Coccidioidomycosis: What a long strange trip it's been.

The recorded history of coccidioidomycosis began in 1892 with the report of the illness of Domingo Escurra by Alejandro Posadas followed by a description of the first North American cases by Rixford and Gilchrist. Originally considered a protozoan, William Ophüls determined that Coccidioides was a fungus and that the lungs were the apparent initial site of infection. During the 1930s, both Gifford and Dickson determined that a self-limited illness, Valley Fever, was caused by the same fungus that caused the often fatal coccidioidal granuloma. Charles Smith, over a period of approximately 2 decades, comprehensively described the clinical and geographic epidemiology of coccidioidomycosis in California. Demosthenes Pappagianis continued this work after Smith's death. In 1957, one year before Marshall Fiese published his masterful monograph on coccidioidomycosis, the use of the first effective agent for the therapy of coccidioidomycosis, amphotericin B, was reported. This was followed by descriptions of its appropriate clinical use by William Winn and by Hans Einstein, among others. The development of the much less toxic azole antifungal agents greatly simplified therapy in many cases, but much of the management of patients with coccidioidomycosis still relies more on art than science. The search for the "Holy Grail" - a vaccine capable of preventing this disease-continues.

Development and validation of a quantitative real-time PCR assay for the early diagnosis of coccidioidomycosis.

A new real-time polymerase chain reaction (RT-PCR) assay based on a Coccidioides genus-specific molecular beacon probe was developed for the detection of coccidioidomycosis and validated with tissues from animal models and clinical samples. The assay showed high analytic reproducibility (r(2) > 0.99) and specificity for cultured strains (100%); the lower limit of detection was 1 fg of genomic DNA/µL of reaction. Fungal burdens in the organs of mice infected with Coccidioides posadasii strain Silveira were more accurately quantified by RT-PCR compared to colony-forming unit for all tissues. The RT-PCR assay was positive for 97.7% of spleen and 100% of liver or lung. Progression of infection in all organs was similar by both methods (P > 0.05). The sensitivity of the assay also was 100% for paraffin-embedded samples and samples from patients with positive cultures. Our RT-PCR assay is effective for the diagnosis and monitoring of Coccidioides infection, and its use also avoids the biohazard and time delay of identifying cultures in the clinical setting.

Influence of 17ß-estradiol on gene expression of Paracoccidioides during mycelia-to-yeast transition.

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 (17ß-estradiol, E(2)) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E(2) inhibits M-to-Y transition. METHODOLOGY: We assessed temporal gene expression in strain Pb01 in the presence or absence of E(2) 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. E(2)-regulated clones were sequenced to identify genes and biological function. PRINCIPAL FINDINGS: E(2)-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 E(2) 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 E(2)-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 E(2), 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 E(2). CONCLUSION: This study characterizes the effect of E(2) at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E(2) may be working through signaling genes that regulate dimorphism.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

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.

Safety, tolerance, and efficacy of posaconazole therapy in patients with nonmeningeal disseminated or chronic pulmonary coccidioidomycosis.

BACKGROUND: Coccidioidomycosis can be difficult to treat with available therapies, particularly in patients with progressive or disseminated disease. Posaconazole is a new azole antifungal with potent activity against Coccidioides species, the causative agent of coccidioidomycosis. METHODS: Twenty patients with chronic pulmonary or nonmeningeal disseminated coccidioidomycosis were enrolled in a multicenter trial to study the safety and tolerability of posaconazole therapy, with efficacy as a secondary end point. Patients received posaconazole (400 mg/day) in capsule formulation for up to 6 months. Safety was evaluated on the basis of the occurrence of adverse events. A satisfactory efficacy response was defined as a >or=50% reduction in the Mycoses Study Group score from baseline. RESULTS: Seventeen (85%) of 20 patients had a satisfactory response to treatment. The median duration of treatment was 173 days. Paired baseline and end-of-treatment culture results for Coccidioides species were available for 4 patients, all of whom converted from being positive to being negative for Coccidioides species. Relapse was experienced by 3 of 9 patients who did not receive antifungal therapy during the follow-up period. In general, posaconazole therapy was well tolerated, with 12 of 20 patients reporting adverse events that were possibly or probably related to treatment. The most common adverse events were dry mouth (in 5 patients [25%]) and headache (in 3 patients [15%]). CONCLUSIONS: Courses of posaconazole therapy that were up to 6 months in duration were well tolerated in patients with coccidioidomycosis. Although this study was limited by the number of patients enrolled, it clearly demonstrates that posaconazole shows promise in the treatment of patients with coccidioidomycosis and warrants additional investigation in a full-scale clinical trial.

Scedosporium apiospermum soft tissue infection successfully treated with voriconazole: potential pitfalls in the transition from intravenous to oral therapy.

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.

Cytokine and inducible nitric oxide synthase mRNA expression during experimental murine cryptococcal meningoencephalitis.

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 beta1, 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 alpha, tumor necrosis factor alpha (TNF-alpha), and iNOS were detected as early as day 1 postinfection, with TNF-alpha rising by approximately 30-fold and iNOS increasing by approximately 5-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 beta 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.

Resistance mechanisms in clinical isolates of Candida albicans.

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.