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Detection of human P-glycoprotein-like molecule in azole-resistant Candida albicans from HIV+ patients.

Stringaro A, Molinari A, Calcabrini A, Arancia G, Ceddia PG, Cianfriglia M, Poloni F, Mondello F, Angiolella L, De Bernardis F, Cassone A.

Laboratorio di Ultrastrutture, Istituto Superiore di Sanita, Rome, Italy.

Azole resistance in Candida albicans may be due to several mechanisms. It has been demonstrated that C. albicans possesses sequences with a high degree of homology with the human MDR-1 gene coding for P-glycoprotein (P-gp), belonging to the ATP-binding cassette transporter (ABC) superfamily and responsible for the multidrug resistance (MDR) in tumor cells. On this basis, the expression and intracellular localization of human P-gp-like molecule in C. albicans strains showing different sensitivity to fluconazole were investigated by flow cytometry and immunoelectron microscopy. Post-embedding immunolabeling revealed that monoclonal antibody (mAb) MM4.17, which recognizes an external epitope of human P-gp, reacted with both fluconazole-sensitive (3153 and CO 23-1) and fluconazole-resistant (AIDS 68 and CO 23-2, isolated from AIDS patient and in vitro drug-selected, respectively) strains of C. albicans. However, the resistant strains displayed a number of MM4.17-reactive epitopes much higher than the drug-sensitive ones. The C. krusei ATCC 6458 strain, whose resistance is not mediated by the presence of ABC transporters, was not reactive at all with mAb MM4.17. The specificity of the immunolabeling was confirmed by a competitive inhibition assay performed by using phage clone particles capable of mimicking the MM4.17-reactive epitope. The flow cytometric analysis confirmed a higher level of intracytoplasmic P-gp expression in azole-resistant strains of C. albicans. Both cyclosporin A and verapamil, which are well-known MDR inhibitors, strongly reduced the MICs for fluconazole and itraconazole of the tested azole-resistant AIDS 68 strain, while they did not influence the MICs of either the sensitive 3153 strain of C. albicans or the ATCC 6458 strain of C. krusei. Overall, our data suggest the existence of a P-gp-like drug efflux pump in C. albicans that may participate in the mechanisms of azole-resistance of this fungus.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12363014&dopt=Abstract fluconazole Diflucan



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Evaluation of differential gene expression in fluconazole-susceptible and -resistant isolates of Candida albicans by cDNA microarray analysis.

Rogers PD, Barker KS.

Departments of Clinical Pharmacy. Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. drogers utmem.edu

The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1, which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3, the ergosterol biosynthesis gene ERG2, and the cell stress genes CRD2, GPX1, and IFD5 were found to be upregulated. Several genes, such as the mitochondrial aldehyde dehydrogenase gene ALD5, the glycosylphosphatidylinositol synthesis gene GPI1, and the iron transport genes FET34 and FTR2 were found to be downregulated. Further study of these differentially regulated genes is warranted to evaluate how they may be involved in azole resistance. In addition to these novel findings, we demonstrate the utility of microarray analysis for studying the molecular mechanisms of drug resistance in pathogenic organisms.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12384344&dopt=Abstract fluconazole Diflucan



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Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection.

Lewis RE, Kontoyiannis DP, Darouiche RO, Raad II, Prince RA.

University of Houston College of Pharmacy, Houston, Texas 77030, USA. rlewis uh.edu

The activity of five simulated antifungal regimens for eradication of catheter-related bloodstream Candida infection was evaluated with an in vitro pharmacodynamic model. Single-lumen central venous catheters were colonized with Candida species by sequentially incubating central venous catheters in plasma and then in growth medium (RPMI plus morpholinepropanesulfonic acid) containing a standardized suspension (10(5) CFU/ml) of Candida albicans, Candida glabrata, or slime-producing Candida parapsilosis. Colonized central venous catheters were then placed in a one-compartment pharmacodynamic model where five antifungal regimens (plus control) were simulated: amphotericin B, 1.0 mg/kg every 24 h; amphotericin B, 0.5 mg/kg every 24 h; fluconazole, 400 mg every 24 h; fluconazole, 800 mg every 24 h; and voriconazole, 4 mg/kg every 12 h. During exposure to the simulated clinical regimens, samples were serially removed from the model over 48 h for quantitation of viable organisms. All antifungal regimens suppressed fungal counts by both peripheral and catheter sampling versus control (P = 0.001). Overall, antifungal activity ranked amphotericin B (1 mg/kg) > amphotericin B (0.5 mg/kg) > or = voriconazole > fluconazole (800 mg) > or = fluconazole (400 mg). No regimen, however, completely eradicated (by culture and electron microscopy) central venous catheter colonization. Regrowth was noted in the model during therapy against C. glabrata and C. parapsilosis but was not associated with an increase in the MICs for the isolates. Lack of in vitro antifungal activity against biofilm-encased organisms appeared to be the primary reason for mycological failure of antifungal regimens in the model.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12384356&dopt=Abstract fluconazole Diflucan



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Effects of fluconazole singly and in combination with 5-fluorocytosine or amphotericin B in the treatment of cryptococcal meningoencephalitis in an intracranial murine model.

Hossain MA, Mukherjee PK, Reyes G, Long L, Ghannoum MA.

Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH 44106-5028, USA.

In this study we developed a highly reproducible intracranial murine model of cryptococcosis. Mice (Balb/c, 5-7 weeks old) were challenged intracranially and treated with intermediate (30 mg/kg) or high (90 mg/kg) dose fluconazole, and amphotericin B (0.75 mg/kg), administered singly or in combination with flucytosine (100 mg/kg). Survival and brain CFU analyses were performed. Effect of fluconazole prophylaxis was also determined. Our data show that the developed model mimics clinical signs of cryptococcal meningitis. In single treatment, fluconazole (30 mg/kg) was more efficacious than amphotericin B or flucytosine (P < 0.0001). Combination treatment led to significantly increased anticryptococcal activity, which was highest for high dose fluconazole + flucytosine (P < 0.0001). However, no significant difference was observed between high dose fluconazole treatment with and without flucytosine (P >0.05). Fluconazole prophylaxis led to a significant decrease in brain CFU. In conclusion, high dose fluconazole administered post-infection, or as prophylaxis, may be highly efficacious in the treatment and prevention of meningoencephalitis.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12420852&dopt=Abstract fluconazole Diflucan



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In vitro susceptibilities of cerebrospinal fluid isolates of Cryptococcus neoformans collected during a ten-year period against fluconazole, voriconazole and posaconazole (SCH56592).

Yildiran ST, Fothergill AW, Sutton DA, Rinaldi MG.

Department of Pathology, University of Texas Health Science Center, San Antonio, Texas, USA.sinasit gata.edu.tr

We investigated the in vitro susceptibilities of 213 cerebrospinal fluid isolates of Cryptococcus neoformans isolated from 192 patients through a 10-year period, 1990-99, against fluconazole, voriconazole and posaconazole (SCH56592) by using the NCCLS (National Committee for Clinical Laboratory Standards) macrodilution method, M27-A. The overall MICs50 and MICs90 of fluconazole, voriconazole and posaconazole were found to be 2 and 8 micro g ml-1, <or=0.125 micro g ml-1 (both), and <or=0.015 and 0.06 micro g ml-1, respectively. The MIC ranges, MICs50, and MICs90 of three triazoles were also determined according to the defined year category (1990-94, 1995, 1996, 1997 and 1998-99). The MICs50 and MICs90 of each triazole remained almost unchanged and did not exhibit any sign of an upward shift during a decade (1990-99). However, a tendency for development of possible cross-resistance between the three triazoles was observed only in 1996. Meanwhile, the individual relapsing isolates (n:21) from 20 patients exhibited same, higher or, suprisingly, lower MICs, particularly against fluconazole and posaconazole.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12421285&dopt=Abstract fluconazole Diflucan



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Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole.

Niimi M, Nagai Y, Niimi K, Wada S, Cannon RD, Uehara Y, Monk BC.

Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan. niimi nih.go.jp

Candida glabrata is an increasingly important cause of opportunistic fungal infection of humans and appears to be intrinsically resistant to the triazole antifungal fluconazole. However, the mechanisms responsible for reduced susceptibility to azole drugs are not understood. Fluconazole exposure rapidly induced expression of a 169-kDa protein band in plasma membrane fractions of C. glabrata cells. Mass spectrometry of trypsin-digested peptide fragments showed that the induced protein band comprised the ATP binding cassette-type drug efflux transporter CgCdr1p. CgCdr1p was also functionally overexpressed in S. cerevisiae and similarly identified by mass spectrometry. A 61-kDa protein band in the plasma membrane fraction from C. glabrata was also induced by fluconazole exposure. Mass spectrometric peptide fingerprinting identified this band as lanosterol 14alpha-demethylase, the enzyme in the ergosterol biosynthesis pathway targeted by fluconazole. The rapid induction of a multidrug efflux pump and/or overproduction of lanosterol 14alpha-demethylase are mechanisms that could make C. glabrata appear intrinsically resistant to fluconazole. Mass spectrometric fingerprint analysis of SDS-PAGE separated plasma membrane fractions combined with heterologous hyper-expression provides a convenient method for protein identification and functional evaluation of induced proteins, even in an organism where the genome sequence database is incomplete.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12458010&dopt=Abstract fluconazole Diflucan



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Frequency and clinical significance of bloodstream infections caused by C albicans strains with reduced susceptibility to fluconazole.

Munoz P, Fernandez-Turegano CP, Alcala L, Rodriguez-Creixems M, Pelaez T, Bouza E.

Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario "Gregorio Maranon", Madrid, Spain. pmunoz micro.hggm.es

Reduced susceptibility to fluconazole (RSF) is relatively common in non-albicans Candida isolates and in Candida albicans recovered from HIV-infected patients with relapsing Candida stomatitis or esophagitis. However, little clinical data on bloodstream infections caused by C. albicans with RSF is available. We analyzed 116 episodes of C. albicans fungemia detected over an 11-year period. Four patients (3.4%) had a blood isolate of C. albicans with RSF. Fluconazole MICs were 16 (3 SDD strains) and 128 microg/ml (1 resistant strain), respectively. Three of the patients were HIV (+) and the fourth was a liver transplant recipient. All of them had been previously treated with an azole compound. The liver recipient had breakthrough fungemia while being treated with 400 mg of preemptive fluconazole despite having an MIC of 16 microg/ml. Fluconazole clinical failure was documented in two of the remaining three cases. Only five other patients with C. albicans fungemia caused by fluconazole-resistant strains (>or=64 microg/ml) are described in the literature. Candida albicans fungemia produced by strains with RSF is still uncommon. It should be suspected in patients previously treated with azole agents or with breakthrough fungemia. In our experience, fluconazole remains a safe option for the treatment of most C. albicans fungemias, although surveillance seems advisable.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12458123&dopt=Abstract fluconazole Diflucan



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Antifungal susceptibility of South African oral yeast isolates from HIV/AIDS patients and healthy individuals.

Blignaut E, Messer S, Hollis RJ, Pfaller MA.

Department of Pathology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.

The in vitro antifungal susceptibility profile of 589 oral yeast isolates from HIV/AIDS patients and healthy South Africans was determined against amphotericin B, nystatin, 5-fluorocytosine (5-FC), clotrimazole, miconazole, ketoconazole, itraconazole and fluconazole. The broth microdilution method of the National Committee on Clinical Laboratory Standards was used and MIC(50) and MIC(90) determined. A 100% susceptibility to fluconazole was observed among the 466 isolates of Candida albicans. Among C. krusei, the second most common isolate, only 2.6% of isolates were susceptible to fluconazole and itraconazole. Despite the lack of previous exposure to antifungal agents, very little difference was observed in the antifungal profile between the South African isolates and isolates from the United States (U.S.), Canada and South America. South Africa has a particularly high incidence of HIV-infection and oral candidiasis is the most common oral complication in these patients. This study provides important baseline data as the isolates were collected prior to fluconazole being made freely available to HIV/AIDS patients attending government health clinics.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12458124&dopt=Abstract fluconazole Diflucan