BRIEF REPORT

The purpose of the present study was to characterize BK

BK Virus Infection in Hematopoietic viremia in a large cohort of HSCT recipients who underwent
Stem Cell Transplant Recipients: longitudinal plasma sampling. Specifically, we sought to deter-
Frequency, Risk Factors, and mine the risk factors for and the incidence, duration, and quan-
titative aspects of BK viremia. We also correlated BK viremia
Association with Postengraftment

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with hemorrhagic cystitis that occurred after platelet engraft-
Hemorrhagic Cystitis ment, thereby eliminating the confounding influence of low
platelet counts and the residual effects of chemotherapy.
Veronique Erard,1 Barry Storer,1 Lawrence Corey,1,2,3 Patients and methods. The study population was a pro-
Jennifer Nollkamper,1 Meei-Li Huang,1 Ajit Limaye,2,3 spective cohort of 132 consecutive adult patients who received
and Michael Boeckh1,3
1
non–T cell–depleted allogeneic or autologous HSCT in 1998.
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center,
and Departments of 2Laboratory Medicine and 3Medicine, This cohort represented an unselected, contemporaneous pop-
University of Washington, Seattle ulation of recipients of myeloablative HSCT. The entire cohort
was retrospectively evaluated by testing for BK viremia, as well
Blood samples from 132 consecutive hematopoietic stem cell as by reviewing the clinical records of the patients to search
transplant recipients were obtained and tested weekly for BK for evidence of hemorrhagic cystitis. Because adenovirus in-
virus DNA by use of quantitative real-time PCR. Forty-four fection has also been proposed to contribute to the develop-
patients (33%) developed BK viremia at a median of 41 days ment of hemorrhagic cystitis, the laboratory records of patients
(range, 9–91 days) after transplantation. Patients with hem- with hemorrhagic cystitis were reviewed for information in-
orrhagic cystitis that occurred after platelet engraftment had dicating the presence of adenovirus in urine, as determined by
higher levels of viremia than did patients without hemor- culture and direct fluorescent antibody assay, from 7 days prior
rhagic cystitis (median, 9.7 ⫻ 10 3 vs. 0 copies/mL; P p .008) to hemorrhagic cystitis until the last day of the event. Testing
and patients with hemorrhagic cystitis that occurred before for adenovirus was performed by clinicians for clinical diag-
platelet engraftment (median, 9.7 ⫻ 10 3 vs. 0 copies/mL; nostic reasons. Testing of urine samples for BK virus was only
P p .0006). BK viremia also was strongly associated with occasionally performed in 1998, when the present study took
postengraftment hemorrhagic cystitis in a time-dependent place. The study received institutional review board approval.
analysis (P p .004). Plasma samples were collected on a weekly basis, starting
from the day of transplantation until approximately day 100
after transplantation. The samples were frozen at ⫺80C and
BK viruria, which is diagnosed on the basis of PCR-based de-
were stored for future analysis. The plasma samples of patients
tection of BK virus DNA in urine samples, occurs in virtually
with hemorrhagic cystitis, which were drawn at the time of the
all hematopoietic stem cell transplant (HSCT) recipients and,
event, were analyzed by PCR for adenovirus DNA.
thus, has a low predictive value for the development of BK
PCR conditions were reported elsewhere [4]. The primers
virus disease in HSCT recipients [1–3]. In contrast, quantitative
and probe that were used for the detection of BK virus were
PCR-based detection of BK virus DNA in plasma samples is
BK-F (AAA TGC CTT AAT CTA AGC TGA CAT AG), BK-R
recognized as a sensitive and specific screening test for BK virus
(GCA AGG AAT GGC CTA TTT GTT CCA AA), and BK-
nephropathy in renal transplant recipients [4, 5]. Viremia has
Probe (FAM-TGC AAG GGC AGT GCA CAG AAG GCT-
been shown to be predictive of clinical disease caused by cy-
TAMRA). Two assays were performed for the detection of ad-
tomegalovirus (CMV) or adenovirus after HSCT [6–8].
enovirus DNA; one assay detects subgroups A and F (Adeno-AF
PCR), and one detects subgroups B,C, D, and E (Adeno-BCE
Received 15 June 2004; accepted 10 August 2004; electronically published 19 November PCR). The primers that were used for the first assay were
2004.
Reprints or correspondence: Dr. Michael Boeckh, Fred Hutchinson Cancer Research Center,
HexonA-F (CCG GKC TGG TGC AAT TCG), HexonA-R1
Program in Infectious Diseases, 1110 Fairview Ave. N. D3-100, PO Box 19024, Seattle, WA (CGATCCACGGGCACAAA), HexonF-1 (TGTTYGAAGTTTT
98109-1024 (mboeckh@fhcrc.org).
CGACGTYGT), and HexonF-2 (SAGGTAGACGGCCTCGA-
Clinical Infectious Diseases 2004; 39:1861–5
 2004 by the Infectious Diseases Society of America. All rights reserved.
TGA); the probes that were used were HexonA-P (FAM-CCAC-
1058-4838/2004/3912-0022$15.00 GGACACCTACTTCACCCTGGG-TAMRA) and HexonF-P

BRIEF REPORT • CID 2004:39 (15 December) • 1861

(FAM-CGCATCCACCAGCCSCACC-TAMRA). For the sec- patients for whom ⭓3 samples were available. Death was con-
ond assay, the primers were HexonB-1 (TGGACATGACYTTT- sidered to be a competing risk for all outcomes and for esti-
GAGGTGGAT), HexonB-2 (CGTCGAARACTTCGAARAGA mation of cumulative incidence. The Mann-Whitney U test
AGA), HexonE-1 (TTAACCACCACCGCAATGC), HexonE-2 was used to compare the maximum viral loads of patients who
(TGGATGTGGAATG GCACGTA), PentonC-F (TCGACACC- had postengraftment hemorrhagic cystitis with the maximum
ACCCGTGTGTAC), and PentonC-R (TGCTG TGGTCGTTC- viral loads of patients who had preengraftment hemorrhagic
TGGTAGTT); the probe sequences were HexonB-P (FAM- cystitis or who did not have hemorrhagic cystitis. All P values
CCATGGA TGAGCCCACCCTGCTTT-TAMRA), HexonE-P are based on likelihood ratios and are 2-sided. Statistical sig-
(FAM-TACCGCTCCATGTCCT GGGCA-TAMRA), and Pen- nificance was determined at an a level of .05.
tonC-P (TGGACAACAAGTCAACGG ATGTGGCA). The con- Results. Of the 132 HSCT recipients, 38% received an HLA
centration of purified plasmid DNA was measured by optical matched, related transplant; 14% received a mismatched, re-

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density at 260 nm (OD260), and the number of plasmid DNA lated transplant; 29% received an unrelated transplant; and 19%
copies was then calculated. The known copy number of plas- received an autologous transplant. For patients who received
mids containing BK virus DNA was linearized by digestion with treatment with cyclophosphamide, prophylaxis against hem-
BamHI. A 10-fold dilution series of the BamHI-digested plas- orrhagic cystitis included forced diuresis and mercaptoethane–
mids that contained BK virus DNA was used as the standard sodium sulphonate therapy [12]. Thirty-four patients (26%)
curve for the real-time PCR assays for BK virus. died within 100 days after transplantation.
PCR products of adenovirus serotypes 1 (group C), 4 (group Hemorrhagic cystitis was observed in 19 (14%) of the 132
E), 7 (group B), 11 (group B), 12 (group A), and 40 (group HSCT recipients, at a median of 9 days (range, 1–84 days) after
F) were cloned using the Invitrogen TOPO TA cloning kit. The transplantation. In univariate analysis, hemorrhagic cystitis was
concentration of each plasmid DNA was measured by the associated with a lack of platelet engraftment (hazard ratio, 8.2;
OD260, and the number of copies was then calculated. The 95% CI, 1.4–4.8; P p .03). Sixteen patients (12%) developed
known copy number of plasmid DNA was then linearized by hemorrhagic cystitis before platelet engraftment at a median of
HindIII digestion, and a 10-fold dilution series was made. Be- 7 days (range, 1–16 days) after transplantation. Three patients
cause the Adeno-AF PCR assay detects the plasmids from ad- (2%) developed postengraftment hemorrhagic cystitis, all cases
enovirus serotype 12 and adenovirus serotype 40 with equal of which were categorized as grade 3, at a median of 45 days
efficiency, we used the 10-fold dilution series of plasmid- (range, 41–84 days) after transplantation. Two of them were
containing PCR product from adenovirus serotype 12 as the receiving a high dose of corticosteroids for graft-versus-host
standard for the Adeno-AF PCR assay. Because the Adeno-BCE disease at the time of hemorrhagic cystitis; the third patient
PCR assay detects the plasmids from adenovirus serotypes 1, received an autologous transplant.
4, 7, and 11 with equal efficiency, we used the 10-fold dilution A total of 919 plasma samples from 132 patients were an-
series of plasmid-containing PCR product from adenovirus se- alyzed for BK virus by quantitative PCR, with a median of 7
rotype 7 as the standard for the Adeno-BCE PCR assay. The samples (range, 1–17 samples) analyzed per patient. BK virus
method used for CMV PCR and human herpesvirus 6 PCR was detected in the blood samples of 44 (33%) of the patients,
has been described elsewhere [9, 10]. with a median of 3 consecutive positive samples (range, 1–11
Hemorrhagic cystitis was graded according to the following samples) detected per patient. BK viremia occurred at a median
criteria: grade 1, microscopic hematuria; grade 2, macroscopic of 41 days (range, 9–91 days) after transplantation. Prolonged
hematuria; grade 3, macroscopic hematuria with clots; and viremia that lasted for 11 month occurred in 16 patients (12%),
grade 4, macroscopic hematuria that requires instrumentation and prolonged viremia that lasted for 12 months occurred in
for the evacuation of clots and/or that causes renal dysfunction 8 patients (6%). The median value of the maximum viral load
[11]. “Platelet engraftment” was defined as a platelet count of was 4.3 ⫻ 10 3 copies/mL (range, 4.3 ⫻ 101 to 3.7 ⫻ 10 6 copies/
120 ⫻ 10 9 platelets/L that was maintained without transfusion mL). The number of patients for whom results remained pos-
support for 7 consecutive days. itive at each week after the first positive result was obtained
The incidence of BK viremia was estimated using the cu- for a sample (figure 1). The results of univariate analysis of
mulative incidence. The risk factors for BK viremia and hem- pretransplantation and posttransplantation variables for any oc-
orrhagic cystitis were evaluated as time-dependent variables by currence of BK viremia and for persistent BK viremia are sum-
use of proportional hazards regression analysis, as was BK vi- marized in table 1.
remia as a risk factor for hemorrhagic cystitis. The analysis of Of 16 patients with preengraftment hemorrhagic cystitis, 1
risk factors for BK viremia was performed for any occurrence patient developed BK viremia the day before hemorrhagic cys-
of BK viremia, as well as for persistent BK viremia, which was titis. All 3 patients with postengraftment hemorrhagic cystitis
defined by positive results for ⭓50% of plasma samples among received positive results of tests for BK virus in plasma at a

1862 • CID 2004:39 (15 December) • BRIEF REPORT

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Figure 1. A , The absolute number of patients for whom results of PCR for BK virus were positive at each week after the positive result was
obtained for a sample. B, The proportion of patients with positive samples among patients with available samples. N values indicate the number of
patients tested.

median of 10 days (range, 1–25 days) before the event. Of these only factors associated with BK viremia. However, we were
3 patients, 1 patient had PCR performed for the detection of unable to demonstrate that the presence of CMV DNA in
BK virus in urine samples at the time of the event, and the plasma or pp65 antigenemia after transplantation was associ-
results were positive. Urine samples from all 3 patients were ated with BK viremia; thus, the correlation between CMV sero-
tested for adenovirus, and the results were negative. Univariate status and BK viremia does not have an obvious biological
time-dependent analysis showed a strong association between explanation, and it requires further validation.
BK viremia and postengraftment hemorrhagic cystitis (P p In contrast to the results of studies of other viral infections
.004). The maximum levels of BK viremia in patients with among HSCT recipients, factors affecting cellular immunity,
postengraftment hemorrhagic cystitis (median, 9.7 ⫻ 10 3 cop- such as lymphopenia, the use of corticosteroids, and graft-
ies/mL; range, 1.8 ⫻ 10 3 to 2.4 ⫻ 10 5 copies/mL) were higher versus-host disease, were not significantly associated with BK
than those in patients without hemorrhagic cystitis (median, 0 viremia in the present study. This finding suggests that, in
copies/mL; range, 0 to 3.7 ⫻ 10 6 copies/mL; P p .008) and addition to impairment of the immune system, other—as yet
those in patients with preengraftment hemorrhagic cystitis (me- unknown—factors contribute to the dissemination of BK virus
dian, 0 copies/mL; range, 0 to 2.7 ⫻ 10 4 copies/mL; P p infection.
.0006). An association between hemorrhagic cystitis in HSCT recip-
Discussion. The present large longitudinal study of 132 ients and the presence of BK virus DNA in plasma has been
patients showed that 33% of patients had BK virus that was examined by Leung et al. [1]. In their study, BK viremia was
quantifiable in plasma. Univariate analysis of risk factors for not consistently associated with hemorrhagic cystitis. Hemor-
any occurrence of BK viremia or for persistent BK viremia rhagic cystitis was associated with low platelet counts; therefore,
identified CMV seropositivity and the underlying disease as the in the present study, we focused our analysis on postengraft-

BRIEF REPORT • CID 2004:39 (15 December) • 1863

 Table 1. Assessment of risk factors for BK viremia among 132 hematopoeitic stem
cell transplant recipients.

Persistent
a
BK viremia BK viremia
(44 events) (20 events)
Risk factor HR (95% CI) Pb HR (95% CI) Pb
CMV serostatus
Of patient .03 .06
Negative (n p 75) 1.0 (Referent) 1.0
Positive (n p 57) 1.9 (1.1–3.4) 2.3 (1.0–5.7)
Of donor and recipient .007 .03

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R⫺ and D⫺ (n p 59) 1.0 (Referent) 1.0
R+ or D+ (n p 73) 2.3 (1.2–4.4) 2.9 (1.1–8.1)
HHV-6 infection .75 .07
No (n p 59) 1.0 (Referent) 1.0
Yes (n p 73) 0.9 (0.4–18) 2.6 (1.0–6.9)
Cumulative dose of steroids,c
per 10-mg/kg increase 1.1 (1.0–1.2) .19 1.2 (1.0–1.4) .08
Treatment regimen received
Cyclophosphamide .39 .06
And TBI 1.3 (0.6–3.2) 2.5 (0.9–6.9)
And busulfan 1.7 (0.8–3.7) 0.7 (0.1–3.4)
Other (n p 50) 1.0 (Referent) 1.0
Diagnosis .06 .02
Acute leukemia or lymphoma
Greater than first remission (n p 53) 1.0 (Referent) 1.0
In first remission (n p 11) 0.3 (0.1–1.4) 0.3 (0.0–2.3)
CML-CP (n p 30) 0.5 (0.2–1.1) 0.3 (0.1–1.0)
Otherd (n p 38) 0.4 (0.2–0.9) .06 0.2 (0.0–0.7) .02

NOTE. Only results with a P value of !.1 are shown. Additional risk factors that were tested but
were not found to be significant included the sex of the transplant recipient, the source of the cells
(peripheral blood stem cells vs. bone marrow), the donor type (autologous vs. allogeneic), the use of
antithymocyte globulin, acute graft-versus-host disease, plasma cytomegalovirus viremia (pp65 110
leukocytes and 150 leukocytes), or positive PCR results (11000 copies/mL and 110,000 copies/mL) for
blood samples, or the lack of lymphocyte engraftment. CML-CP, chronic myeloid leukemia–chronic
phase; D, donor; HD, Hodgkin disease; HHV-6, human herpesvirus 6; HR, hazard ratio; MM, multiple
myeloma; NHL, non-Hodgkin lymphoma; R, recipient; TBI, total body irradiation.
a
Analysis was performed for patients who had at least 3 plasma samples available and for whom
⭓50% of the PCR results for those samples were positive.
b
Overall test of association for each category of risk factor.
c
Based on average.
d
Includes scleroderma, solid tumor, myelodysplastic syndrome, aplastic anemia, and multiple
myeloma.

ment hemorrhagic cystitis to avoid the potential confounding viremia. We speculate that BK virus reactivation originates in
role of thrombocytopenia in the occurrence of hemorrhagic the urinary tract, leading to secondary BK viremia.
cystitis. BK viremia was highly correlated with hemorrhagic The present study has strengths and limitations. Its
cystitis in patients with sustained platelet engraftment; however, strengths include the large sample size, which represents the
we were unable to adjust for others factors because of the small largest cohort to date; the unselected and consecutive nature
number of cases. Although the difference in viral loads between of the patient cohort; weekly sampling; and the use of a val-
patients with postengraftment hemorrhagic cystitis and patients idated quantitative assay. Its limitations include the retro-
with preengraftment hemorrhagic cystitis or patients without spective determination of hemorrhagic cystitis; the lack of
hemorrhagic cystitis was highly significant, the wide range of detection of BK virus in the urine samples obtained from
viral loads in each group limited the clinical usefulness of this some patients; and the small number of cases of postengraft-
measurement. ment hemorrhagic cystitis, which prevented us from per-
Further studies are needed to determine the origin of BK forming multivariate statistical modeling.

1864 • CID 2004:39 (15 December) • BRIEF REPORT

 In conclusion, BK virus is present in the blood of one-third 3. Apperley JF, Rice SJ, Bishop JA, et al. Late-onset hemorrhagic cystitis
associated with urinary excretion of polyomaviruses after bone marrow
of HSCT recipients within the first 100 days after transplan- transplantation. Transplantation 1987; 43:108–12.
tation. A substantial proportion of viremic patients had per- 4. Limaye AP, Jerome KR, Kuhr CS, et al. Quantitation of BK virus load
sistent viremia that lasted for weeks. There is a strong statistical in serum for the diagnosis of BK virus–associated nephropathy in renal
transplant recipients. J Infect Dis 2001; 183:1669–72.
association between BK viremia and postengraftment hemor-
5. Nickeleit V, Klimkait T, Binet IF, et al. Testing for polyomavirus type
rhagic cystitis. Given the small number of cases of posten- BK DNA in plasma to identify renal-allograft recipients with viral
graftment hemorrhagic cystitis, a larger cohort study or a case- nephropathy. N Engl J Med 2000; 342:1309–15.
control study is required to control for possible confounders. 6. Lion T, Baumgartinger R, Watzinger F, et al. Molecular monitoring of
adenovirus in peripheral blood after allogeneic bone marrow trans-
plantation permits early diagnosis of disseminated disease. Blood
2003; 102:1114–20.
Acknowledgments 7. Meyers JD, Ljungman P, Fisher LD. Cytomegalovirus excretion as a

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predictor of cytomegalovirus disease after marrow transplantation: im-
We thank Chris Davis, Rachel Carter, and Peter Choe, for database portance of cytomegalovirus viremia. J Infect Dis 1990; 162:373–80.
services; Jim Reith, for editorial assistance; and Terry Stevens-Ayers, for 8. Wolf DG, Spector SA. Early diagnosis of human cytomegalovirus dis-
assistance in data analysis. ease in transplant recipients by DNA amplification in plasma. Trans-
Financial support. Swiss National Science Foundation (grant PBLAB– plantation 1993; 56:330–4.
100602), Joel Meyers Endowment Fund, and the National Institutes of 9. Zerr DM, Gupta D, Huang ML, Carter R, Corey L. Effect of antivirals
Health (grants CA15704 and CA18029). on human herpesvirus 6 replication in hematopoietic stem cell trans-
Potential conflicts of interest. All authors: no conflicts. plant recipients. Clin Infect Dis 2002; 34:309–17.
10. Boeckh M, Huang M, Ferrenberg J, et al. Optimization of quantitative
detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin
References Microbiol 2004; 42:1142–8.
11. Leung AY, Mak R, Lie AK, et al. Clinicopathological features and risk
1. Leung AY, Suen CK, Lie AK, Liang RH, Yuen KY, Kwong YL. Quan- factors of clinically overt haemorrhagic cystitis complicating bone mar-
tification of polyoma BK viruria in hemorrhagic cystitis complicating row transplantation. Bone Marrow Transplant 2002; 29:509–13.
bone marrow transplantation. Blood 2001; 98:1971–8. 12. Schuchter LM, Hensley ML, Meropol NJ, Winer EP. 2002 Update of
2. Arthur RR, Shah KV, Baust SJ, Santos GW, Saral R. Association of BK recommendations for the use of chemotherapy and radiotherapy pro-
viruria with hemorrhagic cystitis in recipients of bone marrow trans- tectants: clinical practice guidelines of the American Society of Clinical
plants. N Engl J Med 1986; 315:230–4. Oncology. J Clin Oncol 2002; 20:2895–903.

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