Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.
Neither fluconazole nor itraconazole showed statistically superior efficacy in nonmeningeal coccidioidomycosis, although there is a trend toward slightly greater efficacy with itraconazole at the doses studied.
SUMMARYIL-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 C56Bl/6 IL-10 2/± (KO) and wild-type (WT) C57Bl/6 mice by i.v. administration of 1 Â 10 5 26 Â 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/mm 2 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.
The immune events that take place in the central nervous system (CNS) during cryptococcal infection are incompletely understood. We used competitive reverse transcription-PCR to delineate the time course of the local expression of mRNAs encoding a variety of cytokines and inducible nitric oxide synthase (iNOS) during progressive murine cryptococcal meningoencephalitis and assessed the CNS inflammatory response using immunohistochemistry. Interleukin 18 (IL-18), transforming growth factor 1, and IL-12p 40 mRNAs were constitutively expressed in the brains of infected and uninfected mice; IL-2 mRNA was not detected at any time. Increased levels of transcripts corresponding to IL-1␣, tumor necrosis factor alpha (TNF-␣), and iNOS were detected as early as day 1 postinfection, with TNF-␣ rising by ϳ30-fold and iNOS increasing by ϳ5-fold by day 7. Each remained at these levels thereafter. IL-4, IL-6, and gamma interferon transcripts were detected on day 5, and IL-1 and IL-10 transcripts were detected beginning on day 7. Once detected, each remained at a relatively constant level through 28 days of infection. This cytokine profile does not suggest a polarized Th1 or Th2 response. Immunohistochemistry did not reveal inflammatory infiltrates before day 7, despite the presence of cryptococci. Intraparenchymal abscesses with inflammatory cells in their peripheries were found beginning on day 10. The infiltrates were comprised primarily of cells expressing CD4, CD8, or CD11b; low numbers of cells expressing CD45R/B220 were also present. The persistence of Cryptococcus observed in the CNS may result from an ineffective immune response, perhaps owing to an insufficient anticryptococcal effector function of endogenous glial cells resulting from competing pro-and anti-inflammatory cytokines. These data detail the immune response in the brain and could be important for the future design of specific immunomodulatory therapies for this important opportunistic infection.
An immunocompromised patient with an invasive soft tissue infection due to Scedosporium apiospermum was successfully treated with voriconazole and surgical debridement. After transition from intravenous to oral therapy, successive adjustments of the oral dose were required to achieve complete resolution. For soft tissue infections due to molds characterized by thin, septate hyphae branching at acute angles, voriconazole should be considered a first-line antifungal agent. The potential usefulness of plasma voriconazole levels for guiding optimal therapy should be investigated. CASE REPORTA 58-year-old woman who had been treated chronically for Behçet's disease with prednisone, at doses ranging from 10 to 40 mg per day over 10 years, developed pain, swelling, and erythema of the left wrist. These symptoms were felt by her rheumatologist to represent an unusually severe exacerbation of arthritis, and a single dose of infliximab at 3 mg/kg was given intravenously. In addition, prednisone was increased from 20 to 100 mg per day, and amoxicillin-clavulanic acid at 875 mg twice daily was empirically started. Due to continued pain and erythema, the patient underwent exploratory surgery of her left wrist 10 days later at a local community hospital. Intraoperatively, thickening and early mucinous degeneration of the tenosynovium of an extensor tendon was noted from the wrist to the distal end of the third metacarpal. Surgically obtained tissue grew rare methicillin-sensitive Staphylococcus aureus but was negative for fungi and mycobacteria based on both cultures and histopathology. Antibiotic therapy was changed from oral amoxicillin-clavulanic acid to intravenous oxacillin at 2 g every 4 h, and her oral prednisone dosage was reduced gradually from 100 mg daily to 20 mg daily over the following week.The patient's symptoms continued to progress, however, with erythema spreading from the dorsum of the left wrist to the elbow. At 4 weeks after the exploratory surgery, the patient was admitted to Stanford University Medical Center. Oxacillin was discontinued, and intravenous vancomycin, given as 1 g every 12 h, was initiated. The following day, surgical exploration and debridement of the left wrist and forearm were performed, and necrosis involving multiple extensor tendons of the wrist was noted intraoperatively. Specimens from her wrist joint and tendons were sent for bacterial, mycobacterial, and fungal cultures. Small, gray colonies of a mold grew in a culture derived from an extensor tendon. Early examination of this mold revealed thin, septate hyphae branching at acute angles. On the basis of this morphology, intravenous voriconazole was initiated at a loading dose of 6 mg/kg every 12 h for two doses, followed by 4 mg/kg given every 12 h; prednisone was continued at 20 mg daily. Two days later, diminished pain and erythema of the patient's forearm and elbow were noted. The mold was ultimately identified as Scedosporium apiospermum. The patient showed continued improvement over a 10-day course of intravenous voriconazole...
Background Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E2) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E2 inhibits M-to-Y transition.MethodologyWe assessed temporal gene expression in strain Pb01 in the presence or absence of E2 at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E2-regulated clones were sequenced to identify genes and biological function.Principal FindingsE2-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E2 belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E2-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E2, whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E2.ConclusionThis study characterizes the effect of E2 at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E2 may be working through signaling genes that regulate dimorphism.
Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25 % of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.Abbreviations: gDNA, genomic DNA; PCA, principal components analysis.The GenBank accession numbers (dbGSS id) for the DNA sequences reported in this paper are FI778766-FI779357.The microarray data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO series accession number GSE15511 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15511). INTRODUCTIONParacoccidioidomycosis is the most prevalent systemic mycosis in Latin America and is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Mackinnon, 1970;Wanke & Londero, 1994). The infection originates with ...
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