MALDI-TOF MS monitoring of PBMC activation status in sepsis

International audienceBACKGROUND:MALDI-TOF mass spectrometry (MS) on whole cells enables the detection of different cell types and cell activation. Here, we wondered whether this approach would be useful to investigate the host response in sepsis.METHODS:Peripheral blood mononuclear cells (PBMCs) from patients with severe sepsis and healthy donors were analyzed with MALDI-TOF MS. PBMCs from healthy donors were also stimulated with lipopolysaccharide, peptidoglycan, CpG oligonucleotides, polyinosinic polycytidylic acid, and with heat-inactivated bacteria. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software and matching scores between each spectrum from patients and averaged spectra from the database were calculated.RESULTS:We show that the MALDI-TOF MS signature of PBMCs from septic patients was specific, compared with healthy controls. As the fingerprints observed in patients may be related to PBMC activation, PBMCs from healthy controls were stimulated with cytokines, soluble Pathogen-Associated Molecular Patterns (PAMPs) and heat-killed bacteria, and we created a database of reference spectra. The MALDI-TOF MS profiles of PBMCs from septic patients were then compared with the database. No differences were found between patients with documented infection (n = 6) and those without bacteriological documentation (n = 6). The spectra of PBMCs from septic patients matched with those of interferon-γ- and interleukin-10-stimulated PBMCs, confirming that sepsis is characterized by both inflammatory and immunoregulatory features. Interestingly, the spectra of PBMCs from septic patients without documented infection matched with the reference spectrum of PBMCs stimulated by CpG oligonucleotides, suggesting a bacterial etiology in these patients.CONCLUSIONS:Despite the limits of this preliminary study, these results indicate that the monitoring of functional status of PBMCs in peripheral blood by whole cell MALDI-TOF MS could provide unique opportunities to assess disease progression or resolution in clinical settings

Defective Granuloma Formation in Elderly Infected Patients

International audienceGranulomas are compact structures formed in tissues by the immune system in response to aggressions. Thein vitroformation of granulomas using circulating mononuclear cells is an innovative method to easily assess the immune response of patients. Monitoring the efficiency of mononuclear cells from patients to form granulomasin vitrowould help improve their therapeutic management. Circulating mononuclear cells from 23 elderly patients with sepsis and 24 elderly controls patients were incubated with Sepharose beads coated with either BCG orCoxiella burnetiiextracts. The formation of granulomas was measured over 9 days. Most healthy elderly patients (92%) were able to form granulomas in response to BCG andCoxiella burnetiiextracts compared to only 48% of infected elderly patients. Undernutrition was significantly associated with impaired granuloma formation in healthy and infected patients. Granulomas typically comprise epithelioid cells and multinucleated giant cells, however, these cells were not detected in samples obtained from patients unable to form granulomas. We also found that the impairment of granuloma formation was associated with reduced production of tumor necrosis factor without overproduction of interleukin-10. Finally, all genes specifically modulated in granulomatous cells were down-modulated in patients with defective granuloma formation. TNFSF10 was the only M1 gene markedly upregulated in patients who did not form granulomas. Our study suggest that defective granuloma formation may be a measurement of altered activation of immune cells which can predispose to nosocomial infections in elderly patients

Impaired Granuloma Formation in Sepsis: Impact of Monocytopenia.

Granulomas are a collection of immune cells considered to be protective in infectious diseases. The in vitro generation of granulomas is an interesting substitution to invasive approaches of granuloma study. The monitoring of immune response through the determination of in vitro granuloma formation in patients with severe sepsis may be critical to individualize treatments. We compared the in vitro generation of granulomas by co-culturing circulating mononuclear cells from 19 patients with severe sepsis, 9 patients cured from Q fever and 12 healthy subjects as controls, and Sepharose beads coated either with BCG or Coxiella burnetii extracts to analyze both immune and innate granulomas, respectively. We showed that the great majority of patients with severe sepsis were unable to form granulomas in response to BCG and C. burnetii extracts whereas more than 80% of healthy controls and patients cured from Q fever formed granulomas. We also found that monocytopenia and defective production of tumor necrosis factor were associated with reduced formation of granulomas in patients with severe sepsis even if TNF did not seem to be involved in the defective granuloma formation. Taken together, these results suggest that the deficiency of granuloma formation may be a measurement of altered recruitment and activation of monocytes and lymphocytes in patients with severe sepsis

Risk factors for death in septic shock A retrospective cohort study comparing trauma and non-trauma patients

International audienceThe aim of this study was to compare septic shock directly associated-mortality between severe trauma patients and nontrauma patients to assess the role of comorbidities and age. We conducted a retrospective study in an intensive care unit (ICU) (15 beds) of a university hospital (928 beds). From January 2009 to May 2015, we reviewed 2 anonymized databases including severe trauma patients and nontrauma patients. We selected the patients with a septic shock episode. Among 385 patients (318 nontrauma patients and 67 severe trauma patients), the ICU death rate was 43%. Septic shock was directly responsible for death among 35% of our cohort, representing 123 (39%) nontrauma patients and 10 (15%) trauma patients (P<0.0). A sequential organ failure assessment score above 12 (odds ratio [OR]: 6.8; 95% confident interval (CI) [1.3-37], P=0.025) was independently associated with septic shock associated-mortality, whereas severe trauma was a protective factor (OR: 0.26; 95% CI [0.08-0.78], P=0.01). From these independent risk factors, we determined the probability of septic shock associated-mortality. The receiver-operating characteristics curve has an area under the curve at 0.76 with sensitivity of 55% and specificity of 86%. Trauma appears as a protective factor, whereas the severity of organ failure has a major role in the mortality of septic shock. However, because of the study's design, unmeasured confounding factors should be taken into account in our findings

Additional file 1: of MALDI-TOF MS monitoring of PBMC activation status in sepsis

Figure S1. Comparison between in vitro and in vivo data. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software. The spectra (n = 16) from 2 other patients with gram-negative bacillus bacteremia, the second patients with S. aureus bacteremia and three other patients with undocumented infection were then compared with the averaged spectra of the database. Matching scores between each spectrum from patients and averaged spectra from the database are represented with circles. Horizontal lines represent the medians of matching scores; a value higher than 1.5 was considered significant and allowed confident identification of the activation status of PBMCs. (JPG 320 kb

Rapid diagnostic test and use of antibiotic against methicillin-resistant Staphylococcus aureus in adult intensive care unit

International audienceVentilator-associated pneumonia (VAP) due to methicillin-resistant Staphylococcus aureus (MRSA) is associated with excess mortality and costs. Molecular biology test allows rapid identification of MRSA in sputum with high negative predictive value. We hypothesized that use of a rapid diagnostic test in patients with suspected VAP was associated with reduced use of antibiotics directed against MRSA. This retrospective, observational study was conducted in a polyvalent intensive care unit (ICU) of a university hospital. We compared two periods: before (2007-2010) and after (2010-2015) the implementation of a rapid diagnostic test, which uses RT-PCR to detect pathogens in 60 minutes. The primary endpoint was the effect on the empirical use of anti-MRSA antibiotics. The second endpoint was the effect of this strategy on the cost regarding antibiotic treatment. The first group included 120 suspected VAP (88 patients) and the second group 121 suspected VAP (89 patients). Empirical use of vancomycin and linezolid decreased by 50 % between the two periods. Twenty-seven VAP (22 %) were treated with an anti-MRSA treatment between 2007 and 2010, and 13 (11 %) between 2010 and 2015 (p = 0.04). The mean cost of anti-MRSA treatment by patients in the first group was 63 +/- 223 a,notsign, and 13 +/- 52 a,notsign in the second group (p < 0.001). This study shows that a rapid diagnostic test was associated with reduced use and cost of anti-MRSA antibiotics in patients with suspected VAP. These results should be confirmed by further multicenter prospective studies

Granuloma formation according to lymphopenia or monocytopenia.

<p>Granuloma formation with BCG-coated beads (left) and CB-coated beads (right) was measured during 9 days in PBMCs from patients with severe sepsis. Patients were classified according to lymphopenia (A) and monocytopenia (B). The results are expressed as the percentage of beads entirely covered by PBMCs. The boxplots represent the medians with the first and third quartiles. The whiskers represent the highest value that is within 1.5*IQR. Data beyond the end of the whiskers are outliers and plotted as black points. * p < 0.05 represents the differences between patients with and without monocytopenia.</p

Patient features.

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Sepsis is associated with lack of monocyte HLA-DR expression recovery without modulating T-cell reconstitution after lung transplantation

International audienceBackground: Immunosuppressive strategy targets mainly adaptive immunity after solid organ transplantation. We assessed the influence of early post-operative sepsis on T cell and monocyte reconstitution in anti-thymocyte globulin (ATG)-treated lung transplant recipients. Methods: We retrospectively included recipients who underwent a first lung transplant at our Lung Transplant Center (Marseille, France) between July 2011 and February 2013. Peripheral blood T-lymphocyte subset counts and monocyte HLA-DR (mHLA-DR) expression routinely performed by flow cytometry within 60 days post transplant were analyzed. We compared the immune kinetics of patients who did or did not develop sepsis during the post-operative intensive care unit stay. Results: Among the 37 recipients included, 19 patients (51%) developed at least one episode of sepsis. At the ICU admission, septic recipients had higher SOFA score (9 [7.5-9] versus 6 [4-7]), p = .01), higher primary graft dysfunction score (1.4 1.4 versus 0.3 0.7, p = .008) and more frequent use of ECMO (47% versus 0%, p = .003). Whereas both groups had similar T-lymphocytes reconstitution in the post-operative period, mHLA-DR reconstitution was dramatically affected in septic patients after day 14, median mHLA-DR expression at 2.3 MFI [1.3-3.5] in the septic versus 8.0 MFI [5.1-10.5] in the non-septic group, p = .02. Conclusion: We found that sepsis is negatively correlated with the mHLA-DR expression but not adaptive T cell immune reconstitution. This finding highlights the importance of immunomonitoring after lung transplantation and questions the strategy of a lower immunosuppression therapy in context of sepsis