Pirjo Pärnänen

Antifungal Susceptibility and Virulence Attributes of Bloodstream Isolates of Candida from Hong Kong and Finland

Candida bloodstream infection has dramatically increased in the last decade due to the growing number of immunocompromised populations worldwide. In this study, we evaluated the antifungal susceptibility profiles and virulence attributes of Candida bloodstream isolates (CBIs) derived from Hong Kong and Finland, information which are vital for devising empirical clinical strategies. Susceptibility testing of a wide range of antifungals including fluconazole, itraconazole, voriconazole, ketoconazole, 5-fluorocytosine, amphotericin B and caspofungin was performed. Haemolytic activity and secretion of proteinase of CBIs were also examined. All CBIs derived from Hong Kong were susceptible to all the antifungals tested whilst some CBIs from Finland were resistant to azoles and caspofungin. C. albicans, C. glabrata and C. tropicalis showed higher haemolytic activity whereas C. parapsilosis and C. guilliermondii were non-haemolytic in general. Proteinase activity of the Finland C. albicans isolates was significantly higher than the Hong Kong isolates. Our data provide a glimpse of the possible evolutionary changes in pathogenic potential of Candida that may be occurring in different regions of the world. Therefore, continuous surveillance and availability of local data should be taken into consideration when treating candidemia patients.

Human laminin-332 degradation by Candida proteinases.

BACKGROUND Human laminin-332 (Lm-332) degradation by 12 Candida strains and effects of synthetic proteinase inhibitors [Ilomastat (ILM), EDTA, chemically modified tetracycline-3(CMT-3), CMT-308, synthetic peptide CTT-2, and Pefabloc] were studied. MATERIALS AND METHODS Laminin-332 was incubated with sonicated cell fractions and 10 times concentrated cell-free fractions of reference and clinical strains of C. albicans, C. dubliniensis, C. guilliermondii, C. glabrata, C. krusei, and C. tropicalis. Proteolysis, pH effects, and inhibitors were analyzed by fluorography and zymography. RESULTS Cell fractions of all species except C. guilliermondii and cell-free fractions of C. albicans, and C. dubliniensis showed 20-70 kDa gelatinases at pH 5.0 and 6.0. At pH 7.6, C. glabrata, C. krusei, and C. tropicalis cell fractions and C. tropicalis cell-free fractions showed 55-70 kDa gelatinases. CMT-3, CMT-308, and CTT-2 inhibited Candida gelatinases slightly better than Pefabloc, ILM, and EDTA. No Candida fractions degraded Lm-332 at pH 7.6, but at pH 5.0, 100 kDa bands were generated by cell fractions of C. dubliniensis and C. tropicalis; C. albicans and C. glabrata clinical strains; and C. guilliermondii reference strain. C. krusei reference strain yielded three 100-130 kDa bands. C. albicans, C. dubliniensis, and C. tropicalis reference and clinical strains cell-free fractions generated 100 kDa band. CONCLUSIONS Laminin-332 degradation is pH-dependent and differences exist between studied Candida strains. Lm-332 degradation can exert functional disturbances on basement membrane integrity, possibly aiding Candida cell invasion into tissues. Certain synthetic matrix metalloproteinase inhibitors (CMTs, CTT) can inhibit Candida proteinases and may be therapeutically useful in future.

Prevalence and antifungal drug sensitivity of non-albicans Candida in oral rinse samples of self-caring elderly

AIM To assess the prevalence and antifungal drug sensitivity of non-albicans Candida (NAC) species in elderly outpatients. MATERIALS AND METHODS We investigated oral rinse samples of 194 self-caring elderly population (mean age 83 years) with emphasis on background factors for harbouring NAC. Susceptibility of Candida species to antifungal drugs was determined using standard methodology. Multiple logistic regression analysis was performed taking positive NAC count as the dependent variable and a number of known Candida risk factors as independent variables. RESULTS Prevalence of candidal carriage of the population was 78.4%, of which 0.5% of the subjects were NAC positive. Candida dubliniensis was the most prevalent NAC species, followed by Candida glabrata and Candida parapsilosis. The NAC positive elderly were more often edentulous with dental prostheses or had fewer teeth than Candida albicans-positive or yeast-negative subjects. Dental caries slightly increased the risk for having NAC strains (odds ratio 1.08), whilst greater age appeared to lower the risk (odds ratio 0.77). Candida species were susceptible to the commonly used antifungal agents in general, but with considerable variation among species. Occasionally, some NAC exhibited lower antifungal susceptibility. CONCLUSION The possibility of oral reservoirs of NAC strains which are resistant to common antifungals should be noted in elderly outpatients.

Effect of amine fluoride-stannous fluoride preparations on oral yeasts in the elderly: a randomised placebo-controlled trial.

OBJECTIVES Oral yeast infections are an emerging problem among medically compromised and frail elderly. Antifungal drug resistance is also increasing because of an increase in non-albicans Candida strains in these populations. We therefore set out to study, in the randomised-controlled trial setting if the use of a topical amine fluoride-stannous fluoride combination (AmF-SnF2) could control oral Candida growth in the elderly. The hypothesis was based on earlier findings showing that in vitro this combination had antifungal efficacy. METHODS A total of 194 nursing home residents were randomised to receive either the test mouth rinse and toothpaste or a placebo twice daily for 8 months. Of these, 136 completed the trial. Saliva samples were taken using the oral rinse method, cultivated and the strain level identified with routine microbial methods. Compliance and use of preparations was assessed by a nurse. RESULTS Significantly at the end of the trial, less mucosal lesions were observed in the test group in comparison to controls. Total bacterial count decreased in both the groups during the trial. Candida albicans was the most prevalent strain detected both at baseline and 8 months later. Only a few subjects carried non-albicans strains. The AmF-SnF2 did not significantly affect mean oral Candida counts, but median Candida counts were reduced in the AmF-SnF2 group while an increase was seen in the placebo group. However, the differences observed were not statistically significant. Compliance among the regular elderly users slightly increased during the trial for both the groups. CONCLUSION The number of subjects with high Candida counts decreased in the AmF-SnF2 group. Hence, the fluoride combination might be useful as a support therapy for oral candidiasis. Prevalence of non-albicans Candida strains was low in this population.

Laminin-511 and fibronectin degradation with Candida yeast.

BACKGROUND The invasion mechanism of Candida yeast is still partly unknown. In this study, we tested the ability of different commensal Candida yeast to degrade two basement membrane and extracellular matrix proteins: laminin-511 (Lm-511) and plasma fibronectin. METHODS Human Lm-511 was produced by an immortal keratinocyte cell line, labelled with (35)S-methionine and immunoprecipitated from the growth medium with monoclonal antibodies. Human plasma fibronectin was purified from plasma samples of blood donors. Sonicated yeast cells and concentrated yeast cell growth media were incubated with Lm-511 in different pH values and the degradation was detected by fluorography. Fibronectin degradation by yeast was visualized by sodium dodecyl-sulphate polyacrylamide gel electrophoresis. RESULTS The reduced 220 kDa fibronectin monomers were found to be degraded at pH 7.8 by 10x concentrated growth media of most strains tested and at pH 3.0 the degradation was more pronounced. Sonicated cell fractions of C. tropicalis and C. parapsilosis caused degradation of plasma fibronectin at pH 7.8. Instead, none of the tested Candida cell fractions degraded Lm-511 under these conditions. CONCLUSIONS It seems that cleavage of different laminin isoforms by Candida yeast is a laminin-specific process. The ability to cleave human plasma fibronectin is species- and pH-dependent but not hyphal-dependent and also this degradation may affect epithelial integrity.

A novel Candida glabrata cell wall associated serine protease.

We set out to identify the Candida glabrata cell wall attached proteases which may play a role as virulence factors in candidosis, particularly in the immunocompromized host. We studied a clinical C. glabrata strain T-1639, which was isolated from a patient from the Helsinki University Central Hospital. With non-reducing 2-D electrophoresis using parallel fluorogenic gels and mass spectrometry we identified a novel appr. 25 kDa (192 aa in length) cell wall located protease with an estimated pI of 7.6. The LC-MS/MS peptides matched with the ORF of predicted C. glabrata CBS138 cell wall protein Cwp1.2p/pI 7.7/212 aa (http://cbi.labri.fr/Génolevures/[NCBI access 49525604, UniProt access Q6FTZ7]), which is an ortholog to Saccharomyces cerevisiae cell wall protein Cwp1p (UniProt access P28319). The novel serine protease was released by β-1,3-glucanase treatment from the cell wall. In contrast to previous predictions this protease has an enzymatic function instead of being merely a structural cell wall protein. The protease showed gelatinolytic activity and was inhibited by PMSF, a known serine protease inhibitor. Further characterization of the protease may give insight to its role in infections caused by C. glabrata and possibly aid in the development of new kinds of antifungal drugs.

The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases

The analysis of the disease-specific oral and systemic biomarkers in saliva and oral fluids (i.e., mouth rinse, gingival crevicular fluid (GCF), and peri-implantitis fluid (PISF)) is demanding. Several hosts and microbial factors may influence their expression, release, and levels. The type of saliva/oral fluids utilized for the diagnostics affects the analysis. High sensitivity and specificities together with sophisticated methods and techniques are essential for valuable outcome. We describe here recently developed practical, convenient, inexpensive, noninvasive, and quantitative mouth rinse and PISF/GCF/chair-side/point-of-care (PoC) lateral-flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyser) to detect, predict, and monitor successfully the course, treatment, and prevention of periodontitis and peri-implantitis, respectively. The tests have been independently and successfully validated to differentiate periodontal and peri-implant health and disease in Finland, Germany, Netherland, Sweden, Turkey, Nigeria, Malawi, and USA. The clinical use of salivary/oral fluid biomarkers to identify oral and systemic conditions requires additional studies utilizing these noninvasive screening, diagnostic, and preventive aMMP-8 PoC/chair-side technologies.

The effects of Candida proteinases on human proMMP‐9, TIMP‐1 and TIMP‐2

Matrix metalloproteinase (MMP)‐9 activity is controlled by the balance between MMP‐9 and its major tissue inhibitor of metalloproteinases (TIMPs). We hypothesised whether Candida proteinases may affect local tissue inflammatory processes by modifying these molecules. The effects of sonicated cells and concentrated growth media of six Candida species on MMP‐9, TIMP‐1 and TIMP‐2 were tested. Incubated samples were analysed by Western blot and detected by enhanced chemoluminescence techniques. The residual activity of degraded TIMP‐1 was evaluated by a casein degradation assay. The proteinase activity of the microbial strains was also assessed by a fluorimetric assay, and the action of inhibitors on MMP‐14 and Candida parapsilosis Cp2 was demonstrated. Cell fractions of both strains of C. parapsilosis exerted a weak ability to convert 92‐kDa proMMP‐9 to 86‐kDa active form. Cell fractions of both strains of Candida albicans, C. parapsilosis Cp2, Candida glabrata reference strain, and both strains of Candida krusei fragmented TIMP‐1 (28 kDa) to a 24‐kDa species, which associated with reduced inhibitory activity on MMP‐9 caseinolysis. Our findings indicate that Candida can participate in tissue inflammation by modifying the host’s MMP‐9 and their inhibitors. A rapid fluorimetric assay can be adapted for Candida proteinases.

The Effect of Fermented Lingonberry Juice on Candida glabrata Intracellular Protein Expression

Lingonberries have a long traditional use in treating fungal infections on mucosal membranes, but very little is known about the exact antifungal mechanisms. We tested the effects of fermented lingonberry juice on Candida glabrata intracellular protein expression. A Candida glabrata clinical strain was grown in the presence of fermented lingonberry juice (FLJ). Also the effect of lowered pH was tested. Intracellular protein expression levels were analyzed by the 2D-DIGE method. Six proteins detected with ≥1.5-fold lowered expression levels from FLJ treated cells were further characterized with LC-MS/MS. Heat shock protein 9/12 and redoxin were identified with peptide coverage/scores of 68/129 and 21/26, respectively. Heat shock protein 9/12 had an oxidized methionine at position 56. We found no differences in protein expression levels at pH 3.5 compared to pH 7.6. These results demonstrate that FLJ exerts an intracellular stress response in Candida glabrata, plausibly impairing its ability to express proteins related to oxidative stress or maintaining cell wall integrity.