La TD-GC×GC-HRTOFMS pour investiguer la fibrose pulmonaire chez des patients

Comprehensive two-dimensional gas chromatography has a great potential for exhaled breath analysis. The increased peak capacity and sensitivity, provided by the combination of two capillary columns of different stationary phases by means of a modulator, enable the chromatographic separation and detection of thousands of compounds from a complex matrix. For this reason, we carried out an exploratory study on SSc. Basically, breath samples were collected in 5L Tedlar® bags. Volatiles contained in the sampling bag were then transferred onto Tenax®GR/Carbopack™B thermal desorption tubes and finally released and separated into a Pegasus GC-HRT 4D through a mid-polar Rxi-624SilMS as first column (dimension) and a polar Stabilwax as second dimension. The exhaled breath of 32 patients and 30 healthy subjects was therefore analyzed. The high resolving power of this approach and the use of statistical models enabled the identification of 16 compounds discriminating SSC patients from healthy ones. However, further investigations had to be held to reach a better disease classification. In fact, the biomarkers highlighted here could be related to the scarring of the lungs making these non-specific to SSCs. The second phase of the study aims to go deeper in patient stratification. Three groups were investigated: 50 SSC patients, 50 SSC-fibrosis patients and 50 ILD ones. The samples were collected at Maastricht medical center and CHU of Liège. All samples were then analyzed in the OBiACHem lab. Currently, a classification model is under construction to stratify patients based on their fibrosis status

BREATHOMICS APPROACH TO INVESTIGATE SYSTEMIC SCLEROSIS USING THERMAL DESORPTION AND COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY HIGH-RESOLUTION TIME-OF-FLIGHT MASS SPECTROMETRY

Systemic sclerosis (SSc) is a chronic and heterogenous auto-immune disease of unknown origin characterized by fibrosis, inflammation, vascular damages, and involvement of internal organs. Organ involvement appears at the early stage of the disease[1,2]. Interstitial lung disease (ILD) is one of the most common types of pulmonary involvement, responsible for the disease severity, and leading to high morbidity and mortality. One of the challenges in SSc remains the early diagnosis of patients with a high risk of disease progression driving mortality[3]. There is an unmet need for biological markers enabling SSc early diagnosis, prognosis, disease progression monitoring, and improving patients’ classification for more targeted therapies. Ideally, new diagnostic methods for SSc should be simple, fast, accurate, and cost-effective. Comprehensive two-dimensional gas chromatography (GC×GC) has a great potential for exhaled breath analysis. The increased peak capacity and sensitivity of GC×GC, provided by the combination of two capillary columns of different stationary phases by means of a modulator, enable the chromatographic separation and detection of thousands of compounds from a complex matrix[4]. For this reason, we carried out an exploratory study on SSc[5]. Basically, breath samples were collected in 5L Tedlar® bags. Volatiles contained in the sampling bag were then transferred onto Tenax®GR/Carbopack™B thermal desorption tubes (Markes International Ltd., Llantrisant, UK) and finally released and separated into a Pegasus GC-HRT 4D (LECO Corporation, St Joseph, MI, USA) through a mid-polar Rxi-624SilMS (30 m × 0.25 mm × 1.4 μm) as first column (dimension) and a polar Stabilwax (2 m × 0.25 mm ×0.5μm) as second dimension. The exhaled breath of 32 patients and 30 healthy subjects was therefore analyzed. The high resolving power of this approach and the use of statistical models enabled the identification of 16 compounds discriminating SSC patients from healthy ones[5]. However, further investigations had to be held to reach a better disease classification. In fact, the biomarkers highlighted here could be related to the scarring (fibrosis) of the lungs making these non-specific to SSCs. The second phase of the study aims to go deeper in patient stratification. Three groups were investigated: 50 SSC patients, 50 SSC-fibrosis patients and 50 ILD ones. The samples were collected at Maastricht medical center and CHU of Liège. All samples were then analyzed in the OBiACHem lab. Currently, a classification model is under construction to stratify patients based on their fibrosis status. [1] E. Zanatta, V. Codullo, J. Avouac, Y.A.-J. bone spine, undefined 2020, Elsevier (2019). [2] O. Bonhomme, B. André, F. Gester, … D. de S.-, undefined 2019, Academic.Oup.Com (n.d.). [3] J. Guiot, M. Henket, B. Andre, M. Herzog, N. Hardat, M.S. Njock, C. Moermans, M. Malaise, R. Louis, Clin. Epigenetics 12 (2020). [4] D. Zanella, J. Focant, F.A. Franchina, Anal. Sci. Adv. 2 (2021) 213–224. [5] D. Zanella, J. Guiot, P.-H. Stefanuto, L. Giltay, M. Henket, F. Guissard, B. André, M. Malaise, J. Potjewijd, F. Schleich, R. Louis, J.-F. Focant, Anal. Bioanal. Chem. 2021 41314 413 (2021) 3813–3822

Breathomics to monitor interstitial lung disease associated with systemic sclerosis

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SYSTEMIC SCLEROSIS: CAN BREATHOMICS MONITOR INTERSTITIAL LUNG DISEASE?

Systemic sclerosis (SSc) is an autoimmune disease causing inflammation, blood vessel damage, and collagen deposition. Interstitial lung disease (ILD) is common in SSc, leading to significant mortality. Early detection of SSc-ILD is crucial, but current biomarkers are limited. Our previous research identified distinct volatile organic compound (VOC) patterns in SSc patients' breath. This study aims to establish standardized breath analysis procedures, and assess VOCs' potential in predicting SSc-ILD. Two expert medical centers, the University Hospital of Liège (CHU), Belgium, and Maastricht University Medical Center (MUMC+), the Netherlands, collaborated in studying and recruiting 21 SSc patients and 21 SSc-ILD patients. Nine VOCs were identified as discriminatory between SSc and SSc-ILD, outperforming traditional blood markers. The statistical model based on these markers achieved an AUC of 0.82, accuracy of 85%, sensitivity of 77% and a specificity of 100% for indentifying ILD phenotype, comparable to traditional lung function tests. A correlation was also observed between the functional respiratory parameters (i.e., DLco and FVC% predicted value) and the VOCs. Additionally, our study confirmed the potential of four terpenes, observed in both studies, in distinguishing SSc patients. Methodological SOPs for multi-center studies were developed and validated. This study demonstrates the potential of breath analysis and in particular markers discovery in understanding SSc metabolic changes and could aid prompt ILD treatment. Future research will explore VOC changes in early-stage SSc-ILD and treatment response. This study paves the way for improved diagnosis and management of SSc-ILD

Personalized bacteriophage therapy outcomes for 100 consecutive cases:a multicentre, multinational, retrospective observational study

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127–0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage–antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363.</p

GC×GC-HRTOFMS for untargeted screening of exhaled breath

In the galaxy of Omics Science, breathomics (i.e., exhaled breath analysis) knows an exponential growth. The full characterization of the breath composition and the understanding of the origin of these chemicals could have a gigantic impact for clinical research. Indeed, the potential of using non-invasive breath testing for patient status evaluation could be a game changer in disease monitoring and personalized medicine. Following that goal, there are numerous applications under development from cancer detection to diet adjustment. However, the complete characterization of the exhaled breath composition represents a complex analytical quest. From the exhaustive and reliable sampling, through robust untargeted analysis, to validated markers identification, the playground is large and require the development of new analytical workflows. To ensure the robustness of the entire process, every step must be carefully optimized and controlled with a robust QA/QC system. In order to resolve the complex composition of such samples, a powerful analytical technique is required. To obtain an untargeted screening of exhaled breath sample comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC-MS) is a go to analytical solution. GC×GC-MS provides high separation resolution and orthogonal compounds identification metrics. Indeed, the retention times combined to the MS fragmentogram and potentially to the high-resolution MS provide strong identification confidence, which can reach level 2 MSI (metabolomics standard initiative) classification in a single analysis. The organic and biological analytical chemistry group (OBiAChem) from Liège University has been dedicating a lot of effort to tackle the challenges on the road of robust breath test. In this webinar, we will cover the different technological aspects of exhaled breath research including sampling, analysis, data processing, and QA/QC. In addition, we will cover some clinical applications and discuss the transferability of breath testing for large scale population screening

Recent advances in medical applications of GC×GC

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