Cryptococcosis in liver and kidney transplantrecipients receiving anti-thymocyte globulin oralemtuzumab

F.P. Silveira1, S. Husain1, E.J. Kwak1,P.K. Linden2, A. Marcos3, R. Shapiro3,P. Fontes3, J.W. Marsh3, M. de Vera3,K. Tom3, N. Thai3, H.P. Tan3, A. Basu3,K. Soltys3, D.L. Paterson1

1Division of Infectious Diseases, 2Department of Critical CareMedicine, 3Thomas E. Starzl Transplantation Institute,University of Pittsburgh Medical Center, Pittsburgh,Pennsylvania, USA

F.P. Silveira, S. Husain, E.J. Kwak, P.K. Linden, A. Marcos, R. Shapiro,P. Fontes, J.W. Marsh, M. deVera, K.Tom, N.Thai, H.P.Tan, A. Basu,K. Soltys, D.L. Paterson. Cryptococcosis in liver and kidney transplantrecipients receiving anti-thymocyte globulin or alemtuzumab.Transpl Infect Dis 2007: 9: 22^27. All rights reserved

Abstract: Rabbit anti-thymocyte globulin (ATG) and alemtuzumabhave been used for induction or preconditioning and for the treatmentof acute rejection in organ transplant recipients in many centers. Suchregimens may lead to a substantial decline in the CD4 lymphocyte countto levels seen in other population groups at high risk of cryptococcosis.In view of this, we examined the impact of such therapy on thecumulative incidence of cryptococcosis among liver and kidneyrecipients. A total of 834 liver and 727 kidney transplants wereperformed during the study period. Seven hundred and eighty-onepatients did not receive ATG or alemtuzumab; 646 received 1 dose ofeither drug, and 134 patients received 2 doses of either drug.Thecumulative incidence of cryptococcosis was 0.26% (2/781) among thosewho did not receive ATG or alemtuzumab; 0.3% (2/646) among thosewho received only1dose, and 2.24% (3/134) among those who received 2doses (P 5 0.03).There were 5 cases of cryptococcosis in liver recipientsand 2 in kidney recipients.There were 3 cases of cryptococcalmeningitis, 3 of pneumonia, and 1 of disseminated disease.The 2kidney recipients had meningitis. Diagnosis occurred at a median of255 days (range 7^517 ) after transplantation.The mortality rate was14.2%.We conclude that the use of 1 dose of ATG or alemtuzumab is notassociated with an increased cumulative incidence of cryptococcosis,but that those patients receiving 2 doses are at increased risk.

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Many transplant centers have used rabbit anti-thymocyteglobulin (ATG;Thymoglobulins) and alemtuzumab (Cam-path-1Hs) for induction or preconditioning before organtransplantation or for the treatment of acute rejection. Rab-bit ATG is a polyclonal antibody, and alemtuzumab is ahumanized monoclonal antibody directed against CD52, acell surface antigen expressed on B and T lymphocytes,monocytes and NK-cells.The use of ATG and alemtuzumabas preconditioning may be associated with the develop-ment of partial tolerance, a lower incidence of rejection ep-isodes, and a reduced dependence on maintenanceimmunosuppression and steroids (1^11).

However, these products cause a profound lymphoiddepletion that canbe persistent and have an impact on the de-velopment of opportunistic infections. Immunologic recon-stitution studies performed in patients receiving ATG oralemtuzumab for the management of rheumatoid arthritis,B-cell chronic lymphocytic leukemia, bone marrow trans-plantation, and solid organ transplantation have demonstrat-ed that the most profound effects are on the CD41 Tlymphocyte subpopulation.The CD41 T lymphocyte countremains less than 50% of baseline at 12 months (1^6).Cryptococcosis is a significant opportunistic fungal in-

fection in immunocompromised patients, including organtransplant recipients and those with acquired immunode¢-ciency syndrome (AIDS). It has been shown to occur in0.26^5% of organ transplant recipients with an overall mor-tality rate of 25^50% (7^9). The primary risk factor for the

Copyright r Blackwell Munksgaard 2006

Transplant Infectious Disease . ISSN 1398-2273

Presented in part at the 45th Interscience Conference on Antimicrobial Manage-ment and Chemotherapy (ICAAC),Washington, DC, December 16^19, 2005.

Key words: cryptococcosis; anti-thymocyte globulin;alemtuzumab; organ transplantation

Correspondence to:David L. Paterson, Division of Infectious Diseases, 3601 FifthAvenue Suite 3A, Pittsburgh, PA 15213, USATel: 1 412 648-6401Fax: 1 412 648-6399E-mail: PatersonD@dom.pitt.edu

Received 24 September 2005, revised 21 December 2005, 12January 2006, accepted for publication 24 January 2006

DOI: 10.1111/j.1399-3062.2006.00149.xTranspl Infect Dis 2007: 9: 22^27

22

occurrence of cryptococcosis in organ transplant recipientsis presumably the immunosuppressive therapy used to pre-vent allograft rejection, especially steroids (10^13).Studies more than 30 years ago on renal transplant recip-

ients treatedwith equine anti-lymphocyte serum suggestedthat such patients had an increased risk of fungal infection,including cryptococcosis (14, 15).For these reasons, the aim of the current study is to deter-

mine the impact of the use of ATG and alemtuzumab on thecumulative incidence of cryptococcal infection amongliver and kidney transplant recipients at the University ofPittsburgh Medical Center.

Patients and methods

All patients who received a kidney or liver transplant at theUniversity of Pittsburgh Medical Center from October 1,2000 to January 31, 2005, were identi¢ed.The patients weredivided into 3 groups: 1) those who did not receive ATG oralemtuzumab, 2) those who received only 1 dose of ATGor alemtuzumab, and 3) those who received � 2 doses ofATG and/or alemtuzumab.The receipt of ATG or alemtuzumab as induction therapy

was determined by protocol. All kidney transplant recipi-ents received neither ATG nor alemtuzumab from October2000 to July 2001, ATG from July 2001 to October 2002, andalemtuzumab from October 2002 onwards. A second doseof ATG or alemtuzumab was only given as therapy for bi-opsy-con¢rmed acute cellular rejection, in patients unre-sponsive to 1 or more boluses of methylprednisolone. Thedoses of ATG and alemtuzumab were 5^10 mg/kg/doseand 30 mg/dose, respectively. In rare instances 60 mg dos-es of alemtuzumab were used.When used for precondition-ing, these medications were given pre- or intraoperatively,before implantation of the graft.Patients who did not receive ATG or alemtuzumab re-

ceived tacrolimus and steroids as maintenance immuno-suppression. Patients who received ATG or alemtuzumabreceived tacrolimus monotherapy.Tacrolimus troughs weregenerally maintained at 5^10 mcg/mL in all liver patients.All cases of cryptococcosis among these patients, that

occurred until January 31, 2005, were identi¢ed from thecomputerized microbiology database as a quality improve-ment project aimed at optimizing prophylaxis in transplantrecipients. The project was approved by the institution’sTotal Quality and Patient Safety Council.The diagnosis of cryptococcosis was made if there was a

positive cryptococcal antigen in the serum or cerebrospinal£uid (CSF) or a positive culture for Cryptococcus species.Disseminated cryptococcosis was de¢ned as the involve-ment of at least 2 organ systems.

The electronic medical records of the patients withcryptococcosis were reviewed and data was recorded. Dataavailable in the electronic medical records include demo-graphic information, admission history and physical ex-amination, dictated progress notes, laboratory andmicrobiology data, pathology reports, radiology reports,medications dispensed by pharmacy, operative reports,discharge summaries, and expiration summaries. The oc-currence of acute rejection and its treatment was also re-corded.Antifungal prophylaxis in kidney transplant recipients

was limited to oral nystatin. Prior to January 2004, livertransplant recipients at high risk for invasive candidiasisreceived £uconazole or low-dose conventional or lipidformulation amphotericin B. From January 2004, orally ad-ministered voriconazole (200 mg twice per day) was givento liver transplant recipients while they were in the trans-plant intensive care unit. All liver recipients received enter-al nystatin at 2 million units/day.Universal cytomegalovirus (CMV) prophylaxis with oral

ganciclovir or valganciclovir was administered to all kid-ney transplant recipients. Weekly CMV antigenemia wasmonitored in the liver transplant recipients and CMV infec-tion was treated preemptively.

Statistical analysis

Variables analyzed included age, gender, type of trans-plant, use of ATG or alemtuzumab, extent of cryptococcaldisease, time from transplant to cryptococcosis, time fromATG or alemtuzumab to cryptococcosis, and titer of serumand CSFcryptococcal antigen.Datawere analyzedwith SigmaStat (version 3.0) and Sta-

ta (version 9.0). Continuous data were presented as medianand range and categorical data as proportions.To compare the cumulative incidence of cryptococcosis be-

tween the 3 groups of patients, a 2 � 3 F|sher’s exact test wasused. A P-value less than 0.05 was considered significant.

Results

A total of 834 liver and 727 kidney transplants were per-formed at the University of Pittsburgh Medical Center be-tween October 1, 2000 and January 31, 2005. Seven hundredeighty-one patients did not receive ATG or alemtuzumab,646 patients received 1 dose of ATG or alemtuzumab, and134 patients received 2 doses of ATG and/or alemtuzumab.No patient received more than 2 doses of either ATG or

Silveira et al: Cryptococcosis after tolerogenic protocols

Transplant Infectious Disease 2007: 9: 22^27 23

alemtuzumab. Table 1 shows the distribution of patientsamong the 3 groups.The cumulative incidence of cryptococcal disease was

0.26% (2/781) among the patients who did not receive ATGor alemtuzumab, 0.3% (2/646) among those who receivedonly 1 dose of ATG or alemtuzumab, and 2.24% (3/134)among those who received 2 doses of ATG or alemtuzumab(P5 0.03). When we evaluated the liver transplant recipi-ents separately, the association between a higher degree ofimmunosuppression and a higher cumulative incidence ofcryptococcal infection continued to be observed: the cumu-lative incidence was 0.3% for the patients who did not re-ceive ATG or alemtuzumab, 1.22% for those who received asingle dose of ATG or alemtuzumab, and 3.5% for the pa-tients who received 2 doses of ATG and/or alemtuzumab(P5 0.04). The only renal transplant recipients who devel-oped cryptococcosiswere thosewho received 2 doses of alem-tuzumab.This is summarized inTable 2.There were 7 cases of cryptococcosis identi¢ed; 5 among

cadaveric liver recipients and 2 in living-related kidney re-cipients. One of the kidney recipients underwent pancreastransplantation followingkidney transplantation.The medi-an age of the patients was 53 (range 47^72) years, and 71.4%(5/7 ) were males. All patients lived in the same geographicarea (Western Pennsylvania,West V|rginia, and Ohio).The 2 patients who did not receive anti-lymphocyte ther-

apy were receiving steroids, with a mean daily dose ofprednisone of 13 mg. Only 1 of the 5 patients who receivedanti-lymphocyte therapy was on chronic steroid therapy,with a daily dose of 15 mg dose of prednisone.Three of 7 patients had cryptococcal meningitis, 3 of 7

had cryptococcal pneumonia, and 1 of 7 had disseminateddisease. The 2 kidney transplant recipients only had men-

ingitis, while pneumonia was the most common diseasemanifestation among the liver recipients, occurring in60% of the liver recipients with cryptococcosis.Only 2 of 7 patients had an episode of acute rejection

within the 12 months preceding the diagnosis of cryp-tococcosis. One of these patients received ATG as inductionimmunosuppression followed by alemtuzumab for thetreatment of acute rejection. Alemtuzumab therapy waspreceded by a pulse of methylprednisolone. The other re-ceived alemtuzumab for induction immunosuppressionand later for the treatment of acute rejection.Systemic antifungal prophylaxis was administered to

only 1 liver recipient following transplantation, and it con-sisted of voriconazole for a total of 4 days, immediately af-ter surgery. None of the patients were receiving systemicantifungal prophylaxis at the time of diagnosis. Two pa-tients were on oral nystatin at the time of diagnosis.The CD4 1 T-cell count was available for only 1 patient,

who had received 2 doses of alemtuzumab. F|fty-four daysafter his last dose of alemtuzumab, when cryptococcal dis-ease was diagnosed, his CD4 1 T-cell count was only 8.Serum cryptococcal antigen was performed in 5 of 7 pa-

tients, with the exception of 1patient with disseminated dis-ease and another with cryptococcal meningitis. It waspositive in 3 of 5 patients. Only 1 of 3 patients with cryp-tococcal pneumonia had a positive result. The serum cryp-tococcal antigen was positive in the 2 patients withcryptococcal meningitis who had it performed, althoughthe titers were low, 1:4 and 1:32. CSF cryptococcal anti-gen was performed in 5 patients, with the exception of1 patient with disseminated disease and another with crypt-ococcal pneumonia. It was positive in all 3 patients withcryptococcal meningitis, with titers of 1:8, 1:256, and 1:1024.The diagnosis of cryptococcal disease was made at a me-

dian of 204 (range 7^517 ) days after transplantation and ata median of 204 (range 54^268) days after the last dose ofATG or alemtuzumab. Table 3 shows the characteristics ofthe cryptococcosis episodes.All the patients with cryptococcal meningitis were start-

ed on therapy with amphotericin B lipid complex and 5-£u-cytosine. The patients with cryptococcal pneumonia weretreated with £uconazole. The patient with disseminateddisease died before any therapy could be initiated. One ofthe patients with cryptococcal meningitis required place-ment of aventriculo^peritoneal shunt. Adverse events withantifungal therapy were observed in a single patient beingtreated for cryptococcal meningitis with amphotericin Blipid complex and 5-£ucytosine. He developed acute renalfailure caused by the amphotericin B lipid complex as wellas neutropenia and thrombocytopenia induced by the 5 -£ucytosine. The survival rate was 86% (6/7 ). Only thepatient with disseminated disease died. Death occurred be-fore the patient could be started on appropriate therapy.

Receipt of anti-thymocyte globulin or alemtuzumab

Type oftransplant

Number ofpatients

No doses(%)

1 dose(%)

2 doses(%)

Liver 834 643 (77.1) 163 (19.5) 28 (3.4)

Kidney 727 138 (19) 483 (66.4) 106 (14.6)

Total 1561 781 (50) 646 (41.4) 134 (8.6)

Table1

Incidence of cryptococcosis based on the degree of immunosuppression

Type of transplant No doses 1 dose 2 doses

Liver 2/643 (0.3%) 2/163 (1.22%) 1/28 (3.5%)

Kidney 0/138 0/483 2/106 (1.9%)

Total 2/781 (0.26%) 2/646 (0.3%) 3/134 (2.2%)

Table 2

Silveira et al: Cryptococcosis after tolerogenic protocols

24 Transplant Infectious Disease 2007: 9: 22^27

Discussion

Cryptococcus infection results from inhalation of the organ-ism from an environmental source. The initial pulmonaryinfection, usually asymptomatic, is contained by gran-ulomatous in£ammation. The prevalence of Cryptococcusneoformans infection is believed to be high, but cryp-tococcosis as a disease is rare in individuals withoutimpaired immunity, and probably occurs because of reacti-vation rather than primary exposure. Host defense againstCryptococcus is primarily regulated by cell-mediated immu-nity, with CD4 1 T lymphocyte cells being the major e¡ec-tor cells (16).The prevalence of cryptococcosis in AIDS patients has

been reported to be 2.9^13.3% (16^18). The majority ofpatients, 66^85%, present with cryptococcal meningitis(18^20). Risk factors for development of cryptococcal infec-tion have been well studied in this population and a clearcorrelation between CD4 1 T-cell count and the risk ofcryptococcosis has been demonstrated. The risk of cryp-tococcosis is inversely proportional to the CD4 1 T-cellcount, and is greatest at CD4 1 T-cell counts lower than100 (21, 22).The major risk factor for the development of cryp-

tococcosis in transplant patients is the immunosuppres-sion used to prevent allograft rejection (10, 11). The use ofimmunosuppressive protocols that include a precondition-ing regimen with ATG or alemtuzumab have allowed theuse of minimal doses of maintenance immunosuppressionpost transplantationwith steroid avoidance (23, 24). Howev-er, therapy with ATG or alemtuzumab causes a profounddegree of lymphoid depletion, which can last for greaterthan 12 months. A multicenter study evaluating immunecell function testing, measured CD4 1 T-cell counts in theblood of kidney, liver, and pancreas recipientswhowere lessthan 5 years post transplant (25). Some of these patients re-

ceived lymphoid-depleting protocols. In general, the meanCD4 1 T-cell count was 59% of the mean of the healthyadult control population. CD4 1 T-cell counts as low as 10were observed. Studies on the immunologic effects of ale-mtuzumab have shown that the CD4 1 T cells are thelymphocyte subpopulation most severely affected; countsmay be lower than 25% of baseline at 9 months and lowerthan 50% of baseline at 12 months (1^6).Therefore, one canhypothesize that the use of ATG and alemtuzumabmay im-pact the prevalence of cryptococcosis.Alemtuzumab is approved in the United States for thera-

py of B-cell chronic lymphocytic leukemia, but has beenused in other settings including management of other he-matologic malignancies, autoimmune diseases, and organtransplantation. Reported infectious complications associ-ated with its use include reactivation of hepatitis B (26),CMV (27), molluscum contagiosum (28), adenovirus (29),parvovirus B19 (30), and systemic Mycobacterium bovis in-fection (31). A study of fungal infections among pancreasrecipients who received alemtuzumab identi¢ed 3 cases ofcryptococcosis in 121 (2.4%) patients (32). The authors didnot comment on the prevalence rate of cryptococcosis in therecipients who were not treated with alemtuzumab. Recent-ly, a 5-year single-center study of alemtuzumab as inductionin renal transplantation did not show a difference in the in-cidence of fungal infections between the group that receivedalemtuzumab and a contemporaneous control group thatreceived conventional triple immunosuppression (33).However, the number of patients in the study wassmall and therefore the study lacked power to detect such adifference.We observed an increase in the cumulative incidence of

cryptococcosis that was associated with the amount ofATG or alemtuzumab received. There was a significantdifference between the patients who either did notreceive ATG or alemtuzumab or who received only 1 dose,

Characteristics of cryptococcosis episodes

Patient TransplantAnti-thymocyte globulin(ATG) or alemtuzumab Disease site Antigen1

Transplant todiagnosis (days) Outcome

1 Liver No Pneumonia Negative 7 Alive

2 Liver No Disseminated Not done 321 Died

3 Liver ATG and alemtuzumab Pneumonia 1:64 517 Alive

4 Liver 1 dose alemtuzumab Meningitis 1:4 204 Alive

5 Liver 1 dose alemtuzumab Pneumonia Negative 200 Alive

6 Kidney 2 doses alemtuzumab Meningitis Not done 482 Alive

7 Kidney 2 doses alemtuzumab2 Meningitis 1:32 54 Alive

1Serum cryptococcal antigen.2Alemtuzumab as induction for kidney and later for pancreas transplantation.

Table 3

Silveira et al: Cryptococcosis after tolerogenic protocols

Transplant Infectious Disease 2007: 9: 22^27 25

compared with those who received 2 doses. This associa-tion remained when the liver recipients were evaluated sep-arately. A prior study from our institution on the incidenceof cryptococcal disease in liver transplant recipients beforethe use of lymphoid-depleting protocols, identi¢ed an inci-dence of 0.26%, which is similar to the cumulative inci-dence found among our patients who did not receive ATGor alemtuzumab (34). Seventy percent of the cases were ofisolated cryptococcal meningitis.We observed a lower prev-alence of cryptococcosis when compared with most previ-ously reported studies. A possible explanation for this isthat the prevalence of cryptococcosis in any immunosup-pressed population may be dependent on the duration offollow-up. Our patients were followed for a maximum of4 years post transplant. Additionally, we observed a lowermortality rate compared with previous studies, perhaps re-£ecting a greater experience with the disease, resulting inearlier diagnosis and better supportive care.In our study, cryptococcal pneumonia was the most com-

mon disease manifestation among liver transplant recipi-ents, occurring in 3 out of 7 patients with cryptococcosis.Earlier studies, done in patients receiving tacrolimusand steroids, reported an incidence of 6% for isolatedcryptococcal pneumonia (9, 35). Meningitis was the onlymanifestation among the kidney recipients. This is inagreement with prior studies, which showed that men-ingitis is the most common manifestation of cryptococcalinfection in kidney transplant recipients (36, 37 ). However,in our center, from 1990 to 2000, the most common presen-tation of cryptococcal infection was disseminated disease(7 ).We saw no cutaneous disease due to Cryptococcus.Cryptococcal infection occured late, usually 6 months

or longer after transplantation. Receiving ATG or alem-tuzumab did not alter the time to development ofcryptococcosis. The median time to the diagnosis ofcryptococcosis from transplantation was 204 days.Only 1/3 of the patients with cryptococcal pneumonia

had a positive serum cryptococcal antigen. Although thestudy was not meant to evaluate the usefulness of serumcryptococcal antigen in the diagnosis of cryptococcosis in or-gan transplant recipients, these data recon¢rm that serumcryptococcal antigen is not a reliable test to exclude the diag-nosis of isolated pulmonary disease. Cryptococcal pneumo-nia cannot be ruled out in the face of a negative serumcryptococcal antigen (7, 38). Invasive diagnostic proceduresare often necessary in order to make a diagnosis. In ourstudy, this test was negative even in a patient who wasfungemic.To the best of our knowledge, until now there have been

no reports of an increased cumulative incidence of cryp-tococcal disease following ATG or alemtuzumab therapy.We conclude that 2 doses of ATG or alemtuzumab appearto be a risk factor for the development of cryptococcal infec-

tion in organ transplant recipients. If antifungal prophy-laxis was 100% successful, it would be necessary to have42 patients on long-term prophylaxis in order to prevent 1infection among liver or kidney transplant recipients whoreceive at least 2 doses of ATG or alemtuzumab. Althoughthis number needed to treat seems small, considering thatinfection occurred late after transplantation, and the re-sponse to therapy was adequate, with a low rate of adverseevents, it does not seem that antifungal prophylaxis is nec-essarily warranted. It would probably add a considerableamount of cost without a significant increment in bene¢t.However, prospective multicenter studies with systematicmonitoring of CD4 1 T-cell counts may be useful in orderto clarify this question.

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