Membres du jury
Professeur LASOCKI Sigismond | Président Docteur PAILHORIES Hélène | Directeur Docteur RINEAU Emmanuel | Membre Docteur DUMARTINET Benjamin | Membre
Soutenue publiquement le :
2019-2020
THÈSE
pour le
DIPLÔME D’ÉTAT DE DOCTEUR EN MÉDECINE
Qualification en ANESTHESIE-REANIMATION
EVALUATION DE LA PERFORMANCE D’UN KIT DE PCR-MULTIPLEX (CURETIS
®) DANS LE DIAGNOSTIC DES PERITONITES
CHEZ LES PATIENTS EN SOINS INTENSIFS
EVALUATION OF THE PERFORMANCE OF A MULTIPLEX PCR PANEL (CURETIS) IN THE MANAGEMENT OF PERITONITIS IN INTENSIVE
CARE UNITS
Etude PIAC
NICOLLEAU Claire
Né le 28 Avril 1990 à Cholet (49)
Sous la direction de Mme le Docteur PAILHORIES Hélène
ENGAGEMENT DE NON PLAGIAT
Je, soussignée NICOLLEAU Claire déclare être pleinement consciente que le plagiat de documents ou d’une partie d’un document publiée sur toutes formes de support, y compris l’internet, constitue une violation des droits d’auteur ainsi qu’une fraude caractérisée. En conséquence, je m’engage à citer toutes les sources que j’ai utilisées pour écrire ce rapport ou mémoire.
signé par l'étudiant(e) le 15.10.2020
LISTE DES ENSEIGNANTS DE LA FACULTÉ DE SANTÉ D’ANGERS
Doyen de la Faculté : Pr Nicolas Lerolle
Vice-Doyen de la Faculté et directeur du département de pharmacie : Pr Frédéric Lagarce
Directeur du département de médecine : Pr Cédric Annweiler
PROFESSEURS DES UNIVERSITÉSABRAHAM Pierre Physiologie Médecine
ANNWEILER Cédric Gériatrie et biologie du
vieillissement Médecine
ASFAR Pierre Réanimation Médecine
AUBE Christophe Radiologie et imagerie médicale Médecine
AUGUSTO Jean-François Néphrologie Médecine
AZZOUZI Abdel Rahmène Urologie Médecine
BAUFRETON Christophe Chirurgie thoracique et
cardiovasculaire Médecine
BENOIT Jean-Pierre Pharmacotechnie Pharmacie
BEYDON Laurent Anesthésiologie-réanimation Médecine
BIGOT Pierre Urologie Médecine
BONNEAU Dominique Génétique Médecine
BOUCHARA Jean-Philippe Parasitologie et mycologie Médecine
BOUVARD Béatrice Rhumatologie Médecine
BOURSIER Jérôme Gastroentérologie ; hépatologie Médecine
BRIET Marie Pharmacologie Médecine
CAILLIEZ Eric Médecine générale Médecine
CALES Paul Gastroentérologe ; hépatologie Médecine CAMPONE Mario Cancérologie ; radiothérapie Médecine CAROLI-BOSC François-xavier Gastroentérologie ; hépatologie Médecine CHAPPARD Daniel Cytologie, embryologie et
cytogénétique Médecine
CONNAN Laurent Médecine générale Médecine
COUTANT Régis Pédiatrie Médecine
CUSTAUD Marc-Antoine Physiologie Médecine
DE CASABIANCA Catherine Médecine Générale Médecine DESCAMPS Philippe Gynécologie-obstétrique Médecine D’ESCATHA Alexis Médecine et santé au Travail Médecine DINOMAIS Mickaël Médecine physique et de
réadaptation Médecine
DIQUET Bertrand Pharmacologie Médecine
DUBEE Vincent Maladies Infectieuses et
Tropicales Médecine
DUCANCELLE Alexandra Bactériologie-virologie ; hygiène
hospitalière Médecine
DUVAL Olivier Chimie thérapeutique Pharmacie
DUVERGER Philippe Pédopsychiatrie Médecine
EVEILLARD Mathieu Bactériologie-virologie Pharmacie FAURE Sébastien Pharmacologie physiologie Pharmacie
FOURNIER Henri-Dominique Anatomie Médecine
FURBER Alain Cardiologie Médecine
GAGNADOUX Frédéric Pneumologie Médecine
GARNIER François Médecine générale Médecine
GASCOIN Géraldine Pédiatrie Médecine
GOHIER Bénédicte Psychiatrie d'adultes Médecine GUARDIOLA Philippe Hématologie ; transfusion Médecine
GUILET David Chimie analytique Pharmacie
HAMY Antoine Chirurgie générale Médecine
HENNI Samir Chirurgie Vasculaire, médecine
vasculaire Médecine
HUNAULT-BERGER Mathilde Hématologie ; transfusion Médecine IFRAH Norbert Hématologie ; transfusion Médecine
JEANNIN Pascale Immunologie Médecine
KEMPF Marie Bactériologie-virologie ; hygiène
hospitalière Médecine
LACCOURREYE Laurent Oto-rhino-laryngologie Médecine
LAGARCE Frédéric Biopharmacie Pharmacie
LARCHER Gérald Biochimie et biologie moléculaires Pharmacie LASOCKI Sigismond
LEGENDRE Guillaume Anesthésiologie-réanimation
Gynécologie-obstétrique Médecine Médecine
LEGRAND Erick Rhumatologie Médecine
LERMITE Emilie Chirurgie générale Médecine
LEROLLE Nicolas Médecine Intensive-Réanimation Médecine LUNEL-FABIANI Françoise Bactériologie-virologie ; hygiène
hospitalière Médecine
MARCHAIS Véronique Bactériologie-virologie Pharmacie MARTIN Ludovic Dermato-vénéréologie Médecine MAY-PANLOUP Pascale Biologie et médecine du
développement et De la reproduction
Médecine
MENEI Philippe Neurochirurgie Médecine
MERCAT Alain Réanimation Médecine
MERCIER Philippe Anatomie Médecine
PAPON Nicolas Parasitologie et mycologie
médicale Pharmacie
PRUNIER Fabrice Cardiologie Médecine REYNIER Pascal Biochimie et biologie moléculaire Médecine RICHARD Isabelle Médecine physique et de
réadaptation Médecine
RICHOMME Pascal Pharmacognosie Pharmacie
RODIEN Patrice Endocrinologie, diabète et
maladies métaboliques Médecine ROQUELAURE Yves Médecine et santé au travail Médecine ROUGE-MAILLART Clotilde Médecine légale et droit de la
santé Médecine
ROUSSEAU Audrey Anatomie et cytologie
pathologiques Médecine
ROUSSEAU Pascal Chirurgie plastique,
reconstructrice et esthétique Médecine ROUSSELET Marie-Christine Anatomie et cytologie
pathologiques Médecine
ROY Pierre-Marie Thérapeutique Médecine
SAULNIER Patrick Biophysique et biostatistique Pharmacie
SERAPHIN Denis Chimie organique Pharmacie
TRZEPIZUR Wojciech Pneumologie Médecine
UGO Valérie Hématologie ; transfusion Médecine
URBAN Thierry Pneumologie Médecine
VAN BOGAERT Patrick Pédiatrie Médecine
VENIER-JULIENNE Marie-Claire Pharmacotechnie Pharmacie
VERNY Christophe Neurologie Médecine
WILLOTEAUX Serge Radiologie et imagerie médicale Médecine
MAÎTRES DE CONFÉRENCES
ANGOULVANT Cécile Médecine Générale Médecine
BAGLIN Isabelle Chimie thérapeutique Pharmacie
BASTIAT Guillaume Biophysique et biostatistique Pharmacie
BEAUVILLAIN Céline Immunologie Médecine
BELIZNA Cristina Médecine interne Médecine
BELLANGER William Médecine générale Médecine
BELONCLE François Réanimation Médecine
BENOIT Jacqueline Pharmacologie Pharmacie
BIERE Loïc Cardiologie Médecine
BLANCHET Odile Hématologie ; transfusion Médecine
BOISARD Séverine Chimie analytique Pharmacie
CAPITAIN Olivier Cancérologie ; radiothérapie Médecine
CASSEREAU Julien Neurologie Médecine
CHAO DE LA BARCA Juan-Manuel Médecine
CHEVALIER Sylvie Biologie cellulaire Médecine
CLERE Nicolas Pharmacologie / physiologie Pharmacie
COLIN Estelle Génétique Médecine
DERBRE Séverine Pharmacognosie Pharmacie
DESHAYES Caroline Bactériologie virologie Pharmacie
FERRE Marc Biologie moléculaire Médecine
FORTRAT Jacques-Olivier Physiologie Médecine
HAMEL Jean-François Biostatistiques, informatique médicale Médicale HELESBEUX Jean-Jacques Chimie organique Pharmacie
HINDRE François Biophysique Médecine
KHIATI Salim Biochimie et biologie moléculaire Médecine JOUSSET-THULLIER Nathalie Médecine légale et droit de la santé Médecine JUDALET-ILLAND Ghislaine Médecine Générale Médecine KUN-DARBOIS Daniel Chirurgie Maxillo-Faciale et
Stomatologie Médecine
LACOEUILLE Franck Biophysique et médecine nucléaire Médecine
LEBDAI Souhil Urologie Médecine
LANDREAU Anne Botanique/ Mycologie Pharmacie
LEBDAI Souhil Urologie Médecine
LEGEAY Samuel Pharmacocinétique Pharmacie
LE RAY-RICHOMME Anne-
Marie Pharmacognosie Pharmacie
LEPELTIER Elise Chimie générale Pharmacie
LETOURNEL Franck Biologie cellulaire Médecine
LIBOUBAN Hélène Histologie Médecine
LUQUE PAZ Damien Hématologie; Transfusion Médecine MABILLEAU Guillaume Histologie, embryologie et
cytogénétique Médecine
MALLET Sabine Chimie Analytique Pharmacie
MAROT Agnès Parasitologie et mycologie médicale Pharmacie
MESLIER Nicole Physiologie Médecine
MOUILLIE Jean-Marc Philosophie Médecine
NAIL BILLAUD Sandrine Immunologie Pharmacie
PAILHORIES Hélène Bactériologie-virologie Médecine
PAPON Xavier Anatomie Médecine
PASCO-PAPON Anne Radiologie et imagerie médicale Médecine
PECH Brigitte Pharmacotechnie Pharmacie
PENCHAUD Anne-Laurence Sociologie Médecine
PIHET Marc Parasitologie et mycologie Médecine
PY Thibaut Médecine Générale Médecine
RAMOND-ROQUIN Aline Médecine Générale Médecine
RINEAU Emmanuel Anesthésiologie réanimation Médecine
TESSIER-CAZENEUVE
Christine Médecine Générale Médecine
VENARA Aurélien
Chirurgie générale Médecine
VIAULT Guillaume
Chimie organique Pharmacie
PROFESSEURS EMERITES
Philippe MERCIER Neurochirurgie Médecine
Dominique CHABASSE Parasitologie et Médecine Tropicale Médecine
Jean-François SUBRA Néphrologie Médecine
AUTRES ENSEIGNANTS
AUTRET Erwan Anglais Médecine
BARBEROUSSE Michel Informatique Médecine
BRUNOIS-DEBU Isabelle Anglais Pharmacie
CHIKH Yamina Économie-Gestion Médecine
FISBACH Martine Anglais Médecine
O’SULLIVAN Kayleigh Anglais Médecine
Mise à jour au 09/12/2019
REMERC IEMEN T S
A Monsieur le Professeur Sigismond LASOCKI,
Sigismond, merci de me faire l’honneur de présider cette thèse. Votre enseignement, votre soutien et vos conseils depuis ces 5 dernières années m’ont permis d’avancer sereinement dans ma formation. Merci de la patience dont vous avez fait preuve à mon égard non seulement au cours de mon internat mais également ces dernières semaines. Je tacherai d’être à la hauteur de la confiance que vous m’accordez en me permettant de travailler ici.
A Madame le Docteur Hélène PAILHORIES,
C’est un honneur d’avoir pu mener à bien ce projet qui est le vôtre. Vos conseils et votre réactivité m’ont été d’un grand soutien. Merci d’avoir pris le temps de me guider dans ce travail que j’espère pouvoir poursuivre au mieux à vos côtés.
A Monsieur le Docteur Emmanuel RINEAU,
Bien qu’à distance, cela n’enlève rien au plaisir que j’ai de te compter parmi les membres de mon jury. Ce fut un plaisir d’être ton interne. Merci de m’avoir accordé ta confiance. Tu as même su, parfois, me laisser faire mes propres erreurs afin que je puisse toujours progresser et je t’en remercie.
A Monsieur le Docteur Benjamin DUMARTINET,
Benjamin, je te remercie énormément d’avoir accepté, il y a déjà quelque
temps, de faire partie de mon jury. Merci d’être présent à mes côtés, que ce
soit au travail ou à l’extérieur de l’hôpital. Professionnellement parlant, je garde
pour modèle la patience et le calme dont tu fais preuve en, presque, toutes
circonstances. Personnellement, sache que ton inestimable soutien, ton amitié
(et ta capacité à corriger mes fautes d’orthographes) me sont extrêmement
REMERC IEMEN T S
Au Docteur Carole LEMARIE, pour m’avoir également aidée dans ce travail dont vous faites entièrement partie. Merci pour vos conseils et votre disponibilité.A mes frères, pour leur soutien depuis tant d’années. Pour être présents, prendre soin de moi, me protéger et de continuer à m’aimer malgré toutes mes erreurs. Merci d’avoir fait rentrer dans ma vie mes deux belles-sœurs adorées. Je n’ai aucun doute que vous serez fiers de moi et sachez combien cela est réciproque vous concernant.
A mes parents, pour m’avoir permis d’avancer tout en vous sachant à mes côtés. Vous avez su me laisser partir et revenir quand j’en ai eu besoin. Je n’en serais pas là sans votre soutien ni votre patience que j’ai bien souvent mise à l’épreuve.
A mon grand-père, qui bien que ne pouvant pas être présent, m’a soutenu tout au long de ces années.
A l’ensemble des membres de ma famille, et particulièrement Betty, pour avoir été si présente dans ma jeunesse.
A tous les médecins Anesthésiste-Réanimateurs du CHU d’Angers que j’ai croisé au long de mon parcours, merci d’avoir tant contribué à ma formation.
A toute l’équipe de Réanimation et du Réveil de la Pitié-Salpêtrière pour ces 7 mois, qui m’ont permis de progresser professionnellement et humainement. J’ai une pensée toute particulière pour mes co-internes que j’espère revoir bientôt.
A l’ensemble de mes co-internes et amis du CHU d’Angers, travailler et grandir à vos côtés a été une de mes principales motivations et un immense plaisir. J’ai fait de merveilleuses rencontres et je suis ravie de pouvoir continuer à travailler avec beaucoup d’entre vous.
Une parenthèse pour mes co-internes de Réanimation médicale, avec qui j’ai passé 6 excellents mois. J’espère avoir le plaisir de continuer à vous voir en dehors.
A Sarah, pour répondre toujours présente quand j’en ai besoin. Je te souhaite plein de bonheur pour ce qui va suivre.
A Paul, pour me rappeler régulièrement combien un pneumothorax est si vite arrivé et un guide si vite oublié. Ton amitié depuis 5 ans m’est précieuse.
A Esma, pour être présente dans les bons comme les mauvais moments et savoir me motiver quand il le faut.
Merci à tous les deux pour ce que vous faites pour moi au quotidien.
A Alice, pour toute l’aide que tu m’as apportée dans ce travail qui a également été le tien et auquel tu as pleinement participé. Cela n’aurait pas été rendu possible sans ton aide.
A Chachou, ce premier semestre à tes côtés aura marqué non seulement le début de mon internat mais également de notre amitié. Merci de rendre mes jours simplement plus légers.
REMERC IEMEN T S
A Raphaëlle et Benjamin pour l’amitié que vous m’accordez. Merci de me permettre de partager mes passions culinaires réelles et/ou virtuelles.A la sauce Pesto Rosso sans quoi je n’aurais su me nourrir.
A mes Intellectuels, Léa, Lorine, Charlotte et Benjamin, sans qui la fin de cette étape n’aurait pas la même saveur. Cet innocent groupe est devenu un pilier pour moi. Merci de ne, presque, jamais me juger, mais aussi de tolérer que j’écrive la moitié des mots de façon incorrecte et que j’en oublie l’autre moitié.
A Pedro, même si toutes les histoires ne finissent pas forcément comme nous l’avions prévu, celle-ci à marquer ma vie. Je n’en serais pas là sans le soutien dont tu as fait preuve. Merci de m’avoir encouragée dans cette voie, de m’avoir conseillée, rassurée et de continuer à le faire malgré tout encore aujourd’hui. Je garderai comme objectif de devenir un jour la femme et le médecin que tu as cru voir en moi.
Au Docteur Lauren Kline, pour avoir réanimer ce patient d’un coup de poing sternal, permettant ainsi à la fiction de devenir ma vocation.
A ma grand-mère, qui m’a accueillie, nourrit et hébergée à chaque étape importante de ma vie professionnelle. Je te dois énormément pour m’avoir permise d’être dans un tel environnement dans ces moments importants. Merci d’être capable de me faire relativiser chaque fois que je pense ne pas pouvoir surmonter un obstacle.
Liste des abréviations
APACHE Acute Physiologic and Chronic Healt Evaluation ATB Antibiotic therapy
CPE Carbapenemase-Producing Enterobacteriaceae BMI Body Mass Index
ESBL Extended-Spectrum-Seta-Lactamase FN Fasle Negative
FP False Positive
IAI Intra-Abdominal infection ICU Intensive Care Unit
MRSA Methicillin Resistant Staphylococcus aureus NPV Negative Predictive Value
PPV Positive Predictive Value
SAPS II Simplified Acute Physiology Score Se Sensibility
SOFA Sepsis-related Organ Failure Assessment score Sp Specificity
TN True Negative TP True Positive
INTRODUCTION
Complicated intra-abdominal infection is a severe disease and one of the main causes of sepsis in intensive care units (ICU) (1). It is associated with a high mortality, ranging from 29 to 64%
according to studies (2–5). Early and effective empirical antibiotic therapy, along with early surgical treatment, is needed to improve patient prognosis (6,7). Patients receiving inappropriate initial antimicrobial therapy had significantly higher morbidity and mortality rates than patients receiving appropriate therapy. (2)
Nowadays, multidrug-resistance bacteria prevalence is rising worldwide, and can be as high as 64% among strains found in ICU patients with peritonitis (extended-spectrum-beta-lactamase (ESBL), methicillin resistant Staphylococcus aureus (MRSA), and carbapenem-resistant Pseudomonas aeruginosa) (3). In particular, ESBL-producing micro-organism, responsible for intra-abdominal and urinary tract infections, are increasingly found in ICU patients (8). Prevalence of carbapenemase- producing Enterobacteriaceae (CPE) is also dramatically surging, with a prevalence from 1 to 15% in critically ill patients with intra-abdominal infection, depending on the geographic region (5,9). It has been shown that multidrug-resistant bacteria are associated with longer length-of-stay, higher mortality and more treatment failure (10). This impacts empirical antibiotherapy since those patients require broader-spectrum antibiotic regimen. On the other hand, it has been clearly shown those broader antibiotic regimens are responsible for the selection and thus the emergence of antibiotic resistance.
Therefore, rapid identification and susceptibility determination of the pathogens is a major challenge for optimizing treatment for patients with peritonitis in ICUs and allowing subsequent and rapid de-escalation or adaptation with an efficient antibiotic or antifungal therapy, as recommended by international guidelines (11).
Recently, multiplex PCR techniques based on a syndrome-specific approach have been increasingly used in microbiology diagnosis. These PCR kits allow to simultaneously identify numerous
pathogens as well as resistance genes. Its positive impact in respiratory, neurological infections and positive blood cultures diagnosis has already been documented (12–14). Furthermore, it allows antimicrobial therapy de-escalation and clinical decision to be made earlier (15,16), thus potentially resulting in less antibiotic-induced adverse-effects including resistance emergence.
The Curetis® Unyvero (Curetis®, Holzgerlingen, Germany) intra-abdominal infection (IAI) panel is the first commercialized panel specific to intra-abdominal infections. It is an automated multiplex PCR system which permits the identification of 51 targets (1 universal bacteria gene, 21 bacteria, 5 yeasts, 24 resistance genes) in a short time (4 to 5 hours). These targets include the most common pathogens isolated in intra-abdominal infection : Enterobacteriaceae (Klebsiella, Enterobacter, Escherichia coli), Streptococci, Enterococcus sp, non-fermenting Gram-negative aerobes (Pseudomonas, Acinetobacter) and anaerobic organisms (Bacteroides, Clostridium, Finegoldia), Aeromonas and fungi (Candida) A multicentric study performed on 300 biological samples showed that the Curetis® IIA panel is associated with a sensitivity global of 89,3% and a specificity global of 99,5%
and enables a 17 hours earlier identification delay compared to identification results by conventional culture (19). These results seem encouraging for a regular use of the Curetis® panel. However these data are not correlated to the clinical impact on the patient management, particularly the de-escalation or escalation of the antimicrobial therapy.
Thus, the aim of this study is to evaluate the performances of the CURETIS® IAI panel for the diagnosis of bacterial or fungal peritonitis in peritoneal fluids sampled from patients hospitalized in intensive care units and its clinical impact on the antibiotherapy management.
MATERIALS AND METHODS
This monocentric prospective observational study has been approved by the local ethics committee of the University Hospital of Angers (Approval 2019-23). An informative letter was given to the patient joining the study or to his next of kin, and their consent was obtained before inclusion in the study according to the French law.
Inclusion of the patients
This study took place from February 2019 to May 2020. All the patients hospitalized in the Surgical ICU for a complicated Intra-abdominal infection, and for whom a peritoneal fluid sample was collected and sent to the laboratory of bacteriology were included. The exclusion criteria were a residual volume of peritoneal fluid lower than 600 µL, the refusal for the participation to the study or death of the patient within the 24 hours following admission.
Data collection
Clinical and biological data were collected during ICU stay. This included demographic data (age, gender), medical history of the patient with co-morbid conditions, IGS II score, delay between patient’s admission and surgery or sample collection, the antibiotics introduced and the reason for the changes (end planned, de-escalation or escalation), the presence of intercurrent infections and results of microbiological cultures, treatment failure (defined as the need for re-operation or percutaneous drainage), Sepsis-related Organ Failure Assessment score (SOFA) at the time of sample, duration of mechanical ventilation and duration of catecholamines treatment.
Standard Microbiological analysis
Microbiological cultures were performed in the Laboratory of Bacteriology. Briefly, the peritoneal fluid was plated on a selective ESBL® agar plate (bioMérieux®, Marcy l’Etoile, France) incubated in an aerobic atmosphere, on a selective blood agar plate with colistin and nalidixic acid incubated in an anaerobic atmosphere, on a chromogenic UTI agar plate incubated in an aerobic atmosphere and on a Sabouraud agar plate in an aerobic atmosphere (BD®, Heidelberg, Germany). All the agar plates were incubated at a temperature of 35±2 °C for 5 days. The bacterial identifications were performed with mass spectrometry (MALDI-TOF; Vitek® MS, bioMérieux, Marcy l’Etoile, France). The antimicrobial susceptibility testing was performed following the European Committee on Antimicrobial Susceptibility testing. [20]
Curetis® IAI analysis
The peritoneal fluids were analyzed on the Unyvero® Curetis system with the Intra-Abdominal Infection kit, following the manufacturer instructions.
Data analysis
Data were entered into a spreadsheet and analysed on JASP 12 2 0. Continuous data were presented as means ± ET or medians [Q1-Q3], according to their distribution, normal or not. Categorical data were presented as n(%). The population normality was tested by Shapiro-Wilk test.
In this study, standard culture was considered as the reference method. The results of bacterial
negative (FN). In contrast, a result obtained from the Curetis test and not by the culture was considered as a false positive (FP). Sensitivity (Se), specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were evaluated for all the pathogens identified by the Curetis.
In addition, a clinical expert (intensivist) reviewed the results of PCR tests and defined a theoretical antibiotic regimen according to the results of the Curetis technique and to the medical chart. This analyze was made retrospectively, without knowledge of the antibiotic treatment actually administered to the patient. This theoretical antibiotic regimen was considered as adapted when it covered all the strains identified using the conventional sample cultures. This theoretical antibiotic regimen was then compared with the empirical therapy effectively administered to the patient. A de-escalation of the antimicrobial treatment was considered when a broad spectrum antimicrobial therapy would have been replaced by a narrower-spectrum one following the curetis results, and when this narrower spectrum antibiotic therapy would be effective on the micro-organisms isolated by culture. An adaptation of the antimicrobial therapy was considered when the empirical therapy administered to the patient was ineffective on at least one micro-organism identified on standard culture, and when the curetis technique would allow an early adaptation of the antimicrobial treatment. The actual delay observed for antibiotic therapy de-escalation/adaptation was also compared to the theoretical one (ie using Curetis results). The turnaround time was defined by the time between the surgery and the identification of pathogens (by culture or by M-PCR).
RESULTS
1. Demographical characteristics
Between February 2019 and May 2020 33 patients were recruited and 36 clinical samples were analyzed. The median age was 67.5 years (IQR 57,8 – 73,5), 20 (56%) were men. The main co- morbidities are reported in Table 1. The severity of the patients was important with an elevated SAPS II on admission (48 [IQR 37-55]) and a 19% mortality rate.
Concerning the type of intra-abdominal infection, one was a primary peritonitis, one was a tertiary peritonitis and the other ones were secondary peritonitis . Twenty of them (55%) were nosocomial.
Tableau I : Demographics and baseline clinical characteristics of patients and clinical samples.
Patients Number (%) (n = 33)
Age (years) 67.5 [57 .8-73.5]
Male 20 (56%)
IMC 25.2 [22.1-28.7]
Co-morbid conditions Diabete
Chronic respiratory failure Chronic kidney failure Cancer
3 (8%) 3 (8%) 4 (11%) 15 (41%) Hospitalisation
IGS II at admission SOFA at admission ATB 24h before sample Delay admission-sample (d)
48 [37-55.2]
6 [3-8]
15 (42%) 5 [0.8-9.2]
2. Microbiological results
2.1. Turnaround-time
The median turnaround time of the M-PCR was 7.2 ±2 hours and was 33 ±13.8hours for the culture with a median time for the first antibiotic susceptibility test result at 60hours.
2.2. Results of conventional culture
Among the 36 samples, 32 (89%) were associated with a positive culture result and 91 pathogens were identified by conventional techniques. The most frequently isolated bacteria were Gram positive (n=
27 (29.7%)) represented mostly by Enterococcus group (n =11). Enterobacteriaceae were also in high proportion (n= 26 (28.5%)), with a majority of Escherichia coli (n=15). Anaerobic pathogens (n=17 (18.7%)) were mostly mostly identified as members of Bacteroides fragilis group (n=14). Yeasts represented 17.5% of the pathogens and Candida albicans was the most commonly identified yeast (n= 8). Figure 1.
2.3. Results of M-PCR (Curetis)
Among these 36 samples, 26 (72%) were positive with the Curetis M-PCR and 94 pathogens were identified. Gram positive bacteria were in high ratio (n=30 (31.9%)) with predominance of Enterococcus group (n=16). Enterobacteriaceae (n = 23 (24.4%)) were mostly represented by Escherichia coli (n=16). The anaerobic pathogens were in majority (n= 34 (37.2%)) associated with Bacteroides spp and fragilis (n=26).Four yeast (5.3%) were identified. Figure 2
Figure 1 : repartition of pathogens detected by the conventional culture
5 5
6 5
15 0
5 1
1 0 0 0
7 7 1
1 0 0 0
4 3
8 4
0 1
Staphylococcus aureus Streptococcus spp Enterococcus spp.
Enterococcus faecalis Escherichia coli Enterobacter aerogenes Enterobacter cloacae complex Klebsiella pneumoniae Klebsiella oxytoca Klebsiella variicola Proteus spp.
Aeromonas spp.
Bacteriodes spp./Prevotella spp.
Bacteriodes fragilis group Clostridium difficile Clostridium perfringens/clostri Spp Finegoldia magna Propionibacterium acnes Acinetobacter baumannii complex Pseudomonas aeruginosa Candida spp.
Candida albicans Candida glabrata Candida tropicalis Candida Krusei
REPARTITION of pathogens detected by conventional
culture
Figure 2 : repartition of pathogens detected by the M-MPR
6 4 4
15 1
16 0
1 2
3 0
1 0
13 13 3
4 1
0 0
3 1
2 1 0 0
Staphylocoque Coagulase négative /staph Epidermidis Staphylococcus aureus Streptococcus spp/strepto groupe mileri Enterococcus spp. / E. durans/faescium/avis Enterococcus faecalis Escherichia coli Enterobacter aerogenes Enterobacter cloacae complex Klebsiella pneumoniae Klebsiella oxytoca Klebsiella variicola Proteus spp.
Aeromonas spp.
Bacteriodes spp./Prevotella spp.
Bacteriodes fragilis group Clostridium difficile Clostridium perfringens/clostri Spp Finegoldia magna Propionibacterium acnes Acinetobacter baumannii complex Pseudomonas aeruginosa Candida spp.
Candida albicans Candida glabrata Candida tropicalis Candida Krusei
Repartition of pathogens detected by the M-PCR
2.4. Performance of M-PCR (Curetis)
Regarding the performance of the M-PCR technique, its sensitivity was 61,9 % (IC95% : 51,5-72,3), its specificity was 95,3 % (IC95% : 93,7-96,5), with a positive predictive value of 55,3% (IC95% 44,7- 64,7) and a negative predictive value of 96,4 % (IC95% : 94,7-97,3). Among microorganisms, an heterogeneity of the sensitivity was noticed. The weaker sensitivity was associated with yeast with a result of 19%. The sensitivity was higher for Enterobacteriaceae (82%) than for Gram-positive bacteria (54%). Among Enterobacteriaceae, the sensitivity for Enterobacter cloacae was 20% and was 100%
for Escherichia coli. Concerning Gram-positive bacteria, the sensitivity for Enterococcus faecalis target was 20% and 20% for Streptococcus. The sensitivity for anaerobic pathogens was 88% and 75% for non-fermentative bacteria. The specificity of the M-PCR was between 94 to 100% for all the isolated pathogens, except for Enterococcus (75%) and Bacteroides (72%). The performance for each pathogen is presented in Table 2
2.5. Antibiotic resistance
Concerning antibiotic resistance, the M-PCR detected the only ESBL-producing E. coli isolated in conventional culture but was also associated with a false positive for another blaCTX-M gene. The M-PCR detected the only methicillin-resistant Staphylococcus but was also associated with two false mecA positive results. Concerning the 2 nitroimidazole resistance genes (nim A and B), they were detected by the Curetis. We notice that one vanB gene and one toxine gene tcdB were detected by the Curetis but not identified by conventional culture, due mainly to the absence of specific culture medium. Table3
Table III : Antibiotic resistance and toxine
Antibiotic Resistance Detection by M-PCR Detection by culture
Oxacilline C3G Vancomycine Nitroimidazole
mecA Ctx-M
vanB nimA, nimB
3 2 1 2
1 1 0 2
Toxines tcbB 1 0
3. Theorical impact on antibiotic prescription
The probabilist antibiotherapy was initially adapted in 15 (41,67%) cases , while with the CURETIS the theorical antibiotherapy would be adapted in 19 (52,78%) cases (p<0,0003), in a shorter time (median delay 8.15 [0;32.75] vs 23 [0.53.25] hours theoretical vs clinical, p=0.045). These adaptations of antibiotherapy corresponded to introduction of 2 antibiotics efficient on Staphylococcus epidermidis (linezolid and vancomycin), 1 antibiotic efficient on ESBL Escherichia coli (imipenem) and 1 antibiotic for Pseudomonas aeruginosa (piperacillin-tazobactam). However, in 4 cases (11%), the Curetis could have led to an unnecessary escalation of the antibiotherapy. It concerned 1 changes of amoxicillin/clavulanic acid for piperacillin/tazobactam, 1 imipenem for piperacillin/tazobactam and 2 unnecessary treatment by vancomycin.
In 8 cases (22%), in which initial antibiotherapy was de-escalated, it would have been earlier using the Curetis (median delay of 7 [5.8;11.3] vs 54 [45;73] hours, p=0.03). It corresponds of 6 de-escalation of imipenem for piperacillin/tazobactam, 1 piperacillin/tazobactam for amoxicillin/clavulanic acid and 2 stop of unnecessary linezolid.
About yeast, in 2 cases (5,5%), the Curetis could have led to an earlier antifungal treatment and in 6 cases (17%), the Curetis could have led to an earlier de-escalation/stop of an unnecessary antifungal treatment.
Table II : Performance of multiplex PCR (M-PCR) for the identification of micro-organisms isolated at clinical thresholds
True positive
False positive
False negative
Se( %) Sp (%) PPV (%) NPV (%)
SCN
Staphylococcus aureus
Streptococcus spp.
Enterococcus spp.
Enterococcus faecalis
SUB-TOTAL
3 3 1
6 1 14
3 1 3 9 0 16
2 2 4 0 4 12
60%
60%
20%
100%
20%
54%
90%
97%
90%
70%
100%
90%
50%
75%
25%
40%
100%
46%
93%
94%
88%
100%
89%
92%
Escherichia coli
E. aerogenes
E. cloacae complex
Klebsiella pneumoniae
Klebsiella oxytoca
Klebsiella variicola
Proteus spp.
SUB-TOTAL
15 0 1 1 1 0 0 18
1 0 0 1 2 0 1 5
0 0 4 0 0 0 0 4
100%
- 20%
100%
100%
- - 82%
95%
- 100%
97%
94%
- 97%
98%
94%
- 100%
50%
33%
- 0 78%
100%
- 89%
100%
100%
- 100%
98%
Acinetobacter baumannii complex
Pseudomonas Aeruginosa
SUBTOTAL
0 3 3
0 0 0
0 1 1
- 75%
75%
- 100%
100%
- 100%
100%
- 97%
99%
True positive
False positive
False negative
Se (%) Sp (%) PPV (%) NPV (%)
Aeromonas spp.
Bacteriodes spp./Prevotella spp.
Bacteriodes fragilis
Clostridium difficile
Clostri. perfringens
Finegoldia magna
Propionibacterium acnes
SUB-TOTAL
0 5 7 1 1 0 0 14
0 8 6 2 3 1 0 20
0 2 0 0 0 0 0 2
- 71%
100%
100%
100%
- - 88%
- 72%
79%
94%
91%
97%
- 92%
- 38%
54%
33%
25%
0 - 42%
- 91%
100%
100%
100%
100%
- 99%
Candida spp.
Candida albicans
Candida glabrata
Candida tropicalis
Candida Krusei
SUB-TOTAL
0 2 1 0 0 3
1 0 0 0 0 1
3 6 3 0 1 13
0 25%
25%
- 0 19%
97%
100%
100%
- 100%
99%
0 100%
100%
- NA 75%
91%
82%
91%
- 97%
93%
TOTAL 52 42 32 61,9% 95,3% 55,3% 96,4%
DISCUSSION AND CONCLUSION
This study is, to our knowledge, the first analyze of the potential impact of a M-PCR system on antimicrobial therapy, for intra-abdominal infections. Indeed, some studies have already been conducted for ventilator-associated pneumonia. This study suggests that M-PCR might help improving empiric antibiotic therapy on patients with peritonitis. The probabilist antibiotherapy is significatively better adapted with the Curetis that without (p<0.0003). In 25% of the cases (9/36) it would have permitted a positive clinical impact (2 escalations, 5 de-escalations and 2 escalation plus de-escalation).
Also, when patients have had a de-escalation or escalation of the antimicrobial treatment, it would have happened earlier with the Curetis. Unfortunately, in 6 cases (17%), the clinical impact would have been negative. Accordingly, we consider the clinical impact as mixed.
M-PCR provided a sensitivity of 61.9% and a specificity of 95.3% in comparison to culture.
These results are not as good as the ones reported in the literature. Ciesielczuk et al, in a study on 107 fluid peritoneal samples, brought out a sensitivity of 83.7% and a specificity of 99.5%. This discrepancy in our results might be explained by the fact that the sensitivity was very heterogenous for each pathogen, ranging from 0 (Candida spp and Candida krusei) to 100% The biggest sensitivity differences between our study and Ciesielczuk’s were for Enterococcus spp (20 vs 61%) Streptococcus spp (20 vs 67.9%) and Enterobacter cloacae (20 vs 93.3%). Also, a lack of power could explain the difference so far as, we included 36 samples versus 300 samples. Other explanation would be that only peritoneal samples were included in this study whereas in Ciesielczuk’s ascitic or pancreatic samples were also analyzed. As in our study, most studies reported a better performance of the system for Gram-negative bacteria than for Gram-positive cocci. Unfortunately, Gram-positive cocci are an important target in peritonitis. About Enterococcus, although the sensitivity is good for Enteroccocus spp (100%), it is very
Concerning Anaerobic pathogens, we found a high number of “false positive” (n=20) and not much false negative results. In culture, these pathogens are more difficult to cultivate and to identify, and are mostly characterized on the detailed report as anaerobic flora. Unfortunately, the modification of anti-anaerobic treatment is not a really important in clinic and the clinical impact of the detailed identification of the anaerobic flora is poor.
Concerning yeasts, the sensitivity is very low in our study (19%). In Ciesielczuk’study, the sensitivity was also low but better than in our study (44% vs 0% for Candida albicans). The proportion of yeasts was 39%, which corresponds to the prevalence reported in the literature (up to 40%) (20,21).
Unfortunately, yeasts are pathogens that have to be treated in severe peritonitis as recommended by the Societe Francaise d’Anesthesie Reanimation (SFAR). Nowadays, clinical scores are used to predict the presence of yeast at peritoneal culture and to decide to start a probabilistic anti-fungal treatment.
It seems that these scores have a better sensitivity, up to 60% (28) than the Curetis to predict the presence of yeasts in peritonitis. In our study, the Curetis would have led to stop falsely 4 antifungals treatments and being potentially deleterious.
The interest of the Curetis in the detection of resistance genes is limited in our study, due to the local epidemiology. Indeed, the only confirmed EBSL and MRSA have been confirmed but three false positive have also been reported for these genes. Moreover, no carbapenemase was present in the sample. In the literature (8), the proportion of ESBL-producing E.coli and Klebsiella pneumoniae in peritoneal fluids, were respectively of 14.6% and 42.1%, which is far from our results. The clinical impact of this detection would be more useful in hospitals where the rates of resistance are higher, and should have a key role in the adaptation of the antibiotic treatment. However, the absence of detection of high level cephalosporinase, reduce the impact of the resistance genes PCR results on the adaptation and the de-escalation of the antimicrobial treatment.
The Curetis technique has a positive impact on the adaptation of the antimicrobial treatment.
Indeed, in 11% of the cases, the Curetis would have permitted an early adaptation of the antimicrobial treatment. It would be also useful in order to early de-escalate the treatment in 19%. However, we
see that in 2 cases an inappropriate de-escalation would happened. These cases were identifications in culture of Morganella morganii and Hafnia alvei both resistant to piperacillin/tazobactam, however these pathogens are not in the Curetis panel and could have not been detected. Moreover, even if for 6 patients the theorical antibiotherapy was not adapted (such as the probabilist therapy), the Curetis would have led to a saving antibiotic (carbapenem and piperacillin/tazobactam) which is an important consideration today. Also in 2 cases, an appropriate escalation based on the Curetis could have been realized but do not considered as a positive clinical impact because they also would have been de- escaladed in an inappropriate way.
There would be a clinical impact positive concerning de-escalation but the Curetis has to be used in complement of the conventional culture. One should keep in mind that the theoretical antibiotherapy was proposed by experts and took into account the clinical picture, so that broader-antibiotic spectrum (compared to the results of the curetis) was proposed for the more severe cases.
Others limits of this study are the fact that it is a monocentric study and so extrapolation of results are difficult regarding the ecology of our hospital which did not represent the global national ecology. Also, the clinical outcome on antibiotherapy is determinated by a retrospective review of the clinical data limiting the analyze of the clinical impact of this technique. The main limit is linked to the technique, with some important targets unavailable in the Curetis panel. We identified 6 bacteria (Bifidobacterium spp, Hafnia alvei, Stenotrophomonas maltophilia, Lactobacillus spp, Citrobacter freundi and Morganella morganii) which were not included in the panel of Curetis. In addition, the high price of the kits is a big limiting factor for its use in routine.
In conclusion, the sensitivity and specificity of the Curetis in peritoneal fluid is not as high as expected.
The clinical impact could be positive with a better and earlier adapatation of antibiotherapy with the Curetis than without. However, the lack of sensitivity, particularly concerning yeast and Enterococcus, the lack of target in the panel, the price and restrictive technic, limited this clinical impact in peritonitis.
The Curetis would not be used independently of the conventional culture in treatment of peritonitis but would be interesting in association with the conventional culture for an earlier adaptation.
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31. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/
FIGURE LIST
Figure 1
Repartition of pathogens detected by the conventional culture... 8
Figure 2
Repartition of pathogens detected by the M-MPR... 9
TABLE LIST
Tableau I
Demographics and baseline clinical characteristics of patients and clinical samples... Erreur ! Signet non défini.
Tableau II
Performance of multiplex PCR (M-PCR) for the identification of micro-organisms isolated at clinical thresholds... 13-14
Tableau III
Antibiotic resistance and toxine... 11
TABLE DES MATIERES
ABREVIATIONS ... VII
INTRODUCTION ... 1
MATERIAL AND METHODS ... 3
RESULTS ... 6
1. Demographical characteristics ... Erreur ! Signet non défini. 2. Microbiological results………...Erreur ! Signet non défini.
2.1. Turnaround time ... Erreur ! Signet non défini. 2.2. Results of conventional culture ... 7
2.3. Results of M-PCR ... 7
2.4. Performance of M-PCR ... 10
2.5. Antibiotic resistance ... 10
3. Theorical impact on antibiotic prescription………..11
DISCUSSION AND CONCLUSION ... 15
BIBLIOGRAPHIE ... 19
LISTE OF FIGURES ... 23
LISTE OF TABLES ... 24
TABLE DES MATIERES ... 25
ANNEXES ... I
ANNEXES
- Pathogens included in M-PCR panel
Staphylocoque Coagulase négative /staph Epidermidis Staphylococcus aureus
Streptococcus spp/strepto groupe mileri Enterococcus spp. / E. durans/faescium/avis
Enterococcus faecalis Escherichia coli Enterobacter aerogenes Enterobacter cloacae complex
Klebsiella pneumoniae Klebsiella oxytoca Klebsiella variicola
Proteus spp.
Aeromonas spp.
Bacteriodes spp./Prevotella spp.
Bacteriodes fragilis group Clostridium difficile
Clostridium perfringens/clostri Spp Finegoldia magna
Propionibacterium acnes Acinetobacter baumannii complex
Pseudomonas aeruginosa Candida spp.
Candida albicans Candida glabrata Candida tropicalis
Candida Krusei
- Pathogens non included in M-PCR panel
Bifidobacterium spp.Hafnia alvei
Stenotrophomonas maltophilia Lactobacillus spp.
Citrobacter freundi Morganella morganii
- Toxine and resistance genes included in panel
Toxin B - tcbBShiga Toxin - stx1/2 Oxa - mecA Oxa - mecB Vanco - vanA Vanco - vanB Aminoglycoside - aacA4
C3G - ctxM fosA3 mcr-1 Polypep - nimA Polypep - nimB
FQ - qnrA FQ - qnrB FQ - qnrS Tet - tetA Carba - kpc Carba - oxa-23 Carba - oxa24/40
Carba - oxa-48 Carba - oxa-58 Carba - vim Carba - imp Carba - ndm
NICOLLEAU Claire
Evaluation de la performance d’un kit PCR-Multiplex (CURETIS) dans le diagnostic des péritonites chez les patients en soins intensifs
Mots-clés : Péritonite, PCR multiplex, CURETIS, sensibilité bactérienne, levure, antibiothérapie probabiliste
Evaluation of the performance of a PCR Multiplex panel (CURETIS) in the management of peritonitis in intensive care units.
R É S UM É
Les péritonites sont des pathologies graves, associées à une morbi-mortalité élevée dans les services de soins intensifs. Une antibiothérapie efficace et précoce permet d’améliorer le pronostic des patients. Le panel Curetis® IIA, par technique PCR Multiplex, permet de détecter rapidement un certain nombre de bactéries, levures et gènes de résistance aux antibiotiques. Ce kit pourrait par son rapidité d’identification des pathogènes pourrait permettre la mise en place d’une antibiothérapie efficace plus précoce. Trente-six liquides péritonéaux ont été analysés chez des patientshospitalisés pour péritonites en soins intensifs au CHU d’Angers. Les résultats ont été comparés à la culture conventionnelle. La sensibilité du Curetis était de 61.9%
(IC95 :51.5-72.3) et la spécificité de 95,3% (IC 95%:93,7-96,5%). Les sensibiltés étaient hétérogènes allant de 0% (Candida spp et krusei) à 100%. Avec le Curetis, l’antibiothérapie initiée aurait été significativement plus adaptée que celle introduite de façon probabiliste (p<0.0003) et dans un délai significativement plus court
(p=0.045). Dans cette étude, la sensibilité globale n’est pas aussi bonne qu’attendue et présente une importante hétérogéniéte. Le Curetis permettrait tout de même une antibiothérapie probabiliste mieux adaptée et plus précoce. Cerpendant, le manque de sensibilité, son coût et ses limites techniques en fond un outil difficilement utilisable en pratique courante et son utilisation doit être réalisée en parallèle de la culture
conventionnelle.
AB ST RAC T
Peritonitis are frequent and associated with a high morbi-mortality in ICU patients. An adapted and early antibiotherapy is necessary to improve patient pronostic. The Curetis panel, is able to identify numbers of bacteria, fungi, toxin and antibiotic resistance markers in a short delay.This technic, could permit to introduce an adapted antibiotherapy in a shorter time compare to conventional culture.Thirty-six peritoneal fluids were analyzed by the Curetis and the results were compare to the conventionnal culture. The globlal sensibility of the Curetis was 61.9% (IC95 :51.5-72.3) and global specificity was 95,3% (IC 95%:93,7-96,5%). The sensitivity was heterogeneous among pathogens ranging from 0% (candida spp and krusei) to 100%. With the Curetis, the theorical antibiotherapy would have been significatively better adapted (p<0.0003) and in a shorter time (p=0.045) compare to clinical antibiotherapy really introduced.
In this study, the global sensibility is not as high as expected. It seems that with the Curetis, the antibiotherapy could be better and earlier adapted. However, the price, the lack of sensitivity and restrictive technics are a big limit to the interest. The Curetis would not be
