EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: We sought to assess the incidence of invasive fungal infections and identify the risk factors and outcome of invasive fungal infections in liver transplant recipient.

Materials and Methods: A retrospective analysis was made of 408 patients who received a liver transplant between January 1990 to December 2012 at Başkent University in Ankara, Turkey. Only 305 of 408 patients were included. Demographic and clinical findings were reviewed, and these findings were compared between patients with or without invasive fungal infections.

Results: Ten of 408 liver transplant patients (2.5%) developed invasive fungal infections. Aspergillus was the most common cause of invasive fungal infections (n=8), followed by Candida (n=1), and Cryptococcus neoformans (n=1). Pulmonary involvement was dominant in all patients (n=10), and only 1 patient had disseminated fungal infection (cryptococcosis). The mean time from transplant to invasive fungal infection diagnosis was 32 ± 19.2 days. Most patients with invasive fungal infection (9/10) died. Mean survival time between diagnosis of fungal infection and death was 24.2 ± 27.3 days in all 10 patients. Fungal infections occurred significantly more frequently in patients with older transplant age, diabetes mellitus, cytomegalovirus infection, renal insufficiency. In addition, other risk factors included long stays in the surgical intensive care unit, the overall length of stay in hospital, and having preoperative high creatinine level.

Conclusions: Invasive fungal infections were associated with increased morbidity and mortality among liver transplant recipients, with Aspergillus spp. being the most common pathogen in our series. Because of its high mortality rate, it is important to follow up transplant patients for the development of invasive fungal infections.

Key words : Aspergillosis, Invasive fungal infection, Liver, Risk factor, Transplant

Invasive fungal infections are the most important cause of morbidity and mortality in solid-organ transplant recipients. The incidence of invasive fungal infections has been reported to range between 5% and 42%.^1-4 The most common predisposing factors for invasive fungal infection are immunosuppression, broad-spectrum antibiotics, surgical procedures, disruption of intestinal or bladder mucosa, cyto-megalovirus infection, diabetes mellitus, indwelling catheter placement, prolonged pretransplant dialysis, and chronic liver disease.^1-3,5

The most common fungal infection in solid-organ transplant is candidiasis, followed by aspergillosis and cryptococcosis. The incidence of these fungal organisms shows great variability center to center and according to the transplanted organ. Thus, the incidence of aspergillosis is higher than candidiasis and cryptococcosis in lung transplant recipients.⁶ Although the most common fungal infection is candidiasis (47% of cases), the mortality rate of candidiasis is significantly lower than aspergillosis. Aspergillosis occurs as both a primary and secondary infection with a high mortality of up to 88%. Early diagnosis of invasive fungal infections and treatment in immunosuppressive patients are important because of the high rates of mortality and graft loss.

The aim of this study is 2-fold: First, we investigated the incidence of fungal infections among liver transplant recipients, and second, we attempted to identify risk factors for invasive fungal infections in liver transplant patients.

Materials and Methods

Patients
A total of 408 patients received a liver transplant at Baskent University Hospital between January 1990 and December 2012. Donor management and liver transplant surgery were performed according to accepted standard procedure. The study was approved by the Ethical Review Committee of the institute. All protocols conformed with the ethical guidelines of the 1975 Helsinki Declaration.

Only 305 of 408 patients were included in this study. We categorized patients according to development of invasive fungal infections in 2 groups. Group 1 contained patients with invasive fungal infections (n=10), and group 2 patients were selected from the other liver transplant recipients without invasive fungal infections (n=295) and were accepted as a control group. Superficial fungal infections and cases of asymptomatic colonization were excluded.

Demographic and clinical findings were evaluated including age, sex, primary liver disease, donor features, immunosuppressive treatment, blood drug level, and laboratory characteristic of patients. Hospital records were reviewed retrospectively to identify possible risk factors for developing invasive fungal infections. The length of time in transplant surgery, the duration of stay in the surgical intensive care unit, total hospital stay, preoperative creatinine level, serum bilirubin, and albumin level were recorded. Also, presence of diabetes mellitus, cytomegalovirus infection, and acute or chronic rejection were recorded. The time intervals from transplant to diagnosis of invasive fungal infection and from infection to death also were evaluated.

All patients received intravenous prophylactic treatment with ampicillin plus sulbactam, cefotaxime before and after surgery. In addition for cyto-megalovirus prophylaxis intravenous ganciclovir for 14 days was given to all patients after surgery followed by oral valganciclovir or acyclovir for 6 months, Mycostatin for antifungal prophylaxis and sulfamethoxazole-trimethoprim for Pneumocystis jiroveci prophylaxis for 6 months after surgery. These patients were managed in the intensive care unit for 1 to 46 days postoperatively, and were usually discharged from the hospital without complication 3 to 4 weeks after surgery. The baseline immuno-suppression protocol consisted of tacrolimus or cyclosporine with low-dose steroids, and azathioprine or mycophenolate. Rejection episodes were initially treated with corticosteroid boluses. Refractory patients were treated with sirolimus monoclonal antibody.

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 15.0, IBM Corporation, Armonk, NY, USA). The Mann-Whitney U test was used to compare variables in patients, with or without invasive fungal infections. Results were considered statistically significant when P ≤ .05. For quantitative variables, results are given as mean ± standard deviation (SD) and compared using the Mann-Whitney U tests for variables with a non-normal distribution.

Only 10 patients of total 305 liver recipients (2.5%) showed invasive fungal infection during follow-up. Of 10 patients, 8 were males and 2 were females. Among these 10 patients, 5 patients had cirrhosis (cryptogenic cirrhosis [n=2], alcoholic cirrhosis [n=2], secondary to B viral hepatitis [n=1]), 2 patients had hepatocellular carcinoma, 1 patient had Caroli disease, 1 patient had progressive familial intrahepatic cholestasis type 1, and 1 patient had Wilson disease as the primary liver disease. The mean age at the time of liver transplant was 38.6 ± 18.93 years (range, 6 mo-63 y), and invasive fungal infections occurred at a mean of 32 ± 19.27 days after liver transplant (range, 4-64 d). The graft source was a living-related donor in 7 (70%) and a deceased donor in 3 patients (30%) (Table 1).

The clinicopathological findings of 10 patients are given in Table 2. As shown in Table 2, the most common clinical symptom was fever among 9 patients, followed by cough and dyspnea. Upon radiologic examination, lung infiltrates were seen in 8 patients (80%) and lung nodules were detected in the other patient (10%). Bronchoscopic findings of the patients were increased secretions (n=4), necrotic plaque (n=3), and unremarkable (n=3). Diagnosis of fungal infections was made by positive blood or bronchoalveolar lavage cultures, cerebrospinal fluid cytology, and tissue biopsies. Aspergillus species were the most common cause of infections of invasive fungal infections (n=8), candida glabrata (n=1), and cryptococcus neoformans (n=1). Pulmonary involvement was dominant in all patients (n=10). In addition, 1 patient showed disseminated fungal infection (cryptococcosis) during follow-up.

As shown in Table 2, only 3 of 10 patients had biopsy confirmation for invasive fungal infection (2 biopsies were postmortem lung biopsy). The remaining 7 cases were diagnosed by cytologic examination. Microbiological culture was performed on only 8 patients and 5 patients had positive culture. Galactomannan level was investigated in 3 of 8 patients with invasive aspergillosis, and the level of galactomannan was found to be increased in 1 patient. The remaining patients showed galactomannan within normal limits.

Risk factors for invasive fungal infections
Table 3 shows that patients with older age at the time of transplant had a significantly better risk of developing invasive fungal infection compared with younger patients (P = .04). The total length of stay at the hospital after transplant and the duration of stay at surgical intensive care unit are shown have a statistically significant effect on developing invasive fungal infections. The length of stay in the hospital after transplant was significantly longer in patients with invasive fungal infection than in those patients without invasive fungal infection (P = .012). Similarly, the duration of stay in the surgical intensive care unit was longer in patients with invasive fungal infection compared with patients without invasive fungal infection (P = .001). The mean surgical time was 9.9 hours in the recipients with invasive fungal infections, and 8.87 hours in controls. No statistically significant difference existed between the 2 groups regarding development of invasive fungal infection (P = .146).

Three of 10 patients (30%) had cytomegalovirus infections before liver transplant. Diabetes mellitus preceding the liver transplant occurred in 60% of patients (6/10). The presence of diabetes mellitus and cytomegalovirus infection were had a strong effect on the development of invasive fungal infection compared with patients without diabetes mellitus and cytomegalovirus infection (P ≤ .001 for diabetes mellitus; P = .008 for cytomegalovirus). In addition, patients with the high preoperative creatinine had shown higher incidence of invasive fungal infection compared with patients with low preoperative creatinine (P = .036) (Table 3).

Three of 10 recipients (30%) had renal insufficiency (serum creatinine > 132.6 μmol/L [1.5 mg/dL]) before transplant, and 8 of 295 control patients had renal insufficiency without invasive fungal infections (P < .001). Patients with high levels of preoperative creatinine showed a higher incidence of invasive fungal infection compared with patient with a low level of preoperative creatinine.

One of 10 patients (10%) had an episode of acute rejection before invasive fungal infection, and 103 of 295 control patients had an episode of acute rejection before invasive fungal infection without invasive fungal infections (P > .05). Among 10 patients, chronic rejection was not observed in any patients. One of 10 patients (10%) had a retransplant before invasive fungal infection, and 2 of 295 control patients without invasive fungal infections had a retransplant (P = .03).

No statistically significant differences were found between patients with invasive fungal infections and patients without invasive fungal infections regarding sex, donor type, donor age, surgery time, preoperative albumin level, preoperative bilirubin level, preoperative partial thromboplastin time, and postoperative hepatitis.

As shown in Table 1, there was no statistically significant differences between living-related (31.86 ± 23.14 d) and deceased donors (32.33 ± 8.02 d) regarding the time between liver transplant and invasive fungal infection. In addition, there were no statistically significant differences regarding the time of invasive fungal infection after liver transplant, the type of primary disease, the type of immuno-suppressive drugs, the type of infections agents, and the sex of patients.

The laboratory findings of 10 patients were recorded at the time of diagnosis of invasive fungal infection. Renal insufficiency was present in 50% of the patients (5/10) (mean level of serum creatinine level, 133.484 ± 81.328 µmol/L [1.51 ± 0.92 mg/dL]; urea, 18.951 ± 9.74 mmol/L [53 ± 27.3 mg/dL]. All 10 patients had hepatic failure (mean serum total bilirubin, 181.26 ± 198.36 μmol/L [10.6 ± 11.6 mg/dL]; serum direct bilirubin, 114.741 ± 128.763 μmol/L [6.71 ± 7.53 mg/dL], aspartate aminotransferase was 119.3 ± 153.19 U/L, alanine aminotransferase was 66.4 ± 59 U/L).

All fungal infections were treated with antifungal agents (invasive aspergillosis was treated with amphotericin B, triflocin, and caspofungin; candidiasis was treated with triflocin; disseminated cryptococcosis was treated with fluconazole and voriconazole). Most patients with invasive fungal infections died (9/10). Mean survival time between diagnosis of fungal infection and the death was 24.2 ± 27.3 days (range, 2-68 d). The mortality rate in aspergillosis was 100% with the mean survival of 17.8 ± 22.19 days (range, 2-60 d). Only 1 patient with Candida glabrata infection showed response to antifungal therapy.

Nine of 10 patients with invasive fungal infections, and 67 of 295 control patients, died. The rate of the death was higher (90%) in patients with invasive fungal infection than it was in patients without invasive fungal infection (22.7%; P ≤ .001). In addition, cytomegalovirus infection, diabetes mellitus, acute rejection, and the type of immunosuppressive regimen had no statistical effect on the time of development of invasive fungal infections and survival after diagnosis (P > . 05).

Fungal infections are life-threatening conditions with poor prognosis in liver transplant recipients. Previous studies have shown that 3.6% to 42% of liver transplant recipients develop invasive fungal infections, accounting for 20% to 30% of all major infections in liver transplant recipients.^1,2,7,8 Candida spp. is the most common pathogen, followed by Aspergillus spp. However, the incidence of Aspergillus spp. infections has been higher in recent years, ranging from 1% to 40% in liver transplant recipients.^1,2,5,7-10

Rabkin and associates reported 90 patients with liver transplants between 1994 to 1997. In their study, 35 of 90 recipients (38%) developed invasive fungal infections. Twenty-five of 35 patients had Candida spp., 4 of 35 patients had Aspergillus spp., and 1 of 35 had cryptococcus neoformans. Among their patients, the mean time between transplant and infection was 15 days (range, 4-77 d), and the mean survival time from infection to death was 21 days (range, 3-64 d). Eleven of 35 patients died (31.4%), and 4 of 11 patients died owing to invasive Aspergillus infection.¹

In 1 large series with 352 liver recipients who underwent transplant between 2003 and 2006 reported 42 patients with invasive fungal infection.² Of these patients, 53% showed candidiasis and 40% showed aspergillosis. The remaining 2 patients had Cryptococcus neoformans, and 1 patient showed mycosis infection. Among these patients, the mean time to onset of infection was 13 days after transplant (range, 8-32 d). Only 15 of 42 patients were died because of invasive fungal infections, and the cause of death was because of aspergillosis in 10 patients and candidiasis in 5 cases.²

In our study, the incidence of invasive fungal infections was 2.5% after liver transplant, excluding superficial fungal infections and asymptomatic fungal colonization. In contrast to recent studies, the incidence of invasive fungal infections was found to be lower in our liver transplant recipients. The most common pathogen among our patients was Aspergillus spp. accounting for 80% of all invasive fungal infections, followed by Candida glabrata and Cryptococcus neoformans. In opposition to other studies, the incidence of invasive candidiasis in our liver transplant patients was lower than that in the literature.^1,2,5 Among our 10 patients, mean time between transplant and invasive fungal infections was 32 days (range, 4-64 d). All our patients (8/8, 100%) with invasive aspergillosis and 1 patient with disseminated cryptococcosis died at a mean survival time of 24 days (range, 2-68 d). The overall mortality in liver transplant recipients with invasive aspergillosis was higher than in other species (candidiasis, cryptococcosis), the reported studies gave mortality rates for invasive aspergillosis as 66.7% to 100%.^1,2,7

In the study by Wajszczuk and associates, 90% of all invasive fungal infections occurred during the first 2 months after liver transplant, and all patients with invasive aspergillosis died.⁷ Similarly, in our study, the development of invasive fungal infections in all patients were found during the first 2 months, and all our patients with aspergillosis died.

Cryptococcosis is the third most common occurring invasive fungal infection in solid-organ transplant recipients, usually occurring 16 to 21 months after transplant. It has been reported to develop earlier (in 12 months) in liver and lung transplant patients compared with other solid-organ transplant recipients.¹¹ Cryptococcosis led to disseminated disease or central nervous system involvement in 53% to 72% of solid-organ recipients. Among solid-organ recipients, liver transplanted recipients had a 6-fold increased risk for disseminated disease of cryptococcosis.¹¹ In our study, only 1 of 10 patients with invasive fungal infection had disseminated cryptococcosis at 15 days after transplant; this was earlier than reported in the literature. Similar results with our case was reported in the literature with a mean time interval of 80 days by Pappas and associates and 175 days by Wajszczuk and associates.^7,8 The possible reason for earlier development of invasive Cryptococcus infection in our case can be explained by the presence of diabetes mellitus. Diabetes mellitus is an important risk factor for invasive fungal infection. Diabetes mellitus increases the risk of fungal infection by destroying the function of neutrophils, macrophages, and cellular and humoral immunity. The risk of diabetes mellitus among our patients is high. Most patients with invasive fungal infection (6/10, 60%) had diabetes mellitus, while patients without invasive fungal infection showed 6% diabetes mellitus (18/295); this result was statistically significant.

The length of stay in the surgical intensive care unit and the overall length of stay in the hospital were significant factors associated with the development of fungal infections. In the literature, these durations varied. Rabkin and associates noted 13.7 days for surgical intensive care unit and 30.8 days for total stay in the hospital.¹ Fortún and associates reported a mean length of stay in surgical intensive care unit as 24.6 days in 15 patients with invasive aspergillosis among 260 liver transplant recipients.⁹ In our liver transplant recipients, mean stay in surgical intensive care unit was 11.7 days, and the overall length of stay in the hospital was 41.4 days. Similar results were found in the literature. Patients with longer times in the surgical intensive care unit and hospital had a significantly higher degree of risk of developing invasive fungal infection compared with patients who remained in the hospital for a shorter time.

Invasive fungal infections have been reported to increase with retransplant.^1,7,9,10 Similarly, our study found that fungal infections occur more often in recipients with a retransplant.

There is controversy about the effect of acute and chronic rejection regarding development of invasive fungal infections. Fortún and associates reported that development of chronic rejection is associated with late aspergillosis (100 days after transplant).⁹ Our study found that there was no significant differences between acute rejection and developing invasive fungal infection. We found that acute rejection was not a risk factor for developing invasive fungal infection.

Increased serum creatinine levels increase the risk of developing invasive fungal infections in liver transplant recipients.^1,5,10 Renal failure leading to invasive fungal infections causes functional changes of granulocytes and macrophages.^2,5 Singh and associates found 22 of 26 patients with invasive aspergillosis had renal insufficiency before transplant, with the median serum creatinine level of 265.2 μmol/L (3.0 g/dL).⁵ Similar findings were reported by Rabkin and associates and Rosenhagen and associates.^1,10 They found serum creatinine levels in liver transplant recipients with invasive fungal infections were higher than they were in recipients without invasive fungal infection. We saw that renal insufficiency before transplant (defined as serum creatinine > 132.6 μmol/L [1.5 mg/dL]) was present in 3 of 10 liver recipients with invasive fungal infection and in 8 of 295 controls. With regard to this finding, patients with high preoperative creatinine levels had show a higher incidence of invasive fungal infection. When compared with patients with low preoperative creatinine levels.

Transplant recipients with serum albumin levels < 25g/L have a 10-fold increase in the incidence of life-threatening infections.¹² Singh and associates reported the median serum albumin level was 23 g/L in 26 patients with aspergillosis.⁵ We found no difference in albumin levels before liver transplant, as noted in a study by Wajszczuk and associates.⁷

Older transplant age has been proposed as a predisposing factor for the high incidence of invasive fungal infections. In the literature, the age of liver transplant recipients at the time of transplant varied from 3 to 63 years.^1,2,4,8-10 In some of these reports, age was not found to be a significant factor for developing invasive fungal infections.^1,9,10 In contrast with previous reports, we found significant differences between recipients with and without invasive fungal infections. Liver transplant recipients with an older age at transplant showed a higher incidence of invasive fungal infections compared with patients with a younger age at transplant.

Another risk factor for developing invasive fungal infections in liver transplant recipients is surgical length of time. Wajszczuk and associates found that the recipients with long surgery time (mean, 13.1 h) had an increased risk for developing invasive fungal infections compared with patients with short surgery time (mean, 11.1 h).⁷ The authors concluded that risk of developing invasive fungal infection in liver transplant recipients increased with longer surgery time. In our study, the mean surgical time was 9.9 hours in patients with invasive fungal infections, and 8.87 hours in the patients without invasive fungal infections; no significant differences were found between the groups.

Cytomegalovirus infection is a significant risk factor for invasive fungal infections, especially aspergillosis among liver transplant recipients.^12,13 In a 1997 study by Singh and associates, 6 of 26 patients with aspergillosis had cytomegalovirus infection before invasive fungal infection.5 Similarly, Rosenhagen and associates reported 8 patients with cytomegalovirus infections among their 14 patients with invasive aspergillosis. They found that patients with cytomegalovirus infection had a higher risk of invasive fungal infections compared with those without cytomegalovirus infections.¹⁰ In our study, 3 of 10 patients with invasive fungal infections had cytomegalovirus infection; this was statistically significant (P < .01). In addition to cytomegalovirus infection, other systemic infections with immuno-modulating viruses such as Epstein-Barr virus, human herpesvirus 6, hepatitis viruses B and C, and human immunodeficiency virus may be predisposing factors for developing invasive fungal infections.¹²

These results suggest that presence of diabetes mellitus, cytomegalovirus infection, renal insufficiency (increased serum creatinine levels) before transplant, long length of stay in surgical intensive care unit, overall length of stay in the hospital, older transplant age, and preoperative high creatinine levels may predispose liver transplant recipients to develop invasive fungal infections. To decreased the high mortality rate of invasive fungal infections, early diagnosis is paramount.

Volume: 12 Issue: 1 March 2014 - Supplement - 1

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