J Med Microbiol 54 (2005), 959-964; DOI: 10.1099/jmm.0.45720-0
© 2005 Society for General Microbiology
ISSN 0022-2615
Voriconazole susceptibility of yeasts isolated from the mouths of patients with advanced cancer
Jeremy Bagg1,
M Petrina Sweeney1,
Andrew N Davies2,
Margaret S Jackson1 and
Susan Brailsford3
1Infection and Immunity Section, Glasgow Dental Hospital and School, 378 Sauchiehall Street, Glasgow G2 3JZ, UK 2Department of Palliative Medicine, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, UK 3Department of Oral Microbiology, GKT Dental Institute, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, UK
Correspondence Jeremy Bagg j.bagg{at}dental.gla.ac.uk
Received April 27, 2004
Accepted June 27, 2005
The in vitro activity of voriconazole was compared with those of fluconazole and itraconazole against 270 clinical isolates of yeasts from the mouths of patients receiving palliative care for advanced cancer. A broth micro-dilution assay as described by the National Committee for Clinical Laboratory Standards was employed for determination of MICs. Of the 270 isolates, 206 (76 %) were fluconazole sensitive and 64 were fluconazole resistant. Voriconazole showed more potent activity than either fluconazole or itraconazole, including against some isolates resistant to both fluconazole and itraconazole. However, for fluconazole-resistant isolates, the MICs of itraconazole and voriconazole were proportionally higher than for the fluconazole-susceptible isolates, suggesting cross-resistance. Voriconazole may be a useful additional agent for the management of oral fungal infections caused by strains resistant to fluconazole and itraconazole, but susceptibility cannot be assumed and in vitro MIC determination is recommended prior to its use.
Abbreviation: NCCLS, National Committee for Clinical Laboratory Standards.
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INTRODUCTION
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Many patients receiving palliative care for advanced cancer develop oral fungal infections (Aldred et al., 1991; Clarke et al., 1987; Finlay, 1986) due to the immunocompromised state of such patients. These infections are often seen in conjunction with dry mouth (xerostomia), another frequent complication in hospice patients (Jobbins et al., 1992a; Sweeney et al., 1998). Many patients will receive repeated courses of antifungal medication to deal with recurrent fungal infections. These antifungal drugs are often prescribed without confirmation of the diagnosis of an oral fungal infection by culture. In routine clinical practice, therefore, the identities and antifungal susceptibility profiles of the species involved in the infections are frequently unknown.
It has been suggested that prolonged or repeated exposure to low-dose fluconazole may be associated with emergence of fluconazole resistance among strains of Candida albicans (Johnson et al., 1995; Lopez et al., 2001) and the potential selection of non-albicans species of yeast (Sobel et al., 2001). Since many of the non-C. albicans yeasts, such as Candida glabrata (Arias et al., 1996) and Candida krusei (Berrouane et al., 1996), are inherently less susceptible than C. albicans to fluconazole, such population shifts have treatment implications. Previous studies have reported a diverse oral mycological flora in hospice patients, with a significant proportion of non-C. albicans yeasts (Sweeney et al., 1998; Jobbins et al., 1992b), a result confirmed in a recent study of oral yeast carriage among 120 patients with advanced cancer (Davies et al., 2002). In a recently completed multi-centre study (Bagg et al., 2003), the oral mycological flora of 207 patients receiving palliative care for advanced malignant disease was examined. In total, 194 yeasts were isolated, of which 95 (49 %) were C. albicans. There was a high prevalence of C. glabrata isolates (47) of which 72 % were resistant to both fluconazole and itraconazole by standard National Committee for Clinical Laboratory Standards (NCCLS) susceptibility testing (Bagg et al., 2003).
There is clearly a need to examine new methods for both the prevention and management of fungal infections in patients with advanced cancer. This includes assessment of new antifungal drugs. One such agent is voriconazole, a second-generation azole antifungal, which shows excellent in vitro activity against a wide variety of yeasts and moulds (Johnson & Kauffman, 2003).
It has been reported that voriconazole is active against all Candida species, including C. krusei, strains of C. glabrata that are inherently fluconazole-resistant and strains of C. albicans that have acquired resistance to fluconazole (Johnson & Kauffman, 2003). No breakpoints have yet been agreed for voriconazole, but in general the MICs of voriconazole for C. albicans are 1–2 logs lower than the MICs of fluconazole (Johnson & Kauffman, 2003). However, for some fluconazole-resistant strains of C. albicans, MICs of voriconazole are higher than for fluconazole-susceptible isolates (Johnson & Kauffman, 2003; Ruhnke et al., 1997; Cuenca-Estrella et al., 1999), and for C. glabrata and C. krusei the MICs are higher than for other species, though still in the presumed susceptible range (Johnson & Kauffman, 2003). The potential for drug interactions with voriconazole is high because of its metabolism by CYP450 isoenzymes (Hoffman & Rathbun, 2002), and although it is generally well tolerated, approximately 30 % of patients suffer a reversible disturbance of vision, particularly during the first week of therapy (Johnson & Kauffman, 2003).
Based on current knowledge of the oral mycological flora in patients receiving palliative care, the antifungal spectrum of voriconazole suggests that it may be a valuable adjunct to treatment of oral fungal infections in this group. Accordingly, the aim of the present study was to determine the voriconazole susceptibility of a large collection of well-characterized fungal isolates from the oral cavities of patients with advanced cancer.
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METHODS
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Patients.
Yeast isolates were available from the mouths of 199 patients who were receiving palliative care for advanced cancer. These isolates had been collected and characterized during two previous studies of fungal carriage among randomly selected patients receiving palliative care in the UK (Bagg et al., 2003; Davies et al., 2002).
Whilst all of these patients had previously received the standard forms of treatment appropriate to their particular malignancies, for example surgery, radiotherapy and chemotherapy, such modalities were no longer being pursued with a view to curing their disease. They were receiving, instead, symptom control. Accordingly, all of the patients were under the care of one of three hospices, either as in-patients or day-stay patients, and all were suffering from very advanced disease with limited life expectancies. The most common diagnoses were carcinomas of the breast, bronchus, prostate and large bowel. This large group of patients was therefore demographically representative of those receiving treatment in a palliative medicine setting.
Whilst it would have been interesting to collect detailed information on previous antifungal drug use by each patient, this was not possible. The patients had been treated by a wide range of both primary-care and secondary-care health professionals in many settings during the course of their illness and the reliability of data that could be collected was poor.
Yeast isolates.
A collection of 270 clinical isolates of oral yeasts was available. These yeasts had been isolated from oral swabs and oral rinses of the 199 patients described above. The yeast isolates were identified by germ-tube tests and API 20C AUX and API ID 32C (bioMérieux) profiles. Isolates with indeterminate results were further investigated using species-specific PCR and 26S rRNA gene sequencing. All germ-tube-positive isolates were screened by PCR using Candida dubliniensis-specific and fungal universal primers (Donnelly et al., 1999).
Fluconazole and itraconazole susceptibility testing.
Susceptibility to fluconazole and itraconazole was determined for all isolates by means of a broth micro-dilution assay as described in the NCCLS standard M27A (NCCLS, 1997). For fluconazole, isolates with an MIC of 
8 µg ml–1 were classified as susceptible, those with an MIC of 16–32 µg ml–1 as susceptible dose-dependent and those with an MIC of 
64 µg ml–1 as resistant. For itraconazole, isolates with an MIC of 0.125 µg ml–1 were classified as susceptible, those with an MIC of 0.25–0.5 µg ml–1 as susceptible dose-dependent and those with an MIC of 1 µg ml–1 as resistant. In all cases, if results fell between categories, then the higher category applied (NCCLS, 1997).
Voriconazole susceptibility testing.
An adapted NCCLS method was used as described by Pelletier et al. (2002). Pfizer Global Research & Development kindly supplied voriconazole (UK-109496) as a 99.2 % pure powder. Stock solution (1600 µg ml–1) was prepared by dissolving the powder in DMSO. A serial two-fold intermediary dilution was prepared by adding DMSO and RPMI 1640 to stock solution according to NCCLS M27-A procedures (NCCLS, 1997). After reconstitution with Candida suspension, the voriconazole concentrations ranged from 0.016 µg ml–1 to 8 µg ml–1, with a DMSO concentration of exactly 1 % in each well, including the growth control. The assays were performed in flat-bottomed micro-dilution plates. Individual Candida suspensions were prepared according to NCCLS M27-A methods. After incubation for 48 h at 35 °C, plates were read in a Dynatech MR 5000 microplate reader set at 405 nm. The MIC was defined as the lowest concentration of the drug that gave a 50 % reduction in optical density when compared with the turbidity of the growth control well (Rex et al., 2001).
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RESULTS AND DISCUSSION
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Yeast isolates
The identities of the 270 isolates studied are shown in Table 1. There were a significant number of isolates of non-C. albicans yeasts, reflecting the heterogeneous oral mycological flora in patients with advanced cancer.
Fluconazole, itraconazole and voriconazole susceptibilities
The comparative in vitro susceptibilities of the yeast isolates to the three antifungal agents are shown in Table 2. Overall, 206 (76 %) of the isolates were susceptible to fluconazole (MIC 8 µg ml–1). Twenty-five isolates were susceptible dose-dependent and 39 isolates (14 %) were fully resistant to fluconazole (MIC 64 µg ml–1). The comparative in vitro susceptibilities of the 64 isolates with a fluconazole MIC of > 8 µg ml–1 are shown in Table 3; seven were susceptible to itraconazole, 16 were susceptible dose-dependent and 41 were itraconazole resistant.
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Table 2. Comparative in vitro susceptibilities of 270 yeasts isolated from the mouths of 199 patients with advanced cancer
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Table 3. Comparative in vitro susceptibilities of 64 yeasts that were susceptible dose-dependent or resistant to fluconazole
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Overall, 160 (59 %) of the isolates were susceptible to itraconazole (MIC < 0.125 µg ml–1). Sixty-one isolates were susceptible dose-dependent and 49 isolates (18 %) were fully resistant to itraconazole (MIC 1 µg ml–1). Of the 49 isolates that were resistant to itraconazole, 41 were also resistant to fluconazole and eight were susceptible.
The distribution of the MICs to the three antifungal drugs for the 206 fluconazole-sensitive strains is illustrated in Fig. 1(a). The median values (and interquartile ranges) for fluconazole, itraconazole and voriconazole, respectively, were 0.5 µg ml–1 (0.25–2 µg ml–1), 0.125 µg ml–1 (0.06–0.25 µg ml–1) and 0.016 µg ml–1 (0.016–0.125 µg ml–1). Fig. 1(b) shows the distribution of the MICs to the three antifungal drugs for the 64 fluconazole-resistant strains. The median values and interquartile ranges for fluconazole, itraconazole and voriconazole respectively were 64 µg ml–1 (32–64 µg ml–1), 1 µg ml–1 (0.5–8 µg ml–1) and 1 µg ml–1 (0.125–8 µg ml–1).
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Fig. 1. Distribution of fluconazole, itraconazole and voriconazole MIC values for (a) the 206 fluconazole-sensitive isolates and (b) the 64 fluconazole-resistant isolates. Bars: grey, voriconazole; white, fluconazole; striped, itraconazole.
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In total, 41 (15 %) of the isolates were fully resistant to both fluconazole and itraconazole. Of these, 23 were C. glabrata, 12 were C. albicans, four were Saccharomyces cerevisiae, one was Candida parapsilosis and one was Candida tropicalis. The MICs of voriconazole for these isolates are shown in Fig. 2.
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Fig. 2. MIC values for the 41 isolates of yeast that were resistant to both fluconazole and itraconazole on the basis of the NCCLS breakpoints. Bars: grey, voriconazole; white, fluconazole; striped, itraconazole.
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Antifungals in oral fungal infections
Antifungal drugs are essential tools in the management of oral candidosis among debilitated patients. Fluconazole has been widely used to good effect. It is found in saliva at concentrations comparable to those in blood after single or multiple doses (Debruyne, 1997). Itraconazole, both as capsules and as the cyclodextrin solution, has also been shown to be effective in the treatment of oral candidosis (Cross et al., 2000) and the oral solution generates effective levels in both plasma and saliva (Reynes et al., 1997). However, the increasing incidence of colonization and infection with non-C. albicans yeasts displaying reduced susceptibility to the first generation azoles (fluconazole and itraconazole) has raised concerns for the future (Johnson et al., 1995). In the present study of a group of patients who we believe to be representative of those receiving palliative care for advanced cancer, 24 % of the isolates were not susceptible to fluconazole at standard doses. Consideration of the possible role of newer antifungal drugs in this group of patients is therefore important.
The data presented in this paper indicate that voriconazole is more potent than either fluconazole or itraconazole against yeasts isolated from the mouths of patients with advanced cancer. This finding is in keeping with published work on yeasts from a range of other patient groups and body sites (Cuenca-Estrella et al., 1999; Pelletier et al., 2002; Laverdiere et al., 2002). Of particular relevance is the voriconazole susceptibility of the isolates that were resistant to fluconazole and itraconazole. Many of these strains displayed low MICs to voriconazole, confirming reports by others that this agent is of value in treating some infections involving fluconazole-resistant Candida species (Pelletier et al., 2002; Laverdiere et al., 2002). However, the MICs of voriconazole for fluconazole-resistant isolates were higher than those for fluconazole-susceptible isolates, as reported by others (Cuenca-Estrella et al., 1999).
Whilst no interpretive breakpoints have yet been agreed for voriconazole, the pharmacokinetics of the drug suggest that adequate serum levels are available to treat infecting organisms with MICs of 2 µg ml–1 (Pelletier et al., 2002). Forty-four of the fluconazole-resistant strains had voriconazole MICs of 2 µg ml–1, but 18 of the remaining 20 strains had MICs of 8 µg ml–1. The pharmacokinetics of voriconazole appear suitable for the management of oral candidosis. A recent study showed that the pharmacokinetic profiles for saliva followed a pattern similar to those observed for plasma, with a highly significant correlation between plasma and saliva voriconazole concentrations (Purkins et al., 2002).
C. glabrata in oral fungal infections
The frequent isolation of C. glabrata is worthy of note. Of the 51 isolates of C. glabrata, 34 were fluconazole-resistant, with correspondingly high MICs to voriconazole. C. glabrata therefore constituted 53 % of the 64 fluconazole-resistant yeasts isolated. Although the numbers were small, one other group has reported a relatively lower success rate for voriconazole treatment of C. glabrata infections (Perfect et al., 2003). These data indicate the value of accurate identification of isolates to species level, ideally accompanied by antifungal susceptibility testing, when managing florid oral fungal infections in those with advanced disease.
Biofilms in oral fungal infections
One further issue relevant to the management of oral fungal infections is that the yeasts are present in the form of a biofilm. It is well recognized that antimicrobial agents are often far less effective against sessile, as opposed to planktonic, organisms (Ramage & López-Ribot, 2004). These concepts are now being considered in relation to antifungal drug susceptibility testing (Ramage et al., 2001) and are to be examined in more detail in future work by our group.
Conclusions
In summary, 75 % of the oral isolates were susceptible to fluconazole and this drug is likely to continue as the mainstay of treatment for oral fungal infections in the terminally ill for the foreseeable future. A significant proportion of yeasts showed reduced susceptibility to fluconazole and itraconazole, many of which were inhibited by low concentrations of voriconazole. By contrast, some of the fluconazole-resistant isolates, in particular C. glabrata, demonstrated reduced susceptibility to voriconazole. These data suggest a potential role for voriconazole in management of some refractory oral fungal infections in those with advanced cancer. However, more comprehensive studies of voriconazole are required to determine the correlation between in vitro activity and clinical outcome in vivo.
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ACKNOWLEDGEMENTS
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This study was funded by Pfizer (UK) who also provided the fluconazole and voriconazole free of charge. The Janssen Research Foundation (Belgium) provided the itraconazole free of charge. Professor David Coleman, University of Dublin, is thanked for his help in confirming the identifications of isolates of C. dubliniensis. We would like to acknowledge the support of Professor David Beighton, GKT Dental Institute.
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REFERENCES
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Aldred, M. J., Addy, M., Bagg, J. & Finlay, I. (1991). Oral health in the terminally ill: a cross-sectional pilot survey. Spec Care Dentist 11, 59–62.[Medline]
Arias, A., Arevalo, M. P., Andreu, A., Rodriguez, C. & Sierra, A. (1996). Candida glabrata: in vitro susceptibility of 84 isolates to eight antifungal agents. Chemotherapy 42, 107–111.[Medline]
Bagg, J., Sweeney, M. P., Lewis, M. A. O., Jackson, M. S., Coleman, D., Al Mosaid, A., Baxter, W., McEndrick, S. & McHugh, S. (2003). High prevalence of non-albicans yeasts and detection of anti-fungal resistance in the oral flora of patients with advanced cancer. Palliat Med 17, 477–481.[Abstract/Free Full Text]
Berrouane, Y. F., Hollis, R. J. & Pfaller, M. A. (1996). Strain variation among and antifungal susceptibilities of isolates of Candida krusei. J Clin Microbiol 34, 1856–1858.[Abstract]
Clarke, J. G. M., Wilson, J., Von Haacke, N. P. & Milne, L. J. R. (1987). Oral candidiasis in terminal illness. Health Bull (Edinb) 45, 268–271.
Cross, L. J., Bagg, J., Oliver, D. & Warnock, D. (2000). Serum itraconazole concentrations and clinical responses in Candida-associated denture stomatitis patients treated with itraconazole solution and itraconazole capsules. J Antimicrob Chemother 45, 95–99.[Abstract]
Cuenca-Estrella, M., Díaz-Guerra, T. M., Mellado, E., Monzón, A. & Rodríguez-Tudela, J. L. (1999). Comparative in vitro activity of voriconazole and itraconazole against fluconazole-susceptible and fluconazole-resistant clinical isolates of Candida species from Spain. Eur J Clin Microbiol Infect Dis 18, 432–435.[CrossRef][Medline]
Davies, A. N., Brailsford, S., Broadley, K. & Beighton, D. (2002). Oral yeast carriage in patients with advanced cancer. Oral Microbiol Immunol 17, 79–84.[CrossRef][Medline]
Debruyne, D. (1997). Clinical pharmacokinetics of fluconazole in superficial and systemic mycoses. Clin Pharmacokinet 33, 52–77.[Medline]
Donnelly, S. M., Sullivan, D. J., Shanley, D. B. & Coleman, D. C. (1999). Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences. Microbiology 145, 1871–1882.[Abstract]
Finlay, I. G. (1986). Oral symptoms and candida in the terminally ill. Br Med J (Clin Res Ed) 292, 592–593.
Hoffman, H. L. & Rathbun, R. C. (2002). Review of the safety and efficacy of voriconazole. Expert Opin Investig Drugs 11, 409–429.[CrossRef][Medline]
Jobbins, J., Bagg, J., Addy, M., Newcombe, R. G. & Finlay, I. G. (1992a). Oral and dental disease in terminally ill cancer patients. BMJ 304, 1612. 1612.
Jobbins, J., Bagg, J., Parsons, K., Finlay, I., Addy, M. & Newcombe, R. G. (1992b). Oral carriage of yeasts, coliforms and staphylococci in patients with advanced malignant disease. J Oral Pathol Med 21, 305–308.[CrossRef][Medline]
Johnson, L. B. & Kauffman, C. A. (2003). Voriconazole: a new triazole antifungal agent. Clin Infect Dis 36, 630–637.[CrossRef][Medline]
Johnson, E. M., Warnock, D. W., Luker, J., Porter, S. R. & Scully, C. (1995). Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis. J Antimicrob Chemother 35, 103–114.[Abstract/Free Full Text]
Laverdiere, M., Hoban, D., Restieri, C. & Habel, F. (2002). In vitro activity of three new triazoles and one echinocandin against Candida bloodstream isolates from cancer patients. J Antimicrob Chemother 50, 119–123.[Abstract/Free Full Text]
Lopez, J., Pernot, C., Aho, S., Caillot, D., Vagner, O., Dalle, F., Durnet-Archeray, M. J., Chavanet, P. & Bonnin, A. (2001). Decrease in Candida albicans strains with reduced susceptibility to fluconazole following changes in prescribing policies. J Hosp Infect 48, 122–128.[CrossRef][Medline]
NCCLS (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. NCCLS document M27-A. Wayne, PA: National Committee for Clinical Laboratory Standards.
Pelletier, R., Loranger, L., Marcotte, H. & de Carolis, E. (2002). Voriconazole and fluconazole suceptibility of Candida isolates. J Med Microbiol 51, 479–483.[Abstract/Free Full Text]
Perfect, J. R., Marr, K. A., Walsh, T. J. & 8 other authors (2003). Voriconazole treatment for less-common, emerging, or refractory fungal infections. Clin Infect Dis 36, 1122–1131.[CrossRef][Medline]
Purkins, L., Wood, N., Ghahramani, P., Greenhalgh, K., Allen, M. J. & Kleinermans, D. (2002). Pharmacokinetics and safety of voriconazole following intravenous- to oral-dose escalation regimens. Antimicrob Agents Chemother 46, 2546–2553.[Abstract/Free Full Text]
Ramage, G. & López-Ribot, J. L. (2004). Fungal biofilms and drug resistance. In Pathogenic Fungi: Host Interactions and Emerging Strategies for Control, chapter 11. Edited by G. San Blas & R. A. Calderone. Norwich: Horizon Scientific Press.
Ramage, G., Van de Walle, K., Wickes, B. L. & López-Ribot, J. L. (2001). A standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45, 2475–2479.[Abstract/Free Full Text]
Rex, J. H., Pfaller, M. A., Walsh, T. J. & 8 other authors (2001). Antifungal susceptibility testing: practical aspects and current challenges. Clin Microbiol Rev 14, 643–658.[Abstract/Free Full Text]
Reynes, J., Bazin, C., Ajana, F., Datry, A., Le Moing, J. P., Chwetzoff, E. & Levron, J. C. (1997). Pharmacokinetics of itraconazole (oral solution) in two groups of human immunodeficiency virus-infected adults with oral candidiasis. Antimicrob Agents Chemother 41, 2554–2558.[Abstract]
Ruhnke, M., Schmidt-Westhausen, A. & Trautmann, M. (1997). In vitro activities of voriconazole (UK-109,496) against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients with human immunodeficiency virus infection. Antimicrob Agents Chemother 41, 575–577.[Abstract]
Sobel, J. D., Ohmit, S. E., Schuman, P., Klein, R. S., Mayer, K., Duerr, A., Vazquez, J. A. & Rampalo, A. (2001). The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women. J Infect Dis 183, 286–293.[CrossRef][Medline]
Sweeney, M. P., Bagg, J., Baxter, W. P. & Aitchison, T. C. (1998). Oral disease in terminally ill patients with xerostomia. Oral Oncol 34, 123–126.[CrossRef][Medline]
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INTRODUCTION
METHODS
