MD Emmanuelle Cambau
email: emmanuelle.cambau@aphp.fr

Mario Poljak

Sniffing animals as a diagnostic tool in infectious diseases

Keywords: odour, scent, bacteria, diagnostics, tuberculosis, malaria, volatile organic compounds

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Many microorganisms produce characteristic and identifiable odours, and experienced microbiologists can recognise certain microbial genera and even species by simply sniffing culture plates. Candida spp., Clostridioides difficile (formerly Clostridium difficile), Haemophilus spp., Nocardia spp., Proteus spp., Pseudomonas aeruginosa and anginosus group streptococci are those most recognisable by smell in the laboratory, and this remains helpful in routine in vitro identification of selected pathogens [START_REF] Barkham | Sniffing bacterial cultures on agar plates: a useful tool or a safety hazard?[END_REF]. Some experienced clinicians and nurses can also reliably diagnose patients with infectious mononucleosis by typical breath odour, C. difficile and rotaviral diarrhoea by typical stool smell, bladder infection by ammoniacal urine odour, wound infections caused by P. aeruginosa by "fruity" odour, bacterial vaginosis by "fishy" smell, diphtheria by sweet sweat odour and gas gangrene by typical smell. For example, in a controlled study trained nurses attained a sensitivity and specificity of 55% and 83%, respectively, for diagnosis of C. difficile diarrhoea by smelling patients' stool [START_REF] Burdette | Does the nose know? The odiferous diagnosis of Clostridium difficile-associated diarrhea[END_REF].

Animal sniffing is an innate behaviour, and canine olfactory acuity is over 100,000 times stronger than human acuity [START_REF] Weetjens | African pouched rats for the detection of pulmonary tuberculosis in sputum samples[END_REF]. Animal sniffing (by dog and rats) has been used for centuries for detecting people, and since the twentieth century animals have also detect weapons, bombs, narcotics and food -for instance, at borders and airports. According to a recent literature review, animals have been used as diagnostic tools in various fields of medicine, such as recognising diabetic ketoacidosis in emergency rooms and scent detection of various human tumours and metabolic diseases [START_REF] Bijland | Smelling the diagnosis: a review on the use of scent in diagnosing disease[END_REF].

Although most studies exploring animals' scent detection of human malignant tumours suggested similar or even superior accuracy compared with standard screening methods, the results are not easily generalizable and independent confirmation studies are generally lacking [START_REF] Bijland | Smelling the diagnosis: a review on the use of scent in diagnosing disease[END_REF][START_REF] Guirao | Trained dogs can identify malignant solitary pulmonary nodules in exhaled gas[END_REF]. This review summarises the most explored examples of animal sniffing to diagnose infectious diseases and discusses the potential use of scent-based diagnostic instruments in clinical microbiology. Information was sought through PubMed and extracted from peer-reviewed literature published between January 2000 and September 2019 and from reliable online news. The search terms odour, scent, bacteria, diagnostics, tuberculosis, malaria and volatile compounds were used.

Dogs and C. difficile diarrhoea

To address outbreaks of diarrhoea associated with C. difficile, Bomers et al. used dogs' olfactory sensitivity to rapidly detect patients suffering from C. difficile diarrhoea [START_REF] Bomers | Using a dog's superior olfactory sensitivity to identify Clostridium difficile in stools and patients: proof of principle study[END_REF][START_REF] Bomers | A detection dog to identify patients with Clostridium difficile infection during a hospital outbreak[END_REF]. They trained a beagle to sit when it smelled stool containing toxin-positive C. difficile and remain standing when it did not.

A food reward was used for true positive results. In another study, two dogs (a German shepherd and a border collie-pointer mix) were similarly trained and compared [START_REF] Taylor | Using dog scent detection as a point-of-care tool to identify toxigenic Clostridium difficile in stool[END_REF] (photos and video available at https://academic.oup.com/ofid/article-lookup/doi/10.1093/ofid/ofy179#supplementary-data).

The reward was given in less than 10 seconds. The results obtained separately for each dog showed sensitivity of 77.6% and 92.6%, and specificity of 86.1% and 84.5%, respectively after testing 300 samples, with one-third C. difficile positive [START_REF] Taylor | Using dog scent detection as a point-of-care tool to identify toxigenic Clostridium difficile in stool[END_REF]. However, the interrater reliability between dogs was modest (Cohen's kappa = 0.52) [START_REF] Taylor | Using dog scent detection as a point-of-care tool to identify toxigenic Clostridium difficile in stool[END_REF].

Despite promising results, it is questionable whether sniffing dogs can ever be used routinely and widely for C. difficile detection in patients. The main advantage of dogs over the traditional culturebased diagnostic approach is detection speed (a few minutes versus 1 to 2 days). However, several novel point-of-care C. difficile nucleic acid amplification tests (NAATs) can easily meet this challenge, yielding results in less than 1 hour with much higher sensitivity and specificity than dogs [START_REF] Kraft | A laboratory medicine best practices systematic review and meta-analysis of nucleic acid amplification tests (NAATs) and algorithms including NAATs for the diagnosis of Clostridioides (Clostridium) difficile in adults[END_REF].

Trained dogs can also be used for surveillance of C. difficile. Preliminary internal evaluation of a springer spaniel-based program revealed an overall sensitivity of 92.3% and specificity of 95.4% for both odour recognition and search capability using gauze exposed to C. difficile odour [START_REF] Bryce | Identifying environmental reservoirs of Clostridium difficile with a scent detection dog: preliminary evaluation[END_REF]. Building upon proof-of-concept studies [START_REF] Bomers | Using a dog's superior olfactory sensitivity to identify Clostridium difficile in stools and patients: proof of principle study[END_REF][START_REF] Bomers | A detection dog to identify patients with Clostridium difficile infection during a hospital outbreak[END_REF][START_REF] Taylor | Using dog scent detection as a point-of-care tool to identify toxigenic Clostridium difficile in stool[END_REF][START_REF] Bryce | Identifying environmental reservoirs of Clostridium difficile with a scent detection dog: preliminary evaluation[END_REF], a large canine scent detection program on equipment and environmental surfaces rather than on patients was established at Vancouver General Hospital (Canada) in 2017. A recent article described the operational aspects of this validated program, and findings and lessons learned from 18 months of real-life environmental detection at this care facility [START_REF] Li | Using scent detection dogs to identify environmental reservoirs of Clostridium difficile: lessons from the field[END_REF]. During the study period, 659 clinical areas at the hospital were searched over 115 search days (5.7 clinical areas/day). The program demonstrated promising ability to rapidly triage hospital surfaces and equipment, and canines indicated many places where C. difficile reservoirs lie and challenged the way infection prevention is approached. The results of the study highlighted the impact of cross-transmission not only by healthcare workers, but also by patients and the public, as evidenced by alerts in public washrooms and both patient/family as well as staff lounges and lockers [START_REF] Li | Using scent detection dogs to identify environmental reservoirs of Clostridium difficile: lessons from the field[END_REF].

Dogs and urinary tract infection

Dogs have also been trained to sniff urine for urinary tract infection (UTI) [START_REF] Maurer | Detection of bacteriuria by canine olfaction[END_REF]. A double blind, case-control study was conducted with five dogs (Labrador retrievers and golden retrievers) and handlers enrolling 687 persons. For a third, urine samples were culture-positive with more than 10 5 /ml of bacteria such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus or Enterococcus faecalis. Dogs' sniffing showed a high sensitivity (99.6%) and moderate specificity (91.5%) in comparison to culture, especially for E. coli infection [START_REF] Maurer | Detection of bacteriuria by canine olfaction[END_REF]. Inter-dog reproducibility was not calculated. Dogs reliably detected positive samples, but some wrongly assigned certain negative samples and some refused to smell particular samples.

Giant rats and tuberculosis

The ancient Greeks and Chinese diagnosed tuberculosis by burning patients' sputum and recognising the smell in the fumes [START_REF] Bijland | Smelling the diagnosis: a review on the use of scent in diagnosing disease[END_REF]. More recently, African giant pouched rats (Cricetomys gambianus) have been trained, and several studies have explored their ability to diagnose tuberculosis patients by smelling sputum [START_REF] Weetjens | African pouched rats for the detection of pulmonary tuberculosis in sputum samples[END_REF][START_REF] Edwards | Tuberculosis detection by pouched rats: opportunities for reinforcement under low-prevalence conditions[END_REF][START_REF] Poling | Using giant African pouched rats to detect human tuberculosis: a review[END_REF][START_REF] Reither | Evaluation of giant African pouched rats for detection of pulmonary tuberculosis in patients from a high-endemic setting[END_REF][START_REF] Mahoney | Using giant African pouched rats to detect tuberculosis in human sputum samples: 2010 findings[END_REF][START_REF] Mulder | Accuracy of giant African pouched rats for diagnosing tuberculosis: comparison with culture and Xpert((R)) MTB/RIF[END_REF][START_REF] Mgode | Diagnosis of tuberculosis by trained African giant pouched rats and confounding impact of pathogens and microflora of the respiratory tract[END_REF][START_REF] Ellis | Reproducibility of African giant pouched rats detecting Mycobacterium tuberculosis[END_REF][START_REF]Global tuberculosis report[END_REF][START_REF] Mgode | Pediatric tuberculosis detection using trained African giant pouched rats[END_REF]. The rat studies were mainly conducted by the Belgian non-profit organisation APOPO, which also uses rats to detect landmines. In addition to examination by microscopy after Ziehl-Neelsen staining (94% sensitivity), which is still the main diagnostic tool for tuberculosis in low-income countries [START_REF] Poling | Using giant African pouched rats to detect human tuberculosis: a review[END_REF][START_REF] Reither | Evaluation of giant African pouched rats for detection of pulmonary tuberculosis in patients from a high-endemic setting[END_REF][START_REF] Mahoney | Using giant African pouched rats to detect tuberculosis in human sputum samples: 2010 findings[END_REF]. However, the sensitivity was lower when compared to NAAT (80%) [START_REF] Mulder | Accuracy of giant African pouched rats for diagnosing tuberculosis: comparison with culture and Xpert((R)) MTB/RIF[END_REF] or to culture (60-70%) [START_REF] Reither | Evaluation of giant African pouched rats for detection of pulmonary tuberculosis in patients from a high-endemic setting[END_REF][START_REF] Mgode | Diagnosis of tuberculosis by trained African giant pouched rats and confounding impact of pathogens and microflora of the respiratory tract[END_REF]. The results of the largest reallife study in a paediatric population to date performed on 55,148 Tanzanian presumptive tuberculosis patients showed that rats increased paediatric tuberculosis detection by 67.6% compared to classical smear microscopy (standard of care). However, the relative increase in case detection by rats over smear microscopy decreased with the increase in patients' age (p < 0.0001), due to higher sensitivity of classical smear microscopy in adults in comparison to children [START_REF] Mgode | Pediatric tuberculosis detection using trained African giant pouched rats[END_REF].

In all published studies, sniffing rats appeared to be a good diagnostic tool for diagnosing tuberculosis because settings were mainly highly endemic countries and peripheral medical centres without proper laboratory setups [START_REF]Global tuberculosis report[END_REF]. Because the prevalence of tuberculosis in study populations was very high (10-40%), this maximised sensitivity and minimised the potential impact on specificity. Another important obstacle is a lack of reproducibility between animals; for example, after 12,000 samples were sniffed by 22 different rats, intrarater reliability was high (Yule's Q of 0.9) but interrater agreement was only fair (Krippendorff's alpha 0.15-0.45) [START_REF] Ellis | Reproducibility of African giant pouched rats detecting Mycobacterium tuberculosis[END_REF]. Today, the microbiology diagnosis of pulmonary tuberculosis in low-income counties is mainly organised by the World Health Organization and supported by international funding. In addition to these shortcomings, the cost of training and housing rats should be considered when comparing rat sniffing to other microbiological approaches. Moreover, due to the spread of multi-drug-resistant tuberculosis, susceptibility testing is frequently needed, which cannot be performed using sniffing rats [START_REF]Global tuberculosis report[END_REF].

Rats' olfactory acuity for detecting sputa containing M. tuberculosis catalysed research efforts to identify the biological background of this ability [START_REF] Pai | Tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects[END_REF]. The sputa were comparatively analysed by gas chromatography-mass spectrometry (GC-MS), and volatile organic compounds (VOCs) specific to M. tuberculosis were identified [START_REF] Maclean | A systematic review of biomarkers to detect active tuberculosis[END_REF]. These findings served as the basis of novel "breath tests" developed by several companies for diagnosing tuberculosis [START_REF] Pai | Tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects[END_REF][START_REF] Maclean | A systematic review of biomarkers to detect active tuberculosis[END_REF].

Diagnosing malaria by host odour

Research on humans and in animal models has shown that infection by malaria parasites elicits changes in host odour that influence vector attraction, suggesting that such changes might yield robust biomarkers of malaria status [START_REF] Moraes | Volatile biomarkers of symptomatic and asymptomatic malaria infection in humans[END_REF]. Recently, an extraordinary experimental study was performed to measure the attraction of mosquitoes in the presence of the skin odour of gametocyte carriers [START_REF] Busula | Gametocytemia and attractiveness of Plasmodium falciparum-infected Kenyan children to Anopheles gambiae mosquitoes[END_REF]. Fifty Kenyan children confirmed by standard blood smear testing and NAAT to be negative or positive for Plasmodium falciparum gametocytes stayed in a screenhouse (tent) for 3 hours to concentrate their odour. After the subjects left the screenhouse, the number of laboratoryreared uninfected mosquitoes (Anopheles gambiae) attracted to each screenhouse was measured as a study endpoint using a dual-choice olfactometer assay. Two-to three-fold more mosquitoes were attracted to the screenhouse where a gametocyte carrier had stayed, and this was significantly different from the baseline number of approximately 20 mosquitoes attracted per screenhouse.

Moreover, this number fell to baseline after the infected children were successfully treated with antimalarial agents [START_REF] Busula | Gametocytemia and attractiveness of Plasmodium falciparum-infected Kenyan children to Anopheles gambiae mosquitoes[END_REF].

Further studies explored the biological background and quantitative differences in the production of specific skin VOCs in infected versus parasite-free individuals; the aldehydes heptanal, octanal and nonanal were found to be produced in greater amounts by infected individuals [START_REF] Robinson | Plasmodiumassociated changes in human odor attract mosquitoes[END_REF][START_REF] Busula | Mechanisms of Plasmodiumenhanced attraction of mosquito vectors[END_REF]. A recent study based on extensive collections of skin VOCs from human populations with high rates of malaria found broad and consistent effects of malaria infection on skin VOC profiles, as well as significant divergence in the effects of symptomatic and asymptomatic infections [START_REF] Moraes | Volatile biomarkers of symptomatic and asymptomatic malaria infection in humans[END_REF]. Predictive models based on machine-learning algorithms identified asymptomatic infections with 100% sensitivity, even in the case of low-level infections not detectable by microscopy [START_REF] Moraes | Volatile biomarkers of symptomatic and asymptomatic malaria infection in humans[END_REF].

In addition to skin VOCs, breath VOCs patterns in Malawian children infected with malaria were recently studied [START_REF] Schaber | Breathprinting reveals malaria-associated biomarkers and mosquito attractants[END_REF]. Analyses using thermal-desorption-GC-MS (TC-GC-MS) and predictive models reliably distinguished children with P. falciparum malaria versus parasite-free children using a breath profile consisting of six VOC compounds [START_REF] Schaber | Breathprinting reveals malaria-associated biomarkers and mosquito attractants[END_REF].

These findings as well as further studies described in a recent literature review [START_REF] Stanczyk | Can we use human odors to diagnose malaria?[END_REF] suggest that skin and breath volatile biomarkers may have significant potential for the development of a robust, non-invasive screening assay for detecting malaria infections and differentiation of symptomatic and asymptomatic infections.

Use of scent-based instruments in diagnostic microbiology

Studies using sniffing animals performed to date and summarised in this review have demonstrated that at least some microbial infections produce specific scents in those infected.

Consequently, in addition to sniffing animals, these scents can be explored by various standard or portable instruments using spectrometry, metabolomics and other analytical approaches [START_REF] Pleil | Integrating exhaled breath diagnostics by disease-sniffing dogs with instrumental laboratory analysis[END_REF]. In addition to the tuberculosis and malaria studies described above, further examples of proof-ofconcept and pilot studies are summarised below.

The first spectrometry-based "electronic nose" instruments for microbial detection were developed two decades ago [START_REF] Moens | Fast identification of ten clinically important micro-organisms using an electronic nose[END_REF][START_REF] Zhu | Fast detection of volatile organic compounds from bacterial cultures by secondary electrospray ionization-mass spectrometry[END_REF][START_REF] Devaraj | Profiling of headspace volatiles from Escherichia coli cultures using silicone-based sorptive media and thermal desorption GC-MS[END_REF] and were used in some laboratories well before MALDI-TOF became a standard for microbial identification. Secondary electrospray ionisation-mass spectrometry (SESI-MS) and GC-MS have mainly been used [START_REF] Zhu | Fast detection of volatile organic compounds from bacterial cultures by secondary electrospray ionization-mass spectrometry[END_REF][START_REF] Bos | Volatile metabolites of pathogens: a systematic review[END_REF]. These "electronic nose" or "in vitro sniffing tests" detect VOCs and produce pathogen-specific spectral patterns. Several important bacterial pathogens -E. coli, K. pneumoniae, Streptococcus pneumoniae, S. aureus and P. aeruginosa -show specific spectral patterns of VOCs that allow their differentiation in vitro [START_REF] Bos | Volatile metabolites of pathogens: a systematic review[END_REF]. The publicly available mVOC 2.0 database (http://bioinformatics.charite.de/mvoc), based on an extensive literature search for microbial VOCs, is an outstanding and regularly updated data source [START_REF] Lemfack | mVOC 2.0: a database of microbial volatiles[END_REF].

A recent proof-of-principle study showed the ability of TD-GC-MS to "smell" and differentiate cephalexin-and ciprofloxacin-resistant and sensitive UTI-causing bacteria [START_REF] Smart | Sniffing out resistance -rapid identification of urinary tract infection-causing bacteria and their antibiotic susceptibility using volatile metabolite profiles[END_REF]. It detected 578 peaks at unique retention times from 86 chromatograms of 18 bacterial isolates (E. coli, K.

pneumoniae and P. aeruginosa). The study identified nine compounds that differed significantly between cephalexin-sensitive and resistant isolates, and 22 compounds that differed significantly between ciprofloxacin-sensitive and resistant isolates, at p ≤ 0.05 [START_REF] Smart | Sniffing out resistance -rapid identification of urinary tract infection-causing bacteria and their antibiotic susceptibility using volatile metabolite profiles[END_REF].

Using TD-GC-MS, Koo et al. characterised the in vitro volatile metabolite profile of Aspergillus fumigatus and other pathogenic aspergilli [START_REF] Koo | A breath fungal secondary metabolite signature to diagnose invasive aspergillosis[END_REF]. They prospectively collected breath samples from 64 patients with suspected invasive fungal pneumonia and assessed whether they could discriminate patients with proven or probable pulmonary aspergillosis from patients without aspergillosis.

Detection of α-trans-bergamotene, β-trans-bergamotene, a β-vatirenene-like sesquiterpene or transgeranylacetone identified pulmonary aspergillosis patients with 94% sensitivity (95% confidence interval [CI], 81-98) and 93% specificity (95%CI, 79-98) [START_REF] Koo | A breath fungal secondary metabolite signature to diagnose invasive aspergillosis[END_REF], showing that direct detection of exogenous fungal metabolites in breath can be used as non-invasive, pathogen-specific approach to identifying the microbial cause of pneumonia.

A recent systematic review of the potential role of exhaled breath analysis in diagnosing bacterial pneumonia summarising 18 articles reporting on animal and human studies using organic and inorganic molecules in exhaled breath [START_REF] Van Oort | The potential role of exhaled breath analysis in the diagnostic process of pneumonia -a systematic review[END_REF] concluded that, although VOC profiles could potentially improve the diagnostic process of pneumonia and although some VOCs appeared significantly different in patients with infection versus those without infection, it was difficult to match specific VOCs to a particular bacterial species (e.g. whether 1-propanol is related to the presence of E. coli), and no breath test has been shown to have sufficient diagnostic accuracy for pneumonia. However, recent proof-of-concept studies performed as part of the BreathDx consortium [START_REF] Van Oort | BreathDx -molecular analysis of exhaled breath as a diagnostic test for ventilator-associated pneumonia: protocol for a European multicentre observational study[END_REF] showed that exhaled breath metabolomics measured by TD-GC-MS reveals a pathogen-specific response in a rat pneumonia model for S. pneumoniae and P. aeruginosa [START_REF] Van Oort | Exhaled breath metabolomics reveals a pathogen-specific response in a rat pneumonia model for two human pathogenic bacteria: a proof-of-concept study[END_REF], and in an artificial sputum model from mono-and co-cultures of Enterobacter cloacae and P. aeruginosa [START_REF] Lawal | TD/GC-MS analysis of volatile markers emitted from mono-and co-cultures of Enterobacter cloacae and Pseudomonas aeruginosa in artificial sputum[END_REF].

The VOC-based Aetholab "electronic nose" (The eNose Company, Zutphen, Netherlands), developed to discriminate between infected versus non-infected wounds, showed a sensitivity of 91% (95%CI 76-98) and specificity of 71% (95%CI 55-84) when clinical judgment was used as a reference standard, and a sensitivity of 81% (95%CI 64-91) and specificity of 63% (95%CI 46-77) using culture as a reference standard [START_REF] Haalboom | Differentiation between infected and non-infected wounds using an electronic nose[END_REF].

Conclusions

This review summarised the most explored examples of using sniffing animals to diagnose infectious diseases and discussed the potential use of scent-based instruments in diagnostic microbiology. The results of several studies that used sniffing animals and real life experience in both diagnostic and surveillance settings clearly showed that animal sniffing is valid and useful tool which can provide solution also for immediate future, especially as a screening tool in settings which found this approach acceptable and affordable. However, the ultimate goal is to understand the biological background of this animal ability and identify and characterise the pathogen-specific VOCs in human stool, urine, sputum, skin or exhaled breath that animals are sniffing and recognising. This fundamental VOC knowledge, improvement of sampling methods to collect odours from different parts of the human body and development of scent-based point-of-care instruments could hopefully allow implementation of a range of innovative, rapid and affordable scent-based tests for major human pathogens in more distant future. If so, such point-of-care instruments will likely replace sniffing animals in areas where this approach is in current use and become additional diagnostic tool in settings which have never used scent-based tests for diagnostic and surveillance purpose before.

  Tanzania, where the program started in 2007, APOPO is currently running large-scale tuberculosis research programs in Mozambique and Ethiopia. From 2007 to 2018, more than 550,000 sputum samples were screened by APOPO rats, which detected over 14,000 tuberculosis patients in addition

to standard methods. For detecting tuberculosis, trained rats were introduced into a cage where sputum samples were aligned (training is detailed in

[START_REF] Weetjens | African pouched rats for the detection of pulmonary tuberculosis in sputum samples[END_REF]

; photos and videos available at www.apopo.org). In contrast to dogs used in detecting C. difficile in stool or E. coli in urine (see above), which can smell only up to 10 samples per day due to fatigue, rats can to sniff up to 100 samples in 20 minutes. They were rewarded with banana if they paused at least 5 s to sniff sputum containing Mycobacterium tuberculosis. Real-life overall performances were as good as smear

Acknowledgments: This work was presented in part at the 29th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) held in Amsterdam, the Netherlands, from April 13th to 17th, 2019 as the oral presentation "Use of sniffing animals in diagnostic microbiology" (presenter Emmanuelle Cambau) as part of ECCMID's 2-hour session "@mobilemicrobiologylaboratory". Contribution: M.P. created the concept of the review. Both authors (E.C. and M.P.) participated in collecting, analysing and interpreting the data and writing the manuscript. Both authors approved the final version of the manuscript submitted to CMI.

Transparency declaration

Conflict of interest: The authors have no conflict of interest to disclose.

Funding: No external funding was received for this work.


Dr Alice Corbel
email: a.corbel@chru-nancy.fr

Marc Ladrière

Nicolas Le Berre

Laurent Durin

Hélène Rousseau

Luc Frimat

Nathalie Thilly

Céline Pulcini

Microbiological epidemiology of preservation fluids in transplanted kidney: a nationwide retrospective observational study

Keywords: kidney transplantation, preservation fluid, microbiological analysis, organ procurement procedure, infectious risk UC, urine culture, M, Maastricht (M1, M2, M3), GNF, graft non-function

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Kidney transplantation is considered as the choice treatment for end-stage kidney disease patients [START_REF] Wolfe | Comparison of Mortality in All Patients on Dialysis, Patients on Dialysis Awaiting Transplantation, and Recipients of a First Cadaveric Transplant[END_REF], and requires immunosuppressive therapy [START_REF] Halloran | Immunosuppressive drugs for kidney transplantation[END_REF]. As a result, kidney transplant recipients (KTR) are prone to infectious complications, associated with significant morbidity [START_REF] Bodro | Impact of urinary tract infections on short-term kidney graft outcome[END_REF][START_REF] Graversen | Risk and outcome of pyelonephritis among renal transplant recipients[END_REF][START_REF] Kroth | Acute Graft Pyelonephritis Occurring up to 30 Days After Kidney Transplantation: Epidemiology, Risk Factors, and Survival[END_REF][START_REF] Kinnunen | Secular Trends in Infection-Related Mortality after Kidney Transplantation[END_REF]. Three at-risk periods after transplantation have been described [START_REF] Fishman | Infection in Organ Transplantation[END_REF]: the early post-transplant period in which predominate healthcare-associated infections and donortransmitted infections, the 6-month post-transplant period at high risk for reactivation of latent infections and opportunistic infections, and the late post-transplant period (beyond the sixth month) with an increased risk of community acquired infections [START_REF] Fishman | Infection in Organ Transplantation[END_REF]. Today development of multidrug-resistant bacteria (MDR-B) is a major concern [START_REF] Fishman | Infection in Organ Transplantation[END_REF][START_REF] Holmes | Understanding the mechanisms and drivers of antimicrobial resistance[END_REF][START_REF] Korth | Increased resistance of gram-negative urinary pathogens after kidney transplantation[END_REF][START_REF] Rodrigues Dos Santos | Urinary Tract Infections and Surgical Site Infections due to Carbapenem-Resistant Enterobacteriaceae in Renal Transplant[END_REF][START_REF] Cocco | Incidence of urinary tract infections caused by germs resistant to antibiotics commonly used after renal transplantation[END_REF].

Infectious disease screening of donor is systematically performed [START_REF] Fishman | Donor-derived infection--the challenge for transplant safety[END_REF][START_REF] Fischer | AST Infectious Diseases Community of Practice. Screening of donor and recipient in solid organ transplantation[END_REF][START_REF] Jr | Selecting suitable solid organ transplant donors: Reducing the risk of donor-transmitted infections[END_REF]. The kidney transplant preservation fluid (PF) [START_REF] Latchana | Preservation solutions used during abdominal transplantation: Current status and outcomes[END_REF] can also be a source of healthcare-associated infections.

According to the French Agency of Biomedicine (ABM) 2008 guidelines, kidney transplant PF samples are systematically collected for microbiological (bacterial and mycological) analysis through transplant procedure [START_REF] Jr | Selecting suitable solid organ transplant donors: Reducing the risk of donor-transmitted infections[END_REF] [START_REF] De | Prévention de la transmission de bactéries et d'agents fongiques aux receveurs d'organes[END_REF]. In case of positive fungal PF cultures, the risk of mycotic pseudoaneurysm is well-known and its therapeutic management, as well as the patient's surveillance are well-documented according to an international consensus [START_REF] De | Prévention de la transmission de bactéries et d'agents fongiques aux receveurs d'organes[END_REF][START_REF] Matignon | Outcome of renal transplantation in eight patients with Candida sp. contamination of preservation fluid[END_REF]. However, in case of positive bacteriological PF culture, the infectious risk for KTR is unknown and management of such cultures is not consensual [START_REF] Oriol | Clinical impact of culturepositive preservation fluid on solid organ transplantation: A systematic review and metaanalysis[END_REF]. Previous studies suggest that a significant number of KTR receives antibiotic treatment to prevent infections, but the benefit of such prophylactic therapy has not been demonstrated [START_REF] Jr | Selecting suitable solid organ transplant donors: Reducing the risk of donor-transmitted infections[END_REF][START_REF] Ranghino | Clinical impact of bacterial contamination of perfusion fluid in kidney transplantation[END_REF][START_REF] Yahav | Clinical relevance of preservation-fluid contamination in solidorgan transplantation: a call for mounting the evidence[END_REF]. Microbiological epidemiology of PF cultures is also not well-known. Few single-center, retrospective studies carried out in France, Italy, UK and Canada between 1999 and 2012 reported that the prevalence of positive PF cultures varies widely, ranging from 17.8% to 58.0% [START_REF] Oriol | Clinical impact of culturepositive preservation fluid on solid organ transplantation: A systematic review and metaanalysis[END_REF][START_REF] Ranghino | Clinical impact of bacterial contamination of perfusion fluid in kidney transplantation[END_REF][START_REF] Yansouni | Positive cultures of organ preservation fluid predict postoperative infections in solid organ transplantation recipients[END_REF][START_REF] Audet | Incidence and clinical significance of bacterial and fungal contamination of the preservation solution in liver transplantation[END_REF][START_REF] Sauget | Bacterial contamination of organ graft preservation solution and infection after transplantation[END_REF][START_REF] Bertrand | Clinical and microbial impact of screening kidney allograft preservative solution for bacterial contamination with high-sensitivity methods[END_REF][START_REF] Wakelin | The incidence and importance of bacterial contaminants of cadaveric renal perfusion fluid[END_REF]. Although coagulasenegative staphylococci are the most common microorganism isolated in PF cultures, Enterobacteriaceae, which are more virulent and more frequently multidrug-resistant, tend to have a growing role in recent studies [START_REF] Oriol | Clinical impact of culturepositive preservation fluid on solid organ transplantation: A systematic review and metaanalysis[END_REF][START_REF] Ranghino | Clinical impact of bacterial contamination of perfusion fluid in kidney transplantation[END_REF][START_REF] Sauget | Bacterial contamination of organ graft preservation solution and infection after transplantation[END_REF][START_REF] Bertrand | Clinical and microbial impact of screening kidney allograft preservative solution for bacterial contamination with high-sensitivity methods[END_REF]. This nationwide observational study was aimed to determine the epidemiology of bacterial and fungal agents in kidney transplant PF cultures and identify risk factors associated with positive PF cultures.

METHODS

Data source

Exhaustive clinical information on all kidney transplant procedures carried out in France are routinely and prospectively collected in national databases which are maintained by the ABM.

In this study, data were retrieved from these national databases of kidney transplantations, and retrospectively analyzed. All kidney transplant procedures performed in France between October 1, 2015 and December 31, 2016 were included. We double-checked that we comply with the STROBE reporting guidelines for observational studies [START_REF] Von Elm | The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies[END_REF].

Data collection

Kidney transplantations are performed in 42 university hospital centers in France including 28 adult centers, 9 pediatric centers, and 5 both adult and pediatric centers [START_REF] De | Rapport médical et scientifique du prélèvement et de la greffe en France[END_REF]. Furthermore, 157 healthcare institutions across France are authorized to perform kidney procurement procedures [START_REF]Agence de la Biomédecine -Direction juridique. Liste des établissements de santé autorisés par les Agences régionales de santé[END_REF].

Allocation and distribution of organ are coordinated by the ABM. All data prospectively collected by the ABM are recorded in real time; clinical data are recorded by organ procurement and transplant teams, and microbiological data by the ABM. Since 2008, ABM recommends withdrawing twice 25 mL of PF at the opening of graft container (25mL for mycological analysis and 25 mL for bacteriological analysis. After direct exam, PF is cultured on solid media, aerobic blood culture bottle, anaerobic blood culture bottle and mycological blood culture bottle. [START_REF] De | Prévention de la transmission de bactéries et d'agents fongiques aux receveurs d'organes[END_REF]. Each microorganism was identified and antibiograms are performed following standards of the French Microbiology Society(29). Data reporting is mandatory, guaranteeing the exhaustive nature of information. Data anonymization, consistency and completion are controlled by the data manager of the ABM.

The following data are routinely collected: (i) donor: clinical, biological and imaging parameters; death cause and hospitalization process in intensive care when appropriate; (ii) microbiological records of kidney transplant PF cultures; and (iii) recipient: pre-transplant information, mandatory for the file creation process and registration on the national kidney transplant waiting list, as well as post-transplant information on serious events occurring within the first 30 days after transplantation, such as recipient death and graft non-function (GNF).

Variables selection

All variables of interest and statistical analysis plan were a priori selected by the interdisciplinary scientific committee of the study (kidney transplant specialists, infectious disease physician, epidemiologist and medical expert of the ABM) based on literature data (see supplementary data) [START_REF] Matignon | Outcome of renal transplantation in eight patients with Candida sp. contamination of preservation fluid[END_REF][START_REF] Ranghino | Clinical impact of bacterial contamination of perfusion fluid in kidney transplantation[END_REF][START_REF] Yansouni | Positive cultures of organ preservation fluid predict postoperative infections in solid organ transplantation recipients[END_REF][START_REF] Sauget | Bacterial contamination of organ graft preservation solution and infection after transplantation[END_REF][START_REF] Wakelin | The incidence and importance of bacterial contaminants of cadaveric renal perfusion fluid[END_REF][START_REF] Alangaden | Infectious complications after kidney transplantation: current epidemiology and associated risk factors[END_REF][START_REF] Barba | Recipient and donor risk factors for surgical complications following kidney transplantation[END_REF].

Microbiological analyses of PF cultures included positivity of bacterial and fungal cultures, and if appropriate, isolated agent(s) (only species because susceptibility testing results were unavailable). These analyses were performed in kidney graft PF and contralateral kidney PF when it was also transplanted. Microbiological species were grouped as follows: coagulasenegative Staphylococci, Staphylococcus aureus, Streptococci other than Streptococcus pneumoniae, Enterococci, Enterobacteriaceae, non-fermenting Gram-negative bacilli, Candida, commensal bacteria of skin, naso-laryngeal sphere and gastrointestinal tract flora (such as Actinomyces, Propionibacterium, Clostridium, Haemophilus, Corynebacteriae, Moraxella, Lactobacillus, Bacteroides, Bacillus, Neisseria, Streptococcus pneumonia) and others.

Statistical analyses

Analysis of characteristics for donors, recipients, procurement and transplantation was first descriptive. Data are expressed as frequency and percentages for categorical variables and as mean and standard deviation for continuous variables.

Microbiological analysis (frequency, %) were performed for all positive kidney transplant PF cultures, and across subpopulations (living-donor graft, deceased donor graft, en bloc, and positive contralateral kidney PF cultures). To explore process leading to the positivity of PF cultures, microbiological concordance was calculated according to the positivity or negativity of cultures: (i) between kidney transplant and contralateral kidney PF cultures, (ii) between kidney transplant PF cultures and donor's samples (blood cultures and UC). Kidney transplant PF cultures were defined as concordant with contralateral kidney PF cultures or with samples from donor if the same microbiological species were found. Complete concordance is defined as exactly the same microbiological species found in both donor and PF culture, partial concordance is defined as one or more identical microbiological species found among multiple other species in both PF and donor cultures. Cultures for which the microbiological species were not identified were excluded from the present analysis.

If contralateral kidney wasn't transplanted, no PF culture was performed; in this case, culture was considered negative, representing less than 10% of transplantation according to ABM data [START_REF] De | Rapport médical et scientifique du prélèvement et de la greffe en France[END_REF].

As risk factors associated with positive kidney transplant PF cultures are unknown, the scientific committee selected them based on pathophysiology and their own experience [START_REF] Oriol | Clinical impact of culturepositive preservation fluid on solid organ transplantation: A systematic review and metaanalysis[END_REF][START_REF] Barba | Recipient and donor risk factors for surgical complications following kidney transplantation[END_REF]. The following variables were selected to assess their association with positive kidney transplant PF cultures (bacteriological, and/or mycological): year of transplantation (2015 or 2016), transplant laterality (right, left, en bloc), donor's age, procurement activity of the department (<25 versus ≥25 kidney procurements/year(28)), multiorgan or kidney alone procurement surgery, length of donor stay in intensive care (≤2 versus >2 days -mean), cold ischemia time (≤14 versus >14 hours, mean=16 hours), use of perfusion pump, antibiotic therapy in donor, positive blood culture and/or positive UC in donor, and intestinal perforation during procurement.

Bivariate and multivariable logistic regression models were used to identify factors associated with positive PF cultures. Two distinct analyses were conducted according to clinical status of donor (living versus deceased) because of disparity in donor selection criteria, surgical procedures and graft distribution. Variables found to be significant with a p-value < 0.10 in the bivariate logistic regression were entered into the multivariable logistic regression model for which a p value <0.05 (bilateral) was considered statistically significant. As missing data were observed for only four independent variables and their percentage was always < 5%, missing values were not imputed. So, the analyses were performed on PF cultures for which there was no missing data on these variables. Odds ratios (crude & adjusted) with 95% confidence intervals (95% CI) and p-values were calculated. No collinearity was observed between variables and the Hosmer-Lemeshow goodness of fit p-value was at 0.32. All statistical analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC).

RESULTS

Characteristics of kidney recipients and donors

Over the 15-month study period 4,487 kidney transplant procedures were performed in France, 725 from living-donors (16.2% 725/4,487). Characteristics of kidney transplant recipients, organ donors, procurement and transplantation are detailed in Table 1. Most donors (3,449/4,487-76.9%) had neither bacteremia (negative blood cultures), nor bacteriuria (negative UC) at the procurement time. No living donor had bacteremia or bacteriuria at procurement time. Among the 64.9% (2,423/3,736) of donors who received an antibiotic therapy before procurement, 76.6% (1,856/2,423) of them received monotherapy, the most prescribed medications were beta-lactams, most commonly amoxicillin-clavulanic acid (68%-1,641/2,423, data not shown).

Microbiology results of positive PF cultures

Throughout the study period, 920 (20.5% -920/4,487) KTR received a kidney transplant with positive PF cultures (bacteriological and/or mycological) (Table 2). Among them, 907 (98.6% 907/920) received a deceased-donor kidney transplant. Polymicrobial cultures were found in the majority of positive PF cultures (59.9% -485/810). Coagulase-negative staphylococci (65.8% -533/810) and Enterobacteriaceae (28.0% -227/810) were the most common agents isolated in these positive cultures; in 110 cases, the cultures were reported as positive without details on the identified micro-organism. Living-donor kidney transplant PF cultures were rarely positive (1.8% -13/725) and always with coagulase-negative staphylococci, alone or with other agents.

Microbiological concordance between PF cultures of kidneys from the same donor

When kidney transplant was procured from deceased-donor and contralateral kidney was also transplanted, we could compare with microbiological data of contralateral kidney transplant PF cultures (Table 3). Microbiological concordance between kidney transplant PF and contralateral kidney transplant PF cultures were observed in 72.6% (2,674/3,681) of cases. A concordance between negative PF cultures was observed in 63.7% (2,345/3,681) of cases. In 8.9% (329/3681) of cases positive kidney transplant PF cultures was completely concordant with positive contralateral kidney transplant PF cultures, the great majority of samples were culture-positive with coagulase-negative staphylococci and Enterobacteriaceae. Among them, 11.8% (39/329) cultures were also in concordance with donor samples, largely with UC (33/39 -84.6%). In these concordance cases, microorganisms were mainly Enterobacteriaceae (37/39-94.8%).

Microbiological concordance of kidney transplant PF cultures with donor samples

Comparison of microbiological analyses from donor samples and kidney transplant PF cultures are detailed in Table 4. Concordance between positive kidney transplant PF and positive donor sample cultures was complete in 0.5% (23/4,327) of cases and partial in 0.6% (24/4,327) of cases. Enterobacteriaceae, especially Escherichia coli, were the most common organisms found in both kidney transplant PF cultures and donor sample cultures (complete concordance: 78.3%, partial concordance: 91.7%). When the same pathogens were isolated in kidney transplant PF and donor sample cultures, it was mostly bacteria isolated in donor UC (complete concordance: 82.6%, partial concordance: 83.3%).

Risk factors associated with positive deceased-donor kidney transplant PF cultures (Table 5)

Only

DISCUSSION

Our study shows that 20.5% of KTR are exposed to microorganism via kidney transplant PF.

Coagulase-negative staphylococci and Enterobacteriaceae were the most common organisms isolated in PF cultures. Our findings are consistent with those previously published in singlecenter studies [START_REF] Ranghino | Clinical impact of bacterial contamination of perfusion fluid in kidney transplantation[END_REF][START_REF] Yansouni | Positive cultures of organ preservation fluid predict postoperative infections in solid organ transplantation recipients[END_REF][START_REF] Sauget | Bacterial contamination of organ graft preservation solution and infection after transplantation[END_REF][START_REF] Bertrand | Clinical and microbial impact of screening kidney allograft preservative solution for bacterial contamination with high-sensitivity methods[END_REF][START_REF] Wakelin | The incidence and importance of bacterial contaminants of cadaveric renal perfusion fluid[END_REF]. However, the benefit of a systematic use of prophylactic antibiotic therapy focusing on isolated bacteria is not demonstrated, although this practice is a well-known risk factor for MDR-bacteria emergence [START_REF] Holmes | Understanding the mechanisms and drivers of antimicrobial resistance[END_REF][START_REF] Korth | Increased resistance of gram-negative urinary pathogens after kidney transplantation[END_REF][START_REF] Rodrigues Dos Santos | Urinary Tract Infections and Surgical Site Infections due to Carbapenem-Resistant Enterobacteriaceae in Renal Transplant[END_REF][START_REF] Origüen | Progressive increase of resistance in Enterobacteriaceae urinary isolates from kidney transplant recipients over the past decade: narrowing of the therapeutic options[END_REF][START_REF] Delmas-Frenette | Epidemiology and outcome of antimicrobial resistance to gram-negative pathogens in bacteriuric kidney transplant recipients[END_REF].

PF contamination during procurement seemed to be the major mechanism. Indeed, majority of PF cultures were positive with coagulase-negative staphylococci, illustrating a potential contamination by donor skin flora. Agents present in the gut microbiota, such as Enterobacteriaceae, Enterococci, some Streptococci, and Candida were also frequent, and their presence in kidney transplant PF cultures was likely due to the contamination of graft by intra-abdominal flora at procurement time. Positivity of PF cultures due to same bacteria as found in donor culture was a rare event (1% of cases), mainly involving Enterobacteriaceae isolated from donor urines.

The results of the multivariable analysis support these hypotheses. The presence of intestinal perforation during the procurement procedure was a well-known risk factor for PF contamination with Candida [START_REF] De | Prévention de la transmission de bactéries et d'agents fongiques aux receveurs d'organes[END_REF][START_REF] Matignon | Outcome of renal transplantation in eight patients with Candida sp. contamination of preservation fluid[END_REF]; our study shows that all infectious agents face the same risk. Multiorgan procurement is a long and complex surgical procedure, requiring several surgical teams around the donor, increasing contamination risk and sterile mistakes; en bloc transplant procedure is also a potentially risky procedure (surgical kidney procurement procedure can be different from separated kidney procurement and is not a common surgical procedure). In addition, rare complete microbiological concordance (< 9% of cases) of positive PF cultures of both kidneys from one donor and the very low prevalence of positive PF cultures of living-donor kidney transplant (1.8% versus 24.1% in deceased-donor kidney transplants) support that contamination during organ procurement and/or cantonment is the major factor explaining positive PF cultures. It seems important to reinforce and standardize procedures for limiting PF contamination during organ procurement.

In our study, using a perfusion pump for graft preservation was protective factor of positive PF culture. Several hypotheses may explain this finding: procurement teams might pay more attention to aseptia [START_REF] Tixier | Recommandations techniques pour le prélèvement des organes et des tissus sur donneurs en état de mort encéphalique[END_REF] and maintenance of homogeneous temperature (4°C) over the length of preservation thanks to machine perfusion might limit the potential proliferation of microorganisms.

Another factor associated with a lesser risk for positive PF cultures was donors having received antibiotics. There are no guidelines recommending a systematic perioperative antibiotic prophylaxis as a standard of care in organ donors, except for thoracic organ procurements [START_REF] Tixier | Recommandations techniques pour le prélèvement des organes et des tissus sur donneurs en état de mort encéphalique[END_REF][START_REF] Boulard | Monomicrobial culture of contralateral kidney PF: 214/424 (50.5%) Streptococcci: 38/424 (9.0%) Coagulase-negative staphylococci: 289/424 (68.2%) Staphylococcus aureus: 37/424 (8.7%) Enterococci: 14/424 (3.3%) Enterobacteriae: 80/424 (18.9%) Non-fermenting gram-negative bacilli: 4/424 (0.9%) Candida: 31/424 (7.3%) Other commensal bacteria: 78/424 (18.4%) Others: 72/424 (17%) Negative blood culture and UC: 352/485 (72.6%) Positive blood culture: 22/485 (4.5%) Positive UC: 103/485 (21.2%) Positive blood culture and UC: 8/485[END_REF]. Regarding kidney procurements, we believe it is essential: (i) to limit perioperative contamination, as discussed above; and (ii) to assess impact of PF contamination on the onset of infections in KTR during the early period post-transplant, and on emergence of MDR-bacteria. Systematic antibiotic prophylaxis in donor has a potential interest only if positive PF culture is a confirmed risk factor of infection in KTR, if these infections are frequent or very severe, and overall if benefit-risk balance for antibiotic therapy is clearly demonstrated.

In case of culture positive for Candida or of intestinal perforation, a systematic fungal treatment is recommended in KTR because of the risk for mycotic pseudoaneurysm [START_REF] De | Prévention de la transmission de bactéries et d'agents fongiques aux receveurs d'organes[END_REF][START_REF] Matignon | Outcome of renal transplantation in eight patients with Candida sp. contamination of preservation fluid[END_REF].

There is no evidence of complications related to PF cultures positive for bacteria. In contrast, the risk for MDR-bacteria emergence in case of prophylactic antibiotic therapy is well-proven for the latter situation [START_REF] Fishman | Infection in Organ Transplantation[END_REF][START_REF] Holmes | Understanding the mechanisms and drivers of antimicrobial resistance[END_REF][START_REF] Korth | Increased resistance of gram-negative urinary pathogens after kidney transplantation[END_REF][START_REF] Cocco | Incidence of urinary tract infections caused by germs resistant to antibiotics commonly used after renal transplantation[END_REF][START_REF] Origüen | Progressive increase of resistance in Enterobacteriaceae urinary isolates from kidney transplant recipients over the past decade: narrowing of the therapeutic options[END_REF]. These findings lead us to question the need to systematically share results of bacteriological (not mycological) analysis of PF cultures with transplant teams, because it often leads to an antibiotic prescription (Le Berre et al.

Unpublished).

This study brings original findings and included a large number of observations (national exhaustive data over a 1-year period) from data available for transplant teams in routine clinical practice. Moreover, procurements and microbiological PF cultures were performed using national standardized protocols(29).

However, our work has some limitations. The lack of data related to antibiotic susceptibility profiles limits the validity of our findings on microbiological concordance. Our study also could not assess the impact of positive PF cultures or tailored antibiotic prophylaxis, due to the lack of detailed data on the management and outcomes of KTR.

In conclusion, this large observational nationwide study shows that about a quarter of deceased-donor kidney transplant recipients are exposed to microorganisms via kidney transplant PF. The presence of infectious agents in PF seems to be related to contamination occurring during procurement procedures; there is an urgent need to reinforce and standardize procedures in order to limit such contamination. Further studies are needed to assess the impact of bacterial presence in PF, in order to propose a standardized and evidencebased management in this situation and to limit the risk of MDR-bacteria infections in KTR.

  factors associated with positive deceased-donor kidney transplant PF cultures could be explored. Factors associated with positive living-donor kidney transplant PF cultures could not be explored because of low prevalence of positive PF cultures in this subpopulation (1.8% -13/725). Regarding deceased-donor kidney transplant procedures, 907/3,762 (24.1%) were performed with positive PF cultures kidney transplant. The use of a perfusion pump and donor antibiotic therapy reduced the risk of positive PF cultures by 2.5 (OR=0.4 [0.3-0.5] p<0.0001) and by 1.7 (OR=0.6 [0.5-0.7] p<0.0001) respectively, whereas intestinal perforation increased the risk of positive PF cultures by 4.7 (OR=4.7 [2.2-9.8] p<0.0001). Multiorgan procurement and en bloc transplant procedures also significantly increased the risk of positive kidney transplant PF cultures (Respectively OR=1.4 [1.1-1.7] p=0.0033 and OR=2.5 [1.3-4.9] p=0.028).

TABLE 1

 1 Characteristics of organ transplant recipients, donors, procurement and transplantation

	All	Living donors	Deceased donors
	(N=4487)	(N=725)	(N=3762)

Table 2

 2 Microbiological analysis of the kidney transplant preservation fluid (PF)

	Type of	Positive	Microorganisms isolated in culture-positive PF	Data in donor	Antibiotic therapy in donor	Clinical status of recipient
	sample	kidney						at Day 30
		transplant PF cultures		Positive PF culture group	Negative PF culture group	Positive PF culture	Negative PF culture	Positive PF culture	Negative PF culture
	Positive kidney transplant PF cultures: all grafts	920/4487 (20.5%)	Monomicrobial culture : 407/810 (50.2 %) Streptococcci: 96/810 (11.9%) Coagulase-negative staphylococci: 533/810 (65.8%) Staphylococcus aureus: 98/810 (11.0%) Enterococci: 62/810 (7.7%) Enterobacteriaceae: 227/810 (28.0%) Others: 149/810 (18.3%) Other commensal bacteria: 150/810 (18.5%) Candida: 78/810 (9.6%) Non-fermenting gram-negative bacilli: 43/810 (5.3%)	Negative blood culture and UC: 653/920 (71.0%) 23/920 (2.5%) culture and UC: Positive blood 199/920 (21.6%) Positive blood culture: 45/920 (4.9%) Positive UC:	Negative blood culture and UC 2796/3567 (78.4%) 79/3567 (2.2%) culture and UC: Positive blood 473/3567 (13.3%) Positive blood culture: 219/3567 (6.1%) Positive UC:	Yes: 410/820 (50%)	Yes: 1448/3567 (46.6%)	Death: 6/920 (0.7%) GNF: 32/920 (3.5%)	Death: 27/3082 (0.9%) GNF: 96/3082 (3.1%)
	Positive	13/725	Monomicrobial culture: 8/10 (80%)	Negative blood	Negative blood	Yes: 0/13	Yes: 0/712	Deaths:	Deaths:
	kidney transplant PF cultures: living-donor grafts	(1.8%)	Streptococci: 0/10 (0%) Coagulase-negative staphylococci: 10/10 (100%) Staphylococcus aureus: 0/10 (0%) Enterococci: 0/10 (0%) Enterobacteriae: 0/10 (0%)	culture and UC: 10/10 (100%)	culture and UC: 712/712 (100%)	(0%)	(0%)	0/13 (0%) GNF: 0/13 (0%)	1/712 (0.1%) GNF: 20/ 712 (2.8%)
			Non-fermenting gram-negative bacilli: 0/10 (0%)
			Candida: 1/10 (10%)
			Other commensal bacteria: 0/10 (0%)
			Others: 1/10 (10%)
	Positive	16/41	Monomicrobial culture: 5/14 (35.7%)	Negative blood	Negative blood	Yes: 3/16	Yes: 14/25	Deaths:	Deaths: 1/25
	kidney transplant PF cultures: en bloc	(39.0%)	Streptococci: 0/14 (0%) Coagulase-negative staphylococci: 4/14 (28.6%) Staphylococcus aureus: 0/14 (0%) Enterococci: 4/14 (28.6%) Enterobacteriae: 10/14 (71.4%)	culture and UC: 8/16 (50.0%) Positive blood culture: 0/16 (0%) Positive UC: 7/16	culture and UC: 16/25 (64.0%) Positive blood culture: 2/25 (8%) Positive UC: 7/25	(18.8%)	(56%)	0/16 (0%) GNF: 0/16 (0%)	(4%) GNF: 0/25 (0%)
			Non-fermenting gram-negative bacilli: 0/14 (0%)	(43.8%)	(28%)
			Candida: 0/14 (0%)	Positive blood	Positive blood
			Other commensal bacteria: 1/14 (7.1%)	culture and UC:	culture and UC: 0/25
			Others: 1/14 (7.1%)	1/16(6.3%)	(0%)

Table 3

 3 Microbiological concordance between kidney transplant and contralateral kidney preservation fluid (PF) cultures (deceased-donor kidney grafts excluding en bloc) in 3681 kidney transplant recipients

	Clinical status	n/3681 (%)	Microorganisms isolated in positive PF cultures	Microbiological analysis in donor	Antibiotic therapy in donor	Clinical status of recipient at Day 30
	Positive kidney	329/3681	Monomicrobial culture: 79/329 (24.0%)	Negative blood culture and UC:	Yes: 129/293	Deaths: 2/329
	transplant PF cultures: complete PF cultures contralateral kidney concordance with	(8.9%)	Streptococcci: 55/329 (16.7%) Enterobacteriae: 131/329 (39.8%) Enterococci: 40/329 (12.2%) Staphylococcus aureus: 52/329 (15.8%) Coagulase-negative staphylococci: 206/329 (62.6%)	220/329 (66.9%) 12/329 (3.6%) Positive blood culture and UC: Positive UC: 79/329 (24.0%) Positive blood culture: 18/329 (5.5%)	(44.0%)	(0.6%) GNF: 9/329 (2.7%)
			Non-fermenting gram-negative bacilli: 12/329 (3.6%)
			Candida: 45/329 (13.7%)
			Other commensal bacteria: 67/329 (20.4%)
			Others: 120/329 (36.5%)
	Negative kidney	2345/3681		Negative blood culture and UC:	Yes: 1199/1951	Deaths: 25/2345
	transplant PF	(63.7%)		1716/2345 (73.2%)	(61.5%)	(1.5%)
	cultures: complete concordance with or non-performed) PF (negative culture contralateral kidney culture of			Positive blood culture: 195/2345 71/2345 (0.3%) Positive blood culture and UC: Positive UC: 363/2345 (15.5%) (8.3%)		GNF: 76/2345 (3.2%)
	Positive kidney	522/3681	Monomicrobial culture of kidney transplant PF:	Negative blood culture and UC:	Yes: 255/459	Deaths: 5/522
	transplant PF	(14.2%)	233/457 (51.0%)	382/522 (73.2%)	(55.6%)	(1.0%)
	cultures: discordance PF cultures contralateral kidney with negative		Enterococci: 18/457 (3.9%) Staphylococcus aureus: 37/457 (8.1%) Coagulase-negative staphylococci: 313/457 (65.8%) Streptococcci: 41/457 (9.0%)	10/522 (1.9%) Positive blood culture and UC: Positive UC: 105/522 (20.1%) Positive blood culture: 25/522 (4.8%)		GNF: 22/522 (4.2%)
			Enterobacteriae: 86/457 (18.8%)
			Non-fermenting gram-negative bacilli: 4/457 (0.9%)
			Candida: 32/457 (7.0%)
			Other commensal bacteria: 82/457 (17.9%)
			Others: 76/457 (16.7%)

FUNDING

This work was conducted as part of our routine work and was not funded.

ACKNOWLEDGMENTS

We thank all the ABM and collaborators for providing data. We thank Brigitte Bourdillat for editing the English translation.

COLLABORATORS

ALAMARTINE Eric (Saint Etienne, France), AMROUCHE Lucile (Paris, France), ANGLICHEAU Dany (Paris, France), ATTIAS Philippe (Créteil, France), BACCHETTA Justine (Lyon, France), BADET Lionel (Lyon, France), BARROU Benoit (Paris, France), BAUD HUIN Marc (Nantes, France), BAUDOUIN Véronique (Paris, France), BENBOUZID Sabrina (Paris, France), BENSADOUN Henri (Bordeaux, France), BERGIS Alexandre (Rouen, France), BERNHARD Jean-Christophe (Bordeaux, France), BERTRAND Dominique (Rouen, France), BESSEDE Thomas (Le Kremlin-Bicêtre, France), BILAND Guillaume (Saint Denis, La Réunion, France), BLANCHET Pascal (Pointe à Pitre Abymes, France), BLANOT Stéphane (Paris, France), BOISSIER Romain (Marseille, France), BOTTEREL Françoise (Créteil, France), BOUALI Ourdia (Toulouse, France),

Ethical statement

The study protocol was submitted to the scientific committee of ABM who gave its approval for data transmission on 17/10/2017. An authorization from the National Commission of Informatics and Liberty (CNIL) was not required as French ABM performed data anonymization.

AUTHORS CONTRIBUTION

AC, NLB, LF, NT and CP initiated the study and wrote the first draft of the protocol; all remaining authors reviewed them. HR analyzed the data under the supervision of NT. AC and CP wrote the first draft of the manuscript which was reviewed by all authors.

TRANSPARENCY DECLARATION 1

All authors: no conflict of interest to declare. 2

Table 4

Microbiological concordance between kidney transplant preservation fluid (PF) cultures and samples from all donors (N=4327) a : Only bivariate significant variables (p<0.10) were entered in the multivariate model (n=3499).

Hosmer -Lemeshow goodness of fit p-value = 0.32.

Abbreviations: UC, urine culture; CI, confidence interval; GNF, graft non-function; PF, preservation fluid.