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Candida spp. biofilms can be established on a wide range of materials, including implanted medical devices, and can display a resistant phenotype to antifungal drugs. Several factors, including host and surface properties, may influence the establishment and the development of Candida albicans biofilms on biotic and abiotic surfaces. We therefore selected a collection of C. albicans clinical isolates to evaluate the effect of surface and serum on biofilm attachment and development. Disc coupons from the CDC biofilm reactor were used in a well plate assay to study biofilm production on six different surfaces with or without the addition of serum: polycarbonate, polystyrene, stainless steel, Teflon, polyvinyl chloride or hydroxyapatite. Our results showed that serum increases in vitro C. albicans biofilm formation on a wide range of distinct surfaces including metallic and non-metallic materials, and that roughness and hydrophobicity can modulate C. albicans biofilm formation. These findings were also confirmed by scanning electron microscopy and it revealed the deposition of extracellular material on hyphae attached to a solid surface. Interestingly, adhesion can be significantly increased in the early stages of colonisation when serum is provided as a conditioning film in a surface-dependent manner., (© 2010 Blackwell Verlag GmbH.)

Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).

Candida krusei ATCC 6258 was tested by eight laboratories using 96-well plates containing checkerboard pairwise combinations of amphotericin B (AMB), posaconazole (PSC), caspofungin (CSP), and voriconazole (VRC). The methodology led to reproducible results across the laboratories. All drug combinations yielded MICs lower than the MICs of any two drugs tested singly, and combinations of AMB, PSC, CSP, and VRC were indifferent (no antagonism) by summations of fractional inhibitory concentration.

The DNA probe, Cp3-13, was used in a Southern blot assay for genotyping Candida parapsilosis (CP) from 3 fungemia outbreaks in neonatal intensive care units (NICUs) in the southeastern U.S. Genotyping, in 2 outbreaks, supplied evidence of horizontal transmission of CP. In the third outbreak, bloodstream isolates (BSIs) of 2 genotypes circulated in the NICU, one was shared by a BSI and a healthcare worker's hand culture. A fourth cluster of recurrent episodes of fungemia occurred in outpatients of a children's hospital receiving total parenteral nutrition (TPN) at home. Each child was infected with a different CP genotype which persisted during recurrences. These genotypes were included in a dendrogram from a CDC population-based surveillance for candidemia consisting of 73 clone-corrected Cp3-13 genotypes (overall SAB = 0.36). Analysis revealed a cluster of 11 genotypes (mean SAB = 0.66) including 3 pairs with identical hybridization profiles. A second cluster of 8 genotypes contained clones from 3 outbreaks (mean SAB = 0.76) but no clustering of genotypes specific for neonates was identified. No decreased susceptibility to azole and polyene antifungal agents was detected in this collection of CP. The frequent occurrence of transmission of CP in this vulnerable population underlines the relevance of Cp3-13 subtyping to investigate suspected transmission and persistence of CP strains in the NICU.

The paradoxical growth (PG) of Candida sp. biofilms in the presence of high caspofungin (CAS) concentrations was previously unknown. We sought to characterize the PG at supra-MICs of CAS among clinical Candida sp. isolates grown as biofilms in 96-well polystyrene microtiter plates. The MICs of CAS were determined for 30 clinical Candida sp. isolates (4 Candida albicans, 6 C. tropicalis, 7 C. parapsilosis, 8 C. orthopsilosis, and 5 C. metapsilosis isolates) when they were grown as planktonic cells and biofilms and were defined as the lowest drug concentrations that resulted in a prominent decrease in growth and a 50% reduction in metabolic activity, respectively. PG was defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above the MIC. With the exception of C. tropicalis, all isolates displayed PG more frequently when they were grown as biofilms than when they grown as planktonic cells. PG was undetectable among C. metapsilosis isolates in planktonic cell MIC tests but was present in 100% of the isolates in biofilm MIC tests. The drug concentration and the number of drug dilutions supporting PG were higher for biofilms than for planktonic cells. Microscopic changes in cell morphology were observed among both planktonic and biofilm cells with PG. Specifically, the accumulation of enlarged, globose cells was associated with PG, and we hypothesize that CAS-induced changes in the cell wall composition may be the explanation.

Southern hybridization with the complex probe Ca3 is a well established tool for molecular subtyping of Candida albicans. Multilocus sequence typing (MLST) is a DNA sequence-based subtyping method recently applied to C. albicans and shown to have a high degree of intraspecies discriminatory power. However, its utility for studying the molecular epidemiology of sequential isolates from recurrent disease has not been established. We compared Ca3 Southern hybridization and MLST using seven housekeeping genes (CaAAT1a, CaACC1, CaADP1, CaPMI, CaSYA1, CaVPS13, CaZWF1b) for their ability to discriminate among 37 C. albicans isolates from recurrent cases of oropharyngeal candidiasis (OPC) in ten HIV-positive patients from India and the US. Among the 37 isolates, MLST identified 23 distinct genotypes (index of diversity = 97%); Ca3 Southern hybridization identified 21 distinct genotypes (index of diversity = 95%). Both methods clustered isolates into seven genetically-related groups and, with one exception, isolates that were indistinguishable by MLST were indistinguishable or highly related by Ca3 Southern hybridization. These results demonstrate that MLST performs equally well or better compared to Ca3 Southern hybridization for defining genetic-relatedness of sequential C. albicans isolates from recurrent cases of OPC in HIV-positive patients.

Background: Almost one-third of patients with bloodstream infections with Candida species (candidemia) have onset of disease that occurs outside of the hospital or < or = 2 days after hospital admission (i.e., community-onset candidemia). We compared the characteristics of patients who developed candidemia by the timing of onset of infection., Methods: Incident episodes of candidemia were identified through active, population-based surveillance in Connecticut and in Baltimore and Baltimore County, Maryland, during 1 October 1998-30 September 2000. The molecular subtypes of a sample of 45 Candida parapsilosis isolates were evaluated using Southern blots hybridized with the complex probe Cp3-13., Results: Overall, 356 (31%) of the 1143 incident episodes of candidemia were classified as community-onset disease (occurring < or = 2 days after hospital admission), and 132 (37%) were caused by Candida albicans, 89 (25%) were caused by Candida glabrata, 57 (16%) were caused by C. parapsilosis, and 53 (15%) were caused by Candida tropicalis. Community-onset disease was less likely to be associated with concurrent immunosuppressive therapy, recent surgery, or use of a central venous catheter, compared with inpatient disease. Among patients with community-onset disease, the median time from blood culture to initiation of antifungal treatment was 2.7 days, the 30-day case-fatality rate was 26%, and 262 patients (75%) had been hospitalized at least once in the previous 3 months. Although there were few differences between patients with very recent hospitalization (in the previous 1 month), less recent hospitalization (previous 1-3 months), and no documented past hospitalization, C. parapsilosis was more frequently associated with community-onset disease as hospitalization became more distant. C. parapsilosis strains tended to be unique to the patient, with little similarity found between strain types, on the basis of epidemiologic classification of patients., Conclusion: We report that community-onset candidemia is common and occurs in patients with extensive contact with the health care system. Disease caused by C. parapsilosis tends to involve unique strains.

One hundred seven Candida bloodstream isolates (51 C. albicans, 24 C. glabrata, 13 C. parapsilosis, 13 C. tropicalis, 2 C. dubliniensis, 2 C. krusei, and 2 C. lusitaniae strains) from patients treated with amphotericin B alone underwent in vitro susceptibility testing against amphotericin B using five different methods. Fifty-four isolates were from patients who failed treatment, defined as death 7 to 14 days after the incident candidemia episode, having persistent fever of >or=5 days' duration after the date of the incident candidemia, or the recurrence of fever after two consecutive afebrile days while on antifungal treatment. MICs were determined by using the Clinical Laboratory Standards Institute (formally National Committee for Clinical Laboratory Standards) broth microdilution procedure with two media and by using Etest. Minimum fungicidal concentrations (MFCs) were also measured in two media. Broth microdilution tests with RPMI 1640 medium generated a restricted range of MICs (0.125 to 1 microg/ml); the corresponding MFC values ranged from 0.5 to 4 microg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 microg/ml and 0.06 to 2 microg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 microg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.

Five azole-susceptible Candida glabrata isolates obtained before 1975 became resistant to fluconazole, itraconazole, and voriconazole within 4 days of in vitro fluconazole exposure. This cross-resistance was stable for at least 4 months after removal of fluconazole and was associated with increased CgCDR1 and CgCDR2 expression.

We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

Candida parapsilosis is an important cause of bloodstream infections in the health care setting. We investigated a large C. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. We identified 22 cases of bloodstream infection with C. parapsilosis: 15 confirmed and 7 possible. The factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). Samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. Twenty-six percent of the health care workers surveyed demonstrated hand colonization with C. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the DNA probe Cp3-13. Outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. This largest known reported outbreak of C. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. Recommendations for control measures focused on improving hand hygiene compliance.

Zygoascus hellenicus (Candida hellenica) was isolated from a blood culture from a patient who had received an allogeneic stem cell transplant. The isolate displayed an antifungal susceptibility pattern of decreased susceptibility to fluconazole and itraconazole, high susceptibility to voriconazole, and low susceptibility to caspofungin. The organism was misidentified by a commercial yeast identification system. This is the first reported case of human infection with this rare ascomycetous yeast.

We developed a rapid, sensitive, and reproducible assay to quantify Candida albicans ACT1, CDR1, CDR2, ERG11, and MDR1 mRNA using a two-step reverse transcription and LightCycler real-time PCR (RT-LightCycler PCR) method with sequence-specific hybridization probes. We compared RT-LightCycler PCR with Northern hybridization for quantitative analysis of gene expression in isolates with various fluconazole susceptibilities. Specificity of each LightCycler PCR was verified by LightCycler melting curve analysis and agarose gel electrophoresis of amplified products. Correlation of quantification results between RT-LightCycler PCR and Northern hybridization yielded correlation coefficients of > or = 0.91 for all genes except MDR1 (0.74). In this case, reduced correlation was due to the inability of Northern hybridization to accurately quantify the high MDR1 expression in a susceptible dose-dependent isolate which was shown by RT-LightCycler PCR to overexpress MDR1 >200-fold relative to the other isolates tested. In four isolates, low levels of CDR2 mRNA were detected by RT-LightCycler PCR but were undetectable by Northern hybridization. mRNA quantification by RT-LightCycler PCR correlates with Northern hybridization and offers additional advantages, including increased sensitivity and speed of analysis, along with lower RNA concentration requirements and an increased dynamic range of signal detection.

To determine the incidence of Candida bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients, Candida BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months. Candida albicans comprised 45% of the isolates, followed by C. glabrata (24%), C. parapsilosis (13%), and C. tropicalis (12%). Only 1.2% of C. albicans isolates were resistant to fluconazole (MIC, > or = 64 microg/ml), compared to 7% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 0.9% of C. albicans isolates were resistant to itraconazole (MIC, > or = 1 micro g/ml), compared to 19.5% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 4.3% of C. albicans isolates were resistant to flucytosine (MIC, > or = 32 microg/ml), compared to < 1% of C. parapsilosis and C. tropicalis isolates and no C. glabrata isolates. As determined by E-test, the MICs of amphotericin B were > or = 0.38 microg/ml for 10% of Candida isolates, > or =1 microg/ml for 1.7% of isolates, and > or = 2 microg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of Candida BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.

Objectives: To investigate possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of Candida albicans that displayed the trailing growth phenomenon, and to compare these isolates with bloodstream and mucosal isolates that showed reduced susceptibilities to fluconazole., Methods: Twelve C. albicans isolates-seven trailing and five susceptible dose dependent (SDD) or resistant (R)-were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system., Results: SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates., Conclusions: Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.

A two-laboratory study was performed to evaluate the correlation between the NCCLS M27-A and EUCAST microdilution procedures for antifungal testing of Candida spp. A panel of 109 bloodstream isolates was tested against amphotericin B, flucytosine, fluconazole, and itraconazole. Overall, the agreement was 92% and the intraclass correlation coefficient was 0.90 (P < 0.05).

Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven Candida spp., obtained through active population-based surveillance between 1998 and 2000. Of 429 C. albicans isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118 C. tropicalis isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five Candida spp. tested (C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, and C. parapsilosis). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.

MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by "trailing" growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format. To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole. To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C. albicans isolates which were trailers (M27-A MICs at 24 and 48 h, /=64 microg/ml, respectively; SQM MIC, /=64 microg/ml; SQM MIC, 54 microg/ml). Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate. These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.

The PASCO antifungal susceptibility test system, developed in collaboration with a commercial company, is a broth microdilution assay which is faster and easier to use than the reference broth microdilution test performed according to the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A guidelines. Advantages of the PASCO system include the system's inclusion of quality-controlled, premade antifungal panels containing 10, twofold serial dilutions of drugs and a one-step inoculation system whereby all wells are simultaneously inoculated in a single step. For the prototype panel, we chose eight antifungal agents for in vitro testing (amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, clotrimazole, miconazole, and terconazole) and compared the results with those of the NCCLS method for testing 74 yeast isolates (14 Candida albicans, 10 Candida glabrata, 10 Candida tropicalis, 10 Candida krusei, 10 Candida dubliniensis, 10 Candida parapsilosis, and 10 Cryptococcus neoformans isolates). The overall agreements between the methods were 91% for fluconazole, 89% for amphotericin B and ketoconazole, 85% for itraconazole, 80% for flucytosine, 77% for terconazole, 66% for miconazole, and 53% for clotrimazole. In contrast to the M27-A reference method, the PASCO method classified as resistant seven itraconazole-susceptible isolates (9%), two fluconazole-susceptible isolates (3%), and three flucytosine-susceptible isolates (4%), representing 12 major errors. In addition, it classified two fluconazole-resistant isolates (3%) and one flucytosine-resistant isolate (1%) as susceptible, representing three very major errors. Overall, the agreement between the methods was greater than or equal to 80% for four of the seven species tested (C. dubliniensis, C. glabrata, C. krusei, and C. neoformans). The lowest agreement between methods was observed for miconazole and clotrimazole and for C. krusei isolates tested against terconazole. When the data for miconazole and clotrimazole were removed from the analysis, agreement was >/=80% for all seven species tested. Therefore, the PASCO method is a suitable alternative procedure for the testing of the antifungal susceptibilities of the medically important Candida spp. and C. neoformans against a range of antifungal agents with the exceptions only of miconazole and clotrimazole and of terconazole against C. krusei isolates.

As new antifungal agents are introduced for the treatment of infections caused by yeasts and filamentous fungi (moulds), it is important that reliable methods are available for the in vitro testing of both new and established agents. The ultimate goal of in vitro testing is the prediction of the clinical outcome of therapy. The use of the M27-A procedures that were developed by the US National Committee for Clinical Laboratory Standards (NCCLS) has led to increased interlaboratory agreement of minimum inhibitory concentrations (MICs) for yeasts and has facilitated the establishment of interpretive breakpoints for fluconazole and itraconazole. The clinical relevance and limitations of these breakpoints are discussed elsewhere. The focus of this paper is to review the advantages and disadvantages of the available methods for antifungal susceptibility testing of yeasts and moulds as well as the clinical implications of in vitro antifungal resistance.

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 microg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of /=64 microg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 microg of fluconazole/ml, an 84% reduction after exposure to 4 microg/ml, and 95 and 100% reductions after exposure to 16 and 64 microg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 microg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.

Aspergillus species are the most common causes of invasive mold infections in immunocompromised patients. The introduction of new antifungal agents, and recent reports of resistance emerging during treatment of aspergillus infections, have highlighted the need for standardized methods of antifungal drug susceptibility testing for filamentous fungi. This review describes the methods that are now being developed for the in vitro testing of Aspergillus species, and the results of attempts to correlate in vitro findings with in vivo outcome. The mechanisms and clinical importance of resistance to the different agents used in the treatment of human aspergillosis are discussed. Copyright 1999 Harcourt Publishers Ltd.

Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. We demonstrate that mutations affecting early and late steps in sterol homeostasis modulate the expression of the ERG3 gene: a late step in sterol biosynthesis in yeast. Expression of ERG3 is increased in response to a mutation in the major isoform of HMG CoA reductase which catalyzes the rate-limiting step of sterol biosynthesis. Likewise, mutations in non-auxotrophic ergosterol biosynthetic genes downstream of squalene production (erg2, erg3, erg4, erg5, and erg6) result in an up-regulation of ERG3 expression. Deletion analysis of the ERG3 promoter identified two upstream activation sequences: UAS1 which when deleted reduces ERG3 gene expression 3-4-fold but maintains sterol regulation and UAS2, which when deleted further reduces ERG3 expression and abolishes sterol regulation. The recent isolation of two yeast genes responsible for the esterification of intracellular sterol (ARE1 and ARE2) has enabled us to directly analyze the relationship between sterol esterification and de novo biosynthesis. Our results demonstrate that the absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation.