at 35°C. Although the isolate was identifiedinitially as C. ingens, further studies with the API 20Csystem (Analytab Products, Plainview, N.Y.) confirmed itsidentification as C. lipolytica (5697-82). Several colonies thatwere sampled yielded consistent results indicating that theywere C. lipolytica. Antifungal therapy was not administeredat this time or later in the hospitalization.An intravenous catheter-associated thrombophlebitis of

the right forearm was found on day 28 of hospitalization. Thecatheter was removed, and a Trypticase soy broth (BBLMicrobiology Systems, Cockeysville, Md.) culture inocu-lated with a purulent discharge from the venipuncture siteyielded C. lipolytica. Fever persisted and another set ofblood cultures obtained on day 32 of hospitalization grew C.lipolytica in the BACTEC system. No other organisms werecultured.

Daily physical examinations performed from the time ofdetection offungemia revealed no chorioretinal or cutaneouslesions, no cardiac murmurs, and no hemodynamic instabil-ity. There was no azotemia, and urine cultures showed no C.lipolytica. Although the erythema, swelling, and tendernessof the peripheral thrombophlebitis decreased, fever per-sisted and a third set of blood cultures obtained on day 39yielded C. lipolytica. Thrombophlebitis and fever resolved

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and two more sets of blood cultures were negative. Thepatient was discharged, and no evidence of recurrent ordisseminated infection was found during 7 months of follow-up care.

(ài) Localized infection. A 46-year-old female presentedwith an acute exacerbation of chronic sinusitis. Radiographsof the paranasal sinuses revealed opacifications of the leftmaxillary sinus. A Gram stain of the specimen revealedinflammatory cells, bacteria, and occasional budding yeastsand hyphae. Cultures inoculated with the sinus aspiratematerial on several media, including 5% sheep blood-Tryp-ticase soy agar, Sabouraud glucose agar with chloramphen-icol and gentamicin, and brain heart infusion broth withchloramphenicol and gentamicin yielded Staphylococcusaureus Rosenbach, Pseudomonas aeruginosa (Schroeter)Migula, and C. lipolytica (118-88). Since postnasal dischargecontinued despite antibacterial therapy, ketoconazole (200mg orally) was initiated to treat the fungal component of theinfection. The patient reported improvement of symptomswithin 1 month after ketoconazole therapy was started. C.lipolytica was not recovered again after the completion ofthe course of ketoconazole.

(iii) Colonization. Stool (120-88), oropharyngeal swab(119-88, 121-88), and sputum (7755-83) specimens from fourpatients with no evidence of invasive candidiasis yielded C.lipolytica.

Test organisms. Twenty isolates of C. lipolytica fromvarious sources were provided from collections of the Mary-land State Health Laboratories; the University of MarylandSchool of Medical Technology; and the Laboratories forMycology, Wadsworth Center for Laboratories and Re-search, New York State Department of Health. CletusKurtzman of the Northern Regional Research Center, U.S.Department of Agriculture, provided five isolates of C.ingens (Table 1). These and a single isolate from each of thepatients discussed above were maintained at 4°C on Sab-ouraud glucose agar and serially transferred at monthlyintervals to fresh nutrient medium.

Microbiological studies. For all microbiological studies, aportion of growth from a stock culture of each isolate wastransferred aseptically to fresh Sabouraud glucose agar andincubated for 72 h at 27°C. The morphological features of allisolates were studied by inoculating a portion of each 72-h-old culture onto cornmeal-Tween 80 agar in 100-mm-diameter petri plates. Their assimilation and enzyme profileswere investigated with the API 20C and Yeast-Ident yeastidentification kits (Analytab Products). The commercialproducts were inoculated with colonies from a 72-h-oldculture, and the tests were conducted as directed by themanufacturer. In addition, conventional methods were usedto study the following characteristics of the isolates: ureaseactivity; glucose fermentation; and assimilation of erythritol,galactose, and growth in vitamin-free medium (15).The in vitro susceptibilities on nine isolates to amphoter-

icin B (Fungizone IV; E. R. Squibb & Sons, Princeton, N.J.)were evaluated by a tube dilution procedure (4). A suspen-sion of yeast cells of each isolate was prepared by flooding a

72-h-old Sabouraud glucose agar slant with sterile distilledwater and adjusting the final concentration to 50% transmis-sion at 650 nm with a spectrophotometer (Beckman Instru-ments, Inc., Fullerton, Calif.); this corresponded to 1.8 x107 cells per ml, as determined with a hemacytometer. Aportion of the suspension was then added to each tube of atwofold serial dilution series of amphotericin B ranging from4.0 to 1.9 x 10-3 ,ug/ml. Candida albicans (Robin) Berkhout(Squibb 1539) served as a control for ahl studies.

TABLE 1. Codes and sources of isolates of C. lipolyticaand C. ingens

Organism and Site Source"code no.

C. lipolytica2029-79 Unknown NYSDH2751-82 Sputum NYSDH605-84 Left leg NYSDH565-84 Sputum NYSDHNYS 98A Unknown NYSDHNYS 103 Unknown NYSDH1250-85 Skin rash scrapings NYSDH1542-85 Unknownb NYSDH16-86 Sputum NYSDH1034-86 Bronchial washing NYSDH84M27 Unknown' API 20C84M28 Unknown' API 20C84M29 Unknownc API 20C84M245 Unknown' API 20C85MR89 Unknownc API 20CYB423 Corn processing plant NRRL and ATCC

(ATCC 18942)(Yarrowia lipolytica;type culture)

YB423-3 Unknown NRRL and ATCC 189435697-82 Blood BVAMC7755-83 Sputum BVAMC118-88 Sinus aspirate NYSDH119-88 Oropharynx NYSDH120-88 Stool NYSDH121-88 Oropharynx NYSDH122-88 Unknown NYSDH123-88 Unknown NYSDH124-88 Unknown NYSDH

C. ingensY6053 Unknown NRRLY7796 Unknown NRRLY7797 Unknown NRRLY7930 Piggery waste NRRLY10939 Kamaboko (minced fish) NRRL

" NYSDH, New York State Department of Health; API 20C, AnalytabProducts; NRRL, Northern Regional Research Laboratory (now designatedthe Agricultural Research Service), U.S. Department of Agriculture; ATCC,American Type Culture Collection, Rockville, Md.; BVAMC, BaltimoreVeterans Administration Medical Center.

'Proficiency test isolate; New York State Department of Health.Isolates used as standards in API 20C kit (Analytab Products).

The susceptibilities of the same C. lipolytica isolates toketoconazole (Nizoral; Janssen Pharmaceutica, Piscataway,N.J.) were assessed with an agar dilution series (4, 5). Ayeast cell suspension was prepared as described above, andthe concentration was adjusted to 85% transmission at 530nm with a spectrophotometer; this corresponded to 3.4 x 106cells per ml. A portion of the suspension was added to thesurface of nutrient agar plates containing a twofold serialdilution series of ketoconazole ranging from 40 to 2 x 1i-Op.g/ml. Kluyveromyces marxianus var. marxianus (Hansen)van der Walt (NYS 110A) served as a control for all studies.The assays for amphotericin B and ketoconazole weredeveloped as separate assays (4, 5) in which different inoc-ulum preparation methods were used. Although the wave-lengths and percent transmission for the preparation ofinocula were different for amphotericin B and ketoconazole,the final concentrations of cells measured with a hemacy-tometer were similar.The virulence of the fungemia isolate (isolate 5697-82) was

studied in a murine model of experimental disseminated

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candidiasis. This isolate was preserved (after one subcul-ture) in sterile distilled water at 4°C. A clinical isolate (UM83-7700) of C. albicans (Robin) Berkhout served as thevirulence control. A portion of growth of each test isolatewas aseptically streaked over the surface of a Sabouraudglucose agar plate and grown overnight at 37°C. A singleisolated colony was removed with a sterile loop and asepti-cally transferred to 50 ml of glucose-peptone-yeast extractbroth contained in a 250-ml Erlenmeyer flask and grown in agyrarotary shaker at 120 rpm for 2 h at 37°C. Five millilitersof broth from the flask was then aseptically removed with apipette, transferred to each of four similarly prepared Erlen-meyer flasks, and grown overnight at 37°C. The entire brothvolume of each Erlenmeyer flask was centrifuged at 9,200 xg for 15 min at 4°C. The pellets were suspended and washedthree times in sterile normal saline. The concentration ofcells was adjusted with a hemacytometer to an inoculum10-fold dilution series of 104 to 108 CFU. The concentrationswere confirmed by quantitative colony counts on Sabouraudglucose agar plates inoculated with 10-fold serial dilutions ofeach inoculum concentration.Groups of two male CD-1 mice (Charles River, Breeding

Laboratories, Inc., Wilmington, Mass.) each received 0.1 mlof one of the five 10-fold dilution series of each inoculum ofeach fungus via the lateral tail vein. All mice were housed,maintained, and treated as described in the Guide for theCare and Use of Laboratory Animals (Publication 85-23,National Institutes of Health, Bethesda, Md.). Mice wereeuthanized when they were moribund; the remaining micewere euthanized at 14 days after inoculation. Brain, liver,spleen, and kidneys were examined postmortem and cul-tured quantitatively.

RESULTS

Morphological and physiological studies. C. lipolyticaformed distinctive cerebriform, convoluted, white, firm col-onies on Sabouraud glucose agar (Fig. 1). All C. lipolyticaisolates formed narrow, multibranched, true hyphae oncornmeal-Tween 80 agar. Blastoconidia, which generallydeveloped only after several days of incubation, were foundat the apices and less commonly along the length of thehyphae. In contrast, isolates of C. ingens on the samemedium formed chains of budding yeast cells and broadpseudohyphae without blastoconidia. No true hyphae wereobserved in the cultures of C. ingens on cornmeal-Tween 80agar.As a group, the isolates of C. lipolytica were physiologi-

cally consistent. Ail were urease positive and none fer-mented glucose. In conventional Wickerham assimilationtests, all isolates assimilated glucose, glycerol, and erythri-tol. None utilized galactose, with the exception of oneisolate (isolate 121-88), which was weakly positive for galac-tose assimilation. Growth was negative or weakly positive invitamin-free medium for all isolates with the exception ofone isolate (isolate 120-88), which was positive. Maximumgrowth temperatures fared between 35 and 37°C, with theexception of that for one isolate, which did not grow at 30°C.The isolate that caused fungemia grew at 37°C during threetrials and at 35°C during a fourth trial. Isolates gave either a6-002-100 code or a 6-000-100 code when grown in the API20C system. Both of the API 20C codes indicated that therewas a good likelihood that C. lipolytica was the organism.All isolates were identified with the Yeast-Ident kit as C.lipolytica with biocodes of 2-067-573 (good likelihood; C.lipolytica most probable), 2-067-173 (good likelihood; C.

FIG. 1. Typical colony of C. lipolytica derived from point inoc-ulation and incubated for 6 days at 270C on Sabouraud glucose agarsupplemented with penicillin and streptomycin.

lipolytica most probable), 2-167-573 (good identification forC. lipolytica), and 2-167-173 (acceptable identification for C.lipolytica).

Isolates of C. ingens were physiologically more variablethan those of C. lipolytica. Three of five isolates were ureasepositive. None fermented glucose and none assimilatederythritol, as determined by the conventional Wickerhamassimilation test. Two isolates (isolates Y-7796 and Y-7797)assimilated galactose within 72 h of inoculation in theWickerham test, but the remaining three isolates (isolatesY-7930, Y-6053, and Y-10939) only began to utilize thiscarbohydrate source after 7 to 10 days of inoculation in theWickerham test. While two isolates (isolates Y-7796 andY-7797) could not develop in a vitamin-free broth, the otherthree isolates developed rapidly in this medium. A code of6-040-000 was found with four isolates in the API 20C kit anda code of 6-000-000 was noted with the remaining isolate.Since C. ingens was not included in the API 20C data base,these codes merely indicated variations in the assimilationcapabilities of the test isolates. Similar differences were

observed with the Yeast-Ident system, in which C. ingensalso was not included in the data base. Two isolates (isolatesY-6053 and Y-10939) were identified by the Yeast-Ident kitas Candida tropicalis (Castellani) Berkhout, and one (isolateY-7797), was identified as Candida guillermondi (Castellani)Langeron et Guerra. No corresponding codes could be foundfor the two remaining isolates (isolates Y-7930 and Y-7796).

In vitro antifungal susceptibility. The mean MIC of ampho-tericin B after 24 h of incubation of the nine test isolates ofC. lipolytica was 0.660 ,ug/ml (range, 0.313 to 1.25 ,ug/ml).All test isolates were susceptible to ketoconazole, which hada mean MIC of 0.22 ,ug/ml (range, 0.078 to 0.313 ,ug/ml). TheMICs for each isolate are presented in Table 2.

Virulence. All mice receiving C. albicans died of dissem-inated candidiasis within the 2-week experimental period.Autopsy revealed multiple abscesses caused by C. albicansin brain, liver, spleen, and kidneys. In contrast, all mice

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TABLE 2. In vitro susceptibility of C. lipolytica toamphotericin B and ketoconazole

MIC (,ug/mljAccession no.

Amphotericin B Ketoconazole

118-88 0.313 0.078119-88 0.313 0.313120-88 1.25 0.156121-88 0.625 0.156122-88 0.625 0.313123-88 0.625 0.156124-88 0.313 0.0785697-82 0.625 0.3137755-83 1.25 0.313

inoculated with C. lipolytica survived the experimentalperiod. Two abscesses were found postmortem in the leftkidney of a mouse that received the highest inoculum (108CFU). Cultures inoculated with portions of the kidney tissueyielded C. lipolytica. No fungus was revealed in quantitativecultures of tissue from other organs of this mouse or fromany of the other mice inoculated with C. lipolytica. Since theinfected kidney specimen was processed for quantitativecultures, histopathological studies were not performed.

DISCUSSION

C. lipolytica is a weak pathogen that is associated mostclearly with vascular catheter-related fungemia. The 26isolates of C. lipolytica examined in this study demonstratedconsistent morphological and physiological characteristics.The C. lipolytica isolates could be differentiated easily fromthe more physiologically variable isolates of C. ingens bycolonial morphology, formation of true hyphae on cornmeal-Tween 80 agar, and an inability to assimilate galactose as thesole carbon source. To our knowledge, C. ingens has notbeen described as a human pathogen.The absence of clinically overt deep visceral infection in

the index patient with persistent fungemia and the favorableoutcome without the use of antifungal therapy suggests thatC. lipolytica is a weakly virulent pathogen. This observationis further substantiated by the report of Wherspann andFullbrandt (18), who described another case of fungemiacaused by C. lipolytica. That patient also had fungemiawithout evidence of deep visceral infection, such as endoph-thalmitis, osteomyelitis, arthritis, or hepatic infection. More-over, the absence of mortality and development of only twolesions on one of many visceral organs sampled from miceinoculated with C. lipolytica indicates that this species isweakly virulent.The index case reported here and the other documented

case (18) of C. lipolytica fungemia were associated withintravenous catheter infections. These two cases suggestthat a vascular catheter may be required for the introductionof C. lipolytica into the bloodstream. Although the infectedvenous catheter was removed from the index patient,fungemia persisted. This persistent fungemia was likelycaused by an infected catheter-associated mural thrombus(14, 16, 17). Segmental peripheral venous resection often is

effective in eliminating Candida thrombophlebitis as a

source of candidemia.In our opinion, most cases of vascular catheter-associated

candidemia should be treated with a course of amphotericinB and, when possible, the vascular catheter should beremoved because of the risks of subsequent hematogenous

Candida endophthamitis and othér complications of un-treated candidemia. However, to our knowledge C. lipolyt-ica has not been associated with deep visceral infections,such as hematogenous endophthalmitis or hepatospleniccandidiasis.Given the low virulence of C. lipolytica, one must ques-

tion whether removal of a vascular catheter without theconcomitant administration of amphotericin B would beappropriate management of catheter-associated candidemiacaused by this organism. Infections in both our patient andthe previously reported patient with C. lipolytica fungemia(18) were associated with vascular catheters and neither wasapparently complicated by a deep visceral infection. Never-theless, until more experience is acquired in managing thisinfection, we suggest that catheter-associated C. lipolyticafungemia be managed in a similar manner as that of otherCandida spp. fungemias: a course of systemic antifungaltherapy and removal of potentially infected vascular cathe-ters.Amphotericin B is the cornerstone of treatment of candi-

demia caused by susceptible Candida spp. The dosage andduration of amphotericin B therapy for candidemia dependon the extent of infection and the type of host. The nine C.lipolytica isolates that we tested were found to be suscepti-ble to achievable concentrations of amphotericin B in serum(0.313 to 1.25 ,ug/ml). These concentrations are usuallyachieved when amphotericin B is administered intrave-nously at 0.5 mg/kg per day (1, 9). If fungemia persistsdespite amphotericin B therapy, consideration should begiven to resection of any potentially infected catheter-asso-ciated peripheral venous thrombus.An antifungal azole, however, may be considered in

selected patients as a possible alternative for the treatmentof C. lipolytica infections. All isolates tested in this studywere susceptible to ketoconazole. The patient reported byWherspann and Fullbrandt (18) responded successfully toremoval of the infected venous catheter and administrationof ketoconazole; the patient with sinusitis had an apparentimprovement from ketoconazole therapy. Perhaps localizedmucosal infections caused by C. lipolytica may be amenableto ketoconazole therapy. However, since the correlationbetween in vitro and in vivo effects of ketoconazole are oftenvariable (12) and since therapeutic experience with C. lipoly-tica is limited, such patients treated with ketoconazolerequire careful selection and follow-up. Investigational anti-fungal triazoles, such as fluconazole and itraconazole, alsomay be active against isolates of C. lipolytica and may bemore active than ketoconazole. We suggest that antifungalazoles be considered as a possible alternative to amphoter-icin B in the management of selected patients with C.lipolytica infections.The results of this investigation of C. lipolytica indicate

that (i) C. lipolytica is a weakly virulent pathogen associatedwith vascular catheter infections, (ià) C. lipolytica is lessvirulent in experimental animals than is C. albicans, (iii)isolates of C. lipolytica, are susceptible in vitro to ampho-tericin B as well as to ketoconazole, and (iv) isolates of C.lipolytica demonstrate generally consistent morphologicaland physiological characteristics.

ACKNOWLEDGMENTS

We thank Ed Lapa for assistance in conducting the in vitroantifungal susceptibility studies. We thank Sharon Hansen, MarciaMoody, Frank Witebsky, Cletus Kurtzman, Nancy Hooper, SherrilRoss, and Lynn Still for identifying and providing isolates of C.

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lipolytica for this study. We also thank Philip A. Pizzo and MarkMiller for reviewing the manuscript.

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