New sequence variants in patients affected by amyloidosis show transthyretin instability by isoelectric focusing.

The plasma protein transthyretin (TTR) can aggregate into insoluble amyloid fibrils causing systemic amyloidosis (ATTR amyloidosis) in patients carrying a variant TTR protein. If new variants arise, it is crucial to clarify whether they are disease-associated or benign. In this study, we further functionally characterize three new and unclassified TTR variants (Thr40Asn, Phe64Val and the described but not functionally assessed variant Leu12Val), using a simplified, fast isoelectric focusing (IEF) approach. After validating the system with known TTR variants, we assessed the sera of five patients carrying these new TTR variants in a heterozygous state. All three variants showed aberrant banding patterns that were similar to those of other well-characterized TTR variants, including the common Val30Met variant that causes ATTR amyloidosis. In addition to a clear band corresponding to monomeric wild-type TTR, we observed an additional variant band at the cathodal side of the IEF gel. These results indicate conformational instability of the new Thr40Asn, Phe64Val and Leu12Val variants. Together with the clinical and immunohistological data of these patients and affected family members, as well as the absence of these variants in human genetic mutation databases, our results strongly hint that these variants are amyloidogenic and therefore probably disease-associated. These findings have implications for patient therapy and for genetic counselling of family members.

Aspergillus fumigatus biofilms in the clinical setting.

We discuss in this work the role of Aspergillus biofilms in the clinical setting by reviewing the most recent findings on this topic. Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with typical biofilm characteristics under all static conditions tested, i.e., agar media, polystyrene and bronchial epithelial cells. Under static conditions the mycelial growth is greater than in shaken, submerged conditions. The extracellular matrix (ECM) is composed of galactomannan, α-1,3-glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. Typical biofilm structures were observed in the aspergillomas from two patients and in a murine model of invasive pulmonary aspergillosis. The results indicate that α-1,3-glucans plays a predominant role in the agglutination of the hyphae together in aerial conditions, and that nutrient starvation was responsible for mycelial death in aspergilloma. Melanin was produced during the infection, suggesting that this pigment is necessary for lung tissue invasion. All antifungal drugs are significantly less effective when A. fumigatus is grown under biofilm vs. planktonic conditions. Chronic persistence of a unique genotype of A. fumigatus in the respiratory tract of CF-patients and the presence of an ECM in vivo may have some therapeutical application for aspergillosis. The most appropriate antifungal drug should not be selected only on the basis of its efficiency to kill in vitro grown fungal cells, but also on its ability to penetrate the ECM.

Characteristics of pathogenic fungi and antifungal therapy in cystic fibrosis.

A defective mucociliary clearance facilitates colonization with bacteria and fungal spores in cystic fibrosis patients. Yeasts and molds are cultured from the cystic fibrosis respiratory tract and often their clinical relevance is unknown. Candida spp. are the most commonly isolated yeasts, whereas Aspergillus spp., Scedosporium apiospermum, as well as Exophiala dermatitidis in some countries, are the most frequent molds recovered from respiratory specimens. Molecular biotyping studies have revealed that some fungal genotypes are capable of chronically colonizing the airways. Persistent Aspergillus fumigatus infection is associated with an increased risk of pulmonary exacerbations requiring hospitalization. The prevalence of non-Aspergillus molds may be underestimated due to overgrowth of Pseudomonas and Aspergillus spp. on routine media. Allergic bronchopulmonary aspergillosis is usually treated by oral steroids and an antifungal azole drug. Interactions with the co-medication have to be considered. A small number of antifungal pharmacokinetic studies indicate a high inter-subject variability for itraconazole, voriconazole and posaconazole, and therefore therapeutic drug monitoring is recommended.

Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin.

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic- and biofilm-grown A. fumigatus mycelium after 24 and 48 h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real-time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Effects of caspofungin, Candida albicans and Aspergillus fumigatus on toll-like receptor 9 of GM-CSF-stimulated PMNs.

The possible involvement of Toll-like receptors (TLRs) 1, 2, 4 and 9 in the interaction of antifungal drugs with polymorphonuclear neutrophils (PMNs) in response to Aspergillus fumigatus and Candida albicans as stimuli was investigated. Caspofungin revealed the broadest capacity to enable C. albicans and A. fumigatus to stimulate TLR upregulation, TLR 2 by A. fumigatus and TLRs 4, 9 by C. albicans. Conventional amphotericin B (cAMB) stimulated only A. fumigatus to induce TLRs 2 and 4 upregulation; voriconazole stimulated A. fumigatus and fluconazole C. albicans to induce TLR 9 upregulation. For cAMB, only TLR 9 was upregulated by A. fumigatus, whereas in the case of voriconazole, TLRs 2, 4, 9 were upregulated. Caspofungin revealed the broadest capacity: C. albicans was stimulated to upregulate TLRs at least at one of the concentrations, and A. fumigatus was stimulated to upregulate TLRs 2, 4. TLR 9 was upregulated two to three fold by all antifungal drugs on protein, except for fluconazole at the RNA level. Candida albicans preincubated with caspofungin has additional effects on CD11b expression and IL8 chemotaxis in CpG-DNA-stimulated PMNs. These results indicate a relevant upregulation with a functional relevance of TLR 9 in the presence of C. albicans strains preincubated with caspofungin at three concentrations.

Liposomal amphotericin B eradicates Candida albicans biofilm in a continuous catheter flow model.

The aim of this study was to test whether a Candida albicans biofilm can be eradicated by liposomal amphotericin B (LAMB) at the minimal inhibitory concentration in a novel catheter continuous flow model. After 24-h biofilm formation and a 24-h treatment with LAMB, the growth of the hyphal network was reduced to 20% in comparison with the untreated control, whereas fluconazole and caspofungin remained at an intermediate phase (50%). After 24-h biofilm formation and a 24-h treatment with LAMB, 20% of the surface was covered in biofilm and LAMB caused an uneven surface. For caspofungin and fluconazole, the surface covering was 80%. The extracellular matrix (ECM) of the infected, but untreated catheters had a thickness of 5-20 microm at 24 h and 10-150 microm at 48 h. After 24-h biofilm formation and a 24-h treatment with LAMB, the ECM was virtually cleared with 0 microm ECM. After 24-h biofilm formation and a 24-h treatment with fluconazole, the ECM thickness was comparable to the infected, but untreated catheter at 24 h with 10-25 microm; with caspofungin, the ECM thickness was comparable to the infected, but untreated catheter at 48 h with 10-130 microm. Comparing the blastospores, pseudohyphae and ECM, 0.5 microg mL(-1) LAMB could eradicate Candida biofilm, whereas fluconazole and caspofungin were less effective.

Persistence of Candida species in the respiratory tract of cystic fibrosis patients.

It is still controversial as to whether Candida spp. are transient or persistent colonizers of the respiratory tract of cystic fibrosis (CF) patients. We conducted a prospective study of 56 CF patients over a 30 month period to assess the distribution and persistence of different Candida spp. In vitro antifungal susceptibility testing was performed and the C. albicans isolates were typed with CARE-2 hybridization and other Candida spp. by RAPD-PCR for persistence and transmission. We found that the mean persistence of the most frequent Candida spp. was >or= 9 months. In patients from whom more than 10 isolates were recovered, we noted that at least 30% were genetically related and transmission of C. albicans in siblings was observed. The majority of all isolates were susceptible to all antifungals tested. We concluded that there was long-term persistence of Candida in the respiratory tract of CF patients and that transmission between siblings may be one possible means of acquisition. Whether long-term colonization with Candida strains can contribute to the chronic infection and inflammation in the CF lung requires further investigation.

Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis.

Aspergillus fumigatus is a chronic colonizer of the respiratory tract of patients with cystic fibrosis (CF). A total of 204 A. fumigatus isolates from 36 CF patients from three different medical centers, collected over a period of four months till 9.5 years, were genotyped using the short tandem repeat panel for A. fumigatus (STRAf assay). Four different colonization patterns were observed. Colonization patterns with only unique genotypes were found in 36% of the patients. In contrast 17% of the patients were chronically colonized with a single genotype. The remaining patients showed a predominant genotype or genotypes that succeed each other. In this collection no relation was found between colonization patterns and allergic bronchopulmonary aspergillosis.

Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells.

Aspergillus fumigatus is a leading cause of death in immunocompromised patients and a frequent colonizer of the respiratory tracts of asthma and cystic fibrosis (CF) patients. Biofilms enable bacteria and yeasts to persist in infections and can contribute to antimicrobial resistance. We investigated the ability of A. fumigatus to form biofilms on polystyrene (PS) and human bronchial epithelial (HBE) and CF bronchial epithelial (CFBE) cells. We developed a novel in vitro coculture model of A. fumigatus biofilm formation on HBE and CFBE cells. Biofilm formation was documented by dry weight, scanning electron microscopy (SEM), and confocal scanning laser microscopy (CSLM). The in vitro antifungal activities of seven antifungal drugs were tested by comparing planktonic and sessile A. fumigatus strains. A. fumigatus formed an extracellular matrix on PS and HBE and CFBE cells as evidenced by increased dry weight, SEM, and CSLM. These biofilms exhibited decreased antifungal drug susceptibility and were adherent to the epithelial cells, with fungi remaining viable throughout 3 days. These observations might have implications for treatment of A. fumigatus colonization in chronic lung diseases and for its potential impact on airway inflammation, damage, and infection.

In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections.

Long-term inserted and surgically implanted catheters can be colonised by Candida spp. Candida biofilms in vitro are often resistant to antifungal agents. The aim of this study was to investigate the in vitro activity of micafungin (MFG) against six Candida spp. biofilms on polystyrene (PS) and central venous catheter (CVC) sections. Safranin staining and differential interference contrast microscopy were used to demonstrate biofilm production. MFG activity was determined by the reduction in metabolic activity (%RMA) by tetrazolium reduction assay on both substrates. In vitro, Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida dubliniensis and Candida kefyr produced mature biofilms on PS and CVC sections. MFG was active against C. kefyr (0.5 microg/mL) and C. glabrata (<0.5 microg/mL) on PS. However, MFG displayed resistance (>16 microg/mL) against C. albicans, C. dubliniensis,C. tropicalis and C. parapsilosis. On CVC disks, MFG was active against C. glabrata (1 microg/mL) as well as C. parapsilosis and C. albicans (<0.5 microg/mL). MFG was resistant (>16 microg/mL) against C. dubliniensis, C. tropicalis and C. kefyr. MFG was active in vitro against all six Candida spp. on both substrates. However, MFG could not reduce the metabolic activity completely even at the highest concentration.