Fluconazole-resistant Candida species in the oral flora of fluconazole-exposed HIV-positive patients

Vol. 85 No. 5 May 1998

ORAL A N D MAXILLOFACIAL PATHOLOGY

Editor: Carl M. A l l e n

Fluconazole-resistant Candida species in the oral flora of fluconazole-exposed HIV-positive patients Keith D. Hunter, BSc (Hons), a John Gibson, BDS, MB ChB, FDS(OM)RCPS(Glasg), FFDRCS(Irel), b Peter Lockhart, DDS, c Alan Pithie, MD, BMSc, MB ChB, DTM&H, d and Jeremy Bagg, BDS, PhD, FDSRCS(Ed), FDSRCPS(Glasg), FRCPath, e Glasgow, United Kingdom, and Charlotte, N.C. UNIVERSITY OF GLASGOW, CAROLINAS MEDICAL CENTER, AND RUCHILL HOSPITAL

The purpose of this study was to examine the effect of preceding fluconazole treatment on the oral mycologic flora and on the sensitivity of oral Candida albicans isolates to fluconazole. Saline oral rinses were collected from 89 HIV-positive patients, of whom 48 had been exposed to fluconazole and 41 were fluconazole-naive. The rinses were cultured on Sabouraud's and Pagano Levin agars, and yeasts were identified by standard methods. Fluconazole sensitivity of C. albicans isolates was measured by disk diffusion assay. C albicans was isolated from 69% of patients who had received fluconazole and from 93% of the patients who were fluconazole-naive (p < 0.05). Nine other species of yeasts were also isolated, most commonly C. glabrata. Five patients previously exposed to fluconazole harbored fluconazole-resistant C. albicans, whereas no resistance was detected among the patients who were fluconazole-naive (p < 0.01). Sixteen of the patients who were fluconazole-exposed carried yeasts other than C albicans, compared with only five patients in the fluconazole-naive group (p < 0.01). All of the fluconazole-resistant strains were isolated from patients with low CD4 counts (less than 100 cells/ml) and after lengthy fluconazole exposures. Nevertheless, patients in Charlotte, N.C., who had a greater mean fluconazole exposure time (10.25 _+1.41 months) than patients in Glasgow, UK, (0.65 _+0.18 months; p < 0.005), did not develop significantly more in vitro resistance or species diversity. This study indicates that longterm fluconazole treatment can have significant effects on the yeast flora of the mouth, particularly in a patient with a CD4 count of less than 100 cells/ml. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:558-64)

Patients infected with HIV experience a range of oral manifestations, many of which reflect progressive immunodeficiency. 10ropharyngeal candidosis, which is strongly associated with HIV infection, is the most common oral manifestation. 2 Increased isolation of Candida species correlates well with low CD4 lymphocyte counts and increasing T-cell dysfunction. 3,4 The most important species is Candida albicans, but others such as C. glabrata, C. tropicalis, and C. krusei can cause infection. These organisms are found as The BDA/MIA Elective Awards, the Cross Trust, the Argyll & Clyde Health Board, and the Renfrewshire Educational Trust provided financial assistance with this study. aFinal-year dental student, University of Glasgow. bClinical Lecturer in Oral Medicine, University of Glasgow. cChairman, Department of Dentistry, Carolinas Medical Center. dConsultant Physician in Infectious Diseases, Ruchill Hospital, Glasgow. ~Senior Lecturer in Oral Microbiology, University of Glasgow. Received for publication Aug. 27, 1997; returned for revision Sept. 25, 1997; accepted for publication Dec. 29, 1997. Copyright © 1998 by Mosby, Inc. 1079-2104/98/$5.00 + 0 7/14/88714

558

commensals in the mouths of 40% (range, 20% to 60%) of the healthy population. 5 In HIV-seropositive populations the proportion of patients carrying Candida species varies from 57.5% for stage 1 disease to 87.5% for stage 3 disease. 6 The three main classes of antifungal agents used in the treatment of oral candldosis are the polyenes (such as nystatin and amphotericin), the imidazoles (such as clotrimazole), and the triazoles (such as fluconazole and itraconazole). A topical, nonabsorbable agent-for example, nystatin or clotrimazole--is the initial treatment of choice and is often used to treat a patient's first episode of candidosis when compliance is likely to be good. When such treatment fails, systemic antifungal agents such as the synthetic bis-triazole fluconazole (Diflucan, Pfizer Ltd) are now widely used. Many clinical trials have shown fluconazole to be both safe and effective,7 especially in HIV-associated candidosis. 8 It is usually prescribed at a dosage of 50 to 100 mg once daily, often with a 200 mg loading dose

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Hunter et al. 559

on the first day.9 Greenspan 5 recommended that treatment should be for no less than 14 days, and in many cases it is prescribed for much longer. In some early studies, a high clinical response was found (93%), although only 70% of cases showed a mycologic cure. 1° The rate of relapse is high; in one study 53% of patients showed microbiologic relapse after 2 weeks. 11 Not surprisingly, therefore, fluconazole has also been used as long-term suppressive prophylaxis in patients who have chronic or repeated episodes of candidosis. Fluconazole is given in dosages ranging from 50 mg every other day to 200 mg per day, 12 and it is more effective than clotrimazole in this regard] Fluconazole now commands one fourth of the world market for antifungal drugs. 13 With such a high level of exposure to one antifungal agent, it is hardly surprising that reports of treatment failure and resistance began to appear. The first cases of clinical resistance to fluconazole in HIV-seropositive patients were reported in 1990,14 and since then the number of reports of azole drug resistance has risen. The development of resistance seems to be related to two factors: a low CD4 lymphocyte count (usually less than 100 cells/ml) and prolonged exposure to a systemic azole. 5 In the past, inadequacies in laboratory sensitivity testing to fluconazole resulted in marked differences between laboratory results and clinical findings. However, as laboratory standardization has improved, and especially since the National Committee for Clinical Laboratory Standards were proposed, 15 it has become apparent that increasing resistance in vitro correlates well with clinical treatment failure. 16,17 The resistance in some cases does not seem to be stable. Law et al. 18 found that in vitro storage of highly resis: tant yeasts in the absence of fluconazole resulted in a loss of resistance by some of them, a phenomenon that was also noted clinically. This has been attributed to the removal of the positive selection pressure, although fluconazole-resistant C. albicans has been reported in patients who were HIV-seronegative and azole-naive. 19 A further potentially important mechanism of resistance is the replacement of fluconazole-susceptible C. albicans strains with other species that are intrinsically less fluconazole sensitive--for example, C. glabrata and C. krusei. 2° In addition, Law et al. 18 found that more than 60% of fluconazole-treated patients harbored more than one strain of C. aIbicans. This may account for some of the differences in fluconazole resistance within the C. albicans population. McCullough and Hume 21 found that both a change in

C. albicans strain and an increase in fluconazole resistance within a single strain had roles to play. The level of resistance is now at such a level that many clinicians are calling for the routine susceptibility testing of all isolates from HIV-seropositive patients) 8,22 The aim of this study was to determine the extent of fluconazole resistance in C. albicans isolates from HIV-seropositive patients who had been treated with fluconazole in comparison with a group of fluconazole-naive HIV-seropositive patients. The extent of overgrowth with less sensitive species Was also studied.

MATERIAL AND METHODS Ethical approval for the project was granted by the Institutional Review Board of the CharlotteMecklenberg Hospital Authority (US) and by the Ethics Committee of The West Glasgow Hospital University NHS Trust (UK). All patients gave written informed consent.

Patients Seventy consecutive HIV-seropositive patients attending the Infectious Diseases Clinics at Carolinas Medical Center, Charlotte, N.C., and 22 consecutive patients attending the Infectious Diseases Clinics at Ruchill Hospital, Glasgow, were asked to provide saline oral rinse samples. Three patients declined to participate.

Oral rinse specimens Patients were asked to rinse for 1 minute with 9 ml of 0.1 mol/L phosphate-buffered saline. This was then expectorated into the container provided and submitted for microbiologic examination.

Patient details and oral examination Patient data, including age, length of time since HIV serologic diagnosis, CD4 count, oral problems encountered, and history of fluconazole exposure, were recorded. All information volunteered by the patient was checked for accuracy in the case record. A full oral examination was also undertaken, and the findings were recorded.

Laboratory processing of specimens Inoculation, incubation and colony selection. The rinses were centrifuged at 3000 rpm for 10 minutes; the supernatants were then removed. Each pellet was re-suspended in 1 ml of phosphate-buffered saline. An aliquot (50 gL) of the solution was inoculated onto both Sabouraud's dextrose agar and Pagano Levin agars with a spiral plater (Don Whitley Scientific Ltd,

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560 Hunter et aL

May 1998 l a n e I. Demographic details and fluconazole exposure history of patients studied No. of patients (n = 89) Black (n = 33) Exposure history Fluconazole-exposed Fluconazole-naive

White (n = 54)

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Female

Male

Female

Male

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Total

13 7

6 7

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Shipley, Yorkshire). The plates were then incubated aerobically for 48 hours at 37 ° C. After 48 hours, the plates were examined and the yeast colonies were counted. The Pagano Levin plates were checked for colonies of different colors and morphologic conditions. Each colony type was subcnltured for purity onto Sabouraud's dextrose agar and incubated aerobically at 37 ° C for 48 hours. Identification. Each of the subcultured yeasts was tested for germ tube production. Yeasts producing germ tubes were considered to be C. albicans. Germtube-negative yeasts were identified by sugar assimilation tests, through use of either the API 20 C diagnostic kit (bioMerieux Vitek Inc., Hazelwood, Mo.) or carbohydrate disks on agar. 23 Fluconazole sensitivity testing. The C. albicans isolates were tested for sensitivity to fluconazole with a disk d{ffusion assay (DDA). Three control isolates of C. albicans (provided by Pfizer Ltd) were set up with each run of the assay. One control was sensitive to fluconazole, one was of intermediate sensitivity, and one was resistant. Yeasts were subcultured overnight on Sabouraud's dextrose agar, and a suspension of 4 x 107 organisms per ml was prepared in 2 ml of HR medium (high resolution medium and sodium bicarbonate in 0.2mol/L phosphate buffer at pH 7.5). Check-plates for the yeast concentration were set up with 1 gL of the suspension inoculated onto Sabouraud's dextrose agar and incubated overnight at 37 ° C. Eighty microliters of yeast cell suspension at this cell density were added to 5 ml of double-strength amino acid/mineral solution (highresolution medium and sodium bicarbonate) at 37 ° C and vortex-mixed to ensure an even suspension. Agar base (agar and 0.2mol/L phosphate buffer at pH 7.5) was then melted and cooled to 60 ° C in a waterbath. The cell suspension was added to 5 ml of this agar base, mixed well, and poured into a 90-mm petri dish on a level surface. After the agar had set, a fluconazole paper disk (25gg disk, Mast Laboratories, Bootle, Merseyside, UK) was placed centrally on the surface of each plate, which was then incubated (18 hours; 30 ° C). The

plates were read blind. They were examined against a blue background, and the zone of growth inhibition was measured with calipers. By reference to the controis, each test yeast could then be designated sensitive, intermediate, or resistant to fluconazole.

Statistical methods A patient database was constructed in Microsoft Access Version 7. Statistical analysis was undertaken with Minitab Version 10. Data were analyzed with chisquared tests and two sample t tests to determine differences between the study groups. A difference was considered significant only if the p value was not greater than 0.05.

RESULTS Study population The demographic details and fiuconazole exposure of the study population are shown in Table I. Eightynine patients were sampled, 68 at Carolinas Medical Center, Charlotte, and 21 at Ruchill Hospital, Glasgow. There were no significant differences in the basic patient parameters for the groups studied in Glasgow and Charlotte; the data were therefore analyzed as a single cohort study.

Prevalence of oral candidosis Clinical signs of oral candidosis were present in 29.2% of patients. Pseudomembranous candidosis was present in 13 (14.6%) patients and erythematous candidosis in 11 (12.4%) patients; 3 patients had both types. Angular cheilitis was present in 2 patients, and none had hyperplastic candidosis.

CD4 count and oral candidosis The mean CD4 count in patients with candidosis (past or present) was 173.3 _+ 25 cells/ml; this was in contrast to a mean count of 347 _+ 38 cells/ml in patients who had never suffered candidosis (p < 0.001).

Exposure to antifungal agents Forty-eight (53.9%) of the 89 patients had taken or were currently taking flnconazole. Two patients were

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Table I|. Comparison of various patient parameters between fluconazole-exposed and fluconazole-naive groups Parameter

Fluconazole-exposed group (n = 48)

FluconazoIe-naive group (n = 41)

38.5 + 1.1 51.7 -+ 5.24 138 +.+_22.5 2.88 -+ 0.45 77 16 5

35.1 + 1.1 48.8 -+ 5.6 305 + 35.6* 0.74 -+ 0.28* 92 t 5* O*

Mean age (yr) Mean time HIV seropositive (mo) Mean CD4 count (cells/ml) Mean number of Candida episodes Candida carriage (%) Number harboring non-albicans yeasts Number resistant to fluconazole by DDA *Significant difference between groups; p < 0.00l. tSignificant difference between groups; p < 0,05. *Significant difference between groups; p < 0.01.

taking it for prophylactic suppression of cryptococcal meningitis. Other antifungals to which these patients had been exposed were clotrimazole (eight patients), nystatin (three patients), and itraconazole (six patients). The median daily dose of fluconazole was 100 rag, with four patients taking 200 mg/day. Three patients were taking the drug at a dosage of 100 mg 3 times per week. The average number of courses of fluconazole treatment was 1.56 +_ 0.22 (range, 1 to 10), with mean length of treatment of 8.12 +_ 1.21 months (range, 1 week to 36 months). The mean length of each treatment course was significantly longer in Charlotte (10.25 _ 1.41 months; range, 1 week to 36 months) than in Glasgow (0.65 + 0.18 months; range, 1 week to 2 months) (p < 0.005).

Comparison of fluconazole-exposedand fluconazole-naive groups Table II shows the relevant parameters for the two groups. Mean ages and mean times since HIV serologic diagnosis were similar. The mean CD4 count was much lower in the fluconazole-exposed group (138 +_ 22.5 cells/ml is within the AIDS-defining limit of < 200 cells/ml used in the United States), thus indicating more significant immunosuppression. Patients who had been exposed to fluconazole had, on average, suffered more episodes of oral candidosis than those who had not been exposed. This was probably a reflection of the lower CD4 count and more advanced immunosuppression. The percentage of patients with oral candidosis at the time of examination was the same in each group (29.2%). Many of these patients began, or recommenced, systemic antifungal therapy at this consultation. Most (97.9%) of the fluconazole-exposed patients had suffered from oral candidosis (one patient being treated for cryptococcal meningitis had never had overt oral candidosis), whereas only 43% of the flu-

conazole-naive patients had documented evidence of oral candidosis. Patients in the exposed group had experienced significantly more episodes of oral candidosis than the fluconazole-naive patients (Table II). Within the fluconazole-exposed group there was a significant difference (p < 0.05) in the number of episodes of candidosis between Glasgow (5.1 ___ 1.1) and Charlotte (2.24 _ 0.45).

Mycologic findings Yeasts were isolated from 75 (84.3%) of the patients. Eleven of the 14 patients who had no yeasts in the oral rinse had been exposed to fluconazole (p < 0.05). Overall, ten species of yeast were isolated from the patients. Those isolated from more than one patient are shown in Fig. 1. Other species isolated were C. pseudotropicalis, C. rugosa, C. guiIliermondii, and Hansenula anomala. C. albicans was by far the most common (78.9%) isolate (Fig. 1). C. glabrata in pure culture was isolated from only three patients, all in the fluconazole-exposed group. Fourteen patients (29.2%) in the fluconazoleexposed group and five (12.2%) in the fluconazolenaive group harbored non-albicans yeasts (p < 0.01; Table II). There was no difference in mean CD4 count for these patients between the two groups (126 +_ 52 cells/ml and 152 _+ 88 cells/ml, respectively). Each of two patients in the fluconazole-exposed group, both of whom had CD4 counts of less than 100 cells/ml, harbored three species of yeast. C. albicans isolates from 71 patients (37 fluconazole-naive and 34 fluconazole-exposed) were subjected to the fluconazole DDA. Five of these isolates, four from Charlotte and one from Glasgow, were resistant to fluconazole (Table II and Fig. 2). All were cultured from patients in the fluconazole-exposed group, with a mean length of treatment of 12.8 ___2.9 months. Four of the five patients had CD4 counts less than 100

562 Hunter et aL

ORAL SURGERY ORAL MEDICINE ORAL

PATHOLOGY May 1998

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cells/ml; the fifth had been on continuous fluconazole therapy for 16 months. Nine isolates showed intermediate sensitivity, four in the exposed group and five in the naive group. DISCUSSION

This study has provided further evidence of the emergence of fluconazole-resistant strains of C. albicans in HIV-infected patients receiving long-term treatment with fluconazole. The effect on the oral mycologic flora is also evident from the reduced overall rate of Candida carriage in patients previously treated with fluconazole and from the wider range of species present in the mouths of those patients exposed to the drug. The laboratory determination of fluconazole sensitivity is difficult to standardize. Triollet et al. ~6 found that the disk diffusion method, which was used in the present study, was reproducible and that the diameter of the zone of inhibition of growth correlated well with the minimal inhibitory concentration. They concluded that C. albicans strains with a decreased fluconazole susceptibility in vitro could be predicted by means of the DDA and that it correlated well with clinical resistance. A more recent paper, which compared the DDA with the E-test and the National Committee for Clinical Laboratory Standards method, also provided encouraging results on the use of disk diffusion for determining fluconazole sensitivity, but it suggested that additional tests are needed for strains that are not unequivocally susceptible by this method. ~4

Fig. 2. Fluconazole sensitivity by DDA of C. albicans isolated from fluconazole-exposed and fluconazole-naive groups. Comparative data from our own laboratory have also shown that the DDA is an appropriate in vitro method by which to screen for sensitivity to fluconazole. 25 The argument for routine antifungal drug susceptibility testing in HIV-associated oral candidosis is now compelling. Law et at. 18 recommended that testing be done routinely because of the high prevalence of resistance among the non-albicans Candida species and within C. albicans itself. Quereda et al. 22 were in agreement, citing the fact that most treatment failures were associated with increased minimal inhibitory concentrations of fluconazole among strains that were sensitive when isolated before treatment began. The findings of the present study add weight to the argument. Fluconazole resistance need not be a problem if the yeasts are tested for susceptibility to the anfifungal agents at intervals and if sensible prescribing practices are followed. One fluconazole-resistant strain was isolated from the Glasgow cohort. This suggests that intermittent, short-term use of fluconazole is not a guaranteed way to avoid fluconazole resistance. However, the patient who harbored the resistant strain had the longest exposure time of any patient in Glasgow (2 months). Vuffray et al.26 also found that resistance can occur after multiple single-dose treatments, so intermittent therapy does not seem to be the complete solution in avoiding resistance. The difference between the fluconazole-exposed and the fluconazole-naive patients with respect to the fluconazole sensitivity of C. albicans isolates was striking. No patient in the fluconazole-naive group harbored a resistant C. albicans strain, compared with five resistant strains in the fluconazole-exposed

Hunter et aL 563

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 85, Number 5 patients (10.4%). It should be b o r n e in m i n d that cessation of f l u c o n a z o l e therapy for a period m a y allow for loss of the f l u c o n a z o l e r e s i s t a n c e a n d thus for future use of the drug. 18 Similarly, the level of species d i v e r s i t y s h o w e d a m a r k e d d i f f e r e n c e b e t w e e n the e x p o s e d a n d n a i v e groups. E x p o s u r e to f l u c o n a z o l e provides a positive selection pressure for n o n - a l b i c a n s yeasts, m a n y of w h i c h are intrinsically less sensitive to fluconazole. As well as the m o r e c o m m o n n o n - a l b i cans yeasts, such as C. gIabrata and C. krusei, other, more unusual species were found (including Torulopsis candida, C. rugosa, a n d H. anomala). As reported b y others, 5,11 the e m e r g e n c e of fluconazole resistance was related to both a low C D 4 c o u n t and p r o l o n g e d exposure to a systemic azole drug. F o u r o f the five fluconazole-resistant strains o f C. albicans c a m e f r o m patients w h o had C D 4 counts of less than 100 cells/ml and w h o had b e e n exposed to f l u c o n a z o l e c o n t i n u o u s l y for a n a v e r a g e p e r i o d o f m o r e t h a n i year. A patient at risk is not hard to identify, given the C D 4 c o u n t and the drug history. T h e C D 4 c o u n t also seems to be related to the e m e r g e n c e o f n o n - a l b i c a n s species; the m e a n C D 4 c o u n t o f p a t i e n t s w h o were carrying these species was less than 200 cells/ml. T h e two patients w h o were each carrying three species o f Candida also h a d low C D 4 counts, as did the three patients w h o were h a r b o r i n g o n l y C. glabrata; all of their c o u n t s w e r e less t h a n 100 c e l l s / m l . C e r t a i n species, s u c h as S a c c h a r o m y c e s cerevisiae, h a v e i n h i b i t o r y effects o n l y m p h o p r o l i f e r a t i o n , 27 w h i c h m a y e n h a n c e their survival in the a l r e a d y i m m u n o c o m p r o m i s e d host. This study has s h o w n that l o n g - t e r m exposure to fluc o n a z o l e a m o n g H I V - s e r o p o s i t i v e patients m a y lead both to f l u c o n a z o l e resistance in the C. albicans popu l a t i o n a n d to s e l e c t i o n of n o n - a l b i c a n s species. R e g u l a r l a b o r a t o r y - b a s e d e x a m i n a t i o n s o f the oral m y c o l o g i c flora a n d fluconazole-sensitivity testing of isolated yeasts should be routine practices in the m o n itoring of f l u c o n a z o l e t r e a t m e n t in H I V - s e r o p o s i t i v e patients. The assistance of Drs. S. Jenkins, J. Horton, D. Weinrib, and J. Norton and of Mrs. P. Mulholland, Mrs. M. Jackson, and Mrs. J. Connell is gratefully acknowledged. REFERENCES 1. Greenspan JS, Greenspan D. Oral complications of HIV infection. In: Sande MA, Volberding PA, editors. The medical management of AIDS. 4th ed. Philadelphia: W.B. Saunders; 1995. p. 224-40. 2. Williams DM. EC-Clearinghouse on oral problems related to HIV-infections and WHO collaborating centre on oral manifestations of the immunodeficiency virus: classification and diag-

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HIV-infected patients. Eur J Clin Microbiol Infect Dis 1996; 15: 30-7. Warren NG, Shadony HJ. Yeasts of medical importance. In: Balows A, Hausler WJ, Herrmann KL, Isenberg HD, Shadony HJ, editors. Manual of clinical microbiology. 5th ed. Washington: American Society for Microbiology; 1993. p. 6214. Barry AL, Brown SD. Fluconazole disk diffusion procedure for determining susceptibility of Candida species. J Clin Microbiol 1996;34:2154-7. Jackson MS, Bagg J, Wiggins A, Richardson M. Comparison of four assays for determining fluconazole-sensitivity of Candida albicans [abstract]. Br J Biomed Sci 1996;53:69. Vuffray S, Durussel C, Boerlin R Oropharyngeal candidiasis

resistant to single-dose therapy with fluconazole in HIV infected patients. AIDS 1994;8:708-9. 27. Vartivatian S, Smith CB. Pathogenesis, host reponse, and predisposing factors. In: Bodey GP, editor. Candidiasis: pathogenesis, diagnosis and treatment. New York: Raven Press Ltd.; 1993. p. 59-84.

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CALL FOR REVIEW ARTICLES The January 1993 issue of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics contained an Editorial by the Journal's Editor in Chief, Larry J. Peterson, that called for a Review Article to appear in each issue. These Review Articles should be designed to review the current status of matters that are important to the practitioner. These articles should contain current developments, changing trends, as well as reaffirmation of current techniques and policies. Please consider submitting your article to appear as a Review Article. Information for authors appears in each issue of Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology,

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