Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans.

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

Candida glabrata fungemia in transplant patients receiving voriconazole after fluconazole.

The clinical impact of voriconazole resistance in Candida glabrata is not well described. Five hematopoietic stem cell transplant recipients that developed breakthrough Candida glabrata bloodstream infections while receiving voriconazole are described and the clinical management and susceptibility profiles of their isolates are reported. All patients were markedly immunosuppressed, and in all cases, voriconazole use was preceded by prolonged fluconazole exposure (median 60 days); median voriconazole exposure prior to candidemia was 48 days. Isolates from 4 patients were shown to be resistant to fluconazole and itraconazole when tested in vitro; these same isolates had MICs to voriconazole and posaconazole > or = 2 microg/ml. Clinical failure to voriconazole may result from deficits in host defense, retained infected foci, and adaptation of the organism to environmental pressures, the specific sequence and mechanisms of which warrant further study. Clinicians must maintain a high index of suspicion for these infections in highly susceptible hosts despite voriconazole therapy, particularly when voriconazole use is preceded by prolonged fluconazole exposure.

In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus isolates from transplant and nontransplant patients.

We performed in vitro antifungal checkerboard testing on 12 Aspergillus fumigatus clinical isolates (6 transplant recipients and 6 nontransplant patients) with three antifungal agents (amphotericin B, voriconazole, and caspofungin) and three immunosuppressants (FK506, cyclosporine, and rapamycin). We were not able to detect a difference in calcineurin inhibitor antifungal activity against isolates from transplant recipients and nontransplant patients.

Value of an inhalational model of invasive aspergillosis.

Animal models of invasive aspergillosis have been used for virulence studies and antifungal efficacy evaluations but results have been inconsistent. In an attempt to reproduce human infection, many Aspergillus animal models have utilized a 'pulmonary route' for delivery of conidia, largely through intranasal instillation. However, several radiolabeled particle studies have shown that aerosol delivery is preferable to intranasal instillation to create a more homogenous delivery to the lungs. We hypothesized that an inhalational model would be more robust for studies of invasive aspergillosis pathogenesis and antifungal therapy. We developed an inhalational model of Aspergillus fumigatus infection using a Hinners inhalation chamber and demonstrated by quantitative polymerase chain reaction that this new inhalational model creates a more homogenous murine pneumonia, facilitating analysis of mutant strains and treatment regimens.

NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans.

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b5 reductase and cytochrome b5 were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b5 reductase and cytochrome b5, expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent Km values similar to those found in human liver microsomes and specific activities (Vmax) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b5 in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b5 reductase and cytochrome b5 significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b5 reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b5 reductase and cytochrome b5 in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Thiopurine methyltransferase activity in red blood cells of dogs.

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of thiopurine medications such as azathioprine. In humans, activity varies widely among individuals, primarily because of genetic polymorphisms. Low TPMT activity increases the risk of myelosuppression from azathioprine and 6-mercaptopurine, whereas high TPMT activity is associated with poor drug efficacy. The purpose of this study was to determine whether dogs also show a wide range of TPMT activity. Heparinized blood samples were obtained from 177 dogs associated with a veterinary teaching hospital. Red blood cell (RBC) TPMT activity was measured by means of a modification of a radiochemical method as established for use in people. TPMT activity varied across a 9-fold range (7.9-71.8 U of RBC per milliliter; median, 21.7). Variation in TPMT activity was not associated with age, sex, or neutering status. Giant Schnauzers had much lower TPMT activity (7.9-20 U of RBC per milliliter; median, 13.1; P < .001) than did other breeds, and Alaskan Malamutes had much higher TPMT activity (22.7-71.8 U of RBC per milliliter; median, 36.0; P < .001) than did other breeds. Such variations in TPMT activity in the canine population and within groups of related dogs could affect thiopurine drug toxicity and efficacy in canine patients.

Risk of fungemia due to Rhodotorula and antifungal susceptibility testing of Rhodotorula isolates.

Rhodotorula infections occur among patients with immunosuppression and/or central venous catheters. Using standardized methods (NCCLS M27-A), we determined the antifungal susceptibilities of 10 Rhodotorula bloodstream infection isolates. Patient information was collected for clinical correlation. The MICs of amphotericin B and posaconazole were the lowest, and the MICs of triazoles and echinocandins were higher than those of other antifungal agents.

Scedosporium prolificans osteomyelitis in an immunocompetent child treated with voriconazole and caspofungin, as well as locally applied polyhexamethylene biguanide.

Scedosporium species are increasingly isolated from immunocompromised and immunocompetent patients. Unfortunately, Scedosporium infections are generally resistant to amphotericin B, and Scedosporium prolificans strains are particularly resistant to the antifungal agents now in use. We report here on an immunocompetent child with S. prolificans-associated osteomyelitis successfully treated with debridement, local irrigation with polyhexamethylene biguanide, and the systemic administration of voriconazole and caspofungin despite poor in vitro activity of voriconazole alone against the isolate. We also review the treatments and outcomes of 28 reported cases of osteomyelitis or septic arthritis caused by Scedosporium species in immunocompetent patients.

Inhibition by atovaquone of CYP2C9-mediated sulphamethoxazole hydroxylamine formation.

OBJECTIVE: To determine whether the antiprotozoal drug atovaquone inhibits the cytochrome P(450) (CYP)2C9-mediated metabolism of sulphamethoxazole (SMX) to its potentially harmful hydroxylamine metabolite (SMX-HA) in vitro. METHODS: Generation of SMX-HA from SMX was measured directly using high-performance liquid chromatography in human liver microsomes or expressed CYP2C9*1, with or without preincubation with reduced nicotinamide adenine dinucleotide phosphate, and the inhibition constant (K(i)) for atovaquone was determined. To determine the effect of protein binding in vitro, in some experiments, atovaquone was pre-incubated with serum proteins, followed by filtration. RESULTS: The K(i) for inhibition of SMX-HA formation by atovaquone was 15 microM, which is within clinically attainable total plasma atovaquone concentrations of 45-55 microM. Atovaquone (45 microM) inhibited SMX-HA formation by 39% in human liver microsomes. However, following preincubation of atovaquone with serum proteins, no inhibitory effect by atovaquone was observed, consistent with previous reports of high plasma protein binding for atovaquone. Compared with human liver microsomes, CYP2C9*1 showed an eightfold greater specific activity for SMX-HA generation; as for liver microsomes, CYP2C9*1 activity was inhibited by atovaquone. CONCLUSIONS: Atovaquone is a relatively weak inhibitor of CYP2C9-mediated SMX-HA formation in vitro. However, the effect is not observed in the presence of serum proteins. It is therefore unlikely that atovaquone would significantly inhibit SMX-HA formation in vivo.