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.

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.

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.

Genetic susceptibility to vaginal candidiasis.

To enable future studies on host resistance factors and therapy, inbred and outbred mouse strains were tested for susceptibility to vaginal candidiasis. Groups of mice were given 0.5 mg estradiol 3 days before and 4 days after intravaginal challenge with a suspension of Candida albicans. On day 1 after challenge, a swab was used to quantitate infection in all groups and to assure equivalent infection levels. On day 6, this was repeated and the experiment was terminated. BALB/c, the reference strain in repeated experiments, was susceptible, showing persistent infection with levels of cfu at day 6 falling within a range between a twofold decrease and a fourfold increase in relation to day 1 levels. CD-1 outbred mice were markedly resistant, with day 6 cfu levels showing a 74- to 87-fold decrease with respect to day 1 levels, whereas other outbred strains (CF-1, SW, ICR) were susceptible. A BALB/c substrain (ByJ) was also susceptible. With exception of CBA/J, which showed modest resistance, all inbred strains were similarly susceptible, including DBA/2, AKR/J, C3H/HeN, A/J and C57BL/6. The differences between CD-1 and BALB/c mice were also seen with a second C. albicans isolate. Our results show susceptibility to vaginal candidiasis is independent of the major histocompatibility locus H2 haplotype and any effect ascribable to use of particular commercial mouse suppliers. Differences among mouse strains in susceptibility to C. albicans, as seen in previous studies involving nonvaginal challenge routes, are not reflected in this vaginal candidiasis model; in general, such resistance patterns appear specific to the route of challenge administration. The resistance seen in mouse strain CD-1 is of particular interest in that CD-1 is known to be resistant to endocrine disruption by estrogen. Our results suggest this estrogen insensitivity may have broad-ranging effects on processes other than gametogenesis, including vaginal susceptibility to candidiasis.

fos-1, a putative histidine kinase as a virulence factor for systemic aspergillosis.

In fungi, two-component histidine kinases have various functions including regulation of osmosensitivity, and of cell-wall assembly. Furthermore, one of these proteins, cos-1, has been shown to be important for virulence of Candida albicans. Recently, a putative histidine kinase, fos-1, has been isolated and partially characterized from Aspergillus fumigatus. Here we compare the virulence of a fos-1 deletion strain with that of the parental wild-type strain in a murine model of systemic aspergillosis. Our results show that the fos-1 deletion strain has significantly reduced virulence as compared with the parental wild-type strain. Thus, we propose that the fos-1 two-component histidine kinase is a virulence factor of A. fumigatus.

Correlation of the frequency of petite formation by isolates of Saccharomyces cerevisiae with virulence.

In previous studies on the colony phenotype switching of Saccharomyces cerevisiae, we observed that the least virulent isolates formed greater numbers of petite colonies when grown at body temperature, 37 degrees C. To determine if there is a link between virulence and petite formation, we examined the frequency of spontaneous petite formation for virulent clinical isolates (YJM128, YJM309), an intermediate virulent segregant of YJM128 (YJM145) and avirulent clinical (YJM308) and nonclinical S. cerevisiae (Y55, YJM237) after growth at 37 degrees C. The rank order of increasing frequency of petite formation was YJM128 = YJM145 < YJM309 < Y 55 < YJM308 = YJM237, which is similar to the rank-order of virulence in CD-1 mice. To assess the virulence of petites in vivo, two mouse models, CD-1 and DBA/ 2N, were infected i.v. with 10(7) cfu of either the parental grand or a spontaneously derived petite from one of four isolates previously classified with differing degrees of virulence: YJM128, YJM309, YJM145 and Y55. In both CD-1 and DBA/2N, the mean log10 cfu of grands recovered from the brain was significantly higher than that of the petites (P<0001). Overall, petites were significantly less virulent than the parental strains. However, death of some DBA/2N mice caused by YJM128 petite 1 showed that petites are not totally avirulent. To see if S. cerevisiae isolates form petite colonies in vivo, both mouse models were infected with parental grands of YJM128 and Y55. Recovered colonies were counted and confirmed as grand or petite, and the frequency of petite colonies in the brain, the target organ, correlated with the in vitro results. Overall, these studies show an inverse correlation between the frequency of petite-colony formation and the previously determined virulence of S. cerevisiae in CD-1 mice. Furthermore, petites were significantly less virulent than the parental grands, in most cases, and petites are spontaneously formed in vivo at a frequency inversely correlated to the virulence of the strain.

Experimental paracoccidioides brasiliensis infection in mice: influence of the hormonal status of the host on tissue responses.

We have previously proposed that 17beta-estradiol may be responsible in part for the decreased frequency of clinical paracoccidioidomycosis in females via a blocking of the initial morphological transformation necessary to initiate infection. Here we examined the course of infection in male and female mice in relation to their hormonal status. After pulmonary infection with conidia, normal males showed progressive infection, whereas normal females restricted proliferation and progressive disease. In contrast, castrated animals exhibited lesser capacity to restrict disease progression. Castrated male mice reconstituted with 17beta-estradiol initially restricted proliferation, but showed disease progression later in infection, whereas castrated female mice reconstituted with testosterone were unable to restrict disease. Quantitative histological analyses demonstrated that only normal male and castrated reconstituted mice developed granulomas, which decreased in number and size with time correlating with increasing numbers of CFU in the lungs. Greater numbers of chronic inflammatory foci did not correlate with higher CFU. These results further support a role for 17beta-estradiol during early innate resistance of females to paracoccidioidomycosis.

Efficacy of ravuconazole in treatment of systemic murine histoplasmosis.

Ravuconazole (RCZ) was evaluated for efficacy in comparison to fluconazole (FCZ) and itraconazole (ITZ) in murine models of disseminated histoplasmosis. All regimens tested prolonged survival (P < 0.05 to 0.0001). At equivalent doses of 50 mg/kg of body weight, RCZ and ITZ were equally effective and RCZ was more effective than FCZ (P = 0.02). Clearance of fungal burden from the livers and spleens of mice showed RCZ and ITZ at doses of 50 mg/kg to be efficacious but not curative. These data indicate that RCZ should be studied further.

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.

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.

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.

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.

COS-l, a putative two-component histidine kinase of Candida albicans, is an in vivo virulence factor.

The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.

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.

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.

Efficacy of recombinant gamma interferon for treatment of systemic cryptococcosis in SCID mice.

We have previously shown that gamma interferon (IFN-gamma) is a useful adjunct to therapy of experimental systemic cryptococcosis in normal mice. To better emulate AIDS patients, SCID mice were infected intravenously with Cryptococcus neoformans. Mice received no therapy, 3 mg of amphotericin B (AmB) per kg of body weight, or 10(5) U of IFN-gamma alone (prophylactically and therapeutically or only therapeutically) or with AmB. In the first experiment, >75% of the mice survived. Therapy with AmB alone was efficacious compared to no therapy in all organs. Both regimens of IFN-gamma alone were efficacious in the brain and lungs, and the combination of AmB and IFN-gamma showed significant synergy in the kidneys. AmB alone cured 40% of mice of infection, whereas the combination regimens cured >50% of the mice and 90% of the brain infections. In a second study, IFN-gamma again proved efficacious alone, and when given with AmB its efficacy was improved. Therapeutic IFN-gamma alone was effective only in the liver compared to no therapy, and the combination regimen, although highly effective, showed no significant synergy. In a third experiment, AmB alone or in combination with IFN-gamma prolonged survival compared to no therapy or IFN-gamma alone. The combination regimen showed significant synergy over AmB alone in the brain, liver, kidneys, and lungs. AmB alone cured no mice of infections in more than two organs, whereas AmB in combination with IFN-gamma cured 55% of infections in three or more organs. These results indicate that IFN-gamma has therapeutic efficacy in severely immunodeficient animals, especially in combination with AmB. Significant synergistic activity was noted in all organs except the spleen. Overall, IFN-gamma has utility as an adjunctive therapy against systemic cryptococcosis in the severely immunocompromised host.

Comparative toxicities and pharmacokinetics of intrathecal lipid (amphotericin B colloidal dispersion) and conventional deoxycholate formulations of amphotericin B in rabbits.

The lipid formulation of amphotericin B, Amphotec (ABCD), has not been used intrathecally. After a single intrathecal dose or after four doses, conventionally formulated deoxycholate amphotericin B (AMB) (Fungizone) resulted in higher levels of amphotericin B in the cerebrospinal fluid of rabbits than did ABCD. Clinically and histologically, ABCD was about threefold less toxic than AMB after a single dose and 3- to 30-fold less toxic after multiple dosing. These data are encouraging for the potential use of ABCD as an intrathecal treatment.

Role of IL-10 in invasive aspergillosis: increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis.

IL-10 is associated with a Th2 response, down-regulation of a Th1 response and macrophage activation. We assessed the role of IL-10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56B1/6 IL-10(-/-) (KO) and wild-type (WT) C57B1/6 mice by i.v. administration of 1 x 10(5)-6 x 10(5) conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0.001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0.001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0.03 and < 0.001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL-10 has been shown deleterious in models of fungal infection. Our data indicate IL-10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down-regulation of macrophage responses, in WT compared with KO.