Bone Marrow Transplantation (2004) 33, 425–430 & 2004 Nature Publishing Group All rights reserved 0268-3369/04 $25.00 www.nature.com/bmt

Viral infections following T-cell depleted allogeneic transplants in adults

S Chakrabarti1, H Osman2, KE Collingham2, CD Fegan1 and DW Milligan1

1Departments of Haematology, Birmingham Heartlands Hospital, Birmingham, UK; and 2Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham, UK

Summary: kingdom, 40 366 confirmed enterovirus isolates were reported in a 20-year period (1975–1994), with 60% of Anecdotally, have been reported to cause the isolates being from children less than 5 years old.3 serious complications post BMT, but the exact impact of Given the fact that patients undergoing allogeneic stem cell these in the post transplant period has not been transplantation are deficient in both cellular and humoral reported. We prospectively evaluated stool, urine and immunity for a prolonged period and are susceptible to a throat samples for enteroviruses by viral culture together number of endogenous and exogenous viral infections with relevant body fluids by RT-PCR in 64 allograft during this period, there is scanty data on the incidence of recipients receiving grafts T-cell depleted by Campath- enterovirus infections in this setting. Anecdotal reports 1H, following both conventional and reduced-intensity have implicated that enteroviruses might be responsible for conditioning. Seven patients (10.4%) developed nine serious complications in the post transplant period.4–8 Some episodes of enterovirus infections at a median of 146 days stool surveillance studies have reported on the isolation of post transplant. Four episodes were associated with these viruses in post transplant patients with diarrhoea, but symptomatic illnesses, which could be attributable to none have investigated the incidence and outcome of enteroviruses. There was no mortality directly related to enterovirus infections in a comprehensive manner.9–11 T- enteroviruses. There was no correlation between dose and cell depletion of the graft results in an increase in the mode of Campath-1H use, lymphocyte recovery, IgG and incidence of several viral infections, but whether the same is IgA levels and enterovirus isolation. Although entero- true for enterovirus infections is not known. viruses tended to be more frequent in TBI-based conven- We report the incidence and outcome of enterovirus tional conditioning recipients, the only significant risk infections with respect to the immune reconstitution in 64 factor for enterovirus was unrelated donor graft. adult patients receiving T-cell-depleted grafts following both The low incidence of the severe enterovirus infections conventional and reduced-intensity conditioning (RIC). could have been related to a low lymphocyte count in this cohort in the absence of GVHD, particularly CD4 þ count, which has been implicated in tissue damage in Patients and methods experimental animals. Further studies are needed to define its impact in different allograft settings. We evaluated 64 T-cell-depleted allograft recipients, who Bone Marrow Transplantation (2004) 33, 425–430. were treated in the BMT unit at Birmingham Heartlands doi:10.1038/sj.bmt.1704359 Hospital between May 1997 and September 2001, for Published online 22 December 2003 evidence of enterovirus infections. All the patients were Keywords: enterovirus; stem cell transplantation; T-cell nursed in single rooms with high-efficiency particulate air depletion (HEPA)filters.

Conditioning treatment and T-cell depletion Enteroviruses are small RNA viruses belonging to the Conventional conditioning consisted of cyclophosphamide picornaviradae family with 67 distinct serotypes, causing a or etoposide and total body irradiation or busulphan. The wide range of clinical symptoms.1 The annual prevalence of RIC schedule consisted of fludarabine and melphalan. T- symptomatic enteroviral infections in United States is cell depletion was carried out with Campath (anti-CD52) estimated to be about 10–15 million.2 In the United antibodies (Therapeutic Antibody Centre, Oxford) in vivo or in vitro. All patients also received cyclosporin A as graft- versus-host disease (GVHD)prophylaxis from day À1.

Correspondence: Dr S Chakrabarti, Department of Haematology, University Hospital Birmingham, Edgbaston, Birmingham B15 2TH, Supportive care UK; E-mail: [email protected] Received 20 August 2003; accepted 24 September 2003 Antimicrobial prophylaxis consisted of oral fluconazole, Published online 22 December 2003 ciprofloxacin and oral aciclovir from the beginning of Enterovirus infection in transplant recipients S Chakrabarti et al 426 conditioning treatment until engraftment. Oral cotrimox- The absolute lymphocyte count (ALC)was noted at least azole was initiated when the neutrophil count was greater once in every 4 weeks following discharge for the first 6 than 1.0 Â 109/l. Metronidazole 400 mg thrice daily was months. ALC was also recorded at the first detection of an started from the day of transplant to engraftment for enterovirus isolate. The CD4 þ and CD8 þ T-cell counts anaerobic decontamination, as a part of GVHD prophy- (since August 1999)were measured every 6–8 weeks post laxis. transplant. Immunoglobulin levels (IgG and IgA)were also Patients at risk of CMV disease (recipient or donor measured every 3 months for the first year following the positive for CMV IgG)received pre-emptive therapy transplantation. For statistical analysis, the median with ganciclovir and/or foscarnet, based on a PCR assay.9 CD4 þ , ALC, IgG and IgA levels between 100 and 180 CMV sero-negative patients received blood products days post transplant were calculated, if more than one set from CMV sero-negative donors. None of the patients of values were available during that period. received prophylactic intravenous immunoglobulins (IVIg). Statistical methods

Study design Univariate P-values and odds ratios were calculated from 2 Â 2 contingency tables using Epi info version 6 (CDC, Stool, urine and throat samples were examined by Atlanta). The continuous variables were compared using culture from all patients prior to transplant, and weekly to Mann–Whitney’s nonparametric method. The probability of fortnightly thereafter to 180 days post transplant. In various events was examined by the Kaplan–Meier method addition, the stool and urine samples were also examined and the groups were compared using the log-rank test. by electron microscopy. After 180 days post transplant, only relevant samples from symptomatic patients were examined. Tissue samples from liver, lungs or gut obtained Results ante or postmortem were examined by EM and culture. Incidence of enterovirus infection Laboratory methods. For EM, a 50% v/v suspension of each faecal sample was prepared in phosphate-buffered Enteroviruses were isolated in seven out of 64 patients saline, and placed on EM grids with strengthened formvar (10.4%)at a median of 146 days (range 25–796),with an membranes. The grids were stained with 2.5% phospho- actuarial probability of 14% (95% CI 4–24). Two patients tungstic acid and examined using a Jeol 100CX electron had multiple episodes of enterovirus infection. Coxsackie B microscope with an accelerating voltage of 80 kV. For was isolated in three episodes with type 1 and an culture, 0.2 ml of the supernatant prepared from centrifu- echovirus in one episode each. The enteroviruses isolated gation of a 10% v/v suspension of faeces or urine in virus from the other four episodes were not serotyped. Entero- transport medium was added to monolayers of Rhesus viruses were isolated from the throat sample in five episodes monkey kidney and Hep 2 and human fibroblast cell lines. and from stool in the other four. The median duration of The cells were incubated at 331C for 14 days and examined virus excretion was 2 weeks (range 1–4 weeks). The thrice weekly for cytopathic effects. Enteroviruses were characteristics of the patients developing enterovirus typed using pooled fluorescein-conjugated monoclonal infections have been detailed in Table 1. antibodies (Chemicon International Inc., Temecula, CA, USA). In addition, cerebrospinal fluid and other relevant Outcome of enterovirus infections sterile body fluid samples were examined by an RT-PCR- based assay for the presence of enterovirus, as previously Symptomatic illness attributable to enteroviruses was described.12 documented in four patients (Table 2). All had upper Specimens for bacteriological and fungal investigations respiratory illness with systemic symptoms. One patient were processed using standard methods of microscopy, (patient 6)had protracted , weakness and fatigue culture and sensitivity testing. following an enterovirus infection for which no other cause Isolation of enteroviruses from any surveillance site was was identified. Patient 3 had coxsackie B virus isolated termed as enterovirus infection. Enterovirus disease was from a sputum sample during a respiratory illness, which defined as definite on demonstration of enteroviruses from progressed to severe pneumonia, and CMV was subse- a tissue site, and/or detection in the CSF or other sterile quently detected in a bronchial lavage sample by PCR. body fluids with clinical illness attributable to enteroviruses CMV was not detected in blood samples and tissue in the absence of other . If the viruses were specimens were not available. This patient improved isolated from surveillance samples only, in the presence of a following mechanical ventilation, IVIg and ganciclovir clinical illness explainable by enterovirus infections and not therapy. Another patient (patient 2)had an enterovirus attributable to any other , this was defined as isolated from stool sample with concurrent gut GVHD, 7 probable enterovirus disease. Documented infections at days before she succumbed to a fungal pneumonia. She other sites were also noted. Co-pathogen was defined as also had an episode of seizure at the same time, but the any organism isolated from within 2 weeks before or after CSF was negative for enteroviruses by RT-PCR and detection of enteroviruses or onset of enterovirus-asso- culture. Isolation of enteroviruses from the stool was not ciated symptoms. GVHD was graded according to associated with diarrhoeal illness in any of the other standard criteria. patients. Thus, four episodes of probable enterovirus

Bone Marrow Transplantation Enterovirus infection in transplant recipients S Chakrabarti et al 427 Table 1 Characteristics of patients with and without enterovirus CD4 þ T-cell count and the IgG and IgA levels between infections 100 and 180 days post transplant were similar in patients Patients with Patients P-value* with or without enterovirus infections. However, it is worth enteroviruses without noting that the mean IgG levels were in the normal range (n ¼ 7) enteroviruses and the IgA levels were low normal in both groups. (n ¼ 57) Lymphocytopenia, both absolute and CD4 þ T cells, was Median age(range)33.7 (20–52)38(18–57)0.9 documented in both groups. Gender (male/female)5/2 34/23 0.7 Risk factors for enterovirus infections Diagnosis Acute/chronic leukaemia 3/3 28/15 There was no influence of patient age, gender or underlying Lymphoma/myeloma 1/0 12/2 0.8 disease on the isolation of enteroviruses. In addition, there Donor (unrelated/family)5/2 19/38 0.09 was no impact of the dose and mode of the use of Campath-1H on the isolation of enteroviruses, with 3/33 Conditioning patients receiving Campath-1H in vitro developing enter- Conv/Nonmyelo 6/1 35/22 0.2 ovirus infections, compared to 4/31 in those receiving TBI 6 31 0.18 Campath-1H in vivo (Table 1). Campath-1H Patients undergoing unrelated donor transplantation In vitro/in vivo 34 30/27 0.7 were at a greater risk of enterovirus infections (5/24, probability 28%, 95% CI 6–50)than related donors (2/40, CMV seropositive (yes/no)2/5 33/24 0.2 probability 6.9%, 95% CI 0–14, log rank P ¼ 0.02, CMV reactivation/disease 1/0 16/4 0.7 Figure 1). Enteroviruses tended to be more frequent in patients undergoing conventional conditioning (6/41 vs 1/ GVHD 23 in patients receiving RIC, P ¼ 0.2). Among the recipients Acute 2–4 2 16 0.7 of conventional conditioning, enteroviruses were isolated Chronic 3 1 0.1 only in patients receiving TBI. Overall, there was a similar Neutrophil40.5 Â 109/l 14.5 13.5 0.7 trend towards more enterovirus infections in patients (days post transplant) receiving TBI (6/37, compared to 1/27 in patients receiving non-TBI-based conditioning, P ¼ 0.18). Non-CMV virus 6 34 0.2 Infection conv ¼ conventional, nonmyelo ¼ nonmyeloablative, cmv ¼ cytomegalo- Discussion virus, GVHD ¼ graft-versus-host disease. 2 *w and Fisher’s exact tests were used for categorical variables and Mann– Enteroviruses have been reported to cause fatal complica- Whitney nonparametric tests for continuous variables. tions following allogeneic transplantation.4–9 However, the true impact of infections with enteroviruses in this setting can be estimated only by prospective surveillance studies. disease were documented without any mortality directly Several stool surveillance studies have reported on the attributable to enteroviruses. The only patient (patient 4) isolation of enteroviruses and often implicated them as who had asymptomatic poliovirus type 1 infection was possible pathogens or co-pathogens in diarrhoeal or reported to be in contact with his child, who was receiving respiratory illnesses.9–11 It is worth noting that although live vaccination during that period. Four of the seven enteroviruses are usually transmitted via the faeco-oral patients had other viruses isolated at different time-points route, transmission via respiratory route is also possible.1 post transplant and not as a co-pathogen with enteroviruses Hence, a comprehensive surveillance should include throat (Table 2). Patient 2 had CMV isolated concomitantly as specimens and blood or body fluids from symptomatic sites described above, and patient 6 had HSV isolated from oral as well, as was carried out in this study. swabs during the second episode of enterovirus infection. The overall incidence of enterovirus infections was 10% The nonrelapse mortality and overall survival at 3 years in this adult cohort of T-cell-depleted transplant recipients. among patients developing enterovirus infections were 28.6 Four of the seven infected patients had symptomatic and 42.8%, respectively, compared to 23.7 and 60.8% in infections or probable enterovirus disease. All had upper those not developing these infections (P-values ¼ 0.8 and respiratory illnesses, which were often protracted. One 0.5, respectively). patient experienced prolonged myalgia and fatigue even after cessation of viral isolation, very similar to the chronic Relationship to immune reconstitution fatigue syndrome described following enterovirus infec- tions.1 Coxsackie B virus was isolated from the sputum The ALC was available in all patients in both groups alive sample of one patient who progressed to serious but beyond 100 days. The other immunological parameters nonfatal pneumonic illness, who subsequently had CMV were available in five patients with enterovirus infections isolated from bronchial lavage sample. The exact contribu- and 45 patients without enterovirus infection. The relation- tion of coxsackie B virus and CMV in the genesis of ship between immune reconstitution and development of pneumonia was not certain. Although the stool surveillance enterovirus infections is shown in Table 3. The ALC, studies have often implicated enteroviruses, namely

Bone Marrow Transplantation Enterovirus infection in transplant recipients S Chakrabarti et al 428 Table 2 Characteristics and outcome of patients with enterovirus infections

UPN Age/sex Type of Campath Enteroviruses Onset of Sites GVHD Enterodisease Other virus infection Outcome of transplant dose infection grade enterovirus (days) infection

1 33/M UD CMT 100 mg Coxsackie B 28 Stool 0 None Adeno*,RSV*, Infl* Died- adeno 2 18/ F UD- CMT 100 mg Enterovirus 145 Stool 3 None HSV* Died-fungal pneumonia 3 43/M UD- CMT 100 mg Coxsackie B 505 Sputum 0 Pneumonia CMV Improved (probable) 4 52/M UD- NMT 100 mg Poliovirus 1 28 Throat 0 None Adeno* Improved Echovirus 231 Throat 0 URI Improved 5 46/M MSD CMT 20 mg Enterovirus 146 Stool 0 None Adeno* Improved 6 26/M UD NMT 20 mg Enterovirus 73 Throat 0 URI, chronic Adeno*, PIV* Improved fatigue Enterovirus 796 2 None HSV (post-DLI) 7 33/F MSD CMT 20 mg Coxsackie B 464 Throat 0 URI None Improved

M ¼ male; F ¼ female; MSD ¼ matched sibling donor; UD ¼ unrelated donor; CMT ¼ conventional myeloablative transplant; NMT ¼ nonmyeloablative transplant; DLI ¼ donor lymphocyte infusions; PCRprob ¼ probable; HSV ¼ ; PIV ¼ parainfluenza virus; RSV ¼ respiratory syncytial virus; Infl ¼ influenza. *The viruses were not isolated as co-pathogens, but occurred at different time-points post transplant.

Table 3 Immune reconstitution and enterovirus infections isolated from stool samples during a diarrhoeal episode had GVHD of the gut as well and the role of enterovirus in Patients with Patients without 13 enterovirus infections enterovirus infections causing diarrhoea was not clear. The more serious (mean7standard (mean7standard complications of enterovirus infections, such as CNS and deviation) deviation) cardiac involvement, were not documented in any of the patients. All the CSF, pericardial and pleural fluids Absolute lymphocyte 6587545 6397412 count ( Â 109/l) obtained during this period were subjected to viral culture CD4+ T-cell count 1607110 164795 and RT-PCR, but no enteroviruses were detected. ( Â 109/l) Enteroviruses are generally community-acquired and, Serum IgG (g/l)7.9 73.2 12.179.4 although persistence of viral RNA has been demonstrated 7 7 Serum IgA (g/l)0.9 0.1 0.8 0.2 in chronic medical conditions,14 there is no evidence to *The measurements are the median value of estimations done between day suggest that these viruses can establish latency such as 100 and 180 post transplant. The differences between the two groups were CMV or EBV to reactivate during severe immunosuppres- not significant (P40.05)by Mann–Whitney nonparametric test. sion.14–16 Enteroviruses have been reported to cause protracted and often fatal illnesses in agammaglobulinemic individuals, the humoral immunity being serotype-speci- 1.0 fic.1,16 The role of cellular immunity in enterovirus

0.9 infections is not defined, but might play a role in the containment of the virus during early phase of the 0.8 infection.17 Unlike most other viral infections we have 0.7 previously reported, where correlations between the viral infection and the degree of T-cell depletion and lymphocyte 0.6 P=0.02 reconstitution were established,18–21 we did not find any 0.5 correlation between the extent of T-cell depletion, total and CD4 þ lymphocyte recovery and immunoglobulin subtype 0.4 unrelated donor levels and the occurrence of enterovirus infections. We 0.3 identified unrelated donor transplantation as a risk factor

0.2 for enterovirus infections. Immune reconstitution has been

Probability of enterovirus infection reported to be delayed following T-cell-depleted transplan- 0.1 related donor tation from matched unrelated graft compared to matched 22 0.0 sibling grafts, and could account for this association. 0 100 200 300 400 500 600 700 800 900 1000 However, this needs to be confirmed in larger cohorts. Days post-transplant There was a trend towards less enterovirus infections in Figure 1 The incidence of enterovirus infection following T-cell-depleted patients receiving RIC. Although six out of seven allograft in relation to the donor type (related vs unrelated). enterovirus infections were documented in patients receiv- ing TBI, the study probably lacked the statistical power to identify the association between conditioning therapy coxsackie and echoviruses as a causative agent for and post transplant enterovirus infection. In animal post transplant diarrhoea, we did not find such an models, TBI has been associated with impaired humoral association.8–11 The only patient who had enterovirus immune response possibly related partly to functional

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