Electronic nose analysis of exhaled breath to diagnose ventilator-associated pneumonia.

BACKGROUND: Exhaled breath analysis is an emerging technology in respiratory disease and infection. Electronic nose devices (e-nose) are small and portable with a potential for point of care application. Ventilator-associated pneumonia (VAP) is a common nosocomial infection occurring in the intensive care unit (ICU). The current best diagnostic approach is based on clinical criteria combined with bronchoalveolar lavage (BAL) and subsequent bacterial culture analysis. BAL is invasive, laborious and time consuming. Exhaled breath analysis by e-nose is non-invasive, easy to perform and could reduce diagnostic time. Aim of this study was to explore whether an e-nose can be used as a non-invasive in vivo diagnostic tool for VAP. METHODS: Seventy-two patients met the clinical diagnostic criteria of VAP and underwent BAL. In thirty-three patients BAL analysis confirmed the diagnosis of VAP [BAL+(VAP+)], in thirty-nine patients the diagnosis was rejected [BAL-]. Before BAL was performed, exhaled breath was sampled from the expiratory limb of the ventilator into sterile Tedlar bags and subsequently analysed by an e-nose with metal oxide sensors (DiagNose, C-it, Zutphen, The Netherlands). From further fifty-three patients without clinical suspicion of VAP or signs of respiratory disease exhaled breath was collected to serve as a control group [control(VAP-]). The e-nose data from exhaled breath were analysed using logistic regression. RESULTS: The ROC curve comparing [BAL+(VAP+)] and [control(VAP-)] patients had an area under the curve (AUC) of 0.82 (95% CI 0.73-0.9). The sensitivity was 88% with a specificity of 66%. The comparison of [BAL+(VAP+)] and [BAL-] patients revealed an AUC of 0.69; 95% CI 0.57-0.81) with a sensitivity of 76% with a specificity of 56%. CONCLUSION: E-nose lacked sensitivity and specificity in the diagnosis of VAP in the present study for current clinical application. Further investigation into this field is warranted to explore the diagnostic possibilities of this promising new technique.

Identification of microorganisms based on headspace analysis of volatile organic compounds by gas chromatography-mass spectrometry.

The identification of specific volatile organic compounds (VOCs) produced by microorganisms may assist in developing a fast and accurate methodology for the determination of pulmonary bacterial infections in exhaled air. As a first step, pulmonary bacteria were cultured and their headspace analyzed for the total amount of excreted VOCs to select those compounds which are exclusively associated with specific microorganisms. Development of a rapid, noninvasive methodology for identification of bacterial species may improve diagnostics and antibiotic therapy, ultimately leading to controlling the antibiotic resistance problem. Two hundred bacterial headspace samples from four different microorganisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae) were analyzed by gas chromatography-mass spectrometry to detect a wide array of VOCs. Statistical analysis of these volatiles enabled the characterization of specific VOC profiles indicative for each microorganism. Differences in VOC abundance between the bacterial types were determined using ANalysis of VAriance-principal component analysis (ANOVA-PCA). These differences were visualized with PCA. Cross validation was applied to validate the results. We identified a large number of different compounds in the various headspaces, thus demonstrating a highly significant difference in VOC occurrence of bacterial cultures compared to the medium and between the cultures themselves. Additionally, a separation between a methicillin-resistant and a methicillin-sensitive isolate of S. aureus could be made due to significant differences between compounds. ANOVA-PCA analysis showed that 25 VOCs were differently profiled across the various microorganisms, whereas a PCA score plot enabled the visualization of these clear differences between the bacterial types. We demonstrated that identification of the studied microorganisms, including an antibiotic susceptible and resistant S. aureus substrain, is possible based on a selected number of compounds measured in the headspace of these cultures. These in vitro results may translate into a breath analysis approach that has the potential to be used as a diagnostic tool in medical microbiology.

Population structure of Staphylococcus aureus strains isolated from intensive care unit patients in the netherlands over an 11-year period (1996 to 2006).

The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.

Flucloxacillin, still the empirical choice for putative Staphylococcus aureus infections in intensive care units in the Netherlands.

OBJECTIVES: To determine the usefulness of flucloxacillin as empirical therapy for putative Staphylococcus aureus infections in intensive care unit (ICU) patients in the Netherlands, the antibiotic resistance of S. aureus isolates from ICUs over a 13 year period was investigated. METHODS: From 1996 to 2008, 1146 consecutive S. aureus isolates from ICU patients in 14 large referral hospitals were collected. The susceptibility to relevant antibiotics was determined by microbroth dilution according to CLSI guidelines. RESULTS: Resistance to flucloxacillin was only found in 12 isolates (1%). The resistance to clarithromycin, ciprofloxacin and moxifloxacin showed a significant trend over time, from 4.2% to 10.3%, from 1.0% to approximately 10% and from 0.0% to approximately 5.0%, respectively (P < 0.05). The resistance to penicillin, clindamycin and doxycycline increased over time, from 74% to 75%, from approximately 3.0% in 1996 to 3.2% in 2008 and from 2.2% in 1996 to 8.2% in 2008, respectively (P > 0.05). Resistance to cephalosporins, carbapenems, rifampicin and gentamicin was sporadically observed. No resistance was found to vancomycin, teicoplanin and linezolid. CONCLUSIONS: The empirical choice of flucloxacillin in the case of putative S. aureus infections in patients admitted to ICUs in the Netherlands is still justified.

Time course of cellular enzyme release in dog heart injury.

The transport time of enzyme from heart to plasma was studied in two experimental models. First, the enzyme alanine aminotransferase was slowly infused into the left ventricular wall in open-chest dogs. The half-life for the washout of alanine aminotransferase activity into plasma was 20 +/- 4 minutes (mean +/- SEM, n = 8) and was not different in ischemic and normally perfused tissue. From measurements of arteriovenous differences in alanine aminotransferase activity and left ventricular blood flow, it was concluded that 77 +/- 14% of total enzyme washout from ischemic tissue occurred by direct entry into the bloodstream. The corresponding value for the vascular permeability-surface area product was 264 +/- 55 ml.kg-1.hr-1. For a second model, we studied myocardial enzyme release into plasma after abrupt heart injury induced by 10 minutes of calcium-free coronary perfusion followed by reintroduction of calcium (calcium-paradox mechanism). The half-life for the release into plasma was 1.9 +/- 0.2 hours (mean +/- SEM, n = 6) and was again not influenced by sustained ischemia. Slower washout, as observed for this second model, is consistent with increased interstitial protein space and corresponds to a permeability--surface area product between 135 and 285 ml.kg-1.hr-1. These results were used to calculate the time course of cellular enzyme leakage from the rate of enzyme release into plasma in various forms of heart injury. Significant shifts between the time curves of evolving cellular injury and enzyme release into plasma are observed after 2 hours of ischemia followed by coronary reperfusion, but not after permanent ischemia.

Complete recovery in plasma of enzymes lost from the heart after permanent coronary artery occlusion in the dog.

Plasma activities of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) were measured after permanent coronary artery occlusion in the dog. Cumulative release of enzymes in plasma was calculated from these data by using a previously validated two-compartment model for circulating enzymes. Regional myocardial ischemia was measured by injection of radiolabeled microspheres. After 48 hours, the dogs were killed, and a detailed map of left ventricular enzyme activity was obtained from 108 tissue samples. Cumulative release into plasma of CK and HBD was 96 +/- 20% and 112 +/- 26%, respectively, of the total activities depleted from the heart (mean +/- SD, n = 11). The scatter in these values is inherent to the calculations, and it is concluded that both enzymes are recovered completely in plasma and, thus, can be used as quantitative markers of injury. Discrepancies between this result and earlier reports on the recovery of CK are only partly apparent and can be explained partly by underestimation of the elimination rate of CK from plasma, irregardless of tissue edema and incomplete extraction of enzyme activity from tissue.

Liver damage as a potential source of error in the estimation of myocardial infarct size from plasma creatine kinase activity.

The occurrence of liver damage was investigated in patients with uncomplicated acute myocardial infarction (AMI). Cumulative plasma release of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) was compared with release of alanine aminotransferase (ALT). Up to 48 h after AMI, the appearance of ALT could be fully explained by myocardial ALT release. Thereafter additional release of ALT occurred, indicating liver damage. A possible effect of liver function on the rate of elimination of CK from plasma was studied in the dog. Complete temporary arrest of hepatic blood supply was obtained after previous implantation of a portacaval shunt, ligation of secondary inflows and blockade of retrograde perfusion. Neither these preliminary haemodynamic interventions nor the acute arrest of hepatic blood flow had any effect on the disappearance rate of CK from plasma. It is concluded that some liver damage commonly occurs in patients after AMI. However, this phenomenon does not interfere with the estimation of infarct size because the elimination of CK from plasma is unaltered during total hepatic ischaemia.