Non-albicansoralcandidosis in HIV-positive patients

J. D. Cartledge, J. Midgley and B. G. Gazzard*

Department of HIV and Genitourinary Medicine, St Stephen's Centre, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specimens from HIV-positive patients with oral candidosis were taken for culture, species identification and azole susceptibility testing, which was correlated with treatment outcome. Of 921 specimens, 95 yielded non-albicans species, mainly from patients with low CD4 lymphocyte counts and extensive previous azole exposure. Most non-albicans isolates were from specimens co-infected with Candida albicans, complicating the interpretation of in-vitro susceptibility results, which accurately predicted antifungal failure when the non-albicans species was isolated alone. Eighty-five non-albicans isolates were resistant to fluconazole in vitro. Of 149 courses of azole therapy prescribed, 115 failed to clear non-albicans candidosis clinically. Culture media that discoloured in the presence of non-albicans colonies might, therefore, guide therapy.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oral candidosis frequently occurs in HIV infection and initially is easily treated with azole antifungals.1,2 However, later in disease, failure of standard azole therapy is not uncommon in fluconazole-treated patients. Clinically resistant candidosis is most commonly associated with Candida albicans infection but other species have been isolated. Although some of these non-albicans species have reduced azole susceptibility in vitro, their pathogenic importance has been questioned;3 their significance would be clarified if responses to treatment of patients infected with these organisms could be correlated to their in-vitro susceptibility. We have reviewed our patients with candidosis yielding non-albicans species, determined the in-vitro antifungal susceptibilities of these organisms and related this to clinical response. The susceptibility test chosen4 has been found accurately to predict clinical fluconazole,5 itraconazole6 and ketoconazole 7 failure for HIV-positive patients infected with C. albicans.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HIV-positive patients with oral candidosis gave mouth swabs and rinses for culture, species identification and susceptibility testing. Patients were treated with a 7 day standardized azole regime (ketoconazole 200 mg bd, itraconazole solution 200 mg bd, or fluconazole 100 mg od) and the outcome was assessed on day 7, classified as either success (clearance of all signs and symptoms) or failure (where there was persistent candidosis). Patients receiving hepatic enzyme inducers were excluded from this analysis. Where patients failing after a 7 day course of one azole were given a different agent, the susceptibility of the original isolate was correlated with the result of the second agent also.

Mycology

Species identification.Clinical specimens were inoculated on to Sabouraud dextrose agar and incubated for 48 h at 37°C; sample colonies of each morphology present were selected and identified at species level using commercially available API kits (BioMérieux, Basingstoke, UK), examination for chlamydospore production on Rice Tween Agar (BioMérieux) medium and assessment of germ tube production in horse serum.

Susceptibility testing.The relative growth method of azole susceptibility testing, developed by Odds,7 was employed. In three previous studies response to standardized azole treatment (as above) has been correlated with in-vitro susceptibility to establish the degree of resistance for C. albicans that resulted in failure of a particular drug at the standard dose. Having established the best cut-off points for predicting treatment failure for C. albicans (relative growth of 80% for fluconazole, 68% for itraconazole and 75% for ketoconazole) it was possible in the current study to assess whether the same degree of in-vitro resistance carried the same clinical consequences with non-albicansspecies. Where the non-albicansisolate was mixed with C. albicans on the plate, the relative growth of the non-albicans isolate was used in the assessment of the test's predictive value.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of our 921 clinical isolates identified at species level, 526 (57%) were fluconazole-susceptible C. albicans and 300 (32%) were fluconazole-resistant C. albicans. Ninety-five (10%) clinical specimens from 52 HIV-positive patients with pseudomembranous candidosis yielded non-albicans isolates. A total of 97 non-albicans isolates were grown, as two species yielded a mixture of two different non-albicans species. Candida glabrata was the commonest non-albicans species isolated, followed by Candida krusei (Table I). Most of the specimens yielding a non-albicans isolate also grew C. albicans.


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Table I. Frequency of isolation of each non-albicans species from clinical specimens
 
Patient characteristics

All the patients infected with non-albicansspecies had low CD4 counts (median, 15 cells/mm3; range, 2-230 cells/mm3), and extensive previous azole treatment for recurrent candidosis (median, 24 months; range, 2-94 months). The majority of non-albicans species were isolated from patients with clinical azole resistance and <3 months after first fluconazole failure.

Correlation of in-vitro susceptibility results and clinical response

Twenty-five treatment episodes were assessed for patients whose specimens yielded a pure growth of a non-albicans species, and in 23 of these cases the in-vitro susceptibility correctly predicted clinical response, achieving a sensitivity of 90% and specificity of 100%. Most specimens, however, yielded a mixed infection, often with discordant susceptibility to the treatment used. If the susceptibility of the non-albicans isolate was used to predict outcome for all treatment episodes, including those where mixed infections with discordant susceptibility were isolated, the sensitivity of the test was 93% and the specificity 76%.

All 57 C. glabrataisolates were resistant to fluconazole and ketoconazole in vitro and 12 (22%) were cross-resistant to itraconazole. In general, clinical response followed that suggested by the in-vitro susceptibility result, with 90 of the 95 treatment episodes for patients infected with C. glabrata having the outcome predicted by the susceptibility of that organsism (Table II). Of the 79 treatment episodes where C. glabratawas present as part of a mixed infection, the in-vitro susceptibility of the C. glabrata isolate accurately predicted clinical response in 75 cases. In 22 cases there was disagreement between the susceptibility of the C. glabrataisolate and the co-infecting C. albicans isolate—19 where the former was resistant and the latter susceptible, and three with the reverse situation—clinical outcome followed the result of the C. glabrata in 18 of these 22 cases.


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Table II. Treatment outcome of patients with pure or mixed non-albicans infection (figures for pure alone are in brackets) with non-albicans species resistant (R) or susceptible (S) in vitro to the antifungal given
 
All C. krusei isolates were resistant to fluconazole and ketoconazole but susceptible to itraconazole in vitro. Fluconazole failed in 16 of 20 patients infected with C. krusei, all four where it did work being co-infected with fluconazole-susceptible C. albicans. Itraconazole solution was successful in all four infected with C. krusei who took it (Table II).

Only two specimens yielded pure cultures of other non-albicans species, Candida tropicalisand Candida guillermondi, both with resistance to fluconazole in vitro and both from fluconazole-unresponsive patients (Table II).

Seven isolates of Saccharomyces spp. were resistant to fluconazole in vitro, five to ketoconazole and two to itraconazole. All patients treated with a drug to which the Saccharomyces strain was resistant failed therapy, including four cases where the patient was co-infected with a susceptible C. albicans isolate (Table II). Of the six isolates ofC. tropicalis, three were fully susceptible to azoles in vitro and the remaining three (all resistant to fluconazole in vitro) were from fluconazole-unresponsive patients (Table II).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been suggested that, in the context of HIV-related oral candidosis, isolates of non-albicans species of Candida are commensals, rather than pathogens and that their isolation is of no clinical relevance.3 All the isolates in our study were obtained from patients with symptomatic pseudomembranous oral candidosis, and in 25 cases a non-albicans species was the only fungus grown. In 23 of these 25 cases the patient's response was accurately predicted by the in-vitro azole resistance profile of the non-albicans isolate, further supporting the clinical significance of these organisms. Furthermore, in 31 of 39 cases where a resistant non-albicans isolate was cultured together with a susceptible C. albicans isolate, the patient failed to respond.

Ten percent of the specimens obtained from patients attending our candida clinic yielded a non-albicans species, though since referral to the clinic was more likely if the patient had unresponsive candidosis, this may overestimate the prevalence of these organisms. Of the 100 non-albicans isolates, 88 were fluconazole-resistant, which represented almost one quarter of the total fluconazole-resistant Candida isolates obtained from our whole cohort. The HIV-positive patients infected with non-albicans species of Candida were severely immunosuppressed, had long histories of recurrent candidosis requiring treatment and, in many cases, had candidosis unresponsive to standard azole therapy.

C. glabratawas the commonest non-albicansspecies isolated, then C. krusei, Saccharomycesspp. and C. tropicalis (Table I). With regard to individual species, our findings would support a pathogenic role for C. glabrataandC. krusei,which were detected as pure infections in some of our cases and, when mixed with susceptible C. albicans isolates, were associated with non-response in keeping with their resistance in vitro to the treatment used.

Pure cultures of C. tropicalisand C. guillermondi were isolated from single patients, suggesting a possible, though uncommon, role for these organisms in AIDS-related candidosis. Also, where resistant isolates of Saccharomycesspp.were mixed with susceptible C. albicans, treatment failure was observed in four cases, suggesting that this organism might also be clinically relevant.

The pathogenic role of these non-albicans species can only be suggested from our data, since our clinical specimens may not have detected all of the isolates present and pathogenic C. albicans isolates may have been missed. However, both C. glabrata and C. kruseihave been associated with clinical disease in other patient populations.8,9 Even if the presence of non-albicans species is not causally related to treatment failure, their detection predicts non-response to standard azole therapy. The use of culture media which readily identify non-albicans species by differences in colony colour (e.g. Chromagar (Becton Dickinson, Oxford, UK)) could assist clinical decision-making since 85 of our 95 non-albicans isolates were fluconazole-resistant in vitro. In centres with the potential for antifungal susceptibility testing, the relative growth method4 appears applicable to non-albicans isolates as well as C. albicans with the same cut-off values being predictive of clinical failure of standard azole treatment regimen.


    Notes
 
* Corresponding author. Tel: +44-181-746-8239; Fax: +44-181-746-5611 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Smith, D. G., Midgley, J., Allan, M., Connolly, G. M. & Gazzard, B. G. (1991). Itraconazole versus ketoconazole in the treatment of oral and oesophageal candidosis in patients infected with HIV. AIDS 5, 1367–71.[ISI][Medline]

2 . De Wit, S., Weerts, D., Goosens, H. & Clumeck, N. (1989). Comparison of fluconazole and ketoconazole for oropharyngeal candidiasis in AIDS. Lancet i, 746–7.

3 . Dronda, F., Chaves, F., Alonso-Sanz, M., Laguna, F., Martinez-Suarez, J. V., Rodriguez-Tudela, J. L. et al. (1994). Clinical significance of mixed oropharyngeal candidiasis due to Candida albicans and non-albicans strains in HIV-infected patients. In Program and Abstracts of the Thirty-Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, FL, 1994. Abstract I215, p. 212. American Society for Microbiology, Washington, DC.

4 . Odds, F. C. (1992). Antifungal susceptibility testing of Candida spp. by relative growth measurement at single concentrations of antifungal agents. Antimicrobial Agents and Chemotherapy 36, 1727–37.[Abstract]

5 . Cartledge, J. D., Midgley, J. & Gazzard, B. G. (1996). Relative growth measurement of Candida species in a single concentration of fluconazole predicts the clinical response to fluconazole in HIV-infected patients with oral candidosis. Journal of Antimicrobial Chemotherapy 34, 275–83.[ISI][Medline]

6 . Cartledge, J. D., Midgley, J. & Gazzard, B. G. (1997). Itraconazole cyclodextrin solution: the role of in vitro susceptibility testing in predicting successful treatment of HIV-related fluconazole-resistant and fluconazole susceptible oral candidosis. AIDS 11 ,163 –8.[ISI][Medline]

7 . Cartledge, J. D., Midgley, J. & Gazzard, B. G. (1997). Clinical response to ketoconazole of HIV-related oral candidosis is predicted by Odds's relative growth method of susceptibility testing. Journal of Antimicrobial Chemotherapy 40, 117–9.[Abstract]

8 . Wingard, J. R., Merz, W. G., Rinaldi, M. G., Johnson, T. R., Karp, J. E. & Saral, R. (1991). Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. New England Journal of Medicine 325, 1274–7.[Abstract]

9 . Wingard, J. R., Merz, W. G., Rinaldi, M. G., Miller, C. B., Karp, J. E. & Saral, R. (1993). Association of Torulopsis glabrata infection with fluconazole prophylaxis in neutropenic bone marrow transplant patients. Antimicrobial Agents and Chemotherapy 37 , 1847–9.[Abstract]

Received 12 January 1998; returned 16 March 1998; revised 30 April 1998; accepted 27 July 1998





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