Runx2 mediated Induction of Novel Targets ST2 and Runx3 Leads to Cooperative Regulation of Hypertrophic Differentiation in ATDC5 Chondrocytes.

Knowledge concerning expression and function of Suppression of Tumorigenicity 2 (ST2) in chondrocytes is at present, limited. Analysis of murine growth plates and ATDC5 chondrocytes indicated peak expression of the ST2 transmembrane receptor (ST2L) and soluble (sST2) isoforms during the hypertrophic differentiation concomitant with the expression of the hypertrophic markers Collagen X (Col X), Runx2 and MMP-13. Gain- and loss-of-function experiments in ATDC5 and primary human growth plate chondrocytes (PHCs), confirmed regulation of ST2 by the key transcription factor Runx2, indicating ST2 to be a novel Runx2 target. ST2 knock-out mice (ST2-/-) exhibited noticeable hypertrophic zone (HZ) reduction in murine growth plates, accompanied by lower expression of Col X and Osteocalcin (OSC) compared to wild-type (WT) mice. Likewise, ST2 knockdown resulted in decreased Col X expression and downregulation of OSC and Vascular Endothelial Growth Factor (VEGF) in ATDC5 cells. The ST2 suppression was also associated with upregulation of the proliferative stage markers Sox9 and Collagen II (Col II), indicating ST2 to be a new regulator of ATDC5 chondrocyte differentiation. Runx3 was, furthermore, identified as a novel Runx2 target in chondrocytes. This study suggests that Runx2 mediates ST2 and Runx3 induction to cooperatively regulate hypertrophic differentiation of ATDC5 chondrocytes.

Road and rail traffic noise induce comparable extra-aural effects as revealed during a short-term memory test.

To examine extraaural effects as induced by 20 min of road (ROAD) and 20 min of rail (RAIL) traffic noise with same loudness (75 dBA), a laboratory study was carried out. The study (N = 54) consisted of 28 high and 26 low-annoyed healthy individuals as determined by a traffic annoyance test. To control attention, all individuals performed a nonauditory short-term memory test during the noise exposures. A within-subject design, with phases of ROAD, RAIL, and CALM (memory test only), alternated by phases of rest, was defined. Heart rate (HR), systolic blood pressure (sBP), total peripheral resistance (TPR), as well as three autonomic variables, preejection period (PEP), 0.15-0.4 Hz high-frequency component of HR variability (HF), and salivary stress biomarker alpha amylase (sAA) were measured. In relation to CALM, HR increased (RAIL +2.1%, ROAD +2.5%), sBP tended to increase against the end of noise exposure, PEP decreased (RAIL -0.7%, ROAD -0.8%), HF decreased (RAIL -3.4%, ROAD -2.9%), and sAA increased (RAIL +78%, ROAD +69%). No differences were found between RAIL and ROAD, indicating that both noise stressors induced comparable extraaural effects. Factor annoyance showed significant during CALM. Here a reduced sympathetic drive (higher PEP values) combined with an increased vascular tone (higher TPR values) was found at the high-annoyed subgroup.

Modified culture method detects a high diversity of fungal species in cystic fibrosis patients.

Cystic fibrosis (CF) is one of the most common genetic lung diseases worldwide. The production of sticky viscous mucus leads to enhanced bacterial colonization and infection, but yeasts and filamentous fungi are also found abundantly in the mucus of patients suffering from CF. The role of fungi in the airways of CF patients is still not understood completely. Furthermore, recent investigations have shown that the spectrum of fungi isolated from the airways of CF patients depends strongly on the methods used. In this study, different mycological culture methods were compared: culture with a native inoculum, culture with homogenization of CF sputum, and culture after homogenization and serial dilutions of CF sputum. Altogether, 934 sputum samples from 113 patients were examined from July 2009 through December 2011. A total of 1,744 fungal isolates was recovered; 20 different yeasts and 14 filamentous fungal species were identified. Candida albicans, C. dubliniensis, and C. parapsilosis were the most common species of yeast. For the filamentous fungi, Aspergillus fumigatus was the most common, followed by Scedosporium apiospermum/Pseudallescheria boydii group and A. terreus. Many fungal, species such as Exophiala dermatitidis, Rasamsonia (Geosmithia) argillacea, and others, were isolated only from homogenized sputum samples. The longitudinal data also show that fungal colonization of CF patients is quite stable, even when treated with itraconazole. In conclusion, we recommend homogenizing CF sputa with a mucolyticum, to prepare serial dilutions, and to use appropriate fungal culture media with added antibiotics.

Evaluation of the BD MAX Cdiff assay for the detection of the toxin B gene of Clostridium difficile out of faecal specimens.

In this evaluation study, 180 faecal specimens were tested in parallel with the new BD MAX™ Cdiff assay, the Premier™ Toxins A&B, and toxigenic culture as reference method. BD MAX™ Cdiff presented a high sensitivity (96.0%) and specificity (99.4%) with a negative predictive value of 99.4%.

Resistance patterns of Escherichia coli isolated from sewage sludge in comparison with those isolated from human patients in 2000 and 2009.

For some time now, antibiotic-resistant bacterial strains have been found in the human population, in foods, in livestock and wild animals, as well as in surface waters. The entry of antibiotics and resistant bacterial strains into the environment plays an important role in the spread of antibiotic resistance. The goal of the present study was to monitor the entry of antibiotic resistances into the environment through the contamination of wastewater. To assess the extent of transmission of antibiotic resistances from human sources into the environment, the resistance patterns of Escherichia coli strains isolated from human patients have been compared to those found in strains isolated from sewage sludge. Our results may indicate if resistances to particular antibiotics are more prone than others to spread into the environment. To monitor the increase of specific resistances over time, samples taken in the years 2000 and 2009 were analysed. Our study shows that for some antibiotics a parallel development of resistance patterns has taken place in both patient and environmental samples over time. For other sets of antibiotics, independent developments have occurred in the samples. A clear increase of multi-resistant E. coli strains over time was observed in samples from both sources.

Molecular epidemiology of Pseudomonas aeruginosa in cystic fibrosis patients from Southeast Austria.

Pseudomonas aeruginosa is the major pathogen in the cystic fibrosis (CF) lung, the predominant source of its acquisition, however, is under discussion. In order to study the molecular epidemiology, we evaluated 86 P. aeruginosa isolates from 43 CF patients from southeast Austria. The DiversiLab system was used to identify genetic relationships among the isolates. Antibiotic susceptibilities were tested with a broth microdilution method (Micronaut Merlin). A total of 39 unrelated P. aeruginosa genotypes were found of which 34 were unique to a single patient and one was unique to a sibling pair. We found low rates of resistance for ß-lactams with resistance to piperacillin/tazobactam and ceftazidime ranging from 4 to 6%. Resistance rates for meropenem and ciprofloxacin were 11% and 15%, respectively. The prevalence of multidrug-resistant isolates was 2%. We conclude that the majority of P. aeruginosa isolates from CF patients originate from environmental sources and patient-to-patient spread is very uncommon in our centre.

Antimicrobial activity of gentamicin palmitate against high concentrations of Staphylococcus aureus.

The reduction of implant related infections plays a pivotal role in orthopaedic surgery as an increasing number of people require implants (up to 200,000 per year in the United States (source: Joint Implant Surgery & Research Foundation 2010)). The aim of the current study is to prevent and thus decrease the number of bacterial infections. Both pre and post operative systemic antibiotic treatment and gentamicin containing bone cements (polymethylmethacrylate, PMMA) are commonly used strategies to overcome infections. In this study, the antimicrobial efficacy of gentamicin sulfate loaded bone cement was compared with titan discs coated with a new form of gentamicin, gentamicin palmitate. Adherence prevention, killing rates and killing kinetics were compared in an in vitro model, using Staphylococcus aureus (S. aureus), which together with Staphylococcus epidermidis (S. epidermidis) represents 60% of bacteria found responsible for hip implant infections (An and Friedman, 1996, J Hosp Infect 33(2):93-108). In our experiments gentamicin, which was applied as gentamicin palmitate on the surface of the implants, showed a high efficacy in eliminating bacteria. In contrast to gentamicin sulfate containing bone cements, gentamicin palmitate is released over a shorter period of time thus not inducing antibiotic resistance. Another benefit for clinical application is that it achieves high local levels of active ingredient which fight early infections and minimize toxic side effects. Furthermore, the short term hydrophobic effect of gentamicin palmitate can successfully impede biofilm formation. Thus, the use of self-adhesive antibiotic fatty acid complexes like gentamicin palmitate represents a new option for the anti-infective coating of cementless titan implants.

Performance of new enzyme-linked fluorescent assays for detection of Epstein-Barr virus specific antibodies in routine diagnostics.

BACKGROUND AND AIMS: Indirect fluorescent antibody assays are still the gold standard for the detection of Epstein-Barr-virus-specific antibodies; however, this technique requires several manual steps and an experienced technician. This retrospective study investigated the performance of the new VIDAS(®) enzyme-linked fluorescent assays for automated qualitative detection of EBV VCA IgM, VCA/EA IgG, and EBNA IgG antibodies in routine diagnostics. METHODS: 174 serum samples were tested first with the gold standard. In context with the clinical status, 60 samples IFA IgG/IgM positive and with clinical symptoms compatible with infectious mononucleosis, 26 samples IFA IgG/IgM negative and missing any clinical symptoms and 88 samples with varying IFA status and without any clinical information were defined. In a second step all samples were retested with the new assays. RESULTS: In the overall agreement between VIDAS(®) and IFA for evaluable results, almost perfect agreement was observed (kappa = 0.91; 95% confidence interval (CI), 0.86-0.97). Estimating all indeterminate VIDAS(®) results as discordant or concordant the observed kappa values were 0.71 (CI 0.63-0.79) and 0.93 (CI 0.88-0.98), respectively. With the new assays 45, 22, and 70 identical results were obtained, respectively. Western blot analysis of the discrepant samples showed a quasi similar performance of both assays. CONCLUSIONS: The new VIDAS(®) assays can be an alternative to IFA testing especially in high-throughput laboratories. Full automation of EBV serological diagnostis by the new VIDAS assays is of major importance for routine diagnostic laboratories.

Molecular detection and characterisation of fungal heat shock protein 60.

Heat shock proteins (Hsp) are highly conserved molecules, which are both constitutively expressed and up-regulated in response to various stress conditions. In particular, fungal Hsp60 can act as immunodominant antigens and facilitate powerful immunological properties. A possible cellular heat shock response was investigated in eight fungi (Aspergillus fumigatus, Aspergillus terreus, Penicillium chrysogenum, Cladosporium cladosporioides, Scedosporium apiospermum, Trichophyton mentagrophytes, Candida albicans and Saccharomyces cerevisiae). Fully automated RNA extraction was followed by quantitative real-time RT-PCR targeting fungus-specific Hsp60 mRNA and sequencing of the amplicon. Levels of temperature-dependent gene expression were evaluated and rates of similarity and identity were compared. While Hsp60 mRNA was constitutively expressed in all the samples tested, a temperature-dependent induction was not shown in C. cladosporioides. In the 80-amino acid fragment from the hypothetical protein, 66% of the amino acids were identical, 20% showed a conserved and 8% a semi-conserved substitution. Our findings should contribute to a better understanding of host-pathogen relationship and suggest that fungal Hsp60 under temperature-related stress conditions might act as an immunogenic trigger in orchestrating fungi-related diseases.

Feasibility of testing three salivary stress biomarkers in relation to naturalistic traffic noise exposure.

BACKGROUND AND OBJECTIVES: Stress dependent alterations of the salivary biomarkers alpha-amylase (sAA), salivary chromogranin A (sCgA) and salivary cortisol (sC) have been reported in numerous studies recently. The aim of this pilot study was to investigate the feasibility of testing sAA, sCgA and sC in relation to naturalistic traffic noise exposure in order to monitor a direct stress response in a laboratory setup. METHODS: A total of twenty study participants were exposed to binaurally recorded naturalistic traffic noise samples containing 75 dB (L(A,)eq) for 20 minutes via a loudspeaker system. Saliva was collected directly before and after defined exposure to naturalistic traffic noise. Determination of sAA was performed enzymatically on a Hitachi 912 laboratory analyzer, sCgA was determined by ELISA technique and sC was determined using a RIA assay. RESULTS AND CONCLUSIONS: There was a significant increase of sAA and sC concentrations after traffic noise exposure (p=0.045; p=0.01), whereas for sCgA this was not observed (p=0.48). Measuring of sAA and sC appear to be feasible to investigate direct stress effects in relation to naturalistic traffic noise exposure in a laboratory setup. Considering the small sample size of this pilot study, these observations need to be further proved in a larger explorative study.

ESBL-producing E. coli in Austrian sewage sludge.

The aim of this study was to investigate the degree of contamination of sewage sludge with ESBL-producing Escherichia coli strains and the effectiveness of different sewage sludge treatment methods. Monthly sewage sludge samples were collected between January and September 2009 in 5 different sewage treatment plants and tested for the presence of ESBL E. coli. In addition, the number of colony forming units (CFU) of E. coli and coliform bacteria before and after the different sludge treatment methods (aerobic/anaerobic digestion, lime stabilization, and thermal treatment) was investigated. Of the 72 sewage sludge samples investigated, ESBL-positive E. coli were found in 44 (61.1%) sewage sludge samples. The classification of beta-lactamase groups was carried out in 15 strains resulting in the detection of 2 different groups (CTX-M and TEM) of bla genes. All 15 of them had a CTX-M gene and 4 of these strains furthermore carried a TEM gene. With regard to the CFU of E. coli and coliform bacteria, thermal treatment and lime stabilization following dehydration sufficiently reduced pathogen concentrations. The plants using merely stabilization and dehydration showed an increase of E. coli and coliform bacteria and thus also an increase in ESBL-producing E. coli.

Occurrence and genotyping using automated repetitive-sequence-based PCR of methicillin-resistant Staphylococcus aureus ST398 in Southeast Austria.

In this retrospective study, the occurrence and genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in Austrian MRSA patients was investigated. From 2002 to 2008, 14 MRSA ST398 were detected. First occurrence of MRSA ST398 was already found in 2004. Spa ribotyping assigned 12 isolates to spa type t011 and 1 each to spa type t034 and spa type t1451. Isolated MRSA ST398 was nontypeable by pulsed-field gel electrophoresis (NT-MRSA) using restriction enzyme SmaI; therefore, genotyping was performed using automated repetitive-sequence-based PCR (rep-PCR) on the DiversiLab system. Rep-PCR results assigned 10 (71%) of the 14 MRSA ST398 into 1 cluster with a similarity >95%; there was 1 cluster consisting of 2 isolates with a similarity >99% and 2 unique MRSA ST398 isolates. In conclusion, MRSA ST398 was continuously detected in Southeast Austria; first in 2004 with up to 5 MRSA ST398 isolates in 2008. Automated rep-PCR proved as a reliable technique in determining genetic relatedness of NT-MRSA ST398 and demonstrates clonal spread of MRSA ST398 in the investigated region.

In-vivo Candida biofilms in scanning electron microscopy.

Candida biofilms on indwelling devices are an increasing problem in patients treated at intensive care units. The goal of this study was to examine the occurrence and frequency of these biofilms. A total of 172 catheters were collected from 105 male and 67 female patients (the age range of both patient groups was from 3 weeks to 98 years old). The catheters were incubated on blood agar plates and the resulting yeast colonies were subsequently identified. Furthermore, pieces of catheters were fixed, dried and sputter coated with gold for investigation with scanning electron microscopy (SEM). Yeasts were recovered from significantly more catheters obtained from men than from women (chi(2): n = 67; P < 0.01). In SEM, 56.4% catheters turned out to be positive for biofilm formation. Again catheters from male patients were statistically significant (chi(2): n = 40; P < 0.01) more often positive than those from women. Candida albicans (71.1%) was the most common species isolated from the catheters, followed by C. glabrata (10.3%), C. parapsilosis (8.2%) and C. tropicalis (5.2%). Based on the results of this investigation, the epidemiology of Candida biofilms on indwelling devices seems to be a promising target for future investigations.

Detection of CMV DNA: is EDTA whole blood superior to EDTA plasma?

The early diagnosis of human cytomegalovirus (CMV) infection in immunosuppressed patients has been improved by molecular detection of CMV DNA. In this study, corresponding EDTA whole blood and EDTA plasma specimens were obtained from 42 bone marrow transplant recipients. For detection of CMV DNA, two commercially available assays, the CMV HHV6,7,8 R-gene (ARGENE), and the artus CMV LC PCR Kit (QIAGEN), were employed. The linearity of both assays was determined by using a clinical EDTA whole blood sample with high CMV DNA load. With the CMV HHV6,7,8 R-gene test, CMV DNA was detected in 40 EDTA whole blood and in 19 EDTA plasma samples, while the artus CMV LC PCR Kit test detected CMV DNA in 27 EDTA whole blood and in 30 EDTA plasma samples. In conclusion, EDTA whole blood samples were found to be the superior material when using the CMV HHV6,7,8 R-gene test. However, this benefit may not exist when employing alternative assays.

Submasseteric abscess caused by Mycoplasma salivarium infection.

Mycoplasma salivarium preferentially resides in the human oral cavity. Unlike other Mycoplasma species, M. salivarium has not been regarded as a pathogen, although one case of M. salivarium-caused arthritis in a patient with hypogammaglobulinemia has been reported. We describe the first case of submasseteric abscess caused by M. salivarium.

Characterization and temperature-dependent quantification of heat shock protein 60 of the immunogenic fungus Alternaria alternata.

Heat shock proteins or chaperones are found in mitochondrial and cytosolic compartments of cells. They are responsible for the correct folding of proteins and are up-regulated in reaction to various stressors. In addition, when released or presented on the surface of cells, they may play an important role in inflammatory and immunomodulating processes. To identify and characterize hsp60 in the common environmental mold Alternaria alternata, the fungus was cultivated and incubated at different temperatures to induce a possible heat shock response. Fully automated RNA extraction was followed by quantitative real-time RT-PCR targeting A. alternata specific Hsp60 mRNA and subsequent sequencing of the amplicon. While Hsp60 mRNA was constitutively expressed in all samples tested, a temperature-dependent expression of Hsp60 mRNA was observed. Sequencing revealed an identity of more than 85% to other fungal hsp60, indicating the existence of this protein in A. alternata.

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays.

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.

[WHO Polio Eradication Programme:Status quo and implementation in Austria]. / WHO-Eradikationsprogramm für Poliomyelitis: Status quo und Umsetzung in Osterreich.

In 1988 the 41(th) World Health Assembly declared polio to be worldwide eradicated until the year 2000. Although this ambitious aim could not be reached completely, the yearly worldwide incidence was reduced by 99% and three WHO-Regions were declared polio-free (Americans, West Pacific, Europe). To maintain this status the following measures have to be carried out: polio vaccination, enterovirus surveillance, AFP-surveillance, quality control of laboratories and notification of labs keeping stocks of polio wildvirus. Especially after the Second World War Austria faced severe polio epidemics and thus general and free of charge polio vaccination for children and young adults up to 21 years was started in winter 1961/62 by the Austrian Ministry of Health (MoH). Immediately the yearly incidence dropped from 3.65/100.000 (n = 292) in 1961 to 0.1/100.000 (n = 8) in 1962. Since 1998 all mandatory national measures according the WHO polio eradication programme have been performed. Despite the worldwide success of the programme there are currently still four countries with endemic polio and since November 2006 eleven further countries have faced epidemics due to imported cases. Therefore the 60(th) World Health Assembly in 2007 again pointed to the threat of failing worldwide polio eradication. Currently the Global Polio Eradication Initiative (GPEI) works intensively with the concerned countries to fight against this development.

Comparison of molecular assays for detection and typing of human papillomavirus.

OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation.

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.