Journal of Antimicrobial Chemotherapy (2000) 46, 291-295
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Development of simultaneous resistance to fluconazole in Candida albicans and Candida dubliniensis in a patient with AIDS
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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In this report, we describe a patient with recurrent episodes of oral candidosis who finally suffered from fluconazole-refractory oral and oesophageal candidosis. The patient was monitored for 4 years until his death from AIDS. During the observation period, persistent colonization with both Candida albicans and Candida dubliniensis was observed. From the appearance of the first episode of oral candidosis, the patient was treated with fluconazole for 18 months. The infection became unresponsive to fluconazole 400 mg/day. In vitro susceptibility testing revealed the development of resistance to fluconazole in C. albicans and C. dubliniensis. Molecular typing confirmed the persistence of the same C. albicans and C. dubliniensis strains which developed resistance after up to 3 years of asymptomatic colonization. This observation demonstrates that Candida spp. other than C. albicans may develop resistance to fluconazole in a patient who is repeatedly exposed to the drug.
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Introduction |
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Most cases of oropharyngeal candidosis (OPC) are caused by Candida albicans. OPC refractory to therapy with fluconazole has been reported frequently in human immunodeficiency virus (HIV)-infected patients after long-term use of fluconazole.1 Development of resistance in C. albicans in vitro often correlates with clinical refractory disease and the molecular mechanisms for this phenomenon have been studied intensively.2 Candida dubliniensis, a newly described species, has recently been identified as an important cause of mucosal colonization and infection in HIV-infected individuals.3 Development of resistance to fluconazole has been described for this species in vitro but, in contrast to the situation with C. albicans, has not been associated with refractory disease.4
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Materials and methods |
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Case report
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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Methods
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.5–7 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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Sample data, MICs and DNA subtypes obtained during the 4 year observation period are presented in the Table
. Fluconazole and, to a lesser extent, itraconazole MICs for C. albicans and C. dubliniensis isolates increased gradually over time. In C. albicans, the MIC of itraconazole was 0.09 mg/L for isolate Ca1 (the first C. albicans isolate; see Table for isolate numbering and dates) and 1.56 mg/L for isolate Ca18. Similarly, in C. dubliniensis, the MIC of itraconazole was 0.09 mg/L for isolate Cd1 and 1.56 mg/L for isolate Cd16. Two genotypes were identified for the C. albicans isolates, by both AP-PCR (A1 and A2) and CARE-2 fingerprinting (A and B). However, the second genotype was found in only one isolate (Ca11). Using CARE-2 fingerprinting, isolate Ca15 lacks one band compared with Ca1–3, Ca10, Ca17 and Ca18, probably reflecting microevolution of the same strain; this is a well-known phenomenon in an individual infected with Candida spp. Panels (a) and (b) of the Figure illustrate the patterns obtained with AP-PCR for the C. albicans and C. dubliniensis genotypes. All C. dubliniensis isolates showed the same AP-PCR pattern. Panel (c) of the Figure illustrates the CARE-2 hybridization pattern obtained for selected C. albicans isolates. The C. dubliniensis isolates did not hybridize with the CARE-2 probe, so data are not shown.11
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Discussion |
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In 1995, Sullivan et al.5 described a group of atypical Candida isolates, subsequently named C. dubliniensis, from the mouths of HIV-infected individuals. Many of these patients had a history of recurrent oral candidosis and had received fluconazole therapy. For some clinical isolates from two of 15 patients who were previously exposed to fluconazole, fluconazole MICs were high.4 In one of these two patients, fluconazole-susceptible (MIC 0.5 mg/L) and fluconazole-resistant (MIC 32 mg/L) isolates were recovered 18 months apart. The patient had received treatment with rifabutin, which may have altered the pharmacokinetics of fluconazole and thus contributed to the fluconazole-refractory presentation of candidosis. DNA fingerprinting with the C. albicans-specific probe 27A revealed that the profiles of each isolate were significantly different. In contrast, in the patient described here, DNA fingerprinting with AP-PCR and with the C. albicans-specific probe CARE-2 showed that all isolates of each species, except one fluconazole-susceptible isolate of C. albicans, represented a single genotype. According to the molecular typing results, the same C. dubliniensis strain that colonized the mouth of the patient long before symptomatic candidosis occurred persisted over time and became resistant to fluconazole under repeated exposure to the drug. Induction of fluconazole resistance in C. dubliniensis in vitro is similar to that observed in C. albicans but has not been demonstrated to occur clinically.4,12
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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We thank Clarissa Radecke for excellent technical assistance.
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Notes |
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* Correspondence address. Charité Campus Virchow-Klinikum der Humboldt-Universität, Department of Medicine, Division of Haematology/Oncology, Augustenburger Platz 1, 13353 Berlin, Germany. Tel: +49-30-450-59268; Fax: +49-30-450-59910; E-mail: markus.ruhnke{at}charite.de
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References |
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1 . Maenza, J. R., Merz, W. G., Romagnoli, M. J., Keruly, J. C., Moore, R. D. & Gallant, J. E. (1997). Infection due to fluconazole-resistant Candida in patients with AIDS: prevalence and microbiology. Clinical Infectious Diseases 24, 28–34.[Web of Science][Medline]
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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, 557–67.[Web of Science][Medline]
4 . Moran, G. P., Sullivan, D. J., Henman, M. C., McCreary, C., Harrington, B. J., Shanley, D. B. et al. (1997). Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro. Antimicrobial Agents and Chemotherapy 41, 617–23.[Abstract]
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Received 20 September 1999; returned 7 December 1999; revised 25 January 2000; accepted 23 March 2000
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