[Frequency of detection of soluble antigens of Candida spp. and Aspergillus spp. using serological assays in body fluids of patients of Central Clinical Hospital in Warsaw].

INTRODUCTION: Immunodeficient patients, e.g. transplant recipients, patients treated with corticosteroids, people with AIDS and individuals undergoing prolonged antibiotic therapy are at high risk of invasive fungal infections, especially invasive aspergillosis. Basic method for detection of organ/systemic fungal infection is serological monitoring in body fluids, first of all in serum, bu also in broncho-alveolar lavages (BALF). Proven invasive fungal infection should be diagnosed by culture of the pathogen or histopathological examination of infected tissues, however the detection of soluble fungal antigens in body fluids gives enough information for diagnosis of probable fungal infection, according to European Organization for Research and Treatment of Cancer recommendations, what allows introduction of antifungal therapy. Aim of the study was to asses the frequency of detection of circulation soluble fungal antigens with use of immunoenzymatic techniques in patients hospitalized between 2010 and 2015 in Independent Public Central Clinical Hospital (IPCCH) in Warsaw. Methods: In IPCCH, between 2010 and 2015, 6475 serum samples, taken from 2096 patients, was tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for Candida spp. mannan antigen, and 7745 sera from 2243 patients were tested for galactomannan antigen of Aspergillus spp, as well as 64 samples of BALF. Material was collected mainly from haematopoietic stem cell transplant recipients, hospitalized in Haematology and Oncology Clinics, during their routine pos-transplant monitoring. Testing was performed with use of quantitative (Candida antigen) or semiquantitative (Aspergillus antigen) immunoenzymatic methods (BioRad-Platelia), according to respective protocols. Results: During examined period, increase in number of examinations was observed, starting from 1311 tests performed in 2010, up to 3052 examination in 2015. In 2015 testing for Aspergillus antigen in BALF samples was also introduced, resulting in 64 samples tested. Candida spp. antigen was detected in 171 samples (2,7% of all tested samples), and Aspergillus galactomannan was detected in 645 serum samples (8,4%) and 8 BALF samples (12,5%). Majority of examinations was performed for patients hospitalized in Haematology and Oncology Clinics (72,7%), Blood Vessel Surgery and Transplantology Clinics (3,8%), as well as in patients under care of post-transplantation (8,3%) and haematology (4,2%) out-patients clinics. Conclusions: (i) In the 2015-2015 visible increase in number of fungal antigens examinations was observed, (ii) significant number of examinations was performed in onco-haematological patients (88,7%), what also indicates main risk group, (iii) 8,3% of fungal antigen testing was performed in solid organ transplant recipients, the second risk group for invasive fungal infection.

Aspergillus galactomannan detection in comparison to a real-time PCR assay in serum samples from a high-risk group of patients.

Invasive aspergillosis (IA) is a severe infection with a 70% mortality rate. Aspergillus fumigatus is responsible for over 90% of those infections. The diagnosis of invasive aspergillosis is based on clinical sample culture and detection of fungal hyphae in histopathological examination. Additional tests may include the detection of the galactomannan antigen and of fungal genetic material in serum and bronchoalveolar washings. The present study was to assess the use of these two rapid tests in the diagnosis of invasive aspergillosis: serological one - to detect the galactomannan antigen (ELISA assay), and real-time PCR, and to establish a possible correlation between these two methods.

Staphylococcus aureus nasal carriage in Ukraine: antibacterial resistance and virulence factor encoding genes.

BACKGROUND: The number of studies regarding the incidence of multidrug resistant strains and distribution of genes encoding virulence factors, which have colonized the post-Soviet states, is considerably limited. The aim of the study was (1) to assess the Staphylococcus (S.) aureus nasal carriage rate, including Methicillin Resistant S. aureus (MRSA) strains in adult Ukrainian population, (2) to determine antibiotic resistant pattern and (3) the occurrence of Panton Valentine Leukocidine (PVL)-, Fibronectin-Binding Protein A (FnBPA)- and Exfoliative Toxin (ET)-encoding genes. METHODS: Nasal samples for S. aureus culture were obtained from 245 adults. The susceptibility pattern for several classes of antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The virulence factor encoding genes, mecA, lukS-lukF, eta, etb, etd, fnbA, were detected by Polymerase Chain Reaction (PCR). RESULTS: The S. aureus nasal carriage rate was 40%. The prevalence of nasal MRSA carriage in adults was 3.7%. LukS-lukF genes were detected in over 58% of the strains. ET-encoding genes were detected in over 39% of the strains and the most prevalent was etd. The fnbA gene was detected in over 59% of the strains. All MRSA isolates tested were positive for the mecA gene. LukS-lukF genes and the etd gene were commonly co-present in MRSA, while lukS-lukF genes and the fnbA gene were commonly co-present in Methicillin Sensitive S. aureus (MSSA) isolates. No significant difference was detected between the occurrence of lukS-lukF genes (P > 0.05) and the etd gene (P > 0.05) when comparing MRSA and MSSA. The occurrence of the fnbA gene was significantly more frequent in MSSA strains (P < 0.05). CONCLUSIONS: In Ukraine, S. aureus is a common cause of infection. The prevalence of S. aureus nasal carriage in our cohort of patients from Ukraine was 40.4%. We found that 9.1% of the strains were classified as MRSA and all MRSA isolates tested positive for the mecA gene. We also observed a high prevalence of PVL- and ET- encoding genes among S. aureus nasal carriage strains. A systematic surveillance system can help prevent transmission and spread of drug resistant toxin producing S. aureus strains.

Candida nivariensis in comparison to different phenotypes of Candida glabrata.

The purpose of the study was to establish the prevalence of new Candida glabrata complex species: Candida nivariensis and Candida bracarensis isolated from clinical material, evaluate their phenotypes and the prevalence of gene family encoding extracellular glycosylphosphatidylinositol-linked aspartyl proteases, crucial for C. glabrata virulence. Study material included 224 C. glabrata clinical strains. Candida glabrata phenotypes were identified using CHROMagar Candida medium. Strains were analysed by using C. glabrata-specific PCR for the internal transcribed spacer region to confirmed the identification. To identify C. nivariensis and C. bracarensis strains, the D1/D2 region of 26S rRNA was sequenced. The prevalence of YPS-family proteases genes was detected using standard PCR method. Candida nivariensis amounted about 6% among the total number of C. glabrata strains. Candida nivariensis strains had a white phenotype on chromogenic agar media and assimilated two sugars - trehalose and glucose. Among the 13 C. nivariensis strains, 10 did not present any YPS-family protease genes. Coexistence of all detected YPS-family protease genes was specific for C. glabrata species. This study identified C. nivariensis strains; however, no C. bracarensis strains were identified. The white phenotype of C. nivariensis was confirmed. Most strains of the new species do not present any of the tested YPS genes.

Invasive candidiasis serological diagnosis in solid organ transplant recipients.

Solid organ transplant recipients are at high risk of fungal infections, because of ongoing immunosuppressive treatment. There are three post organ transplant phases: early, intermediate, and late, all of them at risk of Candida infections. Since conventional tests are insufficient, specific secondary diagnostic tests are still being explored. Serological tests are currently the most common choice. The present study was to determine the usefulness of mannan antigen and anti-mannan antibody detection in diagnosing invasive candidiasis in liver or kidney transplant recipients. The levels of mannan and anti-mannan antibodies were assessed with Platelia Candida Ag Plus, and Platelia Candida Ab Plus (Biorad, Marne-la-Coquette, France) commercial tests, according to manufacturer's guidelines. Sixty six serum samples were obtained from 25 patients (9 liver transplant recipients, 7 kidney transplant recipients, and 9 patients prepared for a kidney transplant), 29 serum samples from 15 patients tested positive for mannan antigen. Serum samples were obtained from 14 patients tested positive for anti- mannan antibodies. Fungal antigen detection in blood serum in patients under immunosuppression, especially with neutropenia, suggests that antifungal treatment should be administered. Serological tests, especially mannan and anti-mannan ones, are very useful for confirmation or exclusion of invasive candidiasis in high-risk patients.

An initial evaluation of cytotoxicity, genotoxicity and antibacterial effectiveness of a disinfection liquid containing silver nanoparticles alone and combined with a glass-ionomer cement and dentin bonding systems.

BACKGROUND: Bacterial reinfection of dental cavities remains an unsolved clinical problem. The search for methods enabling the limitation of the bacterial factor has become the fundamental goal of the dental materials research. Silver nanoparticles (AgNPs) are used as disinfection agents. An incomplete polymerization of the polymer resins combined with AgNPs, along with the increase of the release of the unbound monomers, have been found. OBJECTIVES: The aim of this study was to evaluate the vitality of the human dental pulp stem cells (DPSCs) in response to a disinfection agent containing silver and gold nanoparticles (NPs), different bonding systems, glass-ionomer cement (GIC), and their combinations with the disinfection agent. Also, the influence of these materials both on the secretory function of DPSCs and on their antibacterial properties was established. MATERIAL AND METHODS: Cytotoxicity (MTT assay) and genotoxicity (enzyme-linked immunosorbent assay - ELISA) assays were used in the study. Antibacterial features were assessed with the optical density (OD) measurement of the bacteria (Streptococcus mutans, Streptococcus salivarius and Lactobacillus acidophilus) kept in dental materials. RESULTS: The disinfection liquid proved to be biocompatible. However, it relevantly interfered with the total-etch bonding system in terms of vitality, which may have serious clinical implications. Its combination with the self-etching system was biocompatible, yet it impaired the antibacterial action of the system. An enhancement of antibacterial action of GIC with AgNPs was found. CONCLUSIONS: The disinfection liquid and GIC were biocompatible toward the DPSCs in terms of cytotoxicity and genotoxicity. Simultaneous usage of AgNPs with other dental materials did not affect the biocompatibility of the used materials. The disinfection liquid and GIC acted as antibacterial agents against all studied bacteria species. Used together with GIC and the total-etch bonding system, the disinfection liquid seemed to be efficient toward bacteria, yet it relevantly impaired the antibacterial action of self-etching systems.

Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs.

BACKGROUND: We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. METHODS: We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. RESULTS: The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. CONCLUSIONS: These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.

Significance of polymethylmethacrylate (PMMA) modification by zinc oxide nanoparticles for fungal biofilm formation.

The objective of this study was to obtain a material composite with antifungal properties for dentures to be used as an alternative protocol in denture stomatitis treatment and prevention. Denture stomatitis is still a clinical problem in patients particularly vulnerable to this disease. Composites of PMMA and doped ZnO-NPs (weight concentrations, 2.5%, 5%, 7.5%) and PMMA with sprayed solvothermal and hydrothermal ZnO-NPs were tested. The following investigations of newly formed biomaterials were undertaken: influence on Candida albicans solution, biofilm staining, XTT analysis and a quantitative analysis of adhered C. albicans. These studies evidenced the antifungal activity of both nanocomposites PMMA-ZnO-NPs and the efficacy of sputtering of zinc oxide nanoparticles on the PMMA. The study of the biofilm deposition on the surface showed that antifungal properties increase with increasing concentration of ZnO-NPs. The XTT assay in conjunction with testing the turbidity of solutions may indicate the mechanism by which ZnO-NPs exert their effect on the increased induction of antioxidative stress in microorganism cells. The denture base made of the aforesaid materials may play a preventive role in patients susceptible to fungal infections. Based on the results obtained a modified treatment of stomatitis Type II (Newton's classification) complicated by fungal infection was proposed.