a Universitätsklinikum Charité Campus Virchow Klinikum, Abteilung für Innere Medizin, Humboldt Universität zu Berlin, D-13353 Berlin; b Zentrum für Zahnmedizin, Universitätsklinikum Charité, D-13353 Berlin and c Zentrum für Infektionsforschung, Universität Würzburg, D-97070 Würzburg, Germany
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Abstract |
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Introduction |
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Materials and methods |
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A 50-year-old bisexual man with HIV infection diagnosed in 1988 presented with a first episode of OPC in January 1994 after uncomplicated progression of HIV disease. The CD4 count was 2 cells/µL; virus load was not available. Apart from a history of herpes zoster in 1989, no other opportunistic infections had been reported. Therapy with fluconazole 100 mg/day for 2 weeks was clinically successful, but there were frequent recurrences during the follow-up period. In April 1994, disseminated Mycobacterium avium intracellulare infection was diagnosed and appropriate anti-tuberculosis therapy was started (rifabutin, clarithromycin and ofloxacin). The patient received intermittent therapy with fluconazole until July 1994, and after July he was given fluconazole 100 mg/day continuously with no further recurrences until December 1994. Antiretroviral therapy was changed from 3'-azido-3'-deoxythymidine and dideoxycytidine (AZT and ddC) to dideoxyinosine (ddI) single therapy in November 1993. Fluconazole was increased to 200 mg/day because of clinical refractory OPC and, in April 1995, was further increased to 400 mg/day. Finally, iv therapy with liposomal amphotericin B, 3 mg/kg/day, was initiated and the OPC resolved temporarily, but this therapy was poorly tolerated because of fevers and rigors. The patient developed cytomegalovirus (CMV) retinitis and was treated with foscarnet but died in July 1995 as a result of disseminated CMV infection and AIDS-related wasting syndrome (see Table).
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All samples from mouth washes were plated on glucose Sabouraud agar and, after June 1994, on CHROMagar (Mast Diagnostika, Reinfeld, Germany); the plates were incubated for 48 h at 30°C. Identification was based on morphology on CHROMagar (C. dubliniensis is dark green) and rice agar, as well as sugar assimilation with the API32ID system (bioMérieux, Marcy l'Etoile, France) and failure to grow at 45°C as previously described.57 Individual colonies of each C. albicans and C. dubliniensis isolate were picked from the primary culture plate on each sampling occasion and used for antifungal susceptibility testing and DNA subtyping. In vitro susceptibility testing with fluconazole and itraconazole was performed on all candida isolates as described earlier.8 DNA subtyping of all C. albicans and C. dubliniensis isolates was performed with arbitrarily primed PCR (AP-PCR) with primer RP02 (5'-GCGATCCCCA-3') and Southern hybridization of EcoRI-digested genomic DNA with the C. albicans- specific repetitive DNA element, CARE-2.9,10
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Results |
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Discussion |
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Our patient suffered from his first episode of OPC only when C. albicans was cultured together with C. dubliniensis (see Table). Other Candida spp. (mostly Candida krusei) were recovered during fluconazole therapy. There are several possible explanations for these results. First, sampling and identification of Candida spp. may not have been as accurate at the start of the sampling period and a mixed infection may not have been detected. CHROMagar, which can distinguish various Candida spp. and is useful for detecting mixed cultures, was not introduced in our laboratory until mid-1994; before then, colonies were picked arbitrarily for further processing. Second, refractory OPC could have developed because of fluconazole resistance in C. albicans and may not have been caused by C. dubliniensis. Evidence for this comes from the fact that isolation of C. dubliniensis from the mouth was not associated with oral disease over a sampling period of several years before the onset of OPC. Third, the patient may have become infected with a new strain of C. dubliniensis between evaluations, but molecular typing with AP-PCR may not have allowed sufficient discrimination between isolates. Recently, specific probes have been described for C. dubliniensis which may produce better discrimination.13 The fact that the C. dubliniensis isolate from the patient became resistant to fluconazole in vitro, but was not isolated again after increasing the dosage of fluconazole despite the persistence of OPC, suggests that this organism was not the primary pathogen. However, the pathogenic role of C. dubliniensis in fluconazole-refractory mucosal candidosis without co-infection with other yeasts is not yet clearly defined and should be studied in larger cohorts.
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Acknowledgments |
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Notes |
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References |
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2
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White, T. C., Marr, K. A. & Bowden, R. A. (1998). Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clinical Microbiology Reviews 11, 382402.
3 . Coleman, D. C., Sullivan, D. J., Bennett, D. E., Moran, G. P., Barry, H. J. & Shanley, D. B. (1997). Candidiasis: the emergence of a novel species, Candida dubliniensis. AIDS 11, 55767.[ISI][Medline]
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5 . Sullivan, D. J., Westerneng, T. J., Haynes, K. A., Bennett, D. E. & Coleman, D. C. (1995). Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141, 150721.[Abstract]
6 . Schoofs, A., Odds, F. C., Colebunders, R., Ieven, M. & Goossens, H. (1997). Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. European Journal of Clinical Microbiology and Infectious Diseases 16, 296300.[ISI][Medline]
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Pinjon, E., Sullivan, D., Salkin, I., Shanley, D. & Coleman, D. (1998). Simple, inexpensive, reliable method for differentiation of Candida dubliniensis from Candida albicans. Journal of Clinical Microbiology 36, 20935.
8 . Ruhnke, M., Eigler, A., Tennagen, I., Engelmann, E., Geiseler, B. & Trautmann, M. (1994). Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and HIV-infection. Journal of Clinical Microbiology 32, 20928.[Abstract]
9 . Lischewski, A., Ruhnke, M., Tennagen, I., Schönian, G., Morschhäuser, J. & Hacker, J. (1995). Molecular epidemiology of Candida isolates from AIDS patients showing different fluconazole resistance profiles. Journal of Clinical Microbiology 33, 76971.[Abstract]
10 . Sullivan, D. J., Henman, M. C., Moran, G. P., O'Neill, L. R., Bennett, D. E., Shanley, D. B. et al. (1996). Molecular genetic approaches to identification, epidemiology and taxonomy of non-albicans Candida species. Journal of Medical Microbiology 44, 399408.[Abstract]
11 . Morschhäuser, J., Ruhnke, M., Michel, S. & Hacker, J. (1999). Identification of CARE-2 negative Candida albicans isolates as Candida dubliniensis. Mycoses 42, 2932.
12 . Calvet, H. M., Yeaman, M. R. & Filler, S. G. (1997). Reversible fluconazole resistance in Candida albicans: a potential in vitro model. Antimicrobial Agents and Chemotherapy 41, 5359.[Abstract]
13
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Joly, S., Pujol, C., Rysz, M., Vargas, K. & Soll, D. R. (1999). Development and characterization of complex DNA fingerprinting probes for the infectious yeast Candida dubliniensis. Journal of Clinical Microbiology 37, 103544.
Received 20 September 1999; returned 7 December 1999; revised 25 January 2000; accepted 23 March 2000