Loss of in vitro resistance in Candida glabrata following discontinuation of fluconazole prophylaxis in a hematopoietic stem cell transplantation patient

Loss of In Vitro Resistance in Candida glabrata FollowingDiscontinuation of Fluconazole Prophylaxis In a HematopoieticStem Cell Transplantation Patient

Steven D Westbrook1,3,*, Nathan P Wiederhold2,4, Ana C. Vallor2,3, Susann Kotara2, StellaBernardo2,3, Samuel A. Lee2,3, William R. Kirkpatrick2,3, Juan J. Toro2,3, Cesar O.Freytes2,3, Thomas F. Patterson2,3, and Spencer W. Redding1,3

1Department of Dental Diagnostic Sciences, The University of Texas Health Science Center atSan Antonio, TX2Department of Medicine, The University of Texas Health Science Center at San Antonio, TX3South Texas Veterans Health Care System, San Antonio, TX4The University of Texas at Austin College of Pharmacy, Division of Pharmacotherapy, Austin, TX

SUMMARYWe report a case of fluconazole-resistant oropharyngeal colonization caused by a strain ofCandida glabrata that rapidly regained susceptibility once prophylaxis with this agent wasdiscontinued and echinocandin therapy was initiated. Isolates collected before and afterdiscontinuation of fluconazole were confirmed to be isogenic by RAPD analysis. Transcriptionanalysis demonstrated constitutive expression of genes encoding efflux pumps in the isolaterecovered on fluconazole prophylaxis and transient expression in those isolates collected afterfluconazole was discontinued.

INTRODUCTIONFluconazole prophylaxis is used routinely for prophylaxis during hemopoietic stem celltransplantation (HSCT). Emergence of fluconazole resistant Candida glabrata is increasing,necessitating the use of alternative antifungal therapy [1,2]. The development of resistancegenerally occurs following chronic exposure to an antifungal agent. Here, we report therapid loss of fluconazole resistance in isogenic C. glabrata isolates from a HSCT patientfollowing discontinuation of this azole followed by short-term echinocandin use.

A 58 year old male diagnosed with stage III IgG Kappa multiple myeloma was referred tothe South Texas Veterans Health Care System (San Antonio, TX) Bone Marrow TransplantProgram for an autologous HSCT. He had previously received localized radiation therapy tothe spine and three cycles of thalidomide/dexamethasone and one cycle of salvagechemotherapy with bortezomib. The patient had received five months of fluconazole 200

*Correspondent footnote: Address correspondence and reprint requests to: Steven D. Westbrook, The University of Texas HealthScience Center at San Antonio, Department of Dental Diagnostic Sciences, 7703 Floyd Curl Drive, Mail Code 7919, San Antonio, TX78229-3900, Phone: (210) 617-5300 ext 14109, Fax: (210) 567-3303, [email protected].

TRANSPARENCY DECLARATIONSDW, ACV, SK, SB, SAL, WRK, JJT, COF: None to declare N.P.W. has received research support from Pfizer, Schering-Plough,and CyDex Pharmaceuticals. T.F.P. has received research support from Merck, Pfizer, Schering-Plough, and Nektar Therapeutics, hasserved on the speakers bureau for Merck and Pfizer, and as a consultant for Basilea, Merck, Nektar, Pfizer, and Toyama. S.W.R. hasreceived research support from Pfizer, Schering-Plough, and Astellas

NIH Public AccessAuthor ManuscriptMed Mycol. Author manuscript; available in PMC 2010 May 01.

Published in final edited form as:Med Mycol. 2010 May ; 48(3): 557–560. doi:10.3109/13693780903213504.

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mg/day since he was taking dexamethasone as part of his therapy. On admission, thefluconazole dose was increased to 400 mg/day. As part of the University of Texas HealthScience Center at San Antonio Institutional Review Board approved research protocol,weekly oral fungal surveillance samples were collected, consisting of a 15 second swishwith 10 mL of sterile water. Samples were plated on CHROMagar Candida plates foridentification of fungal isolates and resistance screening [3, 4]. On the day oftransplantation, the patient was switched to 50 mg of caspofungin due to an oral surveillanceculture collected 3 days prior to transplantation that indicated colonization with fluconazoleresistant C. glabrata (isolate 8127, MIC 64 µg/mL) as verified by CLSI M27-A3methodology, performed in triplicate [5]. Four days later the patient was switched toMicafungin, dosed 100 mg daily for a 9 day course of prophylaxis. Ten days aftertransplantation, engraftment was reached and antifungal prophylaxis was discontinued.Interestingly, C. glabrata isolates 8140 and 8152 collected 6 and 14 days, respectively, aftertransplantation and after discontinuation of fluconazole were found to be fluconazolesusceptible (MICs of 2 and 8 µg/mL, respectively) using CLSI M27-A3 methodology.

MATERIALS AND METHODSTo determine if these C. glabrata strains were isogenic, random amplification ofpolymorphic DNA (RAPD) analysis using previously described primers (OPA-18, OPE-18,and AP50-1) was utilized [6,7]. RAPD profiles were compared following PCR amplificationwith each other and C. glabrata ATCC 2001, an unrelated reference strain, using genomicDNA extracted from using the Masterpure Yeast DNA Purification Kit (EpicentreBiotechnologies, Madison, WI). Band patterns were visualized on 1.2% w/v agarose gelsilluminated with UV light following ethidium bromide staining. As shown in Figure 1, theband patterns were identical for 8127, 8140, and 8152 with each of the three primers used,and distinct from those of ATCC 2001, confirming the isogenicity of the isolates collectedfrom the patient.

To test for upregulation of genes associated with resistance in C. glabrata, relative geneexpression was measured in triplicate before and after in vitro exposure to fluconazole.Strains were adjusted to a starting inoculum of ~1 × 104 cells/mL and incubated at 37°C inYPD with shaking to ~1 × 106 cells/mL, after which they were exposed to control (sterilewater) or fluconazole 64 µg/mL at 37°C for an additional 12 hours. Cells were harvested bycentrifugation and total RNA extracted using the Yeastar RNA Kit (Zymo Research Corp.,Orange, CA). Reverse transcription to cDNA was performed (GeneAmp RNA PCR Kit;Applied Biosystems, Inc., Foster City, CA). Relative gene expression was determined byreal-time PCR (ABI PRISM 7300 Sequence Detection System) with primers and Taqmanprobes (TAMRA as the 3’ quencher dye) specific for DNA encoding the C. glabrata effluxpumps CDR1 and PDH1, and the transcriptional regulator PDR1. ACT1 served as thehousekeeping gene and was amplified in separate PCR reactions. Relative gene expressionlevels were calculated by the 2−ΔΔCT method [8]. Differences in gene expression levelsbetween cells exposed to fluconazole and control were compared using one-way analysis ofvariance (ANOVA) with Tukey’s post-test for multiple comparisons. A p-value of ≤ 0.05was considered significant.

RESULTS AND DISCUSSIONFor isolate 8127 recovered during fluconazole prophylaxis, the transcription levels of CDR1,PDH1, and PDR1 were elevated without in vitro exposure to this azole (≥ 2.25-foldcompared to ATCC 2001; Figure 2A). However, the expression of these genes was notfurther increased upon exposure to fluconazole. These results suggest constitutiveexpression of these genes affecting fluconazole susceptibility within this isolate. In contrast,

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in vitro exposure to fluconazole increased the expression of these genes in the isolatesrecovered from the patient’s oral wash following the discontinuation of azole prophylaxis(Figure 2B and C). Transcription levels of CDR1, PDR1, and PDH1 were significantlyincreased in isolate 8152 (7.0 ± 2.7, 3.6 ± 0.05, and 2.7 ± 0.4 fold, respectively; p < 0.05)following exposure to fluconazole. In isolate 8140, CDR1 transcription also increased 4.0 ±0.8 fold following exposure to fluconazole, although this did not reach statisticalsignificance. These data suggest a transient and reversible increase in the expression of thesegenes affecting fluconazole activity in the isolates recovered after the discontinuation offluconazole.

To our knowledge, this is the first case report describing rapid loss of fluconazole resistancefollowing discontinuation of this agent. Following detection of the C. glabrata isolateresistant to fluconazole, a clinical decision was made to change antifungal prophylaxis to anechinocandin. Although it is unknown if this switch in prophylaxis regimens resulted in therapid loss of fluconazole resistance, the rapid decrease in MICs to this azole after only 6days is impressive. This loss of resistance is in contrast to previous reports in whichresistance develops over time and has been attributed to gain of function mutations withinthe transcription factor PDR1 [9]. Whether this occurred in our patient cannot be determinedas isolates prior to fluconazole prophylaxis azole are not available. It is unknown if this lossof resistance is clinically significant as the patient was colonized but without oropharyngealcandidiasis. Furthermore, it is unknown if fluconazole could be used for subsequent therapyin this patient. However, this may be problematic as in vitro exposure to fluconazole rapidlyincreased resistant gene expression in these susceptible isolates. Any future use offluconazole in such a patient should include antifungal susceptibility monitoring.

AcknowledgmentsThis work was support in part by Public Health Service Grant DE-18096 from the National Institute of Dental andCraniofacial Research. The authors gratefully acknowledge Jon Maust and Marcos Olivo for their technicalassistance with yeast identification and isolation.

REFERENCES1. Fidel PL Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and

clinical disease with comparison to C. albicans. Clin Microbiol Rev. 1999; 12:80–96. [PubMed:9880475]

2. Pfaller MA, Diekema DJ, Gibbs DL, et al. Results from the ARTEMIS DISK Global AntifungalSurveillance study, 1997 to 2005: an 8.5-year analysis of susceptibilities of Candida species andother yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusiontesting. J Clin Microbiol. 2007; 45:1735–1745. [PubMed: 17442797]

3. Patterson TF, Kirkpatrick WR, Revankar SG, et al. Comparative evaluation of macrodilution andchromogenic agar screening for determining fluconazole susceptibility of Candida albicans. J ClinMicrobiol. 1996; 34:3237–3239. [PubMed: 8940483]

4. Patterson TF, Revankar SG, Kirkpatrick WR, et al. Simple method for detecting fluconazole-resistant yeasts with chromogenic agar. J Clin Microbiol. 1996; 34:1794–1797. [PubMed: 8784592]

5. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; ApprovedStandard – Third Edition. CLSI document M27-A3. Wayne, PA: Clinical and Laboratory StandardsInstitute; 2008.

6. Bautista-Munoz C, Boldo XM, Villa-Tanaca L, Hernandez-Rodriguez C. Identification of Candidaspp. by randomly amplified polymorphic DNA analysis and differentiation between Candidaalbicans and Candida dubliniensis by direct PCR methods. J Clin Microbiol. 2003; 41:414–420.[PubMed: 12517882]



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7. Becker K, Badehorn D, Deiwick S, Peters G, Fegeler W. Molecular genotyping of Candida specieswith special respect to Candida (Torulopsis) glabrata strains by arbitrarily primed PCR. J MedMicrobiol. 2000; 49:575–581. [PubMed: 10847212]

8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitativePCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25:402–408. [PubMed: 11846609]

9. Tsai HF, Krol AA, Sarti KE, Bennett JE. Candida glabrata PDR1, a transcriptional regulator of apleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants.Antimicrob Agents Chemother. 2006; 50:1384–1392. [PubMed: 16569856]



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Figure 1.RAPD gel patterns for C. glabrata isolates 8127, 8140, 8152 and ATCC 2001 (lanes 2–5, inorder) obtained using primers OPA-18 (panel A), OPE-18 (panel B), and AP50-1 (panel C).Lane 1 contains 1 kb DNA ladder in panels A, B and C. DNA shown is within the 0.5–4 kbregion of the ladder.



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Figure 2.Relative gene expression of CDR1, PDR1, and PDH1 in isolates (A) 8127, (B) 8140, and(C) 8152 relative to ATCC 2001 in the presence and absence of fluconazole (FLC).Fluconazole exposure occurred at a concentration of 64 µg/mL for 12 hours. Expressionlevels were normalized using ACT1 as the housekeeping gene.



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Table 1

Primer sequences used for RAPD and gene expression of CDR1, PDR1, PDH1, and ACT1 in C. glabrataisolates. (GenBank Accession Numbers)

Primer Sequence

RAPD

AP50-1 5’-GATTCAGACC

OPA-18 5’-AGCTGACCGT

OPE-18 5’-GGACTGCAGA

CDR1(AF109723)

Forward 5’-CAAACCATACTCCCTTGGCTGTTA

Reverse 5’-GAAGTTGGCCTGGTATTCGATATC

Probe 5’-FAM-GAAGTTGGCCTGGTATTCGATATC-TAMRA-3’

PDR1(AY700584)

Forward 5’-TCGGCGAGGGTAAATTCAAC

Reverse 5’-CCAACTGCGTTTGATTCCTTAAG

Probe 5’-FAM-TACACTAACTGCATCTCCCTTATCGG-TAMRA-3’

PDH1(AF046120)

Forward 5’-TGGGCAACATGCCAACTG

Reverse 5’-AGGTTGGTGAATAGTGCATAAGATTG

Probe 5’-FAM-TGAAGAAACTGGCAAACCACGGACA-TAMRA-3’

ACT1 Forward 5’-AATTGAGAGTCGCCCCAGAA

Reverse 5’-CTGTTAGACTTTGGGTTCATTGGA

Probe 5’-FAM-ACACCCAGTCTTGTTGACCGAGG-TAMRA-3’


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Loss of in vitro resistance in Candida glabrata following discontinuation of fluconazole prophylaxis in a hematopoietic stem cell transplantation patient