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Incidences of Infectious Events in a Renal Transplant Cohort of the German Center of Infectious Diseases (DZIF)

Abstract Background Infectious complications are a major cause of morbidity and mortality after kidney transplantation. Methods In this transplant cohort study at the German Center of Infectious Diseases (DZIF), we evaluated all infections occurring during the first year after renal transplantation. We assessed microbial etiology, incidence rates, and temporal occurrence of these infections. Results Of 804 renal transplant recipients (65.2% male, 51 ± 14 years), 439 (54.6%) had 972 infections within the first year after transplantation. Almost half of these infections (47.8%) occurred within the first 3 months. Bacteria were responsible for 66.4% (645/972) of all infections, followed by viral (28.9% [281/972]) and fungal (4.7% [46/972]) pathogens. The urinary tract was the most common site of infection (42.4%). Enterococcus was the most frequently isolated bacterium (20.9%), followed by E. coli (17.6%) and Klebsiella (12.5%). E. coli was the leading pathogen in recipients <50 years of age, whereas Enterococcus predominated in older recipients. Resistant bacteria were responsible for at least 1 infection in 9.5% (76/804) of all recipients. Viral infections occurred in 201 recipients (25.0%). Of these, herpes viruses predominated (140/281 [49.8%]), and cytomegalovirus had the highest incidence rate (12.3%). In the 46 fungal infections, Candida albicans (40.8%) was the most commonly isolated. Other fungal opportunistic pathogens, including Aspergillus fumigatus and Pneumocystis, were rare. Conclusions Renal allograft recipients in Germany experience a high burden of infectious complications in the first year after transplantation. Bacteria were the predominating pathogen, followed by opportunistic infections such as cytomegalovirus. Microbial etiology varied between age groups, and resistant bacteria were identified in 10% of recipients.

Solid organ transplant recipients require long-term immunosuppression, which puts them at risk for life-threatening infections. Infectious diseases are a major cause of morbidity and mortality after kidney transplantation, especially in the early post-transplant period [1][2][3]. Infectious complications also reduce recipients' quality of life and increase health care costs [2,4,5]. Although infections play a fundamental role in recipient prognosis and allograft survival [6,7], many questions regarding the prevention and early diagnosis of infections remain unanswered, and more research is needed in this area. Post-transplant data are often restricted by a lack of both stringent patient follow-up and standardized definitions of infections. Very few prospective cohort studies of transplant recipients have investigated all infectious complications [8][9][10][11][12], and the occurrence and timeline of all posttransplantation infections have not been studied in a German transplant cohort.
To prevent infection and reduce infection-related morbidity and mortality, we need information on multiple factors. For example, immunosuppressive strategies can reduce the incidence of rejection but increase the recipients' susceptibility to infection [13][14][15]. Furthermore, the availability of efficient prophylaxis may have modified frequencies and temporal infection patterns [1,9,12]. Knowing the timing and frequency of infections in the era of extended donor/recipient criteria, modern immunosuppression, and routine use of prophylaxis is crucial for implementing prevention strategies.
The transplant cohort of the German Center of Infectious Diseases (DZIF) is a unique database that evaluates infections after renal transplantation [16]. In this cohort, clinical data are being collected, together with biosamples from transplant recipients (predominantly renal allograft recipients). In this study, we assessed the microbial etiology, incidence rates, and temporal occurrence of all infections occurring in this cohort.

Study Population and Design
The DZIF transplant cohort is a multicenter prospective cohort study at the German Center for Infection Research [16]. It includes allograft recipients from 5 of the largest German transplant centers (University Hospital Hannover, University Hospital and Renal Center Heidelberg, TU Munich, LMU Munich, and University Hospital Tuebingen). In participating transplant centers, all patients receiving organs were informed about the study and invited to participate.
In the present study, we analyzed data from all adult renal and simultaneous pancreas-kidney allograft recipients aged 18  Study visits were performed immediately before transplantation and at months 3, 6, 9, and 12 after transplantation. We also performed study visits when infections were detected. At each study visit, clinical and laboratory data were collected and entered into a central web-based database. The DZIF transplant cohort monitors data and conducts quality audits regularly, and the study was approved by the DZIF Scientific Steering Committee.

Patient Consent
Ethics approval was obtained from all participating centers (Hannover Medical School Nr 6534, Medical Faculty of the University of Heidelberg Nr S-585/2013, Medical Faculty of the TU Munich Nr 5926/13, LMU Munich Nr 380-15, University Hospital Tuebingen Nr 327/2014BO1), and all participants provided written informed consent.

Outcomes
The primary outcome was the occurrence of clinically relevant infection within the first 12 months after transplantation. Infections were categorized as clinically relevant if the patient complained about specific clinical symptoms (eg, fever, malaise, pain) or if the patient was hospitalized and had antibiotic, antiviral, or antifungal treatment in therapeutic doses. Secondary outcomes were the type of pathogen, timing of infection, and site of infection.

Infectious Events
All infections were identified by trained transplant physicians using paper-based or electronic hospital records and referral  [17][18][19][20].
A urinary tract infection was only included in case of clinical symptoms in addition to a positive urine culture [18]. Antimicrobial resistance was also recorded [21]. Herpes virus and polyomavirus infections were detected by real-time polymerase chain reaction. BK virus above the threshold of 10 4 copies per mL plasma was defined as clinically significant [17]. Fungal infections were reported if they met EORTC/MSG criteria with proven and probable invasive infection [19,20]. We included all proven and probable fungal infections and fungemias. Pneumocystis jirovecii infection was diagnosed by sputum culture and bronchial lavage.

Immunosuppression
Almost all patients received a standard triple-drug combination (comprising a calcineurin inhibitor, mycophenolate, and steroids) together with an interleukin 2 receptor antagonist (basiliximab) to induce immunosuppression.

Prophylaxis and Surveillance Strategy
Prophylaxis and surveillance strategy was suggested to be performed according to KDIGO 2009 guidelines [17]. Standard protocol included antiviral prophylaxis with valganciclovir for all cytomegalovirus (CMV) immunoglobulin G (IgG)-positive recipients and for all recipients of organs from CMV IgG-positive donors for at least 3 months. In case of the high-risk constellation D+/R-, 6 months of prophylaxis was suggested. If the recipient was CMV IgG positive, 3-month valganciclovir prophylaxis was recommended. Pneumocystis jirovecii infection was prevented with trimethoprimsulfamethoxazole for 6 months. Anti-Candida prophylaxis with oral nystatin was provided within the first 1-3 months if .20 mg of methylprednisolone was administered. Urinary tract infection prophylaxis was suggested with trimethoprimsulfamethoxazole for 6 months after transplantation. Screening for BKV was was recommended to be performed monthly for the first 6 months and every 3 months thereafter as well as in case of worsening of allograft function. No routine EBV screening was performed. Urinary tract infection surveillance was routinely performed with urine culture at each visit, in addition to recording clinical symptoms.
Recipients who received organs from bacteremic or fungemic donors had targeted antimicrobial therapy for a period of 7-14 days.
It was assumed that all 5 centers together cover at least 20% of all solid organ transplants in Germany and provide a high number of infectious events, allowing in-depth analysis of infectious complications. The time of observation was defined as the time between transplantation and transplant failure, loss to follow-up, death, or 12 months after the transplantwhichever occurred first. Cumulative infection rates were calculated as the percentage of infected patients during the first year post-transplantation. According to respective data distribution, continuous variables were expressed as means and SDs or as medians and interquartile ranges (IQRs). Categorical variables were presented as numbers and percentages. Continuous variables were compared using the Mann-Whitney U test or Student t test, as appropriate. Crosstabs combined with the chi-square test or Fisher exact test was used to compare categorical variables. Statistical significance was defined as a P value ,.05. We performed logistic regression to evaluate independent risk factors for the occurrence of multiple infections. Significant variables in univariate analysis were introduced in a multivariate model. Cox regression was used to analyze the effect of preceding infections depending on pathogen class (bacterial/viral fungal). Any preceding infection within the first year was considered a timedependent covariate. Highly prevalent pathogens in patients suffering multiple infections were detected by logistic regression, thereby comparing first-year cumulative incidence rates of patients suffering .2 infections/year with those of patients suffering 1 or 2 infection(s)/year. All statistical analyses were performed using IBM SPSS Statistics, version 28.0 for Mac OS X (SPSS, Inc. Chicago, IL, USA).

Renal Allograft Recipients and Incidence of Infections
All adult renal transplant recipients consenting to participate in the DZIF transplant cohort and receiving a transplant in 1 of the 5 recruiting centers between April 2011 and November 2019 (n = 804) were enrolled for our analysis; 65.2% were male, and the mean age was 51 + 14 years (Tables 1 and 2). Forty-two (5%) patients had a simultaneous pancreas-kidney transplantation. Follow-up was terminated early in 39 patients (15 patients died, 15 had transplant failure, and 9 were lost to follow-up). Infections were recorded as the direct cause of death in 6/15 patients (40%; pneumonia due to Pseudomonas in 1 patient, invasive aspergillosis in 2 patients, invasive mucormycosis in 1 patient, bornavirus encephalitis in 1 patient, and an outpatient infection of unknown origin in 1 patient).
Standard prophylaxis included 6-month anti-Pneumocystis prophylaxis with trimethoprim-sulfamethoxazole for all recipients. Anti-Candida prophylaxis (mostly oral nystatin) was given to 70.2% of recipients for 1-3 months. Antiviral prophylaxis (valganciclovir/ganciclovir) was provided to 74.9% of all recipients for at least 3 months.
Nosocomial infections (mostly bacterial urinary tract) predominated in the first months, followed by opportunistic viral infections such as CMV and BK virus (BKV) between months 1 and 5 ( Figure 1). There were 2 peaks of fungal infection. Candida spp. and Aspergillus fumigatus were the leading pathogens in the first 2 months, while Pneumocystis jirovecii occurred after month 4. There was no difference in the incidence of infections between male and female recipients (male 54.2%, female 58.2%; P = .28). However, age affected the incidence of infection, with recipients aged .65 years having an increased risk of bacterial and fungal infection (Tables 1 and  2 Figure 3). The percentage of Pseudomonas aeruginosa infections increased with age, and the rate of infection was almost 4 times higher in recipients .65 years old than in recipients ,50 years old (14.5% vs 3.7%). Pseudomonas infection was also more common in males than in females (6.6% vs 2.2%; P = .007), whereas E. coli was more frequently detected in females (E. coli 17.4% vs 10.9%; P = .010).

Multiple Infections
More than 2 infections per year were detected in 17.0% (137/ 804) of recipients. The prevalence increased with age (25.5% in recipients aged .65 years vs 10.5% in recipients aged ,55 years; P , .001). Univariate analysis revealed that recipient age, deceased donor, donor age, delayed graft function, and the number of postoperative inpatient-days were significantly associated with the occurrence of multiple infections ( Table 6). Multivariate analysis confirmed that recipient age and number of postoperative inpatient-days were independent risk factors for multiple infections. Table 7 shows pathogens that were significantly more prevalent in recipients with .  Other nonenteric gram-neg. bac.

DISCUSSION
Effective management of post-transplant infection relies on prevention, early diagnosis, and specific therapy [15,22]. For this, detailed information on the incidence, microbial etiology, and timeline of infections is crucial. This is the first comprehensive study of a representative German renal transplant cohort comprising .800 patients evaluating all clinically relevant infections detected during the first year after transplantation.
Three infection periods have been reported after solid organ transplantation: Nosocomial infections predominate in the first month, followed by opportunistic infections between months 1 and 6, and community-acquired infections after month 6 [2,14]. We confirmed these results; Enterococcus spp. and Candida spp. infections mainly occurred during the first 30 days, and infections with opportunistic pathogens, like CMV and BKV, were highly prevalent in the intermediate period.
Infections with Pneumocystis jirovecii were rare and occurred mostly in the late period, presumably because routine trimethoprim-sulfamethoxazole prophylaxis was administered for 6 months after transplantation. Only a few community-acquired respiratory infections occurred during the late period-most were detected in the first month after transplantation. This might be because pathogens are typically not evaluated in patients with mild symptoms who do not require hospitalization.
Long-term immunosuppression and antimicrobial therapy increased the incidence of resistant pathogens in transplant patients [23][24][25]. We identified resistant strains in 9.5% of all recipients and in 50% of patients with fungal infections. This  might reflect the high prevalence of multiple infections requiring antimicrobial therapies.
An association between bacterial and fungal infections and resistant strains appears likely. Elderly recipients with prolonged postoperative hospitalization seem to be particularly vulnerable. Early and intensified surveillance is needed. This might also be appropriate for pancreas-kidney allograft recipients, appearing to be susceptible to early Candida infections, or elderly males, who are prone to Pseudomonas aeruginosa. All these predominantly nosocomial pathogens, including the most frequently isolated agent, Enterococcus, were associated with the occurrence of multiple infections. There could be a direct effect due to pathogen-specific mechanisms or an indirect effect as a consequence of frequent hospitalizations and unsuccessful therapies. The interaction of both effects seems likely. The current bacterial spectrum, with Enterococcus and E. coli First-year pathogen incidence rates in patients suffering .  predominating, does not seem to be sufficiently covered by trimethoprim-sulfamethoxazole prophylaxis. Considering the high prevalence of bacterial infections, additional agents are required. Several new immunosuppressive regimens with varying immunosuppressive potential have been developed in the last years for transplant recipients [26,27]. Opportunistic infections have been successfully reduced in these patients through prophylaxis and surveillance [2]. An important effect of these strategies is reducing the prevalence of Pneumocystis jirovecii and CMV infections, both of which peak at 4 months after transplantation. Patients receiving a CMV IgG-seropositive organs receive a 3-or 6-month valganciclovir prophylaxis [17,28]. This is important because more than half of viral infections were herpes viruses, with CMV having the highest incidence rate. A prolonged CMV prophylaxis and/or an intensified preemptive strategy once valganciclovir is stopped could also be beneficial. Indeed, CMV infections remain an important risk factor for graft loss and patient mortality [29][30][31]. Another way to reduce CMV infection would be to test CMV-specific cell-mediated immunity in susceptible patients. This would allow CMV prophylaxis to be discontinued in individual patients [32]. BKV nephropathy is rare but can result in graft loss [33,34]. In the DZIF cohort study, Polyomaviridae were the second most common viral pathogens, in agreement with findings from the Swiss Transplant Cohort [8]. Therapeutic interventions are limited, so strict surveillance, as suggested in the KDIGO guidelines, is needed [17].
Most clinical studies have focused on specific infections and have provided limited information on the global burden of infectious complications after renal transplantation [35][36][37][38][39][40]. This cohort study has collected data of optimal quality based on standardized definitions and quality audits [16], and these data guarantee a comprehensive view of all clinically relevant infections in the first year after renal transplantation. However, the generalizability of our results to other settings might vary depending on the geographic setting and distinct microbial prophylaxis strategy.
In conclusion, renal allograft recipients in Germany experienced a high burden of infectious events during the first year after transplantation. Current prophylactic agents appear efficient in the prevention of opportunistic infections, but thereby neglect common nosocomial infections (eg, Enterococcus spp.). The prevalence of nosocomial infections was high during the first months, and the prevalence of viral opportunistic infections such as CMV and BKV increased thereafter. Invasive fungal diseases and infections by opportunistic bacteria were rare. Many bacterial pathogens were multidrug resistant, supporting the need for specific microbiologic diagnoses and critical use of antibiotics. Age not only affects the frequency but also the temporal pattern and microbial etiology of infections. The occurrence of multiple infections was common and seems to correlate with immunodeficiency. Further research is needed to understand the effects and mechanisms of the frequently involved pathogens, first detected in the present study. Targeting these would be a very effective way to prevent further infections. In all, our observations may help to guide future surveillance strategies. Therefore, further prospective multicenter studies are required to verify and update our results and to pursue novel approaches for prophylactic strategies.

Supplementary Data
Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.