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Microbiology & Humanities Sciences

Team 7: Laurent Papazian, PU-PH

Team composition

 

  1. Laurent PAPAZIAN, PU-PH, HDR
  2. Pascal THOMAS, PU-PH, HDR
  3. Christophe DODDOLI, PU-PH, HDR
  4. Antoine ROCH, PU-PH, HDR
  5. Fabienne BREGEON, MCU-PH, HDR

 

1. Introduction: research topics

 

Topic 1: Identification of new agents of pneumonia

Exhaustive search for agents of nosocomial pneumonias in ICU patients.

Metagenomic study on samples from patients for whom no agent could be identified.

Research of the clinical significance of a seroconversion for Mimivirus.

Incidence, prognosis and risk factors active Cytomegalovirus infection among mechanically-ventilated patients.

 

Topic 2: Experimental model of pneumonia: Pharmacology – Interactions with mechanical ventilation

Effects of the volemic variations on blood pharmacokinetics and lung tissue concentration of caspofongine in a porcine model.

Study of the benefit of a muscle paralysis on the development of ventilator-induced lung injuries in a model of infectious pneumonia.

Effects of halogenated anaesthetics on the alveolar clearance and the pulmonary oedema in a murine model of Pseudomonas aeruginosa pneumonia.

 

Topic 3: Optimization of the management of respiratory infections

Clinical study on the statins’ effects on mortality of the patients presenting with a ventilator-associated pneumonia.

BAL and serum procalcitonine in the diagnosis of ventilator-associated pneumonia.

Etiologies of post-operative pneumonias and adaptation of the antibioprophylaxis in pulmonary surgery.

 

2. Work completed during the four-year plan in progress

 

Topic  1: Identification of new agents of pneumonia

 

1.1 – Description of new agents of pneumonia

Tropheryma whipplei is the etiologic agent of the Whipple disease. By using 16SrDNA and a technique of quantitative PCR, we highlighted the presence of the ADN of Tropheryma whipplei at the level of bronchoalveolar lavage (BAL) in 6 ICU patients. Tropheryma whipplei could thus be an agent of pneumonia, in particular in aspiration pneumonitis (Bousbia S, Papazian L, Auffray JP, Fenollar F, Martin C, Li W, Chiche L, Scola B, Raoult D. Tropheryma whipplei in patients with Pneumonia. Emerg Infect Say. 2010 Feb;16(2):258-63).

1. 2 – Demonstration of the pathogenic character of the unexpected agents of pneumonia

Acanthamoeba polyphaga Mimivirus is a giant virus recently discovered (Scola B et al.. Science 2003; 299, 2033) and sequenced (Raoult D. Science 2004; 306, 1344 –1350). A seroconversion was found among certain ICU patients (Scola et al. Emerging Infect Say 2005; 11:449-452; Shepherd P. et al. Emerging Infect Say 2006; 12: 248-255). By carrying out a matched-paired study, we showed that the patients presenting a seroconversion during their ICU stay were ventilated 7 days more and remained in ICU 10 days more than the patients not presenting a seroconversion to Mimivirus (Vincent A, Scola B, Forel JM, Pauly V, Raoult D, Papazian L Clinical significance of has positive serology for mimivirus in patients presenting has suspicion of ventilator-associated pneumonia. Crit Care Med. 2009 Jan;37(1):111-8).

Cytomegalovirus belongs to the agents henceforth recognized of nosocomial pneumonia. By carrying out weekly antigenemia pp65 and at the time of any suspicion of PAV (with research on BAL), we reported an incidence of 16,1 % of active Cytomegalovirus infections associated with an increased duration of mechanical ventilation and hospitalization but especially with an increased ICU mortality (Chiche L. Crit Care Med. 2009).

 

Topic  2: Experimental model of pneumonia

 

2.1 - Effects of the volemic variations on blood pharmacokinetics and lung tissue concentration of caspofongine in a porcine model.

The caspofongine is a new useful therapeutic agent in mechanically-ventilated ICU patients. Its pharmacokinetics allows a single daily administration. However, little is known about the volume of distribution variations and about its lung tissue penetration. In a hemorrhagic shock model followed by volemic resuscitation, we observed a reduction of 37% of the maximum plasmatic concentration of caspofongine as well as a reduction of 25% of the lung concentration of the caspofongine. (A Roch, Intensive Care Medicine, accepted).

2. 2 - Benefits of a muscle paralysis on the development of ventilator-induced lung injuries in an infectious pneumonia model

Mechanical ventilation constitutes the universal method of life support in patients presenting with an acute respiratory distress syndrome (ARDS). In the pig, we showed that muscle paralysis obtained using neuromuscular blocking agents during a mechanical ventilation of 6h under sedation doubled the surface of the atelectasis. On lung injured by Tween, muscular paralysis does not increase the amount of atelectatic areas. It thus appears that during the early phase of a pulmonary lesion, the use of neuromuscular blocking agents does not promote atelectasis formation. (Roch A. et al. submitted for publication to Crit Care Med).

 

Topic  3: Optimization of the management of respiratory infections

 

3.1 - BAL and serum procalcitonine in the diagnosis of ventilator-associated pneumonia

The determination of the procalcitonine level (PCT) would allow starting antibiotics early in the course of pneumonia. We completed a work aiming at evaluating the interest of both serum and BAL PCT levels in the diagnosis of pneumonia. We do not find any significant difference between PCT (blood and BAL) of the patients presenting a VAP and those not presenting a VAP. The use of usual clinical and bacteriological criteria is more relevant (B Jung et al. Intensive Care Medicine 2010).

3.2 –Lung biopsy in the etiological diagnosis of persistent ARDS

The ARDS is associated with a mortality of approximately 50%. A reversible and cortico-sensitive pulmonary fibrosis can complicate its evolution. However this fibrosis is difficult to distinguish from a nosocomial pneumonia. Also, when the BAL and the rest of the infectious assessment are negative, our team proposed to perform a surgical biopsy among mechanically-ventilated patients with a sustained ARDS. We report our experiment in connection with 100 biopsies which showed that a fibrosis was present only in 53% of the cases but was associated an infection contra-indicating or differing a corticotherapy in about half of these cases. (Papazian L et al. Crit Care Med. 2007).

3.3 –Dilution of the BAL and quantitative cultures

The method of realization of the BAL among ICU patients presenting a suspicion of ventilator-associated pneumonia is not standardized. We completed a work where the dilution of each BAL was studied by the method of urea dilution. 241 LBA were analyzed. Finally, dilution influenced the interpretation of the results of the BAL only among 5 patients. It thus proves that it is not necessary to evaluate the dilution of the BAL to do the diagnosis of VAP (Baldesi O et al. Intensive Care Med. 2009).

3. 4 - Etiologies of post-operative pneumonias

Subjects on "respiratory failure in major thoracic surgery" in particular of infectious origin, in the context of lung cancer, oesophageal cancer and in lung transplantation. It is based on various approaches from animal studies to clinical research: 

  • Respiratory complications in thoracic surgery (Case D et al. Rev Fr Badly Respir 2009).
  • Impact of comorbidities on total morbi-mortality (Breen D et al. Clin Lung Cancer 2007 - Greillier L et al. Clin Lung Cancer 2007).
  • Impact of the surgical techniques on the incidence of respiratory complications (D’j ourno XB, et al. Eur J Cardio-thorac Surg 2005).
  • Impact of neoadjuvants treatments on the incidence of respiratory complications after oesophagectomy. (D’journo XB et al. Eur J Cardiothorac Surg.2008).
  • Impact of a preoperative or preoperative bronchial microbiological colonization on the incidence of respiratory complications. (D’journo XB, et al. Or J Cardiothorac Surg 2008).
  • Impact of the the perioperative inflammatory response on the incidence and the severity of the respiratory complications. (D’journo XB, et al. Eur J Thor Cardiovasc Surg 2010 - Michelet P et al. Anesthesiology 2006 - Michelet P et al. Br J Surg 2009 - Michelet P. et al. Ann Fr Anesth Reanim 2007).
  • Evaluation of the pain management in thoracic surgery by a dorsal epidural analgesia on the incidence of respiratory complications (Michelet P, et al. Chest 2005 - Michelet P et al. Acta Anaesthesiol Scand 2007 –Michelet P. et al. Br J Anaesth 2007;99:396-403).
  • Impact of blood transfusions on the incidence and the severity of the respiratory complications. (Thomas P. et al. Eur Respir J 2007).
  • Diagnosis and treatment of the unexpected respiratory and thoracic infections. (Rolain JM et al. Emerg Infect Dis 2006).

This clinical research area was the occasion of a recognition by the attribution by the University of the Mediterranean of the Trophy of Health 2009 in the section Medical research, and European (ESTS Brompton prize) rewarding the best scientific paper for European Society of Thoracic Surgery in 2009 (Member elect: XB D' Journo).

 

3 - Works in progress and projects

 

Topic  1: Identification of new agents of pneumonias


1. 1 - Detection of new agents of nosocomail pneumonia by differential metagenomic

More than 100 new pathogenic bacterial, viral, parasitic or fungic agents were identified in pneumonias during these last years. We could, these last years, to identify in our group or in collaboration, more than 50 new pathogenic agents (rare or frequent) for the man. However, the cause of severe nosocomial pneumonias remains unknown in more than 30 % of the cases, in spite of extremely thorough investigations. In the current project, we propose to amplify the techniques used to detect the unknown agents of pneumonias. Initially we will test in an exhaustive way the known respiratory pathogenic agents, by culture, serology and PCR. We will further apply to the samples of patients without etiology a new diagnostic method for noncultivable micro-organisms (metagenomic). All DNA fragments but also (after action of a DNase and an enzyme of reverse transcriptase), the RNA present in the samples will be sequenced. This approach is the only one which should be able to make it possible to identify eukaryotic micro-organisms (by elimination of genes of the host) prokaryotics (bacteria and archae) and DNA and RNA viruses.

1. 2 - Detection of new agents of post-operative pneumonias

In the context of a University thesis (Doctorant 3rd year: Xavier Benoit D' Journo), we contribute to the installation of modern and fast tools for per and postoperative detection of bronchial colonization by of PCR methods. This work should make it possible to secondarily identify and thus to treat early the presumably infectious complications of associated with surgery. Within the framework of clinical research, we could also propose a modification of the antibioprophylaxis strategies. The prolongation of this work will be finally to develop simple tools and of current practice, allowing a detection and an early identification of the germs responsible for infectious pneumonias associated the surgery.

1. 3 - Detection of new infectious agents in patients under extra-corporeal membrane oxygenation (ECMO)

An exhaustive study (cultures, PCR) of the micro-organisms found in the BAL of patients presenting a suspicion of ventilator-associated pneumonia during ECMO compared to patients presenting an ARDS not requiring an ECMO will make it possible to compare microbial flora and to evaluate if there are differences. These differences are likely to exist because of severity differences in the lung function impairment.

 

Topic 2: Experimental models of pneumonia


2. 1 - Inhalation of aminosteroids for the treatment of pulmonary infections related to Pseudomonas aeruginosa

Chronic respiratory pathologies such as the cystic fibrosis are the source of inflammation and broncho-pulmonary infections, especially related to Pseudomonas aeruginosa. One of the therapeutic approaches consists with the inhaled antibiotic administration. After having synthesized new aminosteroidal derivatives having an antimicrobial activity, we will test these molecules in a chronic model of pneumonia related to Pseudomonas aeruginosa. This germ is included in agar balls and administered to the rats. The principal objective is the bacterial quantitative lung tissue culture.

2. 2 - Effects of halogenated anaesthetics on the alveolar clearance and the pulmonary oedema in a murine model of Pseudomonas aeruginosa pneumonia

The halogenated anaesthetics gases induce anaesthesia by mechanisms implying the modulation of neuronal ionic channels in particular. However, their effects are not limited to the neurons. In a recent work (Roch 2006), we showed on the other hand that the halogenated gases activate a sodium transport mechanism sensitive to the amiloride through the apical membrane of human pneumocytes in culture. The effects of halogenated on the injured alveolar epithelium are not known. The goal of this work will be to evaluate the effects of the inhalation the halogenated on the alveolar clearance and the oedema in a murine model of pneumonia related to Pseudomonas aeruginosa.

2. 3 - Murine model of chronic pneumonia due to Pseudomonas aeruginosa PAO1 and antibiotic aerosolisation

1st phase: development of the model:

Study 1: description of the model of infectious pneumonia

Study 2: aerosol treatment of infectious pneumonia with Pseudomonas aeruginosa PAO1

4 groups of 10 animals

G1: Pneumonia by inoculation of Pseudomonas aeruginosa PAO1 + twice-daily treatment by 11,4 mg of NV503 during 5 days begun with 48h post-inoculation

G2: begun during the day from the inoculation

G3: pneumonia + begun 48h post-inoculation treatment IV

G4: pneumonia + treatment IV begun I day from the inoculation

Criteria of evaluation: mortality, physiological criteria, histology, pharmacological evaluation, lung tissue culture

2. 4 –Neuro-immunomodulation of the inflammatory response induced by a chronic pneumonia to Pseudomonas aeruginosa PAO1

The vagal system produces neurokinines and acetylcholine.  We highlighted a protective effect of a treatment by an inhibitor of the receptors of the neurokinine substance P (SP) (neurotransmitter with proinflammatory and vasodilatory properties) in a model of noninfectious pneumonia (Brégeon F, et al.. J Physiol.  2010). In addition, the specific cholinergic activation by the intravenous administration of the nicotinic alpha 7 receptors (alpha 7nAchR) protects with respect to the lesions from gram-negative bacterial pneumonia. The effectiveness by inhaled route of these therapeutic agents will be tested in the model of Pseudomonas aeruginosa pneumonia.

2. 5 - Murine model of acute infectious pneumonia

Development of a rat model of aspiration pneumonia by the inhalation of human gastric liquid (as previously developed in rabbit).

Characterization of the model in the rat (lung mechanics, gas exchange, pulmonary and systemic inflammation, histopathology (Dr. H LEPIDI)

1st  in vitro part (collaboration: Pr  J Pugin, Hôpitaux Universitaires de Genève, Switzerland):

Study of the cytotoxic and pro-inflammatory capacitiesof the human gastric liquid obtained in ICU patients in the first 2’-h period of a respiratory failure. Determination of an pro-inflammatory ability of the gastric liquid on pneumocytes cultures (A549 cells) characterized by the production of ICAM, according to dilution. Role of some cytokines as TNF and IL1 by addition of their antagonists in the culture.

2nd part In vivo (MASTER m2 2010-2011, F Xéridat)

Checking of the key role of IL-1 in the aspiration pneumonia inhalation of human gastric liquid

4 groups of anaesthetized rats ventilated using a protective mode

2. 6 - Acute bacterial pneumonia: interactions with mechanical ventilation

Mechanical ventilation favoured lung colonization and infection. The interactions between the mechanical aggression and the infectious aggression can bring into play modifications of the infecting systems of the micro-organism as well as host defences mechanisms. These interactions will be studied in the model of pneumonia due to Pseudomonas aeruginosa.

 

Topic 3: Optimization of the management of respiratory infections


3.1 - Statins and ventilator-associated pneumonia mortality (clinical study)

The statins inhibit HMG-CoA reductase. Very recent data draw from animal studies and human models of sepsis suggest that the statins could be beneficial in the modulation of the inflammatory response to the sepsis. The statins exert an anti-inflammatory effect by modulating the respponse of the monocytes and the lymphocytes T by modifying their interactions with the endothelium. The statins modify also the expression of genes of the inflammation, inducing a reduction of the production of cytokines. It exhibit also an antioxydant action, anti-thrombotic properties as well as an inhibition of the antigens of the major histocompatibility complex of the type II Thus, all these actions are potentially of interest during sepsis. The clinical data are very few. No controlled study has been published. We thus are conducting a randomized multicenter, controlled, double-blind study, statin versus placebo aiming at evaluating the impact on mortality associated with the ventilator-associated pneumonia.  This multicenter work (46 centers) received the financial assistance of a national PHRC (2007). It has just begun. Two ancillary studies are associated: 1°) the pharmacokinetics of the oral simvastatine in mechanically-ventilated patients, and 2°) the evaluation of modifications of the immune system induced by the statins during the course of ventilator-associated pneumonia.

3.2 – Epidemiology of the ventilator-associated pneumonia during the course of severe acute respiratory distress syndrome

Ventilator-associated pneumonia can complicate the course of ARDS. Very few prospective studies evaluated their incidence as well as the impact on the ARDS mortality. An ancillary study from the PHRC ACURASYS consists in prospectively investigating the epidemiology of the bacterial VAP during the ARDS. 339 included patients could thus be evaluated. The assumption is that VAP do not modify the prognosis of ARDS. The microbiological diagnosis will be established by means of BAL under fibroscopy as well as by protected distal catheter sampling.

3.3 –Study of the pathogenicity of Cytomegalovirus among mechanically-ventilated patients

We highlighted the existence of authentic pneumonias due to Cytomegalovirus obtained from lung histological examinations obtained by biopsy among patients under mechanical ventilation. The real impact of the active CMV infections acquired under mechanical ventilation was however poorly studied. The principal objective of this study will be to highlight an increase in the number of ventilator-free days and alive in patients presenting with a positive antigenemia (at least a cell) treated by Gancyclovir by comparison to mechanically-ventilated patients presenting a positive antigenemia receiving a placebo. It will be a multicenter study including about 250 patients. The principal secondary objectives will be mortality at day 60 and the percentage of pneumonias due to CMV (positive BAL by culture or by PCR with a clinical picture compatible) in each of the two groups.

3.4 –Bronchial colonization in thoracic surgery (model of the lung cancer)

The primary objective of the study is to describe in a prospective way, by systematically sampling the respiratory tree at the time from the surgery.  This research will implement original tools of isolation and culture on the one hand, detection and molecular identification on the other hand agents potentially colonizing at the bronchial level (BAL) but also at the parenchymal one (pulmonary biopsy). The secondary objective is to develop specific molecular tools for fast detection of these pathogens (rtPCR) and thus precociously to detect these colonizations to be able to treat them in an adapted way, the objective being to develop a fast kit of detection in a POC (point-of-care).

3.5 –Effect of an oro-pharyngeal decontamination in bronchopulmonary surgery for cancer

The goal of this randomized study is to evaluate the interest of an oro-pharyngeal decontamination on the reduction of postoperative respiratory complications and on hospital mortality. The study will include microbiological multiple samplings (parenchyma and bronchi).

3.6 –Bronchial colonization with Cytomegalovirus of the patients proposed for a pulmonary surgery

Pulmonary collections of biopsies and serums are carried out at the time of the surgery with specific research of CMV (antigenemia pp65, serology, PCR) in the lung and blood (preoperative and postoperative). The goal is to characterize the existence of reactivation or infection at the time of the surgery. The other aspect is to characterize the role of lymphocytes NK in this context.

3.7 –Interaction lung cancer and virus of the mosaic of the plant of tobacco

The objective is to highlight the existence and the viral presence of DNA resulting from the plant of the tobacco in the pulmonary parenchyma of the nicotinic patients and presenting an operated lung cancer. Methodology will include: immunohistochemistry on lung (healthy and tumoral lung) and serology of the operated patients.


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