Modelling acute renal failure using blood and breath biomarkers in rats.

This paper compares three methods for estimating renal function, as tested in rats. Acute renal failure (ARF) was induced via a 60-min bilateral renal artery clamp in 8 Sprague-Dawley rats and renal function was monitored for 1 week post-surgery. A two-compartment model was developed for estimating glomerular filtration via a bolus injection of a radio-labelled inulin tracer, and was compared with an estimated creatinine clearance method, modified using the Cockcroft-Gault equation for rats. These two methods were compared with selected ion flow tube-mass spectrometry (SIFT-MS) monitoring of breath analytes. Determination of renal function via SIFT-MS is desirable since results are available non-invasively and in real time. Relative decreases in renal function show very good correlation between all 3 methods (R²=0.84, 0.91 and 0.72 for breath-inulin, inulin-creatinine, and breath-creatinine correlations, respectively), and indicate good promise for fast, non-invasive determination of renal function via breath testing.

Interference of chlorofluorocarbon (CFC)-containing inhalers with measurements of volatile compounds using selected ion flow tube mass spectrometry.

Selected ion flow tube mass spectrometry (SIFT-MS) is a sensitive technique capable of measuring volatile compounds (VCs) in complex gas mixtures in real time; it is now being applied to breath analysis. We investigated the effect of inhalers containing chlorofluorocarbons (CFCs) on the detection and measurement of haloamines in human breath. SIFT-MS mass scans (MS) and selected ion monitoring (SIM) scans were performed on three healthy non-smoking volunteers before and after inhalation of the following medications: Combiventtrade mark metered-dose inhaler (MDI) (CFC-containing); Ventolintrade mark MDI (CFC-free); Atroventtrade mark MDI (CFC-free), Beclazonetrade mark MDI (CFC-containing); Duolintrade mark nebuliser. In addition, the duration of the persistence of the mass/charge ratios was measured for 20 h. Inhalers containing CFCs generated large peaks at m/z 85, 87, 101, 103 and 105 in vitro and in vivo, consistent with the predicted product ions of CFCs 12, 114 and 11. No such peaks were seen with Duolintrade mark via nebuliser, or CFC-free MDIs. We conclude that measurement of VCs, such as haloamines, with product ions of similar m/z values to the ions found for CFCs would be significantly affected by the presence of CFCs in inhalers. This issue needs to be accounted for prior to the measurement of VCs in breath in people using inhalers containing CFCs.

In vitro SIFT-MS validation of a breath fractionating device using a model VOC and ventilation system.

The measurement of volatile organic compounds in exhaled breath is becoming recognized as a method of disease diagnosis and therapeutic monitoring. The aim of this study was to validate the collection of breath from intubated patients in the intensive care setting. This was done by assembling a system of ventilators and humidification to emulate the human respiratory system. A known concentration of acetone was spiked into the system to simulate alveolar and dead-space 'breath'. Selected-ion flow tube mass spectrometry (SIFT-MS) was used to directly measure gas at two separate points (headspace, distal circuit end) and from Tedlar bags collected using a remote breath fractionator. The mean acetone concentration for headspace, distal circuit end and Tedlar bag concentrations were calculated. The fractionator was effective in separating the early (dead space) and late phases of exhaled breath. Results from the late Tedlar bag samples collected by the remote breath fractionator showed a clear correlation with headspace and distal circuit end acetone concentrations. The collection for remote analysis of breath samples from immobile patients is made possible using the breath fractionator in conjunction with SIFT-MS analysis.

Investigation into the production of 2-Pentylfuran by Aspergillus fumigatus and other respiratory pathogens in vitro and human breath samples.

Our objective was to identify, for diagnostic purposes, potential volatile biomarkers of human microbial pathogens. We analysed the head space of cultures of medically important bacterial and fungal respiratory pathogens for 2-Pentylfuran (2PF) production through the use of Solid Phase Micro Extraction (SPME) and Gas Chromatography/Mass Spectroscopy (GC/MS). Our results confirm that 2PF is consistently produced by Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Aspergillus flavus and to a lesser extent by Aspergillus niger. 2-Pentylfuran was not detected from most of the bacterial strains except for Streptococcus pneumoniae. In human studies, four litre breath samples were collected from patients with cystic fibrosis (CF), with and without colonisation by A. fumigatus and other pathogens, as well as healthy volunteers. 2-Pentylfuran was detected in breath samples collected from 4/4 patients with CF and A. fumigatus colonization, 3/7 patients with CF and no microbiological evidence of A. fumigatus and 0/10 healthy control individuals. These results suggest that 2PF may be a biomarker for lung colonization/infection by fungal pathogens. To our knowledge, this is the first report describing the detection in breath samples of a volatile biomarker of a pathogen resident in the lungs. Breath analysis has the potential of being a non-invasive diagnostic method of detecting respiratory infection including invasive aspergillosis.

Comparison of galactomannan detection, PCR-enzyme-linked immunosorbent assay, and real-time PCR for diagnosis of invasive aspergillosis in a neutropenic rat model and effect of caspofungin acetate.

The performance of different in vitro diagnostic tests for the diagnosis of invasive aspergillosis (IA) was investigated in a transiently neutropenic rat model. Rats were immunosuppressed with cyclophosphamide and then inoculated intravenously with 1.5 x 10(4) CFU Aspergillus fumigatus spores. Animals were then either treated with caspofungin acetate, 1 mg/kg/day for 7 days, or not treated. PCR-enzyme-linked immunosorbent assay (ELISA), real-time PCR, and galactomannan (GM) detection were performed on postmortem blood samples, along with culture of liver, lung, and kidney homogenate. Caspofungin-treated animals showed a decrease in residual tissue burden of A. fumigatus from organ homogenate compared to untreated animals (P < 0.002). PCR-ELISA returned positive results for 11/17 animals treated with antifungal agents and for 10/17 untreated animals. Galactomannan was positive in 8/17 caspofungin-treated animals and 4/17 untreated animals. Real-time PCR was positive in 2/17 treated and 3/17 untreated animals. This study demonstrates that PCR-ELISA is a more sensitive test than either GM detection (P = 0.052) or real-time PCR (P < 0.01) for diagnosis of IA but that any of the three tests may return false-negative results in cases of histologically proven disease. Galactomannan indices from animals treated with antifungal agents showed a trend (P = 0.1) towards higher levels than those of untreated animals, but no effect was observed with PCR-ELISA indices (P = 0.29). GM detection, as previously described, may be enhanced by the administration of caspofungin, but PCR-ELISA appears not to be affected in the same way. We conclude that PCR-ELISA is a more sensitive and reliable method for laboratory diagnosis of IA.

Comparison of PCR-ELISA and galactomannan detection for the diagnosis of invasive aspergillosis.

AIM: To compare PCR with galactomannan antigen detection for the diagnosis of invasive aspergillosis (IA). METHODS: We prospectively collected serial blood samples from haematological patients at risk of IA, and analysed their samples retrospectively for galactomannan (GM) antigen using the Platelia test and for aspergillus DNA using an in-house PCR-ELISA assay. Matched GM and PCR analyses were performed on 263 samples from 25 patients. Patients were classified for potential IA according to international consensus criteria, with five patients classified as positive (four proven, one probable) and 20 classified as negative (seven possible, 13 no evidence IA). RESULTS: All five patients with IA were positive by PCR with positive results in 24 of 82 samples, whereas three of five patients were positive by GM with four of 82 samples being positive. Three of 20 patients without IA were positive by PCR in 18 of 181 samples, whereas corresponding results for GM detection were one of 20 and one of 181, respectively. Adjustment of ELISA cut-off values and/or the requirement for two consecutive samples to be positive generated different results; however, lowering the positivity index (PI) for GM detection to 0.5 did not improve the sensitivity of the assay. Optimal results for PCR detection and GM were: 100% and 60% sensitivity, 85% and 95% specificity, 0.625 and 0.75 positive predictive value, and 1.0 and 0.8 negative predictive value, with a false-positive sample rate of 8 and 0.4%, positive likelihood ratio of 6.66 and 11.99 and negative likelihood ratio of 0 and 0.42, respectively. CONCLUSIONS: This PCR method is very sensitive for the diagnosis of IA but is associated with a moderate rate of false positives; the GM assay exhibited poor sensitivity but high specificity. Further evaluation of PCR assays for the diagnosis of IA and other invasive fungal infections is warranted.

Real-time detection of common microbial volatile organic compounds from medically important fungi by Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS).

We describe a new method, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) for the rapid and sensitive real-time detection and quantification of volatile organic compounds from medically important fungi, grown on a range of laboratory media. SIFT-MS utilises the chemical ionisation reactions of mass-selected ions to characterise volatile organic compounds (VOCs) that are produced as metabolites from fungi. This technique has the distinct advantage over others in that it readily detects low molecular weight, reactive volatiles, and allows for real-time, quantitative monitoring. The fungi examined in this study were Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Mucor racemosus, Fusarium solani, and Cryptococcus neoformans grown on or in malt extract agar, Columbia agar, Sabouraud's dextrose agar, blood agar, and brain-heart infusion broth. Common metabolites (ethanol, methanol, acetone, acetaldehyde, methanethiol, and crotonaldehyde) were detected and quantified. We found the fingerprint of volatiles, in terms of presence and quantity of volatiles to be strongly dependent on the culture medium, both in terms of variety and quantity of volatiles produced, but may form the basis for species specific identification of medically important fungi.