Conventional or molecular measurement of Aspergillus load.

The quantification of organisms is a standard tool. Measurement of a hyphal organism, like Aspergillus, presents difficulties in that it is problematic to define what constitutes a cell. Growth occurs by hyphal tip extension, in which the hypha elongates and a septum is formed behind the tip to divide it in to a separate compartment. However, communication between compartments and streaming of nuclei makes defining a cell of a hyphal organism difficult and the best method for quantification of the hyphal organism remains controversial. Conventional CFU determination of fungal burden in tissue homogenates is readily done by most laboratories, but CFU recovered temporally tend to show minimal increase, and may indicate that mechanical homogenization does not cause significant fragmentation of the hyphae in the tissues. Does the lack of increase in CFU as infection worsens inadequately reflect fungal load in the tissue? Non-culture based assays including chitin determination, quantitative PCR and enzyme immunoassay (EIA) of cell wall constituents, galactomannan or beta-glucan overcome this difficulty in part. However, qPCR and EIA do not indicate viability, may result in false negatives. qPCR assay may represent a significant over-estimation, because it correlates with number of nuclei present; it also requires specialized equipment and reagents. Temporal studies of infection have demonstrated that qPCR and to some extent EIA reflect an increase in fungal burden not shown by CFU. Although qPCR and CFU may not correlate in those types of studies, comparative studies have shown CFU and qPCR do correlate when determining antifungal drug efficacy, where each method can clearly demonstrate differences in fungal burden; EIA methods have also been shown to reflect this difference. Overall, there remains no optimal, single method for determination of fungal load of Aspergillus and it may be that a combination of methods (e.g., CFU and qPCR) should be used.

Molecular epidemiology of global Candida dubliniensis isolates utilizing genomic-wide, co-dominant, PCR-based markers for strain delineation.

The molecular epidemiology of Candida dubliniensis has been studied using large complex DNA probes for Southern analysis and has revealed the existence of distinct genotypes within this species. The aim of the present study was to utilize a PCR-based analysis of molecular co-dominant markers to assess the relatedness of a global and temporally diverse collection of well characterized isolates of C. dubliniensis. Sixty-two C. dubliniensis strains were collected from the authors of previously published studies. Co-dominant PCR-based markers utilizing five separate PCR fingerprints were obtained in the present investigation. Phylogenetic and statistical analyses utilizing permutation tests were undertaken to assess correlations amongst the isolates. Three distinct PCR-groups were observed and there was evidence that strains isolated since 1990 were genotypically more similar to each other than they were to strains recovered prior to 1990.

Genetic characterization of pathogenic Saccharomyces cerevisiae isolates.

Saccharomyces cerevisiae isolates from human patients have been genetically analyzed. Some of the characteristics of these isolates are very different from laboratory and industrial strains of S. cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S. cerevisiae. Most of these clinical isolates are able to grow at 42 degrees, a temperature that completely inhibits the growth of most other S. cerevisiae strains. This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts. The ability to grow at 42 degrees is shown to be polygenic with primarily additive effects between loci. S. cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.

Efficacy of interferon-gamma and amphotericin B for the treatment of systemic murine histoplasmosis.

The number of cases of systemic histoplasmosis has increased substantially in recent years, and improved therapy is needed. We examined the efficacy of immunomodulation with interferon (IFN)-gamma alone or in combination with a suboptimal regimen of amphotericin B for the treatment of primary systemic murine histoplasmosis. In the first study, BALB/c mice were infected with Histoplasma capsulatum G217B and treated with 10(5) U of IFN given every other day either preinfection and postinfection or only postinfection, alone or in combination with amphotericin B. IFN alone given subcutaneously (s.c.) postinfection prolonged survival over untreated controls (P < 0.01), whereas intravenous (i.v.) administration was ineffective. All combination regimens and amphotericin B alone significantly prolonged survival (P < 0.0001). The combination regimens of amphotericin B and IFN i.v. (pre- and postinfection) or IFN s.c. (postinfection) reduced the fungal burden in the liver and spleen; the latter regimen had superior efficacy in the spleen (P < 0.05) to either amphotericin B or IFN alone. After infection with a low-challenge inoculum, IFN given s.c. (pre- and postinfection) alone caused a significant reduction in fungal burden in the spleen (P < 0.001). In an acutely lethal model, combination regimens of IFN s.c. or i.v. and amphotericin B again prolonged survival (P < 0.01-0.001), with amphotericin B plus IFN given s.c. (pre- and postinfection) superior to all regimens (P < 0.05-0.01). This regimen also showed enhanced efficacy in causing the reduction of fungal burden in the spleen (P < 0.05). These results indicate that IFN in combination with AmB shows enhanced efficacy in the treatment of systemic histoplasmosis and support the potential utility of IFN as an adjunctive therapy.

Experimental histoplasmosis in mice treated with anti-murine interferon-gamma antibody and in interferon-gamma gene knockout mice.

Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients. Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H. capsulatum. In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis. The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H. capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals. In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001). GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this. These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H. capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.

A novel heparin-coated hydrophilic preparation of amphotericin B hydrosomes.

Amphotericin B Hydrosomes (AH; Access Pharmaceuticals Inc) are a novel formulation of hydrophilic, heparin-surfaced nanoparticles (mean diameter 105 nm) containing amphotericin B (AmB) designed to target infected sites by local adhesion. AH are cleared in part by a hepatobiliary mechanism, which results in a reduction of AmB concentration in major organs by about 50% in 24 h. In mice with pulmonary blastomycosis, unlike Fungizone (Bristol-Myers Squibb Inc), a deoxycholate micellar formulation of AmB, AH accumulates 3-fold more in infected lungs than normal lungs, between 3 and 24 h post-injection. Histologically, AH accumulates at the sites of lesions. AH is approximately 7-fold less toxic than Fungizone based on acute lethality and histopathological assessment of renal damage. In vitro, AH and Fungizone were equally active against Blastomyces dermatitidis and in vivo they were equivalent in prolonging mouse survival, when compared with equal dosing of AmB. In reducing infectious burdens in vivo, Fungizone was 3-fold more effective than AH on a mg/kg basis of administered AmB. However, AH at 4.8 mg/kg cured 50 to 60% of mice, whereas Fungizone at a near lethal dose of 1.2 mg/kg cured none. The AH formulation of AmB has an improved therapeutic index, relative renal-site avoidance and selective accumulation in infected tissues, which combine to merit additional studies in appropriate fungal models.

Percutaneous injury analysis: consistent categorization, effective reduction methods, and future strategies.

OBJECTIVE: To report the results of an 8-year analysis of percutaneous injuries (PI), to describe interventions to decrease these injuries, and to discuss future prevention strategies. DESIGN: Using consistent methods, 881 percutaneous injury reports were reviewed, categorized, and analyzed from 1986 through 1993. SETTING: A 620-bed acute-care county teaching hospital located in San Jose, California, that is affiliated with Stanford University Medical School, Palo Alto, California. PARTICIPANTS: Employees of Santa Clara Valley Medical Center who reported percutaneous injuries from 1986 through 1993. INTERVENTIONS: Placement of needle disposal containers in all patient care areas, 1987; education, 1987 to present; communication of percutaneous injury analyses to all departments, 1988 to present; and safety product evaluation and purchases, 1991 to present. RESULTS: The total number of PI decreased by 65% (P = .0007) from 1986 through 1993. Recapping injuries decreased from 1986 through 1993 by 88% (P < .0002); interventions that included convenient placement of needle disposal containers and consistent annual education may have contributed to this decrease. Injuries from manipulating intravenous lines or heparin locks decreased in 1992 (P < .03) after purchase of a needleless system for intravenous lines. Injuries from improper disposal or from abrupt patient movement did not decrease significantly over the 8-year period. CONCLUSIONS: This institution has conducted percutaneous injury analysis for 8 years, utilizing consistent reviewers and categorization methods. Successful interventions have reduced recapping injuries, injuries from manipulating intravenous lines/heparin locks, and the overall numbers of PI. The categories of "Improper Disposal" and "Patient Moved Abruptly" present challenges for future reductions, as well as the recently identified problem of staff not using available safety devices or using them improperly.

Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis.

The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus.

Initial isolation of Candida dubliniensis from the Middle East.

Two isolates of Candida dubliniensis were identified from a collection of 30 examined from Israel in a molecular epidemiology study. The 30 isolates were tentatively identified as Candida albicans. The new species, C. dubliniensis, is being reported from new geographic locales. These two isolates, from an Arab and a Druze patient, are the first to be reported from the Middle East.

Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

The contribution of animal models of aspergillosis to understanding pathogenesis, therapy and virulence.

Animal models of aspergillosis have been used extensively to study various aspects of pathogenesis, innate and acquired host-response, disease transmission and therapy. Several different animal models of aspergillosis have been developed. Because aspergillosis is an important pulmonary disease in birds, avian models have been used successfully to study preventative vaccines. Studies done to emulate human disease have relied on models using common laboratory animal species. Guinea pig models have primarily been used in therapy studies of invasive pulmonary aspergillosis (IPA). Rabbits have been used to study IPA and systemic disease, as well as fungal keratitis. Rodent, particularly mouse, models of aspergillosis predominate as the choice for most investigators. The availability of genetically defined strains of mice, immunological reagents, cost and ease of handling are factors. Both normal and immunosuppressed animals are used routinely. These models have been used to determine efficacy of experimental therapeutics, comparative virulence of different isolates of Aspergillus, genes involved in virulence, and susceptibility to infection with Aspergillus. Mice with genetic immunological deficiency and cytokine gene-specific knockout mice facilitate studies of the roles cells, and cytokines and chemokines, play in host-resistance to Aspergillus. Overall, these models have been critical to the advancement of therapy, and our current understanding of pathogenesis and host-resistance.

Studies on itraconazole delivery and pharmacokinetics in mallard ducks (Anas platyrhynchos).

Avian aspergillosis is commonly treated with itraconazole (ITZ). This paper describes two studies using mallard ducks (Anas platyrhynchos). The first study evaluated in vivo release of ITZ from subcutaneously injected controlled-release gel formulations and the second study compared pharmacokinetic parameters for two ITZ oral suspensions. ITZ-A suspension was prepared by mixing contents of commercially available capsules with hydrochloric acid and orange juice. ITZ-B suspension was prepared by dispersing the complex of the drug with hydroxypropyl-beta-cyclodextrin in water. Concentrations of ITZ and its active metabolite, hydroxyitraconazole (OH-ITZ), in plasma and tissue samples were measured using high-performance liquid chromatography. In the second study, drug concentrations in plasma samples were also analyzed using a bioassay. After administration of two ITZ controlled-release formulations, plasma and tissue concentrations of ITZ and OH-ITZ were either very low (< or = 52 ng/mL) or undetectable. Exceptions included skin, subcutaneous fat, and muscle adjacent to the injection site. The drug from ITZ-A and ITZ-B suspensions was absorbed after oral administration. ITZ pharmacokinetic parameters for both suspensions in mallard ducks were similar and the bioassay successfully measured ITZ equivalents in plasma samples from ducks.

Comparative efficacies of amphotericin B lipid complex and amphotericin B deoxycholate suspension against murine blastomycosis.

Amphotericin B as a lipid complex and as a deoxycholate suspension (Fungizone) was tested against murine blastomycosis. All doses of each form prolonged survival (P less than 0.05 to 0.001). Fungizone was more effective than lipid complex at doses of 0.8 mg/kg of body weight. However, lipid complex at 12.8 mg/kg was not toxic and superior in efficacy (P less than 0.001) to 2.0 mg of Fungizone per kg (a toxic dose), and it cleared all animals of infection. Lipid complex is an effective therapy for murine blastomycosis.

Therapeutic efficacy of a liposomal formulation of amphotericin B (AmBisome) against murine blastomycosis.

The efficacy of a liposomal amphotericin B preparation (AmBisome) and a deoxycholate suspension (Fungizone) were compared in a pulmonary model of murine blastomycosis. Male, four-week-old CD-1 mice were infected intranasally with 19,400 or 27,450 cfu of viable Blastomyces dermatitidis yeasts and intravenous therapy begun 4 days later. Groups of ten mice were given various dosages of Fungizone or AmBisome or were untreated. All treatments were given 3 times per week for 2 weeks. Deaths were recorded over 49 days and the number of viable yeasts in the lungs of survivors quantitated by viable counting. Therapy with 1.0 mg/kg/dose of Fungizone or 1.0, 3.0, 5.0, 7.5 or 20.0 mg/kg/dose of AmBisome were equivalent and prolonged survival over controls and lower dosages of each drug. No acute or chronic toxicities were observed with any regimen. Quantitation of residual numbers of yeasts in the lung showed that 70-100% of mice given 7.5 mg/kg or greater of AmBisome were free of infection and were superior to other regimens. At equivalent 1.0 mg/kg dosages Fungizone was superior to AmBisome. Although AmBisome was three-fold less active than Fungizone on a dosage basis, higher curative doses could be given that were not toxic. These data indicate that AmBisome should be further tested in other models and in clinical trials.

Utility of the triazole D0870 in the treatment of experimental systemic coccidioidomycosis.

The new triazole, D0870, was tested for therapeutic efficacy in the treatment of experimental systemic murine coccidioidomycosis. D0870 at 1 or 10 mg kg-1 daily, or 10 or 100 mg kg-1 every other day, and fluconazole at 100 mg kg-1 daily prolonged survival (P < 0.01), but were equivalent to each other. D0870 at 10 mg kg-1 daily or 100 mg kg-1 every other day cured 20 or 30% of mice of residual infection, respectively, and were superior to 100 mg kg-1 of fluconazole daily (P < 0.001). D0870 is approximately 10-fold more active than fluconazole. It is a promising treatment for coccidioidomycosis, which warrants further testing.

Efficacies of amphotericin B lipid complex (ABLC) and conventional amphotericin B against murine coccidioidomycosis.

The comparative activities of two preparations of amphotericin B against Coccidioides immitis were investigated. These preparations were a deoxycholate suspension (conventional amphotericin B) and a lipid-based formulation, amphotericin B lipid complex (ABLC). In-vitro susceptibility testing demonstrated that the MICs of ABLC were < or = 0.25 mg/L and of conventional amphotericin B were 0.5 mg/L for C. immitis. However, conventional amphotericin B was at least four-fold more fungicidal, with a minimum fungicidal concentration of 4.0 vs > 16 mg/L for ABLC. The therapeutic efficacies were tested in murine models of acute systemic coccidioidomycosis. Female CD-1 mice were infected iv with C. immitis arthroconidia to establish high (> 50%) or low (< 50%) mortality models. Therapy with conventional amphotericin B or ABLC was given three times per week for two weeks starting three days post-infection. Controls received no therapy or drug-free diluent only. Survival was tallied up to 49 days post-infection and the fungal cfu counts in spleen, liver, and lungs of all survivors were determined. In the low mortality study all treated mice survived and all therapy regimens reduced infection in all organs. All mice given ABLC 6.6 or 13.2 mg/kg/dose and 80% given ABLC 16.5 mg/kg/dose, as well as 90% given conventional amphotericin B 0.66 mg/kg/dose were free of infection; all controls remained infected. In two high mortality studies, all mice given ABLC 0.66-20 mg/kg/dose or conventional amphotericin B 0.22 or 0.66 mg/kg/dose survived compared with 0-20% of controls. Thirty per cent of uninfected mice given ABLC 20 mg/kg/dose and 40% given conventional amphotericin B 2.0 mg/kg/dose died due to drug toxicity. Mice given ABLC or conventional amphotericin B had lower residual cfu counts of C. immitis in all organs than did controls. Sixty to one hundred per cent of mice given ABLC regimens > or = 6.6 mg/kg/dose were cured, whereas all controls and 50-60% of mice receiving the highest non-toxic conventional amphotericin B regimen (0.66 mg/kg/dose) remained infected. At equal non-toxic amphotericin B doses, conventional amphotericin B was more effective than ABLC in reducing cfu in infected organs.(ABSTRACT TRUNCATED AT 400 WORDS)

Efficacy of the partricin derivative SPA-S-753 against systemic murine candidosis.

The polyene partricin compound SPA-S-753 (Societa Prodotti Antibiotici, Milano, Italy) was assessed in a murine model of systemic candidosis. CD-1 mice were infected iv with Candida albicans and treated iv with SPA-S-753 or amphotericin B. All treatment regimens of SPA-S-753 or amphotericin B were equivalent and significantly prolonged survival compared with controls (P < 0.001). Amphotericin B and SPA-S-753 significantly reduced burdens of C. albicans in the spleen and kidneys. Overall, cure rates were similar, amphotericin B at 1 mg/kg cured three and SPA-S-753 at 10 mg/kg cured four mice of infection in both organs. The efficacy of SPA-S-753 is between equivalent and <10-fold as potent as amphotericin B. These results are encouraging and warrant further studies on SPA-S-753.

Comparative efficacy of amphotericin B colloidal dispersion and amphotericin B deoxycholate suspension in treatment of murine coccidioidomycosis.

The efficacy of a novel sterol-complexed preparation of amphotericin B, amphotericin B colloidal dispersion, was compared with that of deoxycholate-complexed amphotericin B in an acute murine model of systemic coccidioidomycosis. Mice (CD-1, female) were infected intravenously with 180 or 200 arthroconidia of Coccidioides immitis, and intravenous therapy was begun 3 days later. Six doses in various regimens of either preparation were given over 14 days, and deaths were tallied for an additional 35 days. All regimens that were not acutely lethal prolonged the survival of mice over that of controls (P less than 0.001). Quantitative determination of residual burdens of C. immitis in the spleen, liver, and lungs of survivors revealed that the colloidal dispersion was not as effective as the deoxycholate suspension on a milligram-per-kilogram basis. Deoxycholate suspension at 1.3 mg/kg cleared the organs in all mice, whereas colloidal dispersion at 5.0 mg/kg was the lowest dose that cleared organisms from all animals. Lower doses cleared organisms from fewer animals or cleared only selected organs. Deoxycholate suspension was more efficacious than colloidal dispersion in clearing C. immitis from the liver or lungs (P less than 0.05 to 0.01, dose and organ dependent) at identical doses. No overt toxicity was observed in mice treated with colloidal dispersion at 10 mg/kg. In contrast, deoxycholate suspension at 2.0 mg/kg was acutely toxic; 50% of the treated mice died after treatment. The two complexes were not equivalent on a milligram-per-kilogram basis; the deoxycholate suspension was three to four times more efficacious and also greater than 5- to greater than or equal to 8-fold more toxic. Thus, the therapeutic index of the colloidal dispersion complex is greater than that of the deoxycholate complex. The amount of amphotericin B per dose could also be increased when given as a colloidal dispersion to an optimally level. Amphotericin B colloidal dispersion shows promise for the therapy of disseminated coccidioidomycosis and should be tested in other animal models and in humans.

Overview of host defense mechanisms in systemic mycoses and the basis for immunotherapy.

Host defense against systemic mycoses is multifactoral, depending on innate, as well as acquired, mechanisms. Innate resistance mechanisms include intact physical barriers, host proteins, nonspecific inflammatory responses, hormonal status, sex, and genetic make-up. However, the importance of any 1 factor in resistance to systemic fungal infections can vary depending on the causative agent. Macrophages and neutrophils play a critical role in the stasis or killing of these organisms by using the production of oxygen radicals, cationic proteins, nitric oxide (NO), and peroxides or iron deprivation. Although these cells are often ineffective in killing the organisms innately, activation of macrophages and neutrophils during an acquired immune response by the proinflammatory cytokine interferon-gamma as well as colony-stimulating factors increases the capacity of these cells for killing. A strong Th1 response can provide protective immunity, whereas a Th2 response can result in increased disease severity. The importance of native antibodies in resistance to mycoses remains in question.

Efficacy of ravuconazole in treatment of mucosal candidosis in SCID mice.

A model of orogastric candidosis in SCID mice, which mimics disease seen in AIDS patients, was used to evaluate ravuconazole in comparison with fluconazole for treatment. Mice were infected orally with Candida albicans and received either no treatment or oral treatment once daily for 12 days with 1, 5, or 25 mg of ravuconazole per kg of body weight per day, 5 or 25 mg of fluconazole per kg per day, or diluent (10% dimethyl sulfoxide in 0.5% carboxymethyl cellulose). The numbers of C. albicans CFU in the esophagus, stomach, small intestine, and cecum on day 25 in mice given no treatment and diluent were equivalent. Both doses of fluconazole significantly reduced numbers of CFU in all four tissues but were equivalent to each other. Ravuconazole showed dose-responsive improvement of clearance of CFU. Ravuconazole at 25 mg/kg was superior in reduction of numbers of CFU in all tissues to controls or 25 mg of fluconazole per kg and to other regimens in at least three tissues. Fluconazole at 25 mg/kg cured no infection in any tissue, whereas 25 mg of ravuconazole/kg cleared infection in all tissues from 50% of mice. Ravuconazole has good efficacy and the potential to cure mucosal candidosis in the absence of a functional immune response.