Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis

M. Martíneza, J. L. López-Ribota,*, W. R. Kirkpatricka, S. P. Bachmanna, S. Pereaa, M. T. Ruesgab and T. F. Pattersona,c

a Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center, South Texas Centers for Biology in Medicine, San Antonio, TX; c Audie Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA; b Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, Lejona, Vizcaya, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme and increased efflux of drug, but the impact of specific treatment regimens on resistance has not been established. A patient with advanced AIDS was enrolled in a longitudinal study to receive continuous oral fluconazole (FLU) 200 mg/day for the treatment of oropharyngeal candidosis (OPC). Oral cultures were obtained at time of enrolment, during episodes of OPC and quarterly for surveillance. The patient had five symptomatic relapses on continuous FLU during 43 months. All OPC episodes were successfully treated with increasing doses of FLU although increased FLU MICs were detected for C. albicans isolates with progression of time. DNA-typing techniques demonstrated that resistance developed in a persistent strain of C. albicans. Both FLU-resistant and isogenic isolates with reduced susceptibility were detected in the same clinical samples through multiple episodes. Analysis of molecular mechanisms of resistance revealed overexpression of MDR and CDR genes encoding efflux pumps (but not ERG11) in isolates with decreased FLU susceptibility. In addition, the presence of the G464S amino acid substitution in their lanosterol demethylase, affecting its affinity for FLU, was also detected. However, other isogenic, but FLU-susceptible isolates recovered from the same samples did not harbour the mutation, indicating microevolution of yeast populations within the oral cavity. In this patient, the continuous antifungal pressure exerted by FLU resulted in development of resistance of multifactorial nature. Despite their clonal origin, different subpopulations of C. albicans demonstrated distinct resistance mechanisms, including concomitant presence and absence of functional point mutations in ERG11 genes.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The development of antifungal drug resistance in Candida albicans following prolonged exposure to azole antifungals is well established.1–5 In patients with advanced AIDS, oropharyngeal candidosis (OPC) continues to be a common presenting illness associated with significant morbidity. In recent years, oral fluconazole, given its low toxicity, has become the most common form of treatment for symptomatic OPC. The widespread use of fluconazole has, however, led to an increased incidence of clinically resistant OPC due to infection with fluconazole-resistant organisms. The development of clinically resistant OPC can occur by several mechanisms, which include: (i) infection with intrinsically resistant organisms; (ii) selection of resistant organisms secondary to antifungal drug pressure; and (iii) development of resistance in a previously susceptible organism.4,5

Fluconazole resistance in previously susceptible organisms appears to be directly related to the total amount of fluconazole a patient has received. Prior studies have reported that cumulative fluconazole doses of >10 g were significant for the emergence of C. albicans isolates with decreased susceptibility to fluconazole.6,7 While the overwhelming majority of data collected on the development of resistance has come from patients receiving intermittent fluconazole therapy, less is known about patients receiving long-term continuous fluconazole for the prevention of OPC. Prior studies on patients receiving long-term continuous oral fluconazole have demonstrated decreased relapses of OPC when compared with intermittently treated patients.8 However, both treatment strategies have ultimately led to the development of azole-resistant isolates and clinically resistant OPC.7–11

C. albicans, the most frequently implicated aetiological agent of OPC, has been shown to develop resistance to fluconazole through a variety of molecular mechanisms.11–19 Such mechanisms include alterations in the target enzyme (lanosterol-14{alpha}-demethylase), leading to decreased azole binding affinity13,18,20–23 or overproduction,19 thus competitively overcoming the azole effect, as well as overexpression of multiple efflux pumps including major facilitators and ABC transporters.11,12,15,19,24–26 However, it is not known whether patients on continuous azole therapy select for specific resistance mechanisms due to their constant exposure to fluconazole compared with patients who have been treated intermittently. In this study, we describe a patient receiving long-term continuous oral fluconazole for the prevention of OPC. We also describe the unique observation of multiple mechanisms of resistance, including the presence and absence of functional point mutations and overexpression of efflux pumps conveying resistance from the same clinical samples in a clonal strain of C. albicans, demonstrating microevolution of resistance under continuous antifungal pressure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subject

A 51-year-old Latin American male with advanced AIDS (CD4+ count 15 cells/mm3) was enrolled in a prospective study to evaluate fluconazole resistance. The patient was randomized to receive continuous oral fluconazole at 200 mg/day for the prevention of OPC. AIDS defining illnesses at time of presentation were Pneumocystis carinii pneumonia (PCP) and OPC. The patient was enrolled in May 1995 (1 month after the diagnosis of AIDS) and followed longitudinally for 43 months at the University of Texas Health Science Center, San Antonio, TX, USA. Episodes of recurrent OPC were evaluated according to symptoms and documented by cultures taken from the oropharynx. Oropharyngeal culture and clinical evaluation of symptoms were also performed quarterly for surveillance. All recurrent episodes of OPC were treated with 200–800 mg/day oral fluconazole for 7–14 days, as needed to control symptoms.

Collection and identification of clinical isolates

Clinical samples were obtained by swabs taken from the oropharynx and by oral saline rinse. Samples were collected at the time of study enrolment, during episodes of OPC relapse and quarterly for surveillance. Samples were plated on chromogenic media (CHROMagar Candida, Paris, France) with and without fluconazole, as previously described by our group,27,28 in order to maximize detection of resistant isolates and allow presumptive identification at the species level. From these plates, individual colonies were recovered and isolates were then maintained as suspensions in sterile water at room temperature. From these stocks, individual isolates were subcultured on to Sabouraud dextrose agar (Becton Dickinson, Cockeysville, MD, USA). Their identity as C. albicans was confirmed by standard biochemical and microbiological procedures, including carbohydrate assimilation patterns (API 20C; bioMérieux, Marcy-l'Étoile, France), germ tube formation in serum, colour of colonies on chromogenic medium and differential growth at 37 and 42°C.29 Isolates were labelled according to the episode of collection (I, initial; R, relapse; S, surveillance) and allocated an isolate number using the following nomenclature: (episode.isolate #).

Antifungal susceptibility testing and determination of MICs

Evaluation of the initial fluconazole susceptibility of C. albicans isolates was performed according to NCCLS methods by broth macrodilution techniques with endpoints read at 48 h.30 Susceptibility testing was performed at the Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, and susceptibilities to fluconazole were reported as MICs. Breakpoints were defined as: susceptible (MIC >= 8 mg/L), susceptible dose dependent (MIC 16–32 mg/L) or resistant (MIC >= 64 mg/L), in accordance with NCCLS guidelines.30,31 Representative C. albicans isolates with decreasing susceptibilities to fluconazole and spanning the patient's entire clinical course were selected for further analysis against a battery of antifungal agents. Susceptibility testing with itraconazole (Janssen Pharmaceutica, Beerse, Belgium), voriconazole (Pfizer Inc., Sandwich, UK), posaconazole (SCH 56592; Schering Plough, Kenilworth, NJ, USA), ravuconazole (BMS 207147; Bristol-Myers Squibb, Princeton, NJ, USA), caspofungin [Cancidas (MK-0991); Merck & Co., Inc., Whitehouse Station, NJ, USA] and amphotericin B (Bristol-Myers Squibb) was carried out following the NCCLS method M-27A using a broth microdilution procedure and reading of the endpoints at 48 h.

DNA-based typing techniques to assess strain identity

Strain identity was investigated by karyotyping, restriction fragment length polymorphism (RFLP) and fingerprinting analysis using the moderately repetitive probe Ca3 as described previously.15,32 For karyotyping (EK) whole cell yeast DNA in 0.75% agarose plugs was resolved on a 1% agarose gel by contoured clamped gel electrophoresis (CHEF) in 0.5x Tris-Borate-EDTA buffer at 14°C. Running conditions for EK were: (i) 120 s, 4.5 V/cm, 21 h; (ii) 300 s, 4.5 V/cm, 18 h; and (iii) 300 s, 3.4 V/cm, 28 h. RFLP patterns were generated by digestion of genomic DNA with SfiI or EcoRI restriction endonucleases (Boehringer-Mannheim, Indianapolis, IN, USA). Briefly, one-quarter plug containing yeast DNA of each isolate was digested overnight (18 h) with 30 U of SfiI at 50°C or 120 U of EcoRI at 37°C in 300 µL of the appropriate reaction buffer. Samples were then loaded into wells of a 0.8% agarose gel. CHEF was performed in 0.5x Tris-Borate-EDTA buffer at 14°C. Pulse times were ramped from 5 to 35 s for 24 h at 6 V/cm for Sfi-digested DNA or 0.1–0.1 s for 20 h at 4.5 V/cm for EcoRI-digested samples. After separation by pulsed-field gel electrophoresis, EK- and SfiI-digested gels were stained with ethidium bromide and photographed. Following electrophoresis, the materials present in EcoRI-digested gels were transferred to nylon membranes (Nytran; Schleicher and Schuell, Keene, NH, USA) using the Turboblotter apparatus (Schleicher & Schuell). The digested DNA was attached to membranes by UV-crosslinking and the membranes were hybridized with Ca3 probe radioactively labelled by random priming (Random Primers DNA Labeling System, Gibco-BRL, Gaithersburg, MD, USA). The membranes were then washed and exposed to autoradiography film (Du Pont, Wilmington, DE, USA) for 24 h at 70°C. Pictures of the gels or films were scanned using the Adobe Photoshop program (Adobe Systems Inc., Mountain View, CA, USA). Dendrogams were generated for both EK- and SfiI-digested gels using Dendron software (Solltech, Oakdale, IA, USA). Similarity coefficients (SAB) were computed by a formula based on band position only and dendrograms were generated by the hierarchical unweighted pairgroup method with arithmetic averages (UPGMA) cluster algorithm. For preparation of figures, digital images were processed using the Adobe Photoshop program.

Analysis of expression of genes implicated in azole resistance

Isolates from the stocks in water were subcultured on to plates containing Sabouraud-dextrose agar 48 h prior to propagation in YEPD medium (2% yeast extract, 1% peptone, 2% glucose) to mid-logarithmic phase. Total RNA from the different isolates was obtained using the RNAeasy mini kit (Qiagen Inc., Santa Clarita, CA, USA) following the manufacturer's instructions. Equal amounts (c. 5 µg) of RNA as determined by A260 measurements were separated by electrophoresis and subsequently transferred to nylon membranes (Nytran) by means of the Turboblotter apparatus. Probes for ERG11, MDR1 and CDR1 genes were prepared as described before.15 In the case of MDR1 and CDR1, the resulting probes are based in the whole sequence of these genes and may cross-hybridize with other members of these gene families.11,15,19 Probes specific for CDR1 and CDR2 genes were prepared by PCR amplification as described before.15 All probes were labelled by random priming (Random Primers DNA Labeling System), and hybridizations performed using Rapid-hyb buffer (Amersham Life Science Inc., Arlington Heights, IL, USA) following the manufacturer's instructions. After hybridization, blots were washed and exposed to autoradiography film (Du Pont). Nylon membranes were probed sequentially with the different probes following stripping of the previously bound probe.18 For densitometric analysis, autoradiograms were scanned using the Adobe Photoshop program, and signals quantified using Dendron. Relative values were adjusted for differences in sample loading based on quantification of 18S rRNA levels. For preparation of figures, digital images were processed using the Adobe Photoshop program.

DNA manipulations, PCR amplification and sequencing

The ERG11 genes encoding lanosterol 14{alpha}-demethylase, from representative isolates showing decreased susceptibility to fluconazole with progression of time and recovered from each different OPC episode or surveillance culture, were amplified by PCR. Briefly, genomic DNA from the different isolates was extracted using YeaStar Genomic DNA (Zymo Research, Orange, CA, USA) and used as a template for amplification of ERG11 genes. PCR was carried out with high-fidelity two DNA polymerase (Boehringer-Mannheim) using the following primers: 5'-GTTGAAACTGTCATTGATGG (forward) and 5'-TCAGAACACT GAATCGAAAG (reverse). Amplicons were purified with Wizard PCR Preps (Promega, Madison, WI, USA). For selected isolates, the nucleotide sequences of the entire amplified ERG11 genes were determined in both strands by primer elongation with an ABI automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). For all other isolates partial sequences were obtained corresponding to the region expanding nucleotides 1029–1460, coding for important functional domains of the enzyme in its interaction with azole derivatives and for which initial analysis revealed the presence of a point mutation leading to an amino acid substitution resulting in a decreased affinity of the enzyme for fluconazole. Sequence data were compared with a published ERG11 sequence33 using BLAST.34


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical course

The patient's clinical course is depicted in Figure 1Go. He was followed longitudinally for a total of 43 months during which time he remained on continuous oral fluconazole at 200 mg/day. His first documented episode of OPC was his presenting illness at the time of HIV diagnosis and occurred 1 month prior to enrolling in the study. This presenting episode of OPC was treated successfully with topical clotrimazole. The patient had no history of prior antifungal drug therapy.



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Figure 1. Patient's HIV clinical course. OPC relapses R1–R5 are listed above the corresponding month of occurrence. Final HIV viral load was 924 copies/mm3. HAART regimens included use of protease inhibitors (saquinavir and saquinavir/ritonavir). Shaded bars, CD4 count; {diamondsuit}, viral load.

 
While on continuous oral fluconazole, the patient had a total of five documented relapses of symptomatic OPC (R1–R5). The first OPC relapse occurred on study day 43. The cumulative fluconazole dose at that time was 8.6 g. Interval time between relapses R1 and R2 was 13 months. Time to relapse between successive episodes ranged from 7 to 11 months with a median of 9 months. OPC relapses R1 and R2 were both successfully treated with an additional 200 mg/day oral fluconazole for 7 days. However, relapses R3, R4 and R5 each required fluconazole doses of 800 mg/ day for 7 days to resolve symptoms. All episodes of OPC were successfully treated with oral fluconazole. There were no treatment failures.

At study entry, the patient's CD4+ count was 15 cells/ mm3. Testing of HIV viral load was not yet routinely performed and was therefore not known until month 9. The treatment of his HIV was initially begun with AZT alone, but by month 5, DDC was added to his regimen. He remained on dual nucleoside reverse transcriptase inhibitor (NRTI) therapy with AZT and DDC until month 14. During this time, an increase in CD4+ count was observed and a peak value of 78 cells/mm3 was documented. On month 14, the patient was started on highly active antiretroviral therapy (HAART) with AZT/3TC/Saquinavir and he remained on protease inhibitor containing antiretroviral therapy throughout the duration of the study. Following initiation of HAART, his CD4+ count peaked at 222 cells/mm3 and his viral load nadir was 924 copies/mm3. Quantitative HIV RNA did not decrease below 400 copies/mm3 at any time during the study. While on HAART therapy, three episodes (R3–R5) of symptomatic OPC were documented.

Antifungal susceptibility testing of isolates

A total of 116 C. albicans isolates were recovered from cultures obtained during the different episodes of OPC and the surveillance cultures. Initial susceptibility testing to fluconazole demonstrated decreased susceptibility of isolates with progression of time, increasing cumulative dose of fluconazole and successive episodes of OPC (Table 1Go). Thus, FLU MICs for isolates recovered from the initial episode and first relapse were all in the susceptible range. However, isolates recovered during a surveillance culture after the first OPC episode relapse already demonstrated reduced susceptibility to FLU, despite the fact that the patient was asymptomatic. The first truly resistant isolates (FLU MIC >= 64 mg/L) emerged during relapse R3 at which time the cumulative fluconazole dose was 140.6 g. However, throughout the course of the study a mixed population of susceptible and resistant yeasts were present as indicated by FLU MIC values, and interestingly a residual population of susceptible isolates persisted in the oral cavity during an extended period despite the continuous pressure exerted by the antifungal treatment.


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Table 1. Development of fluconazole resistance in C. albicans with progression of time, according to fluconazole (FLU) MICs (mg/L) for multiple clinical isolates recovered during each different OPC episode or from surveillance cultures
 
Results of susceptibility testing to fluconazole and additional antifungal agents, for 12 C. albicans isolates with decreasing susceptibilities to fluconazole and representing each OPC relapse or surveillance culture, are shown in Table 2Go. Included in these analyses were isolates I.1, R1.1 and S1.1, collected early in the patient's clinical course. All were susceptible to fluconazole with MICs ranging from 0.25 to 4 mg/L. Isolates with susceptible dose-dependent fluconazole MICs of 16–32 mg/L were collected at the second, third and fourth OPC relapses. Two isolates, S2.2 and R5.1, demonstrating true resistance to fluconazole (MIC >= 64 mg/L), were collected at the end of the patient's clinical course. Isolate S2.2 was collected concomitantly with isolate S2.1 between relapses R4 and R5 (month 31) at a time when there was no evidence of symptomatic OPC, whereas isolate R5.1 was obtained during relapse R5 (month 41). No fluconazole-resistant isolates were collected prior to month 23, but isolates with decreasing susceptibility to fluconazole were seen as the cumulative dose of fluconazole increased. Following the criteria established by Rex et al.31 for the interpretive breakpoints for antifungal susceptibility testing for fluconazole and itraconazole against C. albicans, all the fluconazoleresistant isolates remained susceptible to itraconazole (MIC <= 1 mg/L), although an increase in the MIC was also observed compared with the initial highly susceptible isolates. Decreased susceptibility to the new third generation triazole derivatives was also observed. In the case of posaconazole and ravuconazole there were up to eight-fold dilution increases in MIC values compared with initial isolates. For voriconazole, elevated MICs were observed in several isolates with up to 14-fold dilution increase in resistance. Results of susceptibility testing of all isolates against caspofungin revealed uniform susceptibility within one dilution despite significant changes in fluconazole susceptibility. The same was also true for amphotericin B.


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Table 2. Antifungal MICs (mg/L) for selected C. albicans isolates recovered during each different OPC episode or from surveillance culture
 
Strain identification

Results of EK of representative isolates demonstrated similar banding patterns and were consistent with the expected banding pattern for C. albicans. One isolate, R2.3, demonstrated one additional band when compared with all other isolates. RFLP analysis of SfiI-digested DNA also revealed similar banding patterns for each isolate of 18–20 bands. Dendrograms obtained from computer-assisted analysis of EK- and SfiI-digested DNA gels revealed a high degree of homology between each isolate (90–100%) denoting isogenicity between all isolates, and indicating that development of resistance occurred in a single persistent strain. DNA fingerprinting with Ca3 probe of membranes containing EcoRI-digested DNA further demonstrated a high degree of isogenicity between isolates, indicating the clonal origin of all isolates analysed (data not shown).

Expression of ERG11, MDR and CDR genes implicated in azole resistance

Expression of genes encoding for lanosterol-14{alpha}-demethylase (ERG11) and drug efflux pumps (MDR, CDR, CDR1, CDR2) of representative clinical isolates are shown in Figure 2Go. Expression of ERG11 remained relatively constant in isolates obtained throughout the patient's clinical course. In one isolate, S1.1, ERG11 was minimally expressed. No significant overexpression of ERG11 was demonstrated for all other isolates despite decreasing fluconazole susceptibility.



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Figure 2. Expression of ERG11, MDR and CDR genes in sequential C. albicans isolates. The panels labelled MDR and CDR represent results using probes based on the entire sequence of the C. albicans MDR1 and CDR1 genes that may cross-hybridize with other members of the Major Facilitator and ABC transporters multigene family, respectively, whereas panels labelled CDR1 and CDR2 represent results of hybridizations using PCR-generated probes highly specific for these two genes. Sequential isolates are labelled across the top of the gel with corresponding episode. Episodes: I, initial; R1–R5 are relapses 1–5, respectively. *, isolate collected during surveillance at a time when there was no evidence of OPC. Fluconazole MICs (mg/L) are denoted below each isolate. The bottom panel shows amounts of 18S rRNA used to standardize the signal levels according to lane loading parameters.

 
Increased expression of MDR and CDR genes coding for efflux pumps was apparent in isolates exhibiting decreasing susceptibility to fluconazole. Fluconazolesusceptible isolates, I.1, R1.1 and S1.1, collected early in the patient's clinical course, showed negligible expression of both MDR and CDR. As fluconazole susceptibility decreased to the susceptible dose-dependent range, as exhibited by the sequential isolate R2.1, up-regulation of MDR was demonstrated but not CDR. This isolate (R2.1) was the first to exhibit a fluconazole MIC of 32 mg/L. The simultaneous maximal expression of both CDR and MDR was demonstrated for isolates R3.2, S2.2 and R5.1, each having fluconazole MICs of 32, 64 and 64 mg/L, respectively. These isolates were collected late in the patient's clinical course. CDR1 and CDR2 genes encoding for specific efflux pumps were also evaluated. The highest expression of both CDR1 and CDR2 was demonstrated in the susceptible dose-dependent isolates (R3.2, R4.1) and the resistant isolates (S2.2, R5.1). Isolates taken early in the patient's clinical course exhibited negligible levels of CDR1 and CDR2 when compared with later isolates.

Point mutations in ERG11 genes

Initial sequencing of ERG11 genes from a susceptible and a resistant isolate (isolates S2.1 and S2.2, recovered from the same culture) indicated that the G1390A nucleotide mutation was present in the ERG11 alleles from the resistant isolate, but absent in the susceptible one. This mutation results in the G464S amino acid substitution in the protein sequence of lanosterol demethylase, which leads to an enzyme with decreased affinity for fluconazole, and possibly other azole derivatives.18,22 The analysis of ERG11 sequences was expanded to a total of 20 representative C. albicans isolates with varying susceptibilities to fluconazole and collected throughout the entire clinical course. Partial sequences of the region expanding nucleotides 1029–1460 of ERG11 coding for important functional domains of the enzyme in its interaction with azole derivatives were determined for all 20 selected isolates. All isolates contained the G1309A nucleotide mutation known to produce the amino acid (AA) substitution V437I but not resulting in decreased antifungal susceptibility.18 In addition, several other point mutations (C1110T, T1140C, T1203C and A1440G) not resulting in AA substitutions were present in all isolates, further corroborating their isogenicity. The G1390A mutation, resulting in G464S AA substitution, was identified in several isolates exhibiting fluconazole MICs of 32 and 64 mg/L (R3.2, R3.3, R3.8, S2.2 and R5.1). These isolates demonstrated fluconazole MICs of 32, 32, 64, 64 and 64 mg/L, respectively. All fluconazole-susceptible isolates (MIC <= 8 mg/L) and susceptible dose-dependent isolates with fluconazole MICs of 16 mg/L did not contain the G464S mutation. Interestingly, susceptible dose-dependent isolate R3.5 (MIC 32 mg/L) and resistant isolate R3.6 (MIC 64 mg/L) also did not contain the G464S mutation. Both isolates were collected during OPC relapse 3, at the same time as G464S mutation-containing isolates R3.2 (MIC 32 mg/L), R3.3 (MIC 32 mg/L) and R3.8 (MIC 64 mg/L) were also obtained.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The impact of specific treatment strategies on the development of azole antifungal drug resistance in OPC is not completely understood. It is known, however, that prolonged exposure to azoles can ultimately lead to clinically resistant OPC and Candida isolates demonstrating decreased susceptibilities to azole antifungals.2–4,7–10,35,36 Given its low toxicity and side-effect profile, fluconazole has become a commonly used agent for the treatment of OPC and in recent years the development of clinically resistant OPC and selection for resistant Candida isolates has increased.2,3,7–10 The development of azole resistance in C. albicans has been demonstrated in patients receiving long-term fluconazole whether given intermittently during times of symptomatic OPC or when given continuously for the prevention of OPC.8–10,35,36 Our group has shown recently that HIV-infected patients receiving continuous oral fluconazole have fewer OPC relapses when compared with intermittently treated patients, and that the development of azole resistance was not significantly influenced by continuous versus intermittent administration of fluconazole but rather by the cumulative dose of fluconazole received.8 Cumulative fluconazole doses of >10 g have been reported as a significant risk factor in the development of fluconazole resistance.6,7 A recent prospective study of refractory mucosal candidosis in patients with advanced AIDS has demonstrated that daily or alternate day fluconazole use was associated with the development of refractory infection.35 Despite these clinical observations, it is not known whether patients receiving continuous oral fluconazole for the prevention of OPC select for specific molecular mechanisms of azole resistance, compared with those treated only intermittently.

Here we have described a patient with advanced AIDS who continued to have symptomatic relapses of OPC while on continuous oral fluconazole. The patient had a total of five OPC relapses, three (R3–R5) while on HAART. Improvement in the patient's overall immune status as demonstrated by a rise in CD4+ number and a reduction of HIV viral load was seen after the initiation of HAART. However, it must be noted that although the patient had a dramatic reduction in HIV viral load, it did not fall below the limits of detection at any time during the study. Also, while on HAART, the patient's CD4+ number peaked at 222 cells/mm3, still well in the range at which OPC can be present. These factors may account, in part, for the symptomatic relapses of OPC in this patient. The relationship between uncontrolled HIV viral load and recurrent OPC has yet to be established, but it is well known that undetectable HIV viral loads with concomitant improvement of immune status have led to an overall decreased frequency of opportunistic infections in HIV-infected patients.37 Also of interest is the fact that this patient was receiving saquinavir and saquinavir/ritonavir combinations as part of his HAART regimen. Protease inhibitors, specifically saquinavir and indinavir, have recently been shown to be active against the secreted aspartyl proteinases (SAPS) of C. albicans used for mucousal attachment.38,39 A recent study evaluating the impact of protease inhibitor therapy on OPC demonstrated a significant decrease in the prevalence of OPC in patients receiving protease inhibitors.40 Interestingly, despite an increase in CD4+ cell number, decrease in HIV viral load, and use of protease inhibitor-containing antiretroviral regimens, the patient continued to have symptomatic relapses of OPC.

All recurrent episodes of OPC were successfully treated with oral fluconazole but required doses as high as 800 mg/ day for resolution of symptoms late in his clinical course. This is in agreement with prior studies from our group and others who have reported the successful treatment of OPC with high dose fluconazole, even in the face of highly resistant C. albicans isolates.3,7,8 The patient did not develop clinically refractory OPC and was not considered a treatment failure.

This description of the molecular mechanisms in our patient is unique for the co-isolation of both susceptible and resistant yeasts in a clonal strain, including the presence and absence of a point mutation conferring resistance to azoles. The initial C. albicans isolate, I.1, collected at the time of study enrolment was fluconazole susceptible. Analysis of molecular mechanisms implicated in azole resistance in this isolate showed only baseline expression of ERG11. As the patient's clinical course continued, isolates with decreased susceptibility to fluconazole were seen, and by the time of the second OPC relapse at study month 14, fluconazole MICs had increased to 8 and 32 mg/L. In these isolates increases in MDR and CDR were detected but expression of ERG11 was unchanged from baseline. The first frank fluconazole-resistant C. albicans isolate (MIC >= 64 mg/L) was recovered at study month 23, by which time the cumulative dose of fluconazole received was c. 140 g. Isolates recovered after the second OPC episode varied in susceptibility to fluconazole, ranging from susceptible to resistant, and were present simultaneously in the oral cavity. There appeared to be a stepwise increase in efflux pump expression as fluconazole susceptibility decreased, but there was essentially little or no change in ERG11 expression. This is in agreement with recent studies which have shown that azole resistance can develop in a stepwise fashion in C. albicans.13,19

The presence of the G464S amino acid substitution in lanosterol-14{alpha}-demethylase was demonstrated in several, but not all, isolates demonstrating fluconazole MICs of 32–64 mg/L. Also, the mutation was not present in isolates exhibiting fluconazole MICs < 32 mg/L. This substitution has been associated with decreased azole binding affinity to the target enzyme and decreased target enzyme catalytic activity.18,22 It is unclear whether this substitution alone can confer fluconazole resistance to this level. When comparing three isolates (R4.1, S2.2 and R5.1), one without the G464S substitution and two with the substitution and all having similar efflux pump expression, the presence of the G464S mutation appeared to contribute additional resistance to fluconazole. Of interest was the presence of both fluconazole dose-dependent susceptible and resistant isolates, which either did or did not harbour the G464S substitution and were collected during the same OPC episode (R3) or surveillance culture (S2).

It has been assumed that the most common scenario for the development of resistance in a strain of C. albicans is that the constant pressure exerted by antifungal treatment selects for the most resistant phenotypes, which arise in a gradual fashion as a result of a combination of different resistance mechanisms.5 This would lead to a uniform population of yeasts, where a single predominant phenotype was ultimately selected over less ‘adapted’ cells. However, this conclusion has been drawn mostly from analysis where only single isolates from each time point were available for study. Thus, the genotypic and phenotypic homogeneity of infecting yeast populations has not been adequately explored to make definitive conclusions about how a clonal population develops resistance under constant antifungal pressure. In this regard it is also conceiv-able that, despite a common clonal origin, different yeast subpopulations may evolve differently and display distinct mechanisms of resistance. Methods that increase detection of subpopulations of yeasts at the time of initial culture, such as our novel agar dilution screening technique,27,28 may provide the basis for a more comprehensive assessment of the mechanisms of resistance in infecting and colonizing yeast populations. For example, our group and others have demonstrated the heterogeneity of resistant phenotypes as yeast populations evolve both in vivo and in vitro.1,16,41,42 By examining mechanisms of resistance in multiple isolates collected at the same time it is possible to assess the biodiversity of C. albicans isolates present in the oral cavity of patients with resistant OPC at a given moment. Here we have presented evidence that C. albicans isolates which are clonal in origin (as determined by DNA-typing techniques) exhibit heterogeneous mechanisms of resistance. At least two different, genetically distinguishable subpopulations of C. albicans, according to the presence or absence of the G464S substitution, co-existed in the oral cavity of this patient over a long period of time. They evolved independently leading to a high degree of heterogeneity in mechanisms of resistance, indicating microevolution of yeast populations with regard to development of azole resistance.

In summary, under continuous azole antifungal drug pressure, development of resistance was of a multifactorial nature in a persistent strain of C. albicans. Mechanisms of resistance included point mutations in the ERG11 genes leading to decreased affinity of the drug for its enzyme target and up-regulation of genes encoding efflux pumps (MDR and CDR). The constant antifungal pressure exerted by continuous oral fluconazole did not select for any one mechanism of azole resistance and produces a picture similar to what has been demonstrated for patients treated intermittently. Multiple molecular mechanisms of azole resistance were present at any given time in C. albicans isolates with a shared clonal origin, giving credence to a complex oropharyngeal environment and demonstrating microevolution of different subpopulations of C. albicans under constant azole exposure.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank T. C. White for his helpful advice and for providing the plasmids containing the ERG11 and MDR sequences. This work was supported by Public Health Service grant 5 R01 DE11381-04A2 from the National Institute of Dental and Craniofacial Research to T.F.P. and a Supplemental Postdoctoral Minority Fellowship Grant to M.M. Additional support was provided by Public Health Service grants R29 AI42401 to J.L.L.-R. and M01-RR-01346 for the Frederic C. Bartter General Clinical Research Center and a grant from Pfizer, Inc. Chromogenic media were provided by CHROMagar Candida (Paris, France). S.P. acknowledges the receipt of a NATO postdoctoral fellowship. M.T.R. is the recipient of a predoctoral fellowship from the Ministerio de Educación y Ciencia, Spain. This work was presented in part at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy held in Toronto, Canada, September 2000.


    Notes
 
* Corresponding author. Tel: +1-210-562-5017; Fax: +1-210-562-5016; E-mail: ribot{at}uthscsa.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 26 April 2001; returned 23 July 2001; revised 10 September 2001; accepted 23 November 2001