Genotype-guided diagnostic reassessment after exome sequencing in neuromuscular disorders: experiences with a two-step approach.

BACKGROUND AND PURPOSE: Next-generation sequencing has greatly improved the diagnostic success rates for genetic neuromuscular disorders (NMDs). Nevertheless, most patients still remain undiagnosed, and there is a need to maximize the diagnostic yield. METHODS: A retrospective study was conducted on 72 patients with NMDs who underwent exome sequencing (ES), partly followed by genotype-guided diagnostic reassessment and secondary investigations. The diagnostic yields that would have been achieved by appropriately chosen narrow and comprehensive gene panels were also analysed. RESULTS: The initial diagnostic yield of ES was 30.6% (n = 22/72 patients). In an additional 15.3% of patients (n = 11/72) ES results were of unknown clinical significance. After genotype-guided diagnostic reassessment and complementary investigations, the yield was increased to 37.5% (n = 27/72). Compared to ES, targeted gene panels (<25 kilobases) reached a diagnostic yield of 22.2% (n = 16/72), whereas comprehensive gene panels achieved 34.7% (n = 25/72). CONCLUSION: Exome sequencing allows the detection of pathogenic variants missed by (narrowly) targeted gene panel approaches. Diagnostic reassessment after genetic testing further enhances the diagnostic outcomes for NMDs.

Enterococcus hirae, Enterococcus faecium and Enterococcus faecalis show different sensitivities to typical biocidal agents used for disinfection.

BACKGROUND: Enterococcus faecium and E. faecalis are known nosocomial pathogens. The bactericidal activity of biocidal agents used for disinfection, however, is determined with E. hirae. AIM: To find out whether E. hirae is a suitable species to evaluate the efficacy of biocidal agents against the clinically relevant species E. faecalis and E. faecium. METHODS: The bactericidal activity was determined in suspension tests according to EN 13727 using E. faecium ATCC 6057, E. faecalis ATCC 47077 and E. hirae ATCC 10541. Glutaraldehyde, ethanol, benzalkonium chloride, peracetic acid and sodium hypochlorite were used with three exposure times per biocide. When major differences in the sensitivity of the three enterococcal species to the respective substance was found, two more replicates were performed. The number of colony-forming units (cfu) was transformed into decimal logarithms. Results from replicate experiments were described with means and standard deviations. FINDINGS: At a 5-min exposure time, E. hirae was found to be more tolerant to 0.2% glutaraldehyde and 0.0125% peracetic acid compared to E. faecium and E. faecalis, whereas it was more susceptible to 40% ethanol and 3% sodium hypochlorite. Only with 0.00125% benzalkoniumchloride (15 min) was the susceptibility of E. hirae between that of E. faecium and E. faecalis. CONCLUSIONS: E. hirae is a suitable species when a bactericidal activity should be determined against enterococci with glutaraldehyde and peracetic acid. E. hirae may not be a suitable species for ethanol or sodium hypochlorite if the bactericidal activity should include the clinical pathogens E. faecium and E. faecalis.

MRSA prevalence rates detected in a tertiary care hospital in Austria and successful treatment of MRSA positive patients applying a decontamination regime with octenidine.

Methicillin-resistant Staphylococcus aureus (MRSA) decontamination regimens predominantly use chlorhexidine bathing in combination with mupirocin nasal ointment. However, resistances in Staphylococcus aureus strains are increasingly common and there is a need of alternative, safe and feasible protocols. This interventional cohort study performed at the Albert Schweitzer Hospital in Graz, Austria, aimed to (1) determine MRSA prevalence at different body sites and (2) assess the efficacy of the decontamination using octenidine-based leave-on products added to existing robust infection control measures. All inpatients of this tertiary care hospital being treated in geriatric medical wards (GWs) and apallic care units (ACUs) were screened for MRSA and decontamination rates were determined after one, two or three decontamination cycles, respectively. At baseline, MRSA was detected in 25 of the 126 patients screened (19.8%). We found MRSA in 13/126 (10.3%) swabs from nasal vestibules, in 12/126 (9.5%) skin swabs, in 11/51 (21.6%) swabs from PEG-stomata or suprapubic catheters and in 8/13 (61.5%) tracheostomata swabs. A maximum of three 5-day decontamination cycles reduced the number of MRSA positive patients by 68.0%. Excluding non-compliant and deceased patients, decontamination reduced MRSA carriage by 93.3% (n = 15). No adverse events related to the applied decontamination regimen occurred. Exclusive screening of the nose might underreport MRSA prevalence rates. In this study, decontamination with octenidine-based leave-on products was safe and effective in a critical patient population.

Impact of Staphylococcus aureus protein A (spa) genetic typing in cases of prosthetic shunt graft infections.

INTRODUCTION: In January 2014 an internal audit was performed at the department of surgery, division of vascular surgery of the Medical University Graz, Austria, to assess the short and long-term outcomes of prosthetic shunt graft implantations performed between December 1998 and December 2013. A 10.8 % explantation rate due to graft infection was detected. The majority of the cases were associated with Staphylococcus aureus. The aim of this study was to clarify whether this constitutes a nosocomial problem. PATIENTS AND METHODS: Between December 1998 and December 2014 a total of 490 prosthetic shunt grafts were implanted. After exclusion of 54 cases, 436 shunts remained for statistical analysis. Genetic analysis (spa typing) was acquired from three new cases with involvement of S. aureus in 2014. The impact of several factors (e.g. sex, institute for dialysis, age, type of prosthesis, implantation surgeon and position of shunt) on the shunt graft infection rate was statistically analyzed. RESULTS: Of the prostheses 14.0 % (61 out of 436) had to be explanted of which 12.4 % (54/436) were due to infection. In 77.8 % (42/54) bacteria were found in blood and/or wound cultures. Staphylococcus aureus was present in 76.2 % (32/42) of the cases with detected bacteria and in all cases was sensitive to methicillin. The infection rate was not significantly dependent on any of the investigated factors; however, the factor "institute for dialysis" had a remarkable p­value of 0.060 with the infection rate ranging from 8.5 % to 18.2 % depending on the institution. Three different S. aureus protein A (spa) types were found: t015, t359, t6265. The detection of three different spa types means that these patients had different sources of S. aureus so that a nosocomial problem is very unlikely. CONCLUSION: Genetic typing of spa is a suitable technique for distinguishing between nosocomial and community acquired sources of prosthetic shunt graft infections.

Neuromuscular complications in cancer.

Cancer is becoming a treatable and even often curable disease. The neuromuscular system can be affected by direct tumor invasion or metastasis, neuroendocrine, metabolic, dysimmune/inflammatory, infections and toxic as well as paraneoplastic conditions. Due to the nature of cancer treatment, which frequently is based on a DNA damaging mechanism, treatment related toxic side effects are frequent and the correct identification of the causative mechanism is necessary to initiate the proper treatment. The peripheral nervous system is conventionally divided into nerve roots, the proximal nerves and plexus, the peripheral nerves (mono- and polyneuropathies), the site of neuromuscular transmission and muscle. This review is based on the anatomic distribution of the peripheral nervous system, divided into cranial nerves (CN), motor neuron (MND), nerve roots, plexus, peripheral nerve, the neuromuscular junction and muscle. The various etiologies of neuromuscular complications - neoplastic, surgical and mechanic, toxic, metabolic, endocrine, and paraneoplastic/immune - are discussed separately for each part of the peripheral nervous system.

Antibiotic resistance patterns of more than 120 000 clinical Escherichia coli isolates in Southeast Austria, 1998-2013.

Antibiotic resistance patterns of more than 120 000 clinical Escherichia coli isolates were retrospectively analysed. Isolates originated from both hospitalized patients and outpatients from the region of southeast Austria from 1998 to 2013. Except for amoxicillin/clavulanic acid, nitrofurantoin and piperacillin/tazobactam, all of the antibiotics analysed showed increasing proportions of resistant isolates over time, which were most prominent for ampicillin (from 25.4% in 1998 to 40% in 2013), cefotaxime (0.1% to 6.7%), ceftazidime (0.3% to 14.2%), ciprofloxacin (4.3% to 16.7%) and trimethoprim/sulfamethoxazole (14.6% to 24.8%). There was a marked increase in extended-spectrum ß-lactamase-positive isolates (0.1% to 6.3%) starting in 2005, with male patients and hospital-related patients showing a higher increase than female patients and outpatients. Proportions of resistant isolates for most antibiotics were generally higher for male patients and hospital-related patients. Amikacin, nitrofurantoin and trimethoprim/sulfamethoxazole showed a marked increase in resistance proportions among male subjects aged 10 to 19 years which were absent for female subjects, indicating a strong modulation potential of host characteristics.

MRI negative meningeal myeloma with abducens nerve palsies responding to intrathecal chemotherapy.

Meningeal involvement of multiple myeloma is rare. A patient with multiple myeloma presented with bilateral abducens nerve palsies. In the MRI neither lytic skull lesions nor meningeal enhancement could be found. The diagnosis was based on CSF studies and cytology. A neurologic remission was achieved with intrathecal chemotherapy.

Procalcitonin fails to predict bacteremia in SIRS patients: a cohort study.

BACKGROUND: Procalcitonin (PCT) has previously been proposed as useful marker to rule out bloodstream-infection (BSI). The objective of this study was to evaluate the sensitivity of different PCT cut-offs for prediction of BSI in patients with community (CA)- and hospital-acquired (HA)-BSI. METHODS: A total of 898 patients fulfilling systemic-inflammatory-response-syndrome (SIRS) criteria were enrolled in this prospective cohort study at the Medical University of Graz, Austria. Of those 666 patients had positive blood cultures (282 CA-BSI, 384 HA-BSI, enrolled between January 2011 and December 2012) and 232 negative blood cultures (enrolled between January 2011 and July 2011 at the emergency department). Blood samples for determination of laboratory infection markers (e.g. PCT) were collected simultaneously with blood cultures. RESULTS: Procalcitonin was significantly (p < 0.001) higher in SIRS patients with bacteremia/fungemia than in those without. Receiver operating characteristic curve analysis revealed an area under the curve (AUC) value of 0.675 for PCT (95% CI 0.636-0.714) for differentiating patients with BSI from those without. AUC for IL-6 was 0.558 (95% CI 0.515-0.600). However, even at the lowest cut-off evaluated (i.e. 0.1 ng/ml) PCT failed to predict BSI in 7% (n = 46) of patients. In the group of patients with SIRS and negative blood culture 79% (n = 185) had PCT levels > 0.1. CONCLUSION: Procalcitonin was significantly higher in patients with BSI than in those without and superior to IL-6 and CRP. The clinical importance of this is questionable, because a suitable PCT threshold for excluding BSI was not established. An approach where blood cultures are guided by PCT only can therefore not be recommended.

Soluble urokinase plasminogen activator receptor predicts mortality in patients with systemic inflammatory response syndrome.

OBJECTIVE: The soluble urokinase plasminogen activator receptor (suPAR) reflects inflammation. However, the prognostic value of suPAR measurements, particularly at the very early onset of systemic inflammatory response syndrome (SIRS), is less well defined. METHODS: The prognostic potential of suPAR levels in patients with SIRS was evaluated. From November 2010 until April 2013, 902 adult patients presenting with SIRS were investigated. Blood samples for laboratory testing of inflammation markers were collected simultaneously with initial blood cultures. suPAR testing was performed using suPARnostic(©) assay. RESULTS: Analyses of receiver operating characteristics curves revealed areas under the curve (AUCs) of 0.818 for predicting overall mortality within 48 h (36/902 patients died), 0.739 for 30-day mortality (117/902 died) and 0.706 for predicting 90-day mortality (151/902 died). AUCs for procalcitonin (0.777, 0.671 and 0.638), interleukin-6 (0.709, 0.593 and 0.569) and C-reactive protein (0.66, 0.594 and 0.586) as well as renal function and age were markedly lower. Using multivariable regression analyses, suPAR levels (P < 0.001) remained significant predictors of 48-h mortality, whereas suPAR levels (P < 0.001) and bacteraemia (P = 0.002 and P = 0.001, respectively) remained significant predictors of 30- and 90-day mortality. Using Kaplan-Meier survival plots, patients with suPAR <9.15 ng mL(-1) at SIRS onset had a clear benefit. CONCLUSION: suPAR plasma level determined at early SIRS is predictive for mortality.

KPC-2 and OXA-48 carbapenemase-harbouring Enterobacteriaceae detected in an Austrian wastewater treatment plant.

Multiresistant Enterobacteriaceae, like carbapenemase-producing strains, have their primary reservoir in medical institutions. They can also be found with increasing tendency in other reservoirs. One possible way for entrance of multiresistant Enterobacteriaceae into the environment is via waste water. The aim of the study was to screen isolates from a wastewater treatment plant for the presence of carbapenemase-producing Enterobacteriaceae. Three isolates harboured carbapenemase genes, one Klebsiella pneumoniae harboured KPC-2 and one K. pneumoniae and one Escherichia coli harboured OXA-48. This is the first report of carbapenemase-harbouring Enterobacteriaceae isolated outside medical institutions in Austria and the first detection of KPC-harbouring K. pneumonia MLST ST 1245.

Virulence and antimicrobial resistance genes in human MRSA ST398 isolates in Austria.

This study determined the genetic background of virulence and resistance genes of MRSA ST398 in Austria. From 2004 up to 2008 a total of 41 human isolates of MRSA ST398 were investigated for virulence and resistance gene patterns using DNA microarray chip analysis. Highly similar virulence gene profiles were found in 29 (70·7%) of the isolates but genes encoding Panton-Valentine leukocidin, enterotoxins, or toxic shock syndrome toxin were not detected. Genes conferring resistance to tetracycline and erythromycin-lincosamide were common as all but one of the isolates exhibited tetM and/or tetK, which are involved in tetracycline resistance, and 12 (29·9%) were positive for ermC, conferring resistance to erythromycin/lincosamide. SplitsTree analysis showed that 40 isolates were closely related. Changes in virulence and resistance gene patterns were minimal over the observed time period.

Comparison of extended-spectrum-ß-lactamase (ESBL) carrying Escherichia coli from sewage sludge and human urinary tract infection.

For many years, extended-spectrum-beta-lactamase (ESBL) producing bacteria were a problem mainly located in medical facilities. Within the last decade however, ESBL-producing bacteria have started spreading into the community and the environment. In this study, ESBL-producing Escherichia coli from sewage sludge were collected, analysed and compared to ESBL-E. coli from human urinary tract infections (UTIs). The dominant ESBL-gene-family in both sample groups was bla(CTX-M), which is the most prevalent ESBL-gene-family in human infection. Still, the distribution of ESBL genes and the frequency of additional antibiotic resistances differed in the two sample sets. Nevertheless, phenotyping did not divide isolates of the two sources into separate groups, suggesting similar strains in both sample sets. We speculate that an exchange is taking place between the ESBL E. coli populations in infected humans and sewage sludge, most likely by the entry of ESBL E. coli from UTIs into the sewage system.

Emergence of carbapenem-resistant Enterobacteriaceae in Austria, 2001-2010.

We report the emergence of carbapenem-resistant Enterobacteriaceae in Austria. Over a 10-year period, carbapenem-resistant Enterobacteriaceae isolates were obtained from 13 hospitalized patients, with the first isolation in the year 2005 and a remarkable increase in the number of involved patients in 2010. Carbapenem-resistant Enterobacteriaceae comprise eight Klebsiella pneumoniae isolates, four Klebsiella oxytoca isolates, and one Escherichia coli isolate. The detected carbapenemases were the metallo-ß-lactamases New Delhi ß-lactamase, VIM and IMP, and the serin-ß-lactamase Klebsiella pneumoniae carbapenemase.

Antimicrobial resistance of Streptococcus pneumoniae in Southeast Austria, 1997-2008.

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide but varies within geographical regions. We conducted a retrospective analysis of resistance in S. pneumoniae over a 12-year period to assess local and temporal trends in antibacterial resistance. From 1997 to 2008, a total of 1814 non-duplicate S. pneumoniae isolates were identified at the Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Austria. Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test. For penicillin, the minimum inhibitory concentration was determined by Etest. Susceptibility was defined according to CLSI interpretive criteria. For penicillin, resistance rates were consistently low at 0.2% over the 12-year study period. An increase in resistance was remarkable for erythromycin (3.5% in 1997; 14.7% in 2008), clindamycin (1.8% in 1997; 10.6% in 2008) and tetracycline (1.8% in 2000; 11.0% in 2008). For trimethoprim/sulfamethoxazole, resistance increased slightly to 9.2% in 2008. Quinolones showed a low resistance rate of 0.2% that persisted over the whole study period. In contrast to previously published national data, resistance to penicillin was observed to remain at a remarkably low and constant level. Although international surveillance programmes have set up sustainable and interlinked data networks, our results suggest that regional surveillance may still be needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

Use of automated repetitive-sequence-based PCR for rapid laboratory confirmation of nosocomial outbreaks.

OBJECTIVE: Rapid and reliable diagnosis of genetic relatedness of clinical isolates in microbiologic laboratory is essential in case of nosocomial outbreak investigation. Most molecular techniques used to type microorganisms are technically demanding and time consuming. Currently repetitive-sequence-based PCR (rep-PCR) technique has been adapted to an automated format on the DiversiLab system (bioMérieux, Marcy l'Etoile, France). Aim of this study was to compare the performance of the DiversiLab system to that of pulsed-field gel electrophoresis (PFGE) in nosocomial outbreaks. METHODS: 122 clinical isolates (28 Methicillin-resistant Staphylococcus aureus (MRSA), 26 Acinetobacter baumannii, 45 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae and 13 ESBL-producing Klebsiella oxytoca) were investigated. 70 isolates originated from six well-documented outbreaks, 52 were non-outbreak isolates. RESULTS: Concordant results for identification of outbreak and non-outbreak MRSA, A. baumannii and ESBL-producing K. pneumoniae strains were achieved with both methods. In the outbreak of ESBL-producing K. oxytoca automated rep-PCR was slightly more discriminatory than PFGE. Rep-PCR identified investigated ESBL-producing K. oxytoca outbreak-strains as indistinguishable or closely related, showing similarity of >90%, while PFGE identified these strains as indistinguishable. CONCLUSION: Automated rep-PCR assays on the DiversiLab system were used for MRSA, A. baumannii and for the first time ESBL-producing Klebsiella spp. and proved as a rapid and reliable method for molecular analysis of nosocomial outbreaks.

Isolation of Gordonia terrae from a patient with catheter-related bacteraemia.

A cornyeform bacterium was isolated from a blood culture from a 24-year-old man with familial hypertrophic non-obstructive cardiomyopathy, chronic abuse of anabolic steroids and prior admission to hospital because of clinical signs of sepsis. 16S rRNA gene analysis unambiguously identified Gordonia terrae.

A holistic approach to MRSA eradication in critically ill patients with MRSA pneumonia.

BACKGROUND: The number of Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia cases is increasing in many European countries. In this observational study in one medical and three surgical ICUs multiple interventions for the treatment and eradication of nosocomial MRSA-pneumonia were used. PATIENTS AND METHODS: Twenty-one critically ill patients (age: 59 +/- 14 years, 15 males/6 females, 18 ventilator-associated, 3 nosocomial, clinical pulmonary infection score > 6 in all patients, APACHE II 18 +/- 5) were enrolled. The patients were treated with a 7-day course of iv linezolid (600 mg bid) plus rifampicin (600 mg bid), endotracheal vancomycin 100 mg qid, thrice daily mouth and throat washing with chlorhexidine 1% fluid and nasal mupirocin ointment, twice daily skin and hair washings with chlorhexidine gluconate 4% and tracheostomy (n = 8) wound care with povidone-iodine spray. Control samples (endotracheal secretions, nose, wound, and pharyngeal swabs) were taken 2, 3, 4, 7 days and 2 months thereafter. Multilobular pneumonia was seen in 16, pleural effusion in 12, and MRSA bacteremia in 4 patients. RESULTS: One patient died during the follow-up period due to cerebral bleeding. In the remaining 20 patients, pneumonia was clinically cured in all patients and all patients were free of MRSA after eradication. Six patients died due to myocardial infarction (n = 3), gram-negative septic shock (n = 2), herpes encephalitis (n = 1) > 7 days after eradication. No MRSA reinfection occurred during the control period. CONCLUSION: We conclude that in patients with MRSA pneumonia an approach using a 7-day course of intravenous linezolid plus rifampicin, intratracheal vancomycin, nasal mupirocin, cutaneous and oropharyngeal chlorhexidin plus povidone-iodine cures pneumonia and is effective for MRSA eradication.

Viridans streptococci in endocarditis and neutropenic sepsis: biofilm formation and effects of antibiotics.

OBJECTIVES: Viridans group streptococci (VGS) are a frequent cause of bacterial endocarditis or sepsis in patients with neutropenia. Endocarditis in particular, is associated with plaque formation on the endocardium and valve leaflets whereas VGS septicaemia in neutropenic patients is caused by the influx of oral flora bacteria through mucositic lesions. This study examined the in vitro potency for biofilm formation of clinical VGS bloodstream isolates, and the effects of antibiotics on these biofilms. METHODS: During the years 1998-2000, 40 VGS bloodstream isolates from 18 patients with endocarditis and 22 patients with severe sepsis and neutropenia were collected. The MICs of penicillin, teicoplanin and moxifloxacin were determined using the microdilution broth method according to NCCLS criteria. Biofilms were grown in microtitre plates, dyed with Crystal Violet, and the mean optical density (OD) was used for quantification. Biofilms were incubated with penicillin, teicoplanin and moxifloxacin at various concentrations starting with the MICs for the respective isolates tested. RESULTS: Isolates from eight out of 18 patients with endocarditis and six out of 22 patients with neutropenia formed biofilms (not significant). For the 14 isolates, the MIC(90)s (range) of penicillin, teicoplanin and moxifloxacin were 0.5 mg/L (0.001-0.5), 0.125 mg/L (0.025-0.125) and 0.5 mg/L (0.05-0.5), respectively. Generally, biofilms persisted although incubated with the antibiotics up to concentrations of 128 x MIC. However, the ODs of biofilms after incubation with an antibiotic were significantly lower than the ODs of biofilms without antibiotic (P<0.05). A significant decrease in the biofilms with increasing antibiotic concentrations was observed for teicoplanin and moxifloxacin, but not for penicillin G. CONCLUSIONS: VGS isolated from patients with endocarditis and patients with sepsis and neutropenia form biofilms. Biofilms persist even when exposed to antibiotics at concentrations up to 128 x MIC. Nevertheless, teicoplanin and moxifloxacin reduced the density of the biofilms at concentrations >/=16 x MIC. Thus, testing the effects of antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis.