Human Herpesvirus 6 in Bronchalveolar Lavage Fluid after Lung Transplantation: A Risk Factor for...

transcript

American Journal of Transplantation 2005; 5: 2982–2991Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2005

doi: 10.1111/j.1600-6143.2005.01103.x

Human Herpesvirus 6 in Bronchalveolar LavageFluid after Lung Transplantation: A Risk Factorfor Bronchiolitis Obliterans Syndrome?

C. Neurohra, P. Huppmanna, H. Leuchtea,

M. Schwaiblmaira, I. Bittmannb, G. Jaegerc,

R. Hatzd, L. Freye, P. Uberfuhrf, B. Reichartf

and J. Behra,∗ for the Munich Lung Transplant

aDepartment of Internal Medicine I, Division of PulmonaryDiseases, Klinikum Grosshadern, bInstitute of Pathology,cDepartment of Virology, Max von Pettenkofer Institutefor Hygiene and Microbiology, dDepartment of Surgeryand Thoracic Surgery, Klinikum Grosshadern,eDepartment of Anesthesiology, Klinikum Grosshadern,fDepartment of Cardiac Surgery, Klinikum Grosshadern,Ludwig-Maximilians University, Munich, Germany∗Corresponding author: Juergen Behr,juergen.behr@med.uni-muenchen.de

Bronchiolitis obliterans syndrome (BOS) is the limit-ing factor to long-term survival after lung transplan-tation. Previous studies suggested respiratory viraltract infections are associated with the development ofBOS. To identify the impact of virus detection in bron-choalveolar lavage (BAL) fluid, we analyzed BAL sam-ples from 87 consecutive lung transplant recipients forhuman herpesvirus (HHV)-6, Epstein-Barr virus, Her-pes simplex virus 1/2, Cytomegalovirus, respiratorysyncytical virus and adenovirus by PCR. Acute rejec-tion, BOS and death were recorded for a mean follow-up time of 3.27 ± 0.47 years. Results of PCR analysisand other potential risk factors were entered into aCox regression analysis of BOS predictors and death.Only acute rejection was a distinct risk factor for BOSof all stages, death and death from BOS. HHV-6 wasdetected in 20 patients. Univariate and multivariateanalysis revealed that HHV-6 was associated with anincreased risk to develop BOS ≥ stage 1 and death,separate from the risk attributable to acute rejection.Identification of HHV-6 DNA in BAL fluid is a potentialrisk factor for BOS. Our results warrant further studiesto elucidate a possible causal link between HHV-6 andBOS.

Key words: Bronchalveolar lavage, bronchiolitis oblit-erans, human herpesvirus 6, lung transplantation,virus infection

Received 7 April 2005, revised 13 July 2005 and ac-cepted for publication 6 August 2005

Introduction

Lung transplantation (LTx) has become an established ther-apeutic option for patients with end-stage lung disease.However infections and obliterative bronchiolitis (OB) re-main the leading causes of limited survival rates comparedto those rates achieved after the transplantation of othersolid organs (1). The clinical description of OB has beentermed bronchiolitis obliterans syndrome (BOS) and is de-fined as graft deterioration by persistent airflow obstructionnot due to confounding conditions (2). Although the ex-act pathogenesis of OB remains unknown, studies indicatethat it begins with epithelial injury of the airways followedby inflammatory reaction, a condition that can be inducedby a variety of factors including viral infections (3, 4). Sev-eral studies have suggested that respiratory viral infectionsincluding Cytomegalovirus (CMV) pneumonia are associ-ated with higher mortality and increased rates of BOS (5,6, 7). In pediatric LTx patients, adenovirus identification intransplanted lungs was associated with respiratory failureand chronic graft loss (8). Similarly, an analysis of adult lungtransplant recipients revealed that patients with respiratorysyncytical virus (RSV) infections are predisposed to high-grade BOS (9). Human herpesvirus (HHV)-6 is increasinglyrecognized as a frequent pathogen after bone-marrow andsolid organ transplantation (10, 11). In a longitudinal study,Jacobs et al. reported a higher mortality rate in patientswith HHV-6 infection early after LTx (12). Epstein-Barr virus(EBV) infection has been implicated as a major risk fac-tor for post-transplant lymphoproliferative disease associ-ated with increased rates of graft failure (13, 14). Althoughherpes simplex virus (HSV) pneumonia is a potential se-vere complication of the early post-transplant period, littleis known about the impact of HSV on long-term survivaland BOS (4, 15).

Polymerase chain reaction (PCR) analysis provides a highlyspecific and sensitive method for viral detection. However,the clinical significance of positive PCR results from bron-choalveolar lavage (BAL) fluid has thus far been controver-sial (16, 17). Especially the contribution of viral pathogensin BAL of lung transplant recipients to long-term graft dete-rioration and survival is still unclear. The aim of the presentstudy was to assess the incidence of CMV, RSV, aden-ovirus, HHV-6, HSV1/2 and EBV in BAL specimen detected

HHV-6 in BAL After Lung Transplantation: A BOS Risk Factor?

by PCR after LTx and to investigate a potential impact onBOS and death.

Methods

Study design

From May 1997 through April 2003, we performed analyses for virus detec-tion by PCR in BAL samples from 87 consecutive lung transplant recipientswho underwent diagnostic bronchoscopy and who were in BOS stage 0.The study was in accordance with recommendations of the local board onmedical ethics at Ludwig-Maximilians-University of Munich.

Follow-up data was collected through June 1, 2004. Date of transplanta-tion, recipient gender, sex, age at transplantation, underlying lung disease,donor organ ischemic time, human leukocyte antigen (HLA) data, serologicCMV donor/recipient status, bronchoscopy and transbronchial biopsy re-sults, pulmonary function test data, immunosuppressive protocol, survivalstatus and cause of death were obtained from medical records and com-puterized databases maintained by physicians and transplant coordinatorsof the Munich Lung Transplant Group.

Standard care of lung transplant patients

University of Munich lung transplant recipient were maintained on tripleimmunosuppression with corticosteroids, tacrolimus or cyclopsorine, andmycophenolate mofetil or azathioprine (18, 19). In cases of drug intolerance,toxicity or recurrent acute rejection, a switch to the alternative immunosup-presion regime was accomplished. Medication dose was adjusted accord-ing to trough levels and immunosuppression was gradually lowered at 12months post-operatively in the absence of recent graft rejection. Donor-positive/recipient-negative LTx patients received oral CMV prophylaxis withganciclovir (1000 mg tid, dose was adjusted for renal impairment) for a pe-riod of 3 months. In all other cases, a viral prophylaxis with acyclovir wasadministered for 3 months. In addition, pre-emptive therapy with ganci-clovir and/or immune globulin was initiated based on positive CMV antigen-emia (monitoring frequency: post-transplant once weekly for 3 months andonce monthly thereafter, COBAS Amplicor CMV Monitor® Test, Hoffman-LaRoche AG).

During the first 3 months after transplantation, patients underwent atleast two bronchoscopies with BAL and transbronchial biopsy. Further-more, clinically-indicated bronchoscopies were conducted for new respira-tory symptoms (e.g. shortness of breath, new radiographic findings, >10%decline from baseline forced expiratory volume in 1 second (FEV1) and hy-poxemia). After the first 3 months, only clinically-indicated and follow-upbronchoscopies to monitor for treatment response were performed. Tissueand BAL fluid from bronchoscopies were analyzed for evidence of rejec-tion and viral, bacterial and fungal infections. Immunosuppression was notaltered and antiviral therapy was not administered based on viral BAL find-ings. In case of diagnosis of acute rejection, bolus treatment with methyl-prednisolone was administered at a dose of 500 mg/day for 3 consecutivedays.

Definitions

Post-transplant baseline FEV1 and the diagnosis of BOS were establishedusing the International Society of Heart and Lung Transplantation definitions(2). Acute rejection was diagnosed using standard histologic criteria accord-ing to the Lung Rejection Study Group (20). Any transbronchial biopsy speci-men with grade of acute rejection ≥A2 was considered positive and treatedin our center. In case of acute rejection A = 1, decision to therapy was basedon clinical status. Donor organ ischemic time for bilateral transplants wasthe mean of right and left lung ischemic times. HLA-A, -B and -DR antigen

mismatches between donor and recipient were determined according tothe recommendations of the European Federation of Immunogenetics (21).Cause of death was determined using available medical records.

Virus-PCR

Virus detection in BAL fluid by PCR for EBV, HSV1/2, CMV, HHV-6-DNAand adenovirus and RSV was performed using standard microbiologic tech-niques in the department of virology (Max von Pettenkofer Institute forHygiene and Microbiology, Ludwig-Maximilians-University, Munich).

Statistical analysis

Statistics were calculated using SPSS software version 12.0.1. for Windows(SPSS Inc., Chicago, IL). The demographic data and outcomes betweengroups were compared using two-sided v 2 test or two-sided Fisher´s exacttests (when expected cell size was less than 5) for categorical variablesand two-tailed Student’s t-tests of independent samples for continuousvariables. To evaluate for an association between potential risk factors andthe development of BOS stages, death and death from BOS initial univari-ate Cox regression analysis was used. Identified variables were includedin subsequent multivariate Cox regression models with restriction to twoco-variables due to limited sample size. Stratification was applied to iden-tified risk factors to adjust for potentially related variables. Results wereconsidered statistically significant at p < 0.05.

Results

Patient cohorts

A total of 87 patients without evidence of BOS were as-sessed for positive or negative PCR reports for HHV-6,EBV, HSV1/2, CMV, RSV or adenovirus from BAL fluid. Thegroup characteristics are summarized in Table 1. The de-mographic and clinical information in PCR negative and cor-responding PCR positive groups were statistically indistin-guishable in terms of length of follow-up time, gender, age,underlying lung disease, immunosuppression regime, is-chemic time, type of transplant, HLA and CMV mismatchesand baseline FEV1 for all viruses.

PCR for HHV-6 in BAL fluid

A total of 90 BAL specimen from 64 patients (mean:1.4 samples/patient; range: 1–4 samples/patient) were ob-tained for HHV-6 detection. HHV-6 PCR was positive in20 patients (31%) at an average time of 204 days aftertransplantation (median 47, range: 4–1058). There was atrend to a higher rate of positive HHV-6 PCR results afteracute rejections (≥A2) with an average delay of 55 days(median 22, range: −102 to +256) without the differencereaching statistical significance (p = 0.08). However theHHV-6 PCR positive group had a significantly higher propor-tion of patients that developed BOS ≥ 1 and death (Table 1(upper panel)). In addition there was a trend suggestingthat HHV-6 detection was associated with BOS stage ≥2 and death from BOS (Table 1 (upper panel)). The aver-age time from positive HHV-6 PCR to BOS ≥ 1 was 358days (median: 367, range: 18–1460) and to death was 566days (median 409, range: 88–628). Positive HHV-6 PCRresults were detected in 9 patients prior to the onset ofBOS. For 3 of these patients, no further HHV-6 data were

American Journal of Transplantation 2005; 5: 2982–2991 2983

Neurohr et al.

Table 1: Group characteristics of lung transplant recipients

HHV6-PCR EBV-PCR

Negative Positive Negative Positiven = 44 (69%) n = 20 (31%) p n = 45 (70%) n = 19 (30%) p

Follow-up (years ± SEM) 3.45 ± 0.38 2.9 ± 0.48 0.11 3.45 ± 0.36 3.60 ± 0.63 0.85Female, n (%) 28 (64%) 9 (45%) 0.18 27 (60%) 8 (42%) 0.27Age (years ± SEM) 44.4 ± 2.0 48.0 ± 2.7 0.27 45.8 ± 2.1 48.7 ± 2.2 0.33Underlying disease, n (%)

COPD 7 (16%) 8 (37%) 0.10 8 (18%) 7 (37%) 0.20CF/Bronchiectasis 9 (21%) 2 (11%) 0.48 8 (18%) 1 (5%) 0.26IPF 7 (16%) 7 (36%) 0.10 12 (25%) 3 (16%) 0.52Other 21 (47%) 3 (16%) 0.07 17 (39%) 8 (42%) 0.77

Immunosuppression, n (%) 0.74 0.30Tacrolimus-group 35 (79%) 15 (75%) 38 (85%) 14 (72%)Cyclosporine-group 9 (21%) 5 (25%) 15 (7%) 5 (28%)

Ischemic time (minutes ± SEM) 309 ± 13 315 ± 18 0.78 310 ± 11 322 ± 20 0.62Type of transplant, n (%) 0.06 0.79

Single 18 (41%) 14 (70%) 24 (53%) 11 (58%)Bilateral 26 (59%) 6 (30%) 21 (47%) 8 (42%)

HLA mismatches, n (%)+ 0.68 0.905–6 15 (40%) 9 (50%) 20 (47%) 6 (43%)3–4 21 (57%) 8 (44%) 20 (47%) 8 (57%)0–2 1 (3%) 1 (6%) 2 (6%) 0 (0%)

CMV mismatch (donor+/recipient–)# 9 (21%) 3 (15%) 0.73 8 (18%) 4 (21%) 0.73Baseline FEV1 (% Pred. ± SEM) 83.1 ± 3.0 76.5 ± 3.7 0.12 80.6 ± 2.8 77.6 ± 4.1 0.55Acute rejection, n (%)

≥A1 19 (44%) 13 (65%) 0.18 19 (42%) 15 (78%) 0.013∗≥A2 14 (32%) 11 (55%) 0.08 15 (35%) 11 (58%) 0.08

BOS ≥ Stage 1, n (%) 8 (18%) 9 (45%) 0.012∗ 7 (16%) 9 (47%) 0.009∗BOS ≥ Stage 2, n (%) 6 (14%) 6 (30%) 0.071 5 (11%) 4 (21%) 0.42BOS = Stage 3, n (%) 6 (14%) 5 (25%) 0.150 4 (9%) 3 (16%) 0.40Death from BOS, n (%) 4 (9%) 5 (25%) 0.071 5 (11%) 3 (16%) 0.68Death, n (%) 8 (18%) 8 (40%) 0.048∗ 9 (20%) 5 (26%) 0.52

available. In the remaining six cases, positive HHV-6 PCRresults were detected after the diagnosis of BOS. The aver-age time between entering BOS 1 and second HHV-6 PCRwas 175 days (median: 219, range 8–281) (Figure 1). HHV-6 PCR data collected after the onset of BOS were neitherpart of the stated total HHV-6 specimen (see above) norincluded in subsequent statistical analysis. The individualcourse from transplant to positive HHV-6-PCR, BOS anddeath is presented in Figure 1.

PCR for EBV in BAL fluid

A total of 78 BAL samples from 64 patients (mean: 1.2 sam-ples/patient, range: 1–3 samples/patient) were collectedfor the detection of EBV. In 19 patients (30%), a positiveEBV-PCR was detected at an average of 208 days aftertransplantation (median 95, range: 7–781). After acute re-jections (≥A1), a significant higher number of positive EBV-PCR results occurred with an average delay of 77 days(median: 58, range: −11 to 204) (Table 1 (upper panel)).For positive EBV detection, there was a statistical signifi-cant association with an increased number of patients withBOS ≥ 1 after an average time of 580 days (median: 413,range: 26–1584) (Table 1 (upper panel)).

PCR for HSV 1/2 in BAL fluid

From 74 patients, 115 BAL specimen for HSV1/2 PCR anal-ysis were obtained (mean 1.55 samples/patient, range:1–6). In 12 patients (16%), a positive PCR result was de-tected after an average of 256 days (median: 56, range:7–993). We found no difference in the incidence of acuterejections, BOS stages or death in patients with or withoutpositive HSV1/2 PCR but an isolated association with deathfrom BOS after an average time of 516 days (median: 513,range: 26–1007) (Table 1 (lower panel)).

PCR for CMV in BAL fluid

A total of 143 BAL samples from 79 Patients were collectedfor CMV detection by PCR (mean 1.8 samples/patient,range: 1–5). At an average of 234 days (median 142, range:18–712) after transplantation, specimens were positive in10 patients (13%). Patients with positive PCR results hadno higher proportion of acute or chronic graft rejection,death or death from BOS compared to the correspondingCMV-PCR negative group (Table 1 (lower panel)).

PCR for RSV in BAL fluid

A total of 71 BAL specimens from 59 patients (33 fe-male, age 46.2 ± 1.7 years, 33 SLTX) were sent to the

2984 American Journal of Transplantation 2005; 5: 2982–2991

Table 1: Continued.

HSV1/2-PCR CMV-PCR

Negative Positive Negative Positiven = 62 (84%) n = 12 (16%) p n = 69 (87%) n = 10 (13%) p

Follow-up (years ± SEM) 3.54 ± 0.32 3.10 ± 0.75 0.57 3.55 ± 0.31 2.50 ± 0.52 0.07Female, n (%) 37 (60%) 7 (58%) 1.00 41 (59%) 5 (50%) 0.74Age (years ± SEM) 46.6 ± 1.5 50.6 ± 3.5 0.30 46.1 ± 1.5 50.4 ± 2.8 0.21Underlying disease, n (%)

COPD 13 (21%) 5 (42%) 0.16 18 (26%) 2 (20%) 1.00CF/Bronchiectasis 10 (15%) 1 (8%) 1.0 9 (14%) 1 (10%) 1.00IPF 14 (23%) 2 (17%) 1.0 14 (20%) 2 (20%) 1.00Other 25 (41%) 4 (33%) 0.73 28 (40%) 5 (50%) 0.45

Immunosuppression, n (%) 1.00 0.15Tacrolimus-group 49 (80%) 9 (80%) 59 (86%) 7 (70%)Cyclosporine-group 12 (20%) 3 (20%) 10 (14%) 3 (30%)

Ischemic time (minutes ± SEM) 308 ± 11 310 ± 26 0.94 305 ± 9 315 ± 30 0.75Type of transplant, n (%) 1.00 1.00

Single 34 (55%) 7 (58%) 39 (57%) 6 (60%)Bilateral 28 (45%) 5 (42%) 30 (43%) 4 (40%)

HLA mismatches, n (%)+ 0.95 0.265–6 23 (42%) 4 (40%) 28 (46%) 3 (33%)3–4 29 (54%) 6 (60%) 32 (52%) 5 (56%)0–2 2 (4%) 0 (0%) 1 (2%) 1 (11%)

CMV mismatch (donor+/recipient–)# 13 (21%) 2 (17%) 1.00 12 (18%) 3 (30%) 0.41Baseline FEV1 (% Pred. ± SEM) 80.84 ± 2.60 77.36 ± 3.43 0.43 78.3 ± 2.4 76.8 ± 6.1 0.82Acute rejection, n (%)

≥A1 32 (52%) 9 (75%) 0.21 39 (56%) 6 (60%) 1.00≥A2 23 (36%) 8 (66%) 0.11 30 (43%) 5 (50%) 1.00

BOS ≥ Stage 1, n (%) 18 (28%) 5 (42%) 0.30 24 (35%) 3 (30%) 1.00BOS ≥ Stage 2, n (%) 12 (19%) 4 (33%) 0.24 16 (23%) 2 (20%) 1.00BOS = Stage 3, n (%) 10 (16%) 4 (33%) 0.20 13 (19%) 1 (10%) 1.00Death from BOS, n (%) 6 (9%) 4 (33%) 0.04∗ 10 (15%) 2 (20%) 0.65Death, n (%) 16 (25%) 5 (42%) 0.28 21 (30%) 4 (40%) 0.72

Definition of abbreviations: BOS = bronchiolitis obliterans syndrome, COPD = chronic obstructive pulmonary disease, CF = cysticfibrosis, IPF = idiopathic pulmonary fibrosis, HHV-6 = human herpesvirus 6, EBV = Epstein-Barr virus, HSV 1/2 = herpes simplex virus,CMV = cytomegalovirus, RSV = respiratory syncytical virus, SEM = standard error of the mean, FEV1 = forced expiratory volume in1 second, +represents 75 (86%) transplant recipients, #represents 81 (93%) transplant recipients, ∗p< 0.05

laboratory for RSV-PCR detection (mean 1.2 sam-ples/patient, range: 1–3). Samples of four patients (6.8%, 3female, age 51.0 ± 3.0, 2 SLTX) were tested positive afteran average time of 215 days after transplantation (median274, range: 88–292). The RSV-PCR positive group showedno higher incidence of acute rejections, BOS stages, deathfrom BOS or death.

PCR for adenovirus in BAL fluid

From 60 patients (35 female, age 46.0 ± 1.8 years,32 SLTX), a total of 72 BAL samples were analyzed byadenovirus-PCR (mean 1.2 samples/patient, range 1–3).In two patients (3.3%, 2 female, 2 DLTX, age 30.0 and34 years respectively), a positive adenovirus-PCR was ob-served at day 15 and day 526 after transplantation, re-spectively. No acute or chronic rejection or death occurredin these two cases during follow-up time (3.01 and 4.16years, respectively).

Concomitance of positive PCR results

In 10 of 19 patients (53%) with positive EBV-PCR, a positiveHHV-6 result was detected at the same time. In 5 (50%)

of these patients, the development of BOS ≥ stage 1 wasobserved with consecutive death from BOS in 3 cases.For 12 patients with positive HSV1/2 data, concomitantpositive HHV-6 specimen were demonstrated in 5 patients(42%) with death from BOS in 2 patients. Three patients(30%) of those with positive CMV samples were retrievedwith positive HHV-6 PCR data. In this group, 2 deaths withan association to BOS in one case were found. In fourpatients with positive RSV-PCR, a positive HHV-6 PCR wasdetected in 2 patients with BOS in one case and death fromBOS in the other patient. We observed no concomitanceof positive HHV-6- and adenovirus-PCR results.

Identification of risk factors for BOS stages and death

Univariate Cox regression analysis was performed to deter-mine whether there were associations between potentialpredictor variables and BOS stages, death and death fromBOS. Due to limited sample size, analysis was not per-formed for adenovirus PCR data. Ischemic time, HLA mis-matches, type of transplant, serologic CMV donor/recipientstatus, immunosuppression regimen and CMV-PCR werenot predictors of these endpoints (Table 2). Univariate

Neurohr et al.

Figure 1: Individual representation

of the interval from lung transplan-

tation to positive HHV-6 PCR, BOS

and death (n = 20 patients). Inter-vals represent time from transplant toBOS stage 1 (grey bar), time to pos-itive HHV-6 PCR in BAL fluid (arrow),time in BOS stages 1, 2 or 3 (corre-sponding grey and black bars), timeof death (black arrowhead) and timeof death from BOS (grey arrowhead).

analysis revealed that age (≥50 years), COPD, acute re-jections and positive BAL-PCR for HHV-6 and EBV weresignificantly associated with BOS stages ≥ 1 (Table 2). Sim-ilar univariate analysis for BOS ≥ stages 2, BOS stage 3,death and death from BOS demonstrated that acute rejec-tions were significant predictors for all endpoints whereasCOPD was significantly associated with death. PositiveHHV-6 PCR was a significant predictor of BOS stages ≥ 2,death and death from BOS. Only for death from BOS, therewas statistical significant association with positive HSV1/2PCR results. Positive EBV-PCR data and age were not sta-tistical significant risk factors for death or death from BOS(Table 2).

To determine if HHV-6 detection in BAL fluid by PCR andCOPD were BOS and death risk factors separate fromacute rejection multivariate Cox regression analysis wasutilized (Table 3). Inclusion of COPD and acute rejection(≥A2) revealed that COPD was not a separate predictor forBOS stages and death.

Positive HHV-6 PCR results and acute rejections (≥A2)were found to be significant predictors of BOS ≥ 1(Table 3). In addition, positive HHV-6 PCR showed a trendtoward a distinct risk factor for BOS ≥ stage 2 and deathfrom BOS in a multivariate regression model with acuterejection (≥A2) as a co-variate (Table 3). Subsequent strati-fication of patients with HHV-6 PCR data in cases with andwithout acute rejection (≥A2) demonstrated that positiveHHV-6 PCR remained an independent significant predictorfor BOS ≥ 1 and death (BOS ≥ 1: hazard ration (HR) = 2.72,95% confidence interval (CI) 1.12–7.29, p = 0.046; death:HR = 3.10, 95% CI 1.39–9.11, p = 0.042).

A multivariate model with acute rejection (≥A2) and age(≥50 years) demonstrated that age (≥50 years) was a dis-tinct predictor of BOS ≥ 1 (age: HR = 2.99, 95% CI 1.32–6.74, p = 0.008; acute rejection: HR = 5.48, 95% CI 2.31–12.02, p = 0.001). When combining positive HHV-6 PCRwith age in one model, HHV-6 PCR remained a significantfactor for BOS ≥ stage 1 (positive HHV-6 PCR: HR = 3.59,

(≥50

8∗2.

045∗

001∗

2∗3.

037∗

8∗6.

003∗

001∗

6∗4.

004∗

5∗2.

7∗3.

023∗

034∗

Neurohr et al.

≥Sta

4–4.

.69–

.61–

.63–

.92–

.81–

001∗

8–10

.29–

017∗

8–16

0∗3.

.27–

014∗

.10–

040∗

9–7.

.83–

4–1.

.17–

045∗

.25–

001∗

6–9.

6∗4.

.21–

026∗

0–17

1∗3.

.26–

047∗

95% CI 1.33–9.71, p = 0.012; age: HR = 2.33, 95% CI0.81–6.66, p = 0.12).

Analysis of EBV-PCR data and acute rejection (≥A2) in-cluded as candidate variables demonstrated that positiveEBV-PCR was not a separate predictor of BOS ≥ stage1 (positive EBV-PCR: HR = 1.41, 95% CI 0.86–2.30, p =0.17; acute rejection: HR = 3.30, 95% CI 1.16–4.60, p =0.003). Similar multivariate analysis revealed that positiveHSV-PCR was not a risk factor for death from BOS dis-tinct from HHV-6 (positive HSV1/2-PCR: HR = 2.94, 95%CI 0.66–7.01, p = 0.16; positive HHV6-PCR: HR = 4.35,95% CI 1.12–6.80, p = 0.042).

In summary, our results demonstrated that positive HHV-6 PCR results were associated with a significant risk forthe subsequent development of BOS ≥ stage 1 and death.This risk was distinct from the risk attributable to otherpredictors, especially acute rejection.

Discussion

BOS has remained the most significant cause of graft fail-ure and main limitation to long-term survival after LTx. Inthe context of previous studies describing BOS risk fac-tors, our data suggest that detection of HHV-6 in BAL fluidis associated with an increased risk of developing BOS ≥stage 1 and death. In addition, although not statisticallysignificant, there was a trend toward an association be-tween HHV-6 and more advanced stages of BOS. A limi-tation in our study was that beyond the first 3 months af-ter transplantation, bronchoscopy was performed for clin-ical indications only. Therefore, patients with more symp-tomatic episodes were more likely to have bronchoscopieswith the risk of selection bias in favor of detecting viralpathogens. However, this present study showed that theserisks from positive HHV-6 PCR results before the devel-opment of BOS were distinct from the risk imputable toestablished BOS risk factors, including acute rejection, forwhich a similar selection bias can be assumed. However,a statistical relationship does not necessarily establish acausal relationship. The potential mechanisms responsiblefor an association between HHV-6 and OB, the histologi-cal confirmation of BOS are unknown. The virus probablyremains latent after primary infection in peripheral bloodmononuclear cells, central nervous systems, liver tissueand salivary glands. It then reactivates upon host immuno-suppression. There have been reports that HHV-6 infectionmay independently induce a state of immunosuppressionby impairing T-cell activation to antigen presentation or re-duce the ability of macrophages to produce interleukin-12upon stimulation with interferon-gamma (22, 23). More-over, HHV-6 has also been demonstrated to increase ad-hesion molecule expression and lymphocyte infiltration inliver allografts (24). Based on these observations, we spec-ulate that molecular interactions between HHV-6 and thehost immune system can initiate both acute and chronic

responses that may trigger the development or accelera-tion of OB progression. In this context, it is important toconsider that the PCR method applied in the present studydid not allow quantitative assessment of HHV-6 viral load inBAL fluid. Therefore, we could not discriminate betweenreplicating HHV-6 due to reactivation or a latent state per-haps as a result of contamination from passage with thebronchoscope.

In agreement with multiple published reports, we foundthat the occurrence of acute rejections was a significantand separate predictor of all BOS stages and consecutivedeath (1, 2, 25). In our analysis, the impact of further poten-tial risk factors like HLA mismatches, age, ischemic timeand underlying disease was not observed to be significantlyassociated with BOS or death. However, due to the num-ber of variables and events considered in this study andthe fact that some confounders are not available, there isa substantial chance for the occurrence of a type II error.Therefore, we cannot rule out that the impact of other fac-tors including further viral pathogens might have been sig-nificant given higher numbers. Accordingly, the evidenceof an association for some of these risk factors has beenestablished mainly from registry studies and they are un-likely to be detected by single center studies due to limitedsample size (5, 26, 27, 28).

HHV-6 has been well studied in bone marrow (BMT), liverand renal transplantation. The virus, which is the causativeagent of exanthema subitum in children was first describedas a novel herpesvirus in 1986 in patients with lympho-proliferative disorders and acquired immunodeficiency syn-drome (29). It is now increasingly recognized as a frequentpathogen after transplantation (11). In BMT HHV-6, reacti-vation has been associated with fever, skin rashes, inter-stitial pneumonia, rejection and obliterative bronchitis (30,31). After renal transplantation, detection of HHV-6 duringthe first 12 weeks was significantly associated with chronicallograft nephropathy within 3 years (32). In pediatric kid-ney transplant recipients, an increased risk for HHV-6 as-sociated acute rejections was found (33). In a study of 200liver transplant recipients, HHV-6 was detected in bloodsamples in 28% of patients and associated with the devel-opment of opportunistic infections and acute rejections,whereas a disease directly attributable to HHV-6 alone oc-curred in only 1% (34).

To our knowledge only two series have specifically in-volved lung transplant recipients. Somewhat astonishing,Michaelides and colleagues did not detect HHV-6 by PCRin any blood samples of 26 LTx patients (35). In a longitu-dinal study of 30 heart–lung and lung transplant recipients,14.5% of all BAL specimens were found to be positivefor HHV-6 by PCR during 100 days with a median delay of18 days after transplantation. Although detection of HHV-6was not significantly associated with any specific clinicalmanifestation including CMV disease or higher incidenceof acute rejection episodes, Jacobs and co-workers ob-

served a significantly higher mortality rate (12). Becausethis carefully conducted study did not analyze BOS as anendpoint, direct comparisons to our study are difficult. Butin accordance with published data, our results support theview that even if detection of HHV-6 in lung is not by itselfsufficient to incriminate the virus as the etiologic agent,it may have a significant contribution to the morbidity andmortality observed after LTx .

Detection of CMV-DNA in BAL fluid was not found to be asignificant risk for BOS in our analysis, a common findingin lung transplant recipients (16, 27, 36). Furthermore, theintroduction of preemptive treatment of CMV disease inseropositive patients and the prophylaxis with ganciclovirin seronegative recipients may have reduced the overallincidence of CMV-related complications (37, 38).

Since no controlled trials of antiviral therapy against HHV-6have been conducted so far, the role of acyclovir and ganci-clovir on treatment and prophylaxis of HHV-6 in our study isdifficult to assess. Although both drugs target the viral DNAploymerase of HHV-6, only ganciclovir proved effective inpreventing HHV-6 reactivation in BMT or stem cell trans-plant recipients (39, 40). However in the present study,CMV mismatch, which determined prophylactic adminis-tration of either ganciclovir or acyclovir, was not identifiedas a risk factor for BOS or death. Moreover, the HHV-6 PCRnegative and positive groups were statistical indistinguish-able in terms of CMV mismatch prevalence, pointing at aprophylaxis independent impact of HHV-6.

Accumulating data support the notion of community-acquired respiratory tract viral (CARV) infections as a poten-tially important risk factor for BOS (5, 6, 9). In contrast, wedid not identify RSV and adenovirus as risk factors for BOSor death. However, the lack of assessment for influenza(flu) and parainfluenza (paraflu) limits the conclusions of ourstudy regarding CARV. Since these two respiratory virusesmight be important for the development of BOS, we can-not exclude that the impact of CARV infections on BOSwas not detected due to inadaequacy of our study proto-col. In accordance with this assumption, the incidence ofpost-transplant patients with positive reports for RSV andadenovirus was lower than previously reported (RSV: 6.8%vs. 12–50%, adenovirus: 3.3% vs. 10–12%, respectively).

We clearly recognize the inherent limitations in our workrelated to availability of data, sample size, study designand selection bias. Nevertheless, this cohort analysis iden-tified a significant association between detection of HHV-6in BAL fluid and BOS. It cannot be excluded that the oc-curence of HHV-6 DNA in BAL samples could simply bea consequence of immunosuppression and not the causeof BOS. However, our results suggest that HHV-6 in thelower respiratory tract of lung transplant recipients maypredispose patients to developing BOS. Since the ma-jor pathogenic role of the virus may be related to an im-munomodulatory effect rather than to directly attributable

Neurohr et al.

clinical manifestations, our observation warrants furtherlong-term studies with complete viral testing performed atall surveillance and clinically-indicated bronchoscopies.

Acknowledgments

The authors thank A. Crispin, M.D., Institut fuer Medizinische Informa-tionsverarbeitung, Biometrie und Epidemiologie for his continuing supportand his excellent statistical assistance and H. Nitschko, M.D., Departmentof Virology, Max von Pettenkofer Institute for Hygiene and Microbiology(both: Ludwig-Maximilians University, Munich, Germany).

References

1. Trulock EP, Edwards LB, Taylor DO et al. The Registry of the In-ternational Society for Heart and Lung Transplantation: TwentiethOfficial adult lung and heart-lung transplant report–2003. J HeartLung Transplant 2003 Jun; 22(6): 625–635.

2. Estenne M, Maurer JR, Boehler A et al. Bronchiolitis obliteranssyndrome 2001: An update of the diagnostic criteria. J Heart LungTransplant 2002; 21(3): 297–310.

3. Vilchez RA, Dauber J, Kusne S. Infectious etiology of bronchiolitisobliterans: The respiratory viruses connection—myth or reality?Am J Transplant 2003; 3(3): 245–249.

4. Cainelli F, Vento S. Infections and solid organ transplant rejec-tion: A cause-and-effect relationship? Lancet Infect Dis 2002; 2(9):539–549.

5. Khalifah AP, Hachem RR, Chakinala MM et al. Respiratory viralinfections are a distinct risk for bronchiolitis obliterans syndromeand death. Am J Respir Crit Care Med 2004; 170(2): 181–187.

6. Holt ND, Gould FK, Taylor CE et al. Incidence and significanceof noncytomegalovirus viral respiratory infection after adult lungtransplantation. J Heart Lung Transplant 1997; 16(4): 416–419.

7. Sharples LD, Tamm M, McNeil K, Higenbottam TW, Stewart S,Wallwork J. Development of bronchiolitis obliterans syndrome inrecipients of heart-lung transplantation—early risk factors. Trans-plantation 1996; 61(4): 560–566.

8. Bridges ND, Spray TL, Collins MH, Bowles NE, Towbin JA. Aden-ovirus infection in the lung results in graft failure after lung trans-plantation. J Thorac Cardiovasc Surg 1998; 116(4): 617–623.

9. Billings JL, Hertz MI, Savik K, Wendt CH. Respiratory virusesand chronic rejection in lung transplant recipients. J Heart LungTransplant 2002; 21(5): 559–566.

10. Buchbinder S, Elmaagacli AH, Schaefer UW, Roggendorf M. Hu-man herpesvirus 6 is an important pathogen in infectious lungdisease after allogeneic bone marrow transplantation. Bone Mar-row Transplant 2000; 26(6): 639–644.

11. Singh N. Human herpesviruses-6, -7 and -8 in organ transplantrecipients. Clin Microbiol Infect 2000; 6(9): 453–459.

12. Jacobs F, Knoop C, Brancart F et al. Brussels Heart and LungTransplantation Group. Human herpesvirus-6 infection after lungand heart-lung transplantation: A prospective longitudinal study.Transplantation 2003; 75(12): 1996–2001.

13. Reams BD, McAdams HP, Howell DN, Steele MP, Davis RD,Palmer SM. Posttransplant lymphoproliferative disorder: Inci-dence, presentation and response to treatment in lung transplantrecipients. Chest 2003; 124(4): 1242–1249.

14. Malouf MA, Chhajed PN, Hopkins P, Plit M, Turner J, Glanville AR.Anti-viral prophylaxis reduces the incidence of lymphoprolifera-tive disease in lung transplant recipients. J Heart Lung Transplant2002; 21(5): 547–554.

15. Smyth RL, Higenbottam TW, Scott JP et al. Herpes simplex virusinfection in heart-lung transplant recipients. Transplantation 1990;49(4): 735–739.

16. Riise GC, Andersson R, Bergstrom T, Lundmark A, Nilsson FN,Olofsson S. Quantification of cytomegalovirus DNA in BAL fluid:A longitudinal study in lung transplant recipients. Chest 2000;118(6): 1653–1660.

17. Bewig B, Haacke TC, Tiroke A et al. Detection of CMV pneumoni-tis after lung transplantation using PCR of DNA from bronchoalve-olar lavage cells. Respiration 2000; 67(2): 166–172.

18. Treede H, Klepetko W, Reichenspurner H et al. Munich and ViennaLung Transplant Group. Tacrolimus versus cyclosporine after lungtransplantation: A prospective, open, randomized two-center trialcomparing two different immunosuppressive protocols. J HeartLung Transplant 2001; 20(5): 511–517.

19. Reichenspurner H, Kur F, Treede H et al. Optimization of the im-munosuppressive protocol after lung transplantation. Transplan-tation 1999; 68(1): 67–71.

20. Yousem SA, Berry GJ, Cagle PT et al. Revision of the 1990 work-ing formulation for the classification of pulmonary allograft rejec-tion: Lung Rejection Study Group. J Heart Lung Transplant 1996;15(1 Pt 1): 1–15.

21. Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for factorsof the HLA system, 2002. Tissue Antigens 2002; 60(5): 407–464.

22. Smith A, Santoro F, Di Lullo G, Dagna L, Verani A, LussoP.Selective suppression of IL-12 production by human her-pesvirus 6. Blood 2003; 102(8): 2877–2884.

23. Flamand L, Gosselin J, Stefanescu I, Ablashi D, Menezes J. Im-munosuppressive effect of human herpesvirus 6 on T-cell func-tions: Suppression of interleukin-2 synthesis and cell proliferation.Blood 1995; 85(5): 1263–1271.

24. Lautenschlager I, Harma M, Hockerstedt K, Linnavuori K, LoginovR, Taskinen E. Human herpesvirus-6 infection is associated withadhesion molecule induction and lymphocyte infiltration in liverallografts. J Hepatol 2002; 37(5): 648–654.

25. Husain AN, Siddiqui MT, Holmes EW et al. Analysis of risk factorsfor the development of bronchiolitis obliterans syndrome. Am JRespir Crit Care Med 1999; 159(3): 829–833.

26. Sharples LD, McNeil K, Stewart S, Wallwork J. Risk fac-tors for bronchiolitis obliterans: A systematic review of re-cent publications. J Heart Lung Transplant 2002; 21(2): 271–281.

27. Chalermskulrat W, Neuringer IP, Schmitz JL et al. Human leuko-cyte antigen mismatches predispose to the severity of bronchi-olitis obliterans syndrome after lung transplantation. Chest 2003;123(6): 1825–1831.

28. Girgis RE, Tu I, Berry GJ et al. Risk factors for the development ofobliterative bronchiolitis after lung transplantation. J Heart LungTransplant 1996; 15(12): 1200–1208.

29. Salahuddin SZ, Ablashi DV, Markham PD et al. Isolation of a newvirus, HBLV, in patients with lymphoproliferative disorders. Sci-ence 1986; 234(4776): 596–601.

30. Nishimaki K, Okada S, Miyamura K et al. The possible involvementof human herpesvirus type 6 in obliterative bronchiolitis after bonemarrow transplantation. Bone Marrow Transplant 2003; 32(11):1103–1105.

31. Yoshikawa T. Human herpesvirus 6 infection in hematopoieticstem cell transplant patients. Br J Haematol 2004; 124(4): 421–432.

32. Tong CY, Bakran A, Peiris JS, Muir P, Herrington CS. The as-sociation of viral infection and chronic allograft nephropathy withgraft dysfunction after renal transplantation. Transplantation 2002;74(4): 576–578.

33. Wade AW, McDonald AT, Acott PD, Lee S, Crocker JF. Humanherpes virus-6 or Epstein-Barr virus infection and acute allograftrejection in pediatric kidney transplant recipients: Greater risk forimmunologically naive recipients. Transplant Proc 1998; 30(5):2091–2093.

34. Humar A, Kumar D, Caliendo AM et al. Clinical impact of humanherpesvirus 6 infection after liver transplantation. Transplantation2002; 73(4): 599–604

35. Michaelides A, Glare EM, Spelman DW et al. Beta-Herpesvirus(human cytomegalovirus and human herpesvirus 6) reactivation inat-risk lung transplant recipients and in human immunodeficiencyvirus-infected patients. J Infect Dis 2002; 186(2): 173–180.

36. Ettinger NA, Bailey TC, Trulock EP et al. Cytomegalovirus infec-tion and pneumonitis. Impact after isolated lung transplantation.Washington University Lung Transplant Group. Am Rev RespirDis 1993; 147(4): 1017–1023.

37. Brumble LM, Milstone AP, Loyd JE et al. Prevention of cy-tomegalovirus infection and disease after lung transplantation:Results using a unique regimen employing delayed ganciclovir.Chest 2002; 121(2): 407–414.

38. Egan JJ, Lomax J, Barber L et al. Preemptive treatmentfor the prevention of cytomegalovirus disease: In lung andheart transplant recipients. Transplantation 1998; 65(5): 747–752.

39. Rapaport D, Engelhard D, Tagger G, Or R, Frenkel N. Antiviral pro-phylaxis may prevent human herpesvirus-6 reactivation in bonemarrow transplant recipients. Transplant. Infect. Dis 2002; 4: 10–16.

40. Tokimasa S, Hara J, Osugi Y et al. Ganciclovir is effective forprophylaxis and treatment of human herpesvirus-6 in allogeneicstem cell transplantation. Bone Marrow Transplant 2002; 29: 595–598.

Human Herpesvirus 6 in Bronchalveolar Lavage Fluid after Lung Transplantation: A Risk Factor for...

Documents