Fluconazole-resistant Candida Albicans Isolates Research Articles

In vitro interactions of nystatin and micafungin combined with chlorhexidine against Candida albicans isolates.

Oral candidiasis has become a growing problem in hospitals worldwide, and the development of antifungal drug resistance in Candida species constitutes a serious concern. This study aimed to evaluate the in vitro efficacy of nystatin, and micafungin with chlorhexidine against fluconazole-resistant and fluconazole-sensitive Candida albicans (C. albicans) isolates. In this experimental-laboratory study, a total of 20 fluconazole-resistant (n=10) and fluconazole-susceptible (n=10) C. albicans strains were obtained from the reference culture collection of the Invasive Fungi Research Center in Mazandaran University of Medical Sciences, Sari, Iran. In vitro combination of nystatin and micafungin with chlorhexidine was performed using a microdilution checkerboard method based on the Clinical and Laboratory Standards Institute guideline. Micafungin had the highest antifungal activity against C. albicans susceptible and resistant strains, with a Geometric mean of (GM) =0.008µg/ml and GM=0.008µg/ml, followed by nystatin with GM=0.06µg/ml and GM=0.042µg/ml and chlorhexidine with GM=0.25µg/ml and GM=0.165µg/ml against C. albicans resistant and sensitive strains, respectively. The interaction of micafungin and nystatin with chlorhexidine showed a synergistic interaction against most C. albicans strains. In addition, no antagonistic interaction was observed between micafungin, nystatin, and chlorhexidine against C. albicans strains. The synergistic interaction of micafungin with chlorhexidine against azole-resistant C. albicans suggests an alternative approach to overcome antifungal drug resistance. However, further studies are needed for in vivo evaluation.

Biofilm Alterations on the Stepwise Acquisition of Fluconazole-resistant Candida Albicans Isolates.

Objectives:By assessing and comparing the phenotypic changes on the stepwise acquisition of fluconazole resistant Candida albicans isolates, we could find and describe the relationship between drug resistance and biofilm formation ability in a series of clonal strains.Methods:We performed antifungal susceptibility of five drugs (fluconazole, itraconazole, voriconazole, caspofungin and amphotericin B) to further verify the antifungal activity of the six isolates in vitro. Then we combined hyphal formation assay, cell surface hydrophobicity test positively related to adherence ability, and biofilm assays in vitro to observe and compare the phenotypic characteristics of our six clonal strains.Results:Biofilm capability is enhanced for four drug- intermediate strains, whereas the initial susceptible strain and the final resistant strain are both poor in adherence, hyphal growth and biofilm formation.Conclusions:It was suggested that the biofilm formation ability were not absolutely related to the degree of fluconazole resistance.

The growth of social media in science: Social media has evolved from a mere communication channel to an integral tool for discussion and research collaboration.

Social media has become popular among scientists to communicate within and beyond their communities. More specialized channels and the ongoing lockdowns further increase their value and use.

Preliminary studies on the antifungal activities of ethanol and hot water extracts of local spices against fluconazole-resistant <i>Candida albicans</i> isolates recovered from clinical samples

This study evaluated the antifungal effect of local spices namely Capsicum annum var. shombo, Capsicum annuum var. atarugu, Capsicum annuum var. Nsukka yellow pepper, Zingiber officinale and Allium sativum against fluconazole-resistant Candida albicans isolates. Hot water and ethanol were used as solvents for extraction. The seed and pericarp extracts from Capsicum annuum alone showed no inhibitory effect against the test organisms. Notwithstanding, whole Capsicum annuum fruit extracts showed variable levels of inhibition against the tested isolates. The minimum inhibitory concentration (MIC) results of hot water extract showed that Capsicum annuum var. atarugu had the lowest MIC of 6.25 mg/ml while Capsicum annuum var. yellow pepper showed the highest MIC of 100 mg/ml. Zingiber officinale showed MIC range of 3.125 mg/ml to 50 mg/ml and Allium sativum an MIC range of 12.5 mg/ml to 50 mg/ml for both hot water extract and ethanol extracts. The least MIC was obtained with Zingiber officinale ethanol extract with an MIC of 3.125 mg/ml. Our finding shows that these spices may be potential sources of antifungal agents. Keywords: Capsicum annum , Zingiber officinale , Allium sativum , Antifungal activity, Nsukka

Extensive ERG11 mutations associated with fluconazole-resistant Candida albicans isolated from HIV-infected patients.

Background and Purpose:Azoles are preferred antifungal agents given their inexpensiveness, limited toxicity, and potentiality of oral administration. However, the extensive use of prophylactic azole therapy for chronic infections, especially in immunocompromised patients, has led to an increase in azole resistance, thereby rising health care costs. Fluconazole resistance is associated with poor clinical outcomes and the emergence of new infections. The present study aimed to investigate the mutations of ERG11 gene in fluconazole-resistant Candida albicans isolates.Materials and Methods:This study was conducted on 80 clinical samples collected from HIV-infected patients with suspected candidiasis in Tagore Medical College Hospital and Government Hospital of Thoracic Medicine, Chennai, India, for a period of 18 months (May 2016-December 2017). The antifungal susceptibility pattern was determined by agar diffusion and broth dilution techniques as per the Clinical and Laboratory Standards Institute guidelines. The ERG11 gene of the known fluconazole-resistant strains of C. albicans was amplified by polymerase chain reaction (PCR). In addition, the samples were subjected to sequencing and mutation analysis. Results:A total of 60 Candida species were isolated from HIV patients and were speciated using standard, conventional, and molecular methods. Candida albicans comprised 28.3% (n=17) of the Candida isolates, 59% (n=10) of which were resistant to fluconazole. Sequencing of the amplified product of ERG11 C. albicans gene isolates showed that they were highly mutated and included many nonsense mutations which were not reported earlier.Conclusion:The molecular characterization of ERG11 gene showed many missense and nonsense mutations. Such mutations, which were unique to the geographical area under investigation, could be concluded to account for the development of resistance to fluconazole.

Efflux pumps genes of clinical origin are related to those from fluconazole-resistant Candida albicans isolates from environmental water.

Efflux pumps coded for by CDR1, CDR2, FLU1 and MDR1 genes could be responsible for the observed resistant phenotypes in azole-resistant Candida albicans from environmental water. This was demonstrated for clinical isolates. The aim of this study was to determine the presence and genetic similarity between efflux pump genes from clinical and environmental C. albicans isolates. Yeasts were isolated and identified using 26S rRNA gene sequencing. Disk diffusion tests were conducted. PCR was used to detect the presence of efflux genes. The fragments were sequenced and subjected to BLAST and subsequent phylogenetic analysis. Thirty seven C. albicans were identified from five selected rivers; Mooi River (19 isolates), Harts River (9 isolates), Marico River (5 isolates), Crocodile River (3 isolates) and Schoonspruit River (1 isolate). All the isolates were completely resistant to azoles. Efflux pump genes were detected in most (≥60%) of the isolates. Phylogenetic analysis showed high sequence similarity between sequences from environmental isolates and clinical isolates. Resistance to the azoles and the detection of efflux pump genes renders these antifungal agents ineffective. This is a major problem, particularly for the immune-compromised sector of the community of the North West Province and warrants further investigation.

In vitro Synergistic Activity of Caspofungin Plus Polymyxin B Against Fluconazole-Resistant Candida glabrata

BackgroundCandida species account for most invasive fungal infections, and the emergence of fluconazole and caspofungin resistance is problematic. Overcoming resistance with synergism between 2 drugs may be useful. In a 2013 in vitro study, caspofungin plus colistin (polymyxin E) was found to act synergistically against fluconazole-resistant and susceptible Candida albicans isolates. The purpose of our study was to extend this finding by evaluating caspofungin plus polymyxin B for in vitro synergy against fluconazole-resistant Candida glabrata isolates. Materials and MethodsA total of 7 fluconazole-resistant C. glabrata bloodstream infection isolates were obtained from 2010–2011. Of these, 2 isolates were also resistant to caspofungin. Minimum inhibitory concentrations (MICs) for caspofungin and polymyxin B were determined by Etest and broth microdilution. Clinical and Laboratory Standards Institute breakpoints were used for fluconazole and caspofungin MIC interpretations. No interpretive guidelines exist for testing polymyxin B against C. glabrata. Synergy testing with caspofungin (1 × MIC) and polymyxin B (½MIC) was performed using a modified bacterial Etest synergy method and time-kill assay. ResultsWith the Etest synergy method, 4 out of 7 isolates showed in vitro synergy and 1 out of 7 showed additivity. The remaining isolates (both caspofungin resistant) showed indifference. Using the time-kill assay, 1 out of 7 isolates showed synergy, 1 showed additivity and the remaining 5 (including both caspofungin-resistant isolates) showed indifference. ConclusionsCaspofungin susceptibility may be required for synergism between caspofungin and polymyxin B. Further synergy testing with caspofungin plus polymyxin B and additional fluconazole-resistant C. glabrata isolates should be performed. In vitro synergy/additivity may or may not correlate with in vivo benefit.

In vitro antifungal activity of essential oils from local plants against fluconazole-resistant oral Candida albicans isolates

Background and objective: Candida albicans is an oral commensal flora that causes opportunistic local and systemic infections in immunocompromied individuals. Fluconazole is frequently used for treating patients with active infections or preventing recurrent infections. The emergence of resistant strains encouraged scientists to search for compounds that have antifungal property and can overcome the usual microbial resistant mechanisms to antimicrobial agents. Essential oils from natural plants have received great interest due to the antimicrobial property of their multiple constituents. The aim of this study was to investigate the antifungal activity of Rosemary and Thyme essential oils against fluconazole resistant oral Candida albicans isolates. Methods: Essential oil from areal parts of Rosemarium officinalis and Thymus vulgaris were obtained by hydrodistillation. Disk diffusion and microbroth dilution methods were followed to test the sensitivity of eight fluconazole resistant oral Candida albicans isolates and one ATCC strain to the extracted Essential oils. Minimum inhibitory concentration and minimum fungicidal concentration were used to evaluate the antifungal potential of the tested essential oils. Results: The results of disk diffusion method using concentrated Essential oils showed marked inhibition of growth around the prepared discs from both Essential oils. However, the recoded diameters was higher in Thyme Essential oil (Mean ± SD: 42.4 ± 6.5) when compared with Rosemary essential oil (Mean ± SD: 11.8 ± 2.8). Serial two fold dilutions of the tested essential oils showed that both essential oils attain their antifungal activities even at very low concentrations. The highest MIC and MFC of Rosemary EO were 3.125% and 6.25%, respectively. Thyme essential oil showed very low MIC and MFC (<1.56%). Conclusion: Thyme and Rosemary essential oils were ideal natural compounds against drug resistant Candida albicans strains.

ERG11 mutations and expression of resistance genes in fluconazole-resistant Candida albicans isolates.

Azole resistance in the pathogenic yeast Candida albicans poses significant challenges for its antibiotic treatment. The conformational change of the target enzyme 14 alpha-demethylase (Erg11p) due to ERG11 gene mutations is one of the mechanisms resulting in the azole resistance. ERG11 of 23 isolates (8 susceptible and 15 resistant) and 6 standard strains of Candida albicans were amplified and sequenced. Nineteen missense mutations were detected. Two mutations, G487T (A114S) and T916C (Y257H), coexisted exclusively in 14 fluconazole-resistant isolates. To identify the resistance mechanisms in the isolates with G487T and T916C mutations, we compared the expression of 5 resistance-related genes in the 14 azole-resistant isolates with those in the susceptible type strain ATCC 10231, Saccharomyces cerevisiae AD/CDR1 and AD/CDR2. The tested values of mRNA transcription of CDR1 and CDR2 were higher than that of control strain, while the semi-quantified Cdr1p values were not higher in all of the 14 resistant isolates. And the data analyzed with t test suggest that both of the differences are significant (P < 0.0005) when the resistant isolates are considered as a whole. Cdr2p was up-regulated in 5 isolates, and down-regulated or even undetectable in the remaining 9 isolates. The transcription of ERG11, MDR1, and FLU1 varied in these isolates. These data suggested that overexpression of the five genes might not be the reason of resistance in the 14 isolates with G487T and T916C, especially in the 5 isolates (GZ09, GZ15, GZ16, GZ58, and 4263) in which neither translation of Cdr1p/Cdr2p nor transcription of ERG11, MDR1, or FLU1 was detected up-regulated. The results suggest that Erg11p conformational change due to the point mutations is most likely responsible for the azole resistance in these isolates.

Green synthesis of silver nanoparticles: Another honor for the yeast model Saccharomyces cerevisiae

Background and Purpose:Microorganism-based synthesis of nanostructures has recently been noted as a green method for the sustainable development of nanotechnology. Nowadays, there have been numerous studies on the emerging resistant pathogenic bacteria and fungal isolates, the probable inability of bacteria and fungi to develop resistance against silver nanoparticles’ (SNPs) antibacterial, antifungal, antiviral and, particularly antibacterial activities. In this study, we aim to use the yeast Saccharomyces cerevisiae model for synthesis of SNPs and to investigate its antifungal activity against some isolates of Candida albicans.Materials and Methods:A standard strain of S. cerevisiae was grown in liquid medium containing mineral salt; then, it was exposed to 2 mM AgNO3. The reduction of Ag+ ions to metal nanoparticles was virtually investigated by tracing the color of the solution, which turned into reddish-brown after 72 hours. Further characterization of synthesized SNPs was performed afterwards. In addition, antifungal activity of synthesized SNPs was evaluated against fluconazole-susceptible and fluconazole-resistant isolates of Candida albicans. Results:The UV-vis spectra demonstrated a broad peak centering at 410 nm, which is associated with the particle sizes much less than 70 nm. The results of TEM demonstrated fairly uniform, spherical and small in size particles with almost 83.6% ranging between 5 and 20 nm. The zeta potential of SNPs was negative and equal to -25.0 (minus 25) mv suggesting that there was not much aggregation. Silver nanoparticles synthesized by S. cerevisiae, showed antifungal activity against fluconazole-susceptible and fluconazole-resistant Candida albicans isolates, and exhibited MIC90 values of 2 and 4 μg/ml, respectively.Conclusion:The yeast S. cerevisiae model demonstrated the potential for extracellular synthesis of fairly monodisperse silver nanoparticles.

Synergistic activity of chloroquine with fluconazole against fluconazole-resistant isolates of Candida species.

The in vitro activity of chloroquine and the interactions of chloroquine combined with fluconazole against 37 Candida isolates were tested using the broth microdilution, disk diffusion, and Etest susceptibility tests. Synergistic effect was detected with 6 of 9 fluconazole-resistant Candida albicans isolates, with Candida krusei ATCC 6258, and with all 12 fluconazole-resistant Candida tropicalis isolates.

Investigation of mutations in Erg11 gene of fluconazole resistant Candida albicans isolates from Turkish hospitals

Widespread use of fluconazole has resulted in resistance in strains of Candida. The aim of our study was to investigate Y132H and other mutations in the ERG11 gene in conferring fluconazole resistance to C.albicans isolates. Seven fluconazole-resistant (R)/susceptible dose-dependent (SDD)/trailing and 10 fluconazole-susceptible (S) isolates were included. Restriction enzyme analysis was performed on all isolates for Y132H mutation and sequence analysis was performed for other mutations in the ERG11 gene. None of our strains had Y132H mutation. One single mutation (D153E, E266D, D116E, V437I) was detected in isolates 348, 533, 644, 1453, 2157, while the others had more than one nucleotide change. D116E and E266D, which were two mutations found in fluconazole R/SDD/trailing isolates with the highest frequency, were also detected in azole S strains. K143R, G464S, G465S and V488I mutations were determined in three of the R/SDD isolates. S412T and R469K mutations were detected only in this group of strains by sequence analysis. Mutations such as K143R, G464S, G465S, V488I, S412T and R469K in the ERG11 gene were determined to be effective mechanisms in our fluconazole R/SDD C.albicans isolates. Other mechanisms of resistance, such as overexpression of ERG11 and efflux pumps and mutations in the ERG3 gene should also be investigated.

Relationship between phospholipase expression and fluconazole resistance in Candida albicans

Objective To explore the relationship between antifungal resistance and expression of a virulence factor phospholipase B1 in Candida albicans. Methods Total RNA was extracted from 15 fluconazole-resistant and 15 fluconazole-sensitive Candida albicans isolates. RT-PCR was performed to detect the expression of phospholipase B1 mRNA. Extracellular and intracellular proteins secreted by Candida albicans cells were concentrated using salting-out method and extracted by P0013B RIPA lysis buffer, respectively followed by Western-blotting analysis to detect the expression of extracellular and intracellular phospholipase B1 protein. Results The relative expression of phospholipase B1 mRNA was 0.6173 ± 0.1090 in fluconazoleresistant Candida albicans isolates, significantly higher than in fluconazole-sensitive isolates (0.2653 ± 0.0935,P ＜ 0.05). Increased expressions of extracellular and intracellular phospholipase B1 protein were noted in fluconazole-resistant Candida albicans isolates compared with fluconazole-sensitive isolates (0.4145 ± 0.2773 vs 0.2720 ± 0.2194, 0.1825 ± 0.1831 vs 0.2653 ± 0.0935, t = 2.703, 3.443, both P ＜ 0.05). Conclusions The expressions of phospholipase B1 mRNA and protein are elevated in fluconazole-resistant Candida albicans isolates, suggesting that the expression of phospholipase B1 may be associated with the occurrence of antifungal resistance in Candida albicans. Key words: Candida albicans; Phospholipase; Resistance; mRNA; Protein

Detection of TAC1 gene point mutations in fiuconazole-resistant Candida albicans isolates with rolling circle amplification.

Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albi-cans isolates with rolling circle amplification (RCA), develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms (SNPs), and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicana isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance. Key words: Candida albicans; Fluconazole; Drug resistance, fungal; Point mutation; Rolling circle amplification

Overexpression of CDR1 and CDR2 Genes Plays an Important Role in Fluconazole Resistance in Candida albicans with G487T and T916C Mutations

This study was designed to investigate potential resistance mechanisms by studying the expression of resistant genes in 14 fluconazole-resistant Candida albicans isolates, with G487T and T916C mutations in the 14alpha-demethylase (ERG11) gene, collected from human immunodeficiency virus uninfected patients and a fluconazole-susceptible control strain. The in vitro susceptibilities of the C. albicans isolates to fluconazole were determined using the broth microdilution method and a disc diffusion assay. Expression of Candida drug resistance (CDR)1, CDR2, ERG11, fluconazole resistance (FLU)1 and multidrug resistance (MDR)1 genes was measured using real-time reverse transcription-polymerase chain reaction and evaluated relative to the expression of the control gene 18SrRNA. The CDR1 and CDR2 genes were upregulated in all the fluconazole-resistant C. albicans isolates, whereas only a few isolates showed high expression of MDR1, FLU1 and ERG11 genes compared with the control strain. In conclusion, overexpression of the CDR1 and CDR2 genes may play an important role in fluconazole-resistant C. albicans with G487T and T916C mutations.

Estudio de la sensibilidad in vitro de aislamientos clínicos de mohos y levaduras a itraconazol y voriconazol

The interest on the in vitro susceptibility to itraconazole has recently increased due the availability of the intravenous formulation. In this study, comparative MICs of this antifungal with voriconazole were carried out in 62 clinical isolates of filamentous fungi and 100 yeasts isolates using the NCCLS microbroth methods described in M38-A and M27-A2 documents. A MIC90 of 0.125 micrograms per ml was observed for itraconazole and voriconazole against Aspergillus fumigatus. Higher susceptibility to itraconazole was found for the filamentous form of Sporotrhix schenckii (p = 0.001). Voriconazole was more effective against Scedosporium apiospermium while Scedosporium prolificans isolates were resistant to both azoles. Some isolates of Rhizopus stolonifer were susceptible to itraconazole and resistant to voriconazole, but without statistical significance. Susceptibility of nine species of Candida was similar for both triazoles used in this study. However, Candida glabrata was more susceptible to voriconazole. Some fluconazole-resistant Candida albicans isolates were susceptible to itraconazole and / or voriconazole. Cryptococcus neoformans was more susceptible to itraconazole than to voriconazole. Itraconazole and voriconazole showed very close in vitro activity against the tested fungal isolated, except against S. schenckii. In spite of this, there were some differences in susceptibility among isolates within the same fungal species.

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains.

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.

In-vitro activity of dicationic aromatic compounds and fluconazole against Cryptococcus neoformans and Candida spp.

We investigated the in-vitro activity of three selected dicationic aromatic compounds for nine clinical isolates of Cryptococcus neoformans and 93 clinical isolates of Candida spp., representing 12 different species, using a broth macrodilution method following NCCLS recommendations. All the clinical isolates were also tested for fluconazole susceptibility. The in-vitro data demonstrate that compounds 39 and 57 have excellent in-vitro activity for all tested strains (MIC 0.19-1.56 mg/L) except Candida pelliculosa. Moreover, compound 39 showed excellent in-vitro fungicidal activity against Candida krusei, Candida glabrata, Candida lusitaniae and Cryptococcus neoformans with MFCs in the range 0.39-6.25 mg/L. Both compounds 39 and 57 showed excellent in-vitro activity against fluconazole-resistant Candida albicans isolates, including a C. albicans strain that contains all known fluconazole-resistant mechanisms. Comparing MIC data from compounds 21, 39 and 57 with fluconazole, we found a statistically significant difference only with compound 39 (P = 0.043). However, comparing MFC data from compounds 21, 39 and 57 with fluconazole, we found statistically significant differences with all three compounds (P < 0.00001). These data indicate the potential antifungal breadth of two bis-benzimidazoles (compounds 39 and 57) as antifungal agents against yeasts. If it can be determined that compounds 39 and 57 are effective and non-toxic in vivo, the prospect of these compounds as clinically useful antifungal agents will be enhanced.

Clinically significant azole cross-resistance in Candida isolates from HIV-positive patients with oral candidosis.

To determine the proportion of fluconazole-resistant Candida albicans isolates that have clinically significant cross-resistance to itraconazole or ketoconazole, that is sufficient to result in failure of these agents at their standard doses (200 and 400 mg daily for 7 days, respectively). Seven hundred C. albicans isolates from HIV-positive patients with oral candidosis underwent susceptibility testing using a relative growth method, for which cut-off values corresponding to clinical drug failure have been established. A total of 431 isolates were fully azole-susceptible and three main resistance patterns were detected: isolates resistant to fluconazole alone (n = 100); isolates resistant to fluconazole and ketoconazole but susceptible to itraconazole (n = 94); and isolates resistant to all three drugs (n = 50). No isolates were consistently resistant to ketoconazole without being fluconazole-resistant, and no itraconazole resistance was detected without ketoconazole resistance. Resistance to fluconazole alone was more common in specimens obtained soon after first clinical fluconazole failure, whereas specimens from patients with a longer history of fluconazole-unresponsive candidosis were more likely to be infected with cross-resistant isolates. Median days of prior azole exposure and cumulative fluconazole dose were significantly less for those with isolates resistant to fluconazole alone than for those with ketoconazole cross-resistant isolates, who had received less azole therapy and smaller cumulative fluconazole doses than those with isolates cross-resistant to all three drugs (although not statistically significant). After the diagnosis of fluconazole-unresponsive candidosis, increasing cumulative doses of itraconazole solution were associated with increasing likelihood of cross-resistance. Clinically significant cross-resistance to other azoles may occur in fluconazole-resistant isolates of C. albicans, although initially most isolates are not cross-resistant and the detection of cross-resistant isolates is associated with a history of greater prior azole exposure. Patients who have been treated for fluconazole-resistant candidosis for longer and with greater cumulative doses of itraconazole solution tend to become infected with increasingly cross-resistant isolates of C. albicans.