Comparison of rapid hybridization-based pathogen identification and resistance evaluation in sepsis using the Verigene® device paired with "good old culture".

Rapid microbial diagnostics is important for septicemic patients. The current gold standard is blood culture with consecutive pathogen identification and antimicrobial susceptibility testing. However, these culture-based methods need at least 48 h.The aim of this study was to compare Verigene® (Nanosphere, Northbrook, IL, USA), a rapid hybridization-based method, with conventional culture-based methods for detection of pathogens and resistance markers from positive blood cultures of septic patients.In 85 of 100 tested blood culture samples (85 %), pathogen identification as well as resistance profile were identical in Verigene and conventional culture. In 4 %, discordant results were observed. In 9 %, conventional culture revealed a pathogen ID or resistance phenotype not included in the Verigene panel. In 2 % no Verigene result was available.In conclusion, Verigene offers the availability of fast and reliable pathogen identification and resistance profile determination, which may result in an earlier start of adequate antimicrobial treatment.

Nosocomial outbreak of extensively drug-resistant Pseudomonas aeruginosa associated with aromatherapy.

An increase of extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) in various clinical specimens among intensive care unit patients (n = 7) initiated an outbreak investigation consisting of patient data analyses, control of adherence to infection control guidelines, microbiologic surveys, and molecular-based studies. XDR-PA was detected in a jointly used aroma-oil nursing bottle for aromatherapy. We implemented the restriction of oil sharing among patients. Hence, the outbreak was controlled successfully.

A point prevalence survey on hand hygiene, with a special focus on Candida species.

BACKGROUND: A 1-day point prevalence study evaluated hand hygiene compliance, yeast colonization, and contamination, focusing on the hands of health care workers (HCWs) and patient-oriented surfaces. METHODS: Hand hygiene compliance was evaluated by applying the direct observation technique and the World Health Organization's compliance program, "My Five Moments for Hand Hygiene." A total of 128 samples from HCWs working in intensive care (n = 11) and intermediate care (n = 2) units and 65 environmental samples from Innsbruck Medical University Hospital were investigated. RESULTS: Hand hygiene compliance was superior for nurses (83.5%) and moderate for medical doctors (45.2%). In general, fungal growth was unique; only 9 of 128 HCW samples and only 4 of 65 environmental samples yielded positive results. The genetic relatedness of yeasts from the same species was investigated by random amplified polymorphic DNA (RAPD) typing. RAPD profiles exhibited the potential for cross-transmission of yeasts. CONCLUSION: In general, the fungal colonization and contamination rate was low, but a high level of hand hygiene compliance was lacking.

Evaluation of a multiplex OnSpot Primer-Extension PCR assay in the diagnosis of sepsis.

For evaluation of the Anagnostics Pathogens DNA xA (PxA) assay in the diagnosis of sepsis 58 blood specimens were tested in comparison with the LightCycler SeptiFast assay and blood culture as gold standard. The PxA assay had a lower sensitivity (0.63 vs. 0.8), but higher specificity (0.83 vs. 0.67) than SeptiFast.

Aspergillus-Specific Lateral-Flow Device and Real-Time PCR Testing of Bronchoalveolar Lavage Fluid: a Combination Biomarker Approach for Clinical Diagnosis of Invasive Pulmonary Aspergillosis.

Clinical experience with the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in hemato-oncology. Invasive pulmonary aspergillosis (IPA) is the most common manifestation, so we evaluated biomarkers in bronchoalveolar lavage (BAL) fluid. An Aspergillus-specific lateral-flow device (LFD), quantitative real-time PCR (qPCR), and the galactomannan (GM) test were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven IPA, 3 had probable IPA, 6 had possible IPA, and 15 patients had no IPA by European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (EORTC/MSG) criteria. The diagnostic accuracies of the tests were evaluated, and pairwise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified to be the most useful for IPA diagnosis. Using the EORTC/MSG criteria, the sensitivities of qPCR and LFD were 100% and the sensitivity of the GM test was 87.5% (GM test index cutoff, >0.8), with the tests having specificities of between 66.7 and 86.7%. The agreement between the results of qPCR and LFD was almost perfect (Cohen's kappa coefficient = 0.93, 95% confidence interval, 0.81 to 1.00). LFD and qPCR combined had a sensitivity of 100% and a specificity of 85.7%. Calcofluor staining and culture of all BAL fluid samples were negative for fungal infection. The median time from the start of mold-active antifungal therapy to the time of collection of BAL fluid was 6 days. Reversing roles and using dual testing by LFD and qPCR to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. All three tests are useful for the diagnosis of IPA in BAL fluid samples. Despite the significant delays between the start of antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remained informative. In particular, the combination of LFD and qPCR allows the sensitive and specific detection of IPA.

Bronchoalveolar Lavage Fluid (1,3)ß-D-Glucan for the Diagnosis of Invasive Fungal Infections in Solid Organ Transplantation: A Prospective Multicenter Study.

BACKGROUND: Prompt diagnosis of invasive fungal infections (IFI) remains a challenge. (1,3)ß-D-glucan detection in bronchoalveolar lavage (BAL) fluid by Fungitell assay aims to further improve upon the test's utility by directly applying it to specimens from the target organ. METHODS: A prospective multicenter analysis of the Fungitell assay was performed on BAL and serum samples obtained from nonselected solid-organ transplantation patients suffering from probable, proven or no IFI according to the revised criteria of the European Organisation for Research and Treatment of Cancer / Mycosis Study Group. RESULTS: Two hundred thirty-three BAL and 109 serum specimens from 135 patients with proven, probable, or no IFI were tested. Based on a 100 pg/mL: cutoff per test sensitivity, specificity, positive and negative predictive values were 79.2%, 38.5%, 27.6%, and 86.3% in BALs and 79.2%, 81.8%, 69.2%, and 83.1% in sera investigated. CONCLUSIONS: The accuracy of the (1,3)ß-D-glucan test is marginal so that its utility as a clinical test for early diagnosis of IFI is questionable in the lung transplant population. Although the high negative predictive value of the Fungitell assay in both, BALs and sera, may support exclusion of pulmonary IFI in solid-organ transplantation patients, the low positive predictive value limits its utility as a screening tool for early diagnosis of IFI.

Rep-PCR and RAPD-PCR fingerprinting of Aspergillus terreus.

We compared two PCR methods for molecular typing the medically important filamentous fungus Aspergillus terreus. In a set of 46 strains investigated we found 19 and 12 different fingerprinting types obtained by random amplified polymorphic DNA PCR (RAPD) and semi-automated repetitive element PCR (rep-PCR), respectively.

Utility of PCR in diagnosis of invasive fungal infections: real-life data from a multicenter study.

Prospective studies addressing the clinical value of broad-range PCR using the internal transcribed spacer region (ITS) for diagnosis of microscopy-negative fungal infections in nonselected patient populations are lacking. We first assessed the diagnostic performance of ITS rRNA gene PCR compared with that of routine microscopic immunofluorescence examination. Second, we addressed prospectively the impact and clinical value of broad-range PCR for the diagnosis of infections using samples that tested negative by routine microscopy; the corresponding patients' data were evaluated by detailed medical record reviews. Results from 371 specimens showed a high concordance of >80% for broad-range PCR and routine conventional methods, indicating that the diagnostic performance of PCR for fungal infections is comparable to that of microscopy, which is currently considered part of the "gold standard." In this prospective study, 206 specimens with a negative result on routine microscopy were analyzed with PCR, and patients' clinical data were reviewed according to the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group. We found that broad-range PCR showed a sensitivity, specificity, positive predictive value, and negative predictive value of 57.1%, 97.0%, 80%, and 91.7%, respectively, for microscopy-negative fungal infections. This study defines a possible helpful role of broad-range PCR for diagnosis of microscopy-negative fungal infections in conjunction with other tests.

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron.

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux ("ATP paradoxon"). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration.

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.

Effect of temperature and pH on the toxicity of aluminium towards two new, soil born species of Arthrobacter sp.

Two coryneform bacteria Arthrobacter sp. PI/1-95 (Arth1) and Arthrobacter sp. PI/3-95 (Arth3) were isolated from forest soil characterized and investigated with respect to their reaction towards aluminium (Al). Sigmoid functions were used to describe dose-response relationships between Al concentration and microbial growth and to calculate EC-values. EC(100) -- indicating a complete inhibition of microbial growth -- varied between 185 microM Al for Arth1 and 11 mM Al for Arth3. A pure pH effect seems probable in connection with the sensitive Arth1 but not with the tolerant species Arth3. Temperature was shown to distinctly increase the toxic effects of Al towards Arth3 whereas only a moderate modification in Al-toxicity was observed with Arth1.