Colonisation des solutions de conservation en transplantation rénale : influence sur les pyélonéphrites aiguës secondaires du greffon

25 mai 2018

Auteurs : F. Encatassamy, A.-S. Valentin, J. Capsec, M. Buchler, F. Bruyère
Référence : Prog Urol, 2018, 6, 28, 322-328




 




Introduction


The prevalence of infection following transplantation may be explained by numerous risk factors, for example immunosuppressive therapies. During the last 30 years, hospitalization due to infection in the 2 years following transplantation, has exceeded that of hospitalization due to acute rejection [1]. This is exemplified in renal transplantation, in which nearly half of the number of hospitalizations are due to infections [2, 3], the majority of which are urinary tract infections (UTI) [4].


Fungal colonization of the conservative liquid has been observed in solid organ transplantation. Furthermore, the frequency of donor-derived candidiasis is estimated to be 1/1000 in renal transplantation and may be caused by a colonized preservative solution (PS) [4]. Several cases have been described in mycology, but the issue of bacterial colonization in renal transplantation has poorly been discussed.


The present study aimed to determine the impact of the bacterial colonization of preservative solution (PS) samples on the development of a graft pyelonephritis in renal transplantation.


Material and methods


All patients who underwent renal transplantation in our center between January 2010 and December 2013 were eligible for inclusion in this study. All the grafts came from cadaveric donors. Exclusion criterion was a lack of information concerning the postoperative follow-up.


Before transplantation, two samples of 20mL (milliliters) of PS were collected by an urologist and analyzed for bacterial or fungal contamination.


Specimens were examined microscopically for the presence of bacterial or fungal agents. Each sample was cultured in a thioglycolate broth and on 2 blood agar plates that were incubated aerobically and anaerobically, respectively, at 37.1°C for 5 days. Cultures were examined daily for the presence of bacterial or fungal colonies until specimens were examined microscopically for the presence of bacterial or fungal agents.


In total, 0.1mL of sample was cultured in a blood agar and chocolate agar that were incubated aerobically, and on blood agar plates that were incubated anaerobically at 37°C for 5 days.


One mL of samples was cultured on two Sabouraud dextrose agar with chloramphenicol slants were incubated at 37°C and 30°C for 30 days.


Cultures were examined daily for the presence of bacterial or fungal colonies.


In parallel, 5mL of samples was cultured on one aerobic and one anaerobic blood culture bottles were seeded with 5mL of samples each and incubated for 5 days in the BacTec FX System.


Finally, 1mL of samples was cultured on CandidaID2 for 5 days at 37°C.


The detection limit was 10CFU/mL, as stated in the 2008 Biomedicine Agency Guidelines [5].


Patients at low immunological risk received basiliximab as an induction, as well as steroids, cyclosporine or tacrolimus and mycophenolate mofetil. High-risk patients received intravenous anti-lymphocyte immunoglobulins, steroids, high-dose intravenous immunoglobulins, tacrolimus and mycophenolate mofetil.


All patients, with the exception of those with allergies, were given an antibiotic prophylaxis with pefloxacin and amoxicillin-clavulanic acid during the transplantation, followed by trimethoprim-sulfamethoxazole from the second day post-transplantation. If cytopenia or cholestasis occurred, the latter was changed to pentamidine. Prophylactic antibiotic therapy was administered for a duration of 4 to 6 months, or until the lymphocyte count reached a minimum threshold of 200/mm3.


The Lich-Gregoir ureterovesical anastomosis was used for 408 patients (96.2%), 9 patients had a pyeloureterostomy reconstruction (2.1%), 1 patient underwent an uretero-colic anastomosis because of his past of enterocystoplasty and 5 had another type oh ureterovesical anastomosis (1.2%). One patient did not have a urinary anastomosis because of an immediate renal graft failure.


Ureteral stenting was done to 423 recipients. The stent was removed after a mean time of 36.5 days after renal transplant.


A follow-up period of 12 months postoperation was performed for all patients, considering a surgical site infection with an implant (ureteral stent) in place [6].


Vascular CT-scan or magnetic resonance angiogram was systematically performed at 3 months postoperation, and pyelonephritis was defined as a positive urine analysis (bacteria≥105CFU/mL), with fever≥38.5°C or graft pain, occurring in the first year after transplantation. Antibiotic sensitivity of bacteria found in the patients' urine was compared with that found in the previously extracted conservative liquid.


Qualitative data was analyzed by Pearson's Chi-squared test and quantitative data by Student test. The normality test was not performed because all of the conditions of the Student test were verified. Association of pyelonephritis to every variable was tested by a multivariate logistic regression. Statistical analyses were performed using JMP v.10.0 software (SAS Institute Inc, Cary, NC, USA). A P -value<0.05 was considered significant.


Results


Four hundred and twenty four patients were operated on for transplantation between January 2010 and December 2013 (Table 1), and among the 424 conservative solutions sent for analysis, 195 (46%) were colonized. Two hundred and twenty nine samples (54%) remain sterile (Figure 1).


Figure 1
Figure 1. 

Flowchart of our retrospective study design.




One hundred and fifty-eight (81%) positive PS samples were monomicrobial, and 37 (21.5%) were multimicrobial. Two hundred and fifty-one microbial agents were isolated from those 195 colonized PS (Table 2).


Candida albicans were found on 5 colonized PS samples (2.6%); these patients were pre-emptively treated with caspofungin, followed by fluconazole. None of them developed systemic candidiasis or mycotic aneurysm. Vascular CT-scan or MRA, systematically performed at 3 months in search of a mycotic aneurism, were strictly normal.


Pyelonephritis was found in 45 patients (10.6%) in a mean time of 136.8 days after transplantation during the follow-up. The bacterial urine culture is summarized in Table 3. Among the 45 patients who developed graft pyelonephritis during the year following their transplantation, 21 had colonized conservative liquid (46.7%). Of the remaining 379 patients, the conservative liquid was colonized in 174 cases (45.9%). This difference was not significant (P =0.697).


Instead, results from the univariate analysis highlighted as risk factors: female gender and antibody induction with an anti-lymphocyte serum (Table 4). Similarly, the results from the multivariate analysis (Table 5) showed that the risk of developing graft pyelonephritis was significantly increased for females (OR=2.557; P =0.0043) and for individuals treated with anti-lymphocyte serum (OR=2.711; P =0.0037).


A total of 17 different bacterial species were identified on the 21 colonized PS samples. Five PS samples (23.8%) were found to be colonized by multiple bacteria: Staphylococcus haemolyticus and Staphylococcus hominis ; Staphylococcus hominis and Streptococcus mitis ; Klebsiella pneumoniae , Clostridium jejuni and Klebsiella pneumoniae ; Clostridium jejuni and Escherichia coli ; Candida albicans and Klebsiella pneumoniae . Only 2 patients had the same microbial agent in their PS sample and in their urine sample (4.4%). These 2 patients, for whom pyelonephritis was associated with E . coli infection, E . coli was also found to be present in their PS sample; however, antibiogram results showed the strains to be different (Table 6).


Discussion


Although bacterial colonization of the conservative liquid is a common occurrence, the exact rate of colonization has yet to be published. In this study we found the preservative solution of 195 grafted patients to be colonized during transplantation (46%). Two hundred and fifty one organisms were isolated from those 195 PS. Coagulase-negative staphylococci were the most frequently isolated microbial agent, suggesting a skin contamination.


The use of a high-sensitivity culture method might increase the prevalence of PS contamination. The high proportion of skin microorganisms, such as Coagulase-negative staphylococci found in the PS samples indicated that in the majority of the cases of positive PS cultures, the contamination occurred during the inoculation process and was not due to infected PS; thus the patients were not treated.


Similar data were previously reported by Bertrand et al. In his study, Gram-positive cocci were found on 70% of the colonized PS, and 52% of them were coagulase-negative staphylococci [7].


The most common cause of infection after renal transplantation are UTIs [8] and pyelonephritis rate is estimated to be between 12 and 20% [9].


The statistical analysis did not suggest a correlation between the conservative liquid colonization and the incidence of pyelonephritis during the postoperative year. Patients found to have Gram-negative bacilli in their preservative liquid were systematically treated with ceftriaxone, followed by an efficient antibiotic selected in response to bacterial susceptibility.


PS contamination with coliform agents is associated with severe complications, from the graft infection to the breakdown. The presence of E . coli in both conservative liquid and urine was found for 2 patients. However, the bacteria were determined to be of different strains. Those found in the PS samples were multisensitive, whereas those found in the urine during pyelonephritis showed a resistance for quinolones (patients A and B) and amoxicillin (patient B). Resistance in these cases may be explained by several factors, such as routine antibiotic prophylaxis after transplantation (trimethoprim-sulfamethoxazole), the antibiotic used to manage the colonization of the conservative liquid (ceftriaxone), or by the presence of a community derived E . coli UTI, for which nowadays, resistance to quinolones varies from 3 to 25% [10]. It can be admitted that a pre-emptive antibiotherapy based on the PS colonization with a high-sensitivity culture method is associated with the emergence of resistances in this population.


Mycotic arteritis or aneurysms secondary to fungal infection are rare but mostly caused by Candida albicans and severe consequences from graft removal to death have been described [11, 12]. No patient contracted an infectious or a vascular complication due to fungal colonization in our study.


The results from the multivariate analysis suggest a significant increase in the risk of developing pyelonephritis for females and for patients treated with anti-lymphocyte serum. Female gender is a well-known risk factor of lower UTI due to the shortness of the urethra, and also appears to be a risk factor for pyelonephritis, as previously described [13, 14].


An additional risk factor identified in our study is patient treatment with anti-lymphocyte serum. Induction therapy blocks T cell activation and antigen recognition at the time of transplantation, particularly in recipients at high immunologic risk of graft rejection, sensitized patients and in recipients from an expanded-criteria donor.


It has been described that antibody induction with anti-lymphocyte serum was associated with significantly increased risks for infectious deaths during the first 6 months after renal transplantation [15]. It is also well known that CMV infection is a common complication after kidney transplantation associated with induction treatment with anti-lymphocyte globulin. A near-significant trend toward higher risk for acute graft pyelonephritis was found for cytomegalovirus infection in Fiorante's model [16]. Moreover, Kamath et al. [17] found a positive association between both events, suggesting that the cytokine-based response to bacterial infection may trigger CMV replication through the generation of the nuclear transcription factor kappa B (NF-&kgr;B). It makes sense that induction with anti-lymphocyte serum, and CMV infection may increase graft pyelonephritis.


Our study confirms that there is no correlation between bacterial colonization of the conservative liquid and the occurrence of graft pyelonephritis.


However, this study may be subject to certain bias, including a potential under-estimation of the rate of pyelonephritis. We found pyelonephritis incidence rate at the end of the 1-year follow-up period to be 10%; lower than previously published rates [13]. This reduced incidence rate could result from several factors. Moreover, post-surgery follow-up was carried out for the majority of patients, however, in case of emergency, patients may have presented at an alternative hospital during the follow-up period, so the findings of this study may be limited as they derive from a single-center. Also, pyelonephritis was determined when the patient presented with a combination of fever, graft pain and positive urinanalysis. However, it may also be associated with an isolated fever. Similarly, we did not included febrile patients without bacteriological proof, thus it is possible that our low incidence rate, compared to previously published data, is the result of under-diagnosis.


The high percentage of PS contaminated by agents from skin flora could be decreased with a standard culture method, but globally, the advances in microbial detection became more and more sensitive recently [18, 19], as in Italy [20], United Kingdom [18] or Spain [21].


Finally, it could be interesting to add a standard culture method in the protocol to remove the microbial agents which come from skin flora, as they do not seem to be dangerous to the transplant.


Further studies should include a greater number of patients, by screening recipients in other centers, add a standard culture method and enlarged pyelonephritis criteria to increase statistical power.


Conclusion


The results of our study suggest that there is no connection between contamination of the solution for preserving organs and an acute graft pyelonephritis occurring during the post-transplant year.


Although most contamination was with agents from skin flora, microorganisms with high potential for morbidity like Candida were found. The results of high-sensivity bacteriology culture of the PS should be interpreted with caution.


Author's contribution


F. Encatassamy: data collection; data analysis; manuscript writing.


F. Bruyère: project development; manuscript editing.


M. Buchler: manuscript editing.


A.S. Valentin: data collection.


J. Capsec: data analysis.


Disclosure of interest


The authors declare that they have no competing interest.


As a medical research involving human subjects, the well-being of the individual research subject took precedence over all other interests, as described by the Declaration of Helsinki of 1975.


Informed consent has been collected from each patient.



Acknowledgements


Not applicable.




Table 1 - Baseline characteristics (n =424).
Transplant recipient age (mean±SD) years  48.3±16.2 
Sex   
Female (%)  160 (37.7%) 
Male (%)  264 (62.3%) 
Cause of renal failure   
Glomerulonephritis  95 (22.4%) 
Polycystic kidney disease  58 (13.6%) 
Diabetes  52 (12.3%) 
Vascular  38 (8.9%) 
Uropathy  25 (5.9%) 
Tubulo-interstitial nephritis  24 (5.7%) 
Unknown  102 (24.1%) 
Other  30 (7.1%) 
History of kidney transplant   
None  370 (87.2%) 
44 (10.4%) 
8 (1.9%) 
2 (0.5%) 
Donor age (mean±SD) years  50.0±25.0 
Use of a perfusion machine   
Yes (%)  82 (19.3%) 
No (%)  342 (80.7%) 
Conservative solution   
Celsior  263 (62.0%) 
IGL1  52 (12.3%) 
Custodiol  46 (10.8%) 
Scot15  23 (5.4%) 
KPS1  5 (1.2%) 
Other  35 (8.3%) 
Cold ischemia time (mean±SD) hours  16.8±7.2 
Warm ischemia time (mean±SD) minutes  58.5±27.3 





Table 2 - Microbial agents isolated from the 208 positive PS.
Organisms  Number of isolates on colonized PS
(n =251) 
Secondary acute graft pyelonephritis
(n =28) 
No pyelonephritis
(n =223) 
Gram-negative bacilli (n %)  40 (19.2%)  10 (4.8%)  30 (14.4%) 
Escherichia coli   16 (7.7%)  4 (1.9%)  12 (5.8%) 
Others  24 (11.5%)  6 (2.9%)  18 (8.6%) 
Gram-positive cocci (n %)  164 (78.8%)  13 (6.2%)  151 (72.6%) 
Coagulase-negative staphylococci   121 (58.2%)  12 (5.8%)  109 (52.4%) 
Staphylococcus aureus   9 (4.3%)  0 (0.0%)  9 (4.3%) 
Streptococcus   18 (8.6%)  1 (0.4%)  17 (8.2%) 
Others  16 (7.7%)  0 (0.0%)  16 (7.7%) 
Gram-positive bacilli (n %)  41 (19,7%)  3 (1.4%)  38 (18.3%) 
Yeasts (n %)  6 (2.9%)  2 (1.0%)  4 (1.9%) 
Candida albicans   5 (2.4%)  2 (1.0%)  3 (1.4%) 
Candida glabrata   1 (0.4%)  0 (0.0%)  1 (0.4%) 
Multimicrobial PS (n %)  37 (17.8%)  6 (2.9%)  31 (14.9%) 





Table 3 - Bacteria or fungi found in urine analysis of the 45 patients suffering from graft pyelonephritis.
Strain  Value (%) 
Escherichia coli   24 (53.3%) 
Enterococcus faecalis   6 (13.3%) 
Klebsiella pneumoniae   5 (11.1%) 
Enterococcus faecium   1 (2.2%) 
Proteus vulgaris   1 (2.2%) 
Enterobacter cloacae   1 (2.2%) 
Salmonella typhymurium   1 (2.2%) 
Candida glabrata   1 (2.2%) 
Multiple strains  5 (11.1%) 





Table 4 - Risk factors of pyelonephritis (univariate analysis).
  No pyelonephritis
(n =379) 
Pyelonephritis
(n =45) 
P  
Sex      0.0011 
Female  133 (35.1%)  27 (60.0%)   
Male  246 (64.9%)  18 (40.0%)   
Preservative solution      0.9233 
Colonized  174 (45.9%)  21 (46.7%)   
Sterile  205 (54.1%)  24 (53.3%)   
Graft on perfusion machine      0.3591 
Yes  71 (18.7%)  11 (24.4%)   
No  308 (81.3%)  34 (75.6%)   
Anti-lymphocyte serum      0.0010 
Yes  163 (43.0%)  31 (68.9%)   
No  216 (57.0%)  14 (31.1%)   
Cold ischemia mean time (minutes)  1017  966  0.3287 
Warm ischemia mean time (minutes)  56.3  55.6  0.7744 





Table 5 - Risk factors of pyelonephritis (multivariate analysis).
  Odds ratio (CI=95%)  P -value 
Colonized preservation fluid  0.880 (0.464-1.669)  0.6965 
Female sex  2.557 (1.343-4.867)  0.0043 
Anti-lymphocyte serum  2.711 (1.384-5.313)  0.0037 





Table 6 - Differences between the Escherichia coli strains on the preservation solutions and the urines.
  Antibiogram of the preservation solution  Antibiogram of the urines 
Patient A     
Quinolones     
Nalidixic acid  Sensitive  Resistant 
Ciprofloxacin  Sensitive  Resistant 
Norfloxacin  Sensitive  Resistant 
Ofloxacin  Sensitive  Resistant 
Patient B     
Bêta-lactam     
Amoxicillin  Sensitive  Resistant 
Quinolones     
Nalidixic acid  Sensitive  Resistant 
Ciprofloxacin  Sensitive  Resistant 
Norfloxacin  Sensitive  Resistant 
Ofloxacin  Sensitive  Resistant 




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