Blood, 1 October 2000, Vol. 96, No. 7, pp. 2355-2357
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Nonmalignant disease associated with human herpesvirus 8 reactivation in patients who have undergone autologous
peripheral blood stem cell transplantation
Mario Luppi,
Patrizia Barozzi,
Thomas F. Schulz,
Raffaella Trovato,
Amedea Donelli,
Franco Narni,
Julie Sheldon,
Roberto Marasca, and
Giuseppe Torelli
From the Department of Medical Sciences, Section of
Hematology, University of Modena, Italy; the Department of
Medical Microbiology and Genitourinary Medicine, University of
Liverpool, Liverpool, United Kingdom.
 |
Abstract |
Fever, cutaneous rash, and hepatitis
for which an infectious cause
was suspecteddeveloped in an Italian patient with non-Hodgkin lymphoma after autologous peripheral blood stem cell (PBSC)
transplantation. Polymerase chain reaction (PCR) with degenerate
primers for the highly conserved DNA polymerase gene of herpesviruses
detected herpesvirus sequences 100% identical to human herpesvirus-8
(HHV-8) in serial cell-free serum samples, collected immediately before or concomitant with the occurrence of clinical symptoms; no other common infections were documented. The presence of the HHV-8 genome (clade C) was confirmed by PCR with HHV-8-specific primers for orf 26 and orf-K1. HHV-8 viremia was undetectable either before transplantation or when the patient was clinically asymptomatic. Semiquantitative PCR analysis showed variations of the viral load correlating with the clinical status. Anti-HHV-8 antibodies were detected before and after transplantation by an immunofluorescence assay for lytic antigens. Active HHV-8 infection may be
associated with nonmalignant illness after PBSC/bone marrow transplantation.
(Blood. 2000;96:2355-2357)
© 2000 by The American Society of Hematology.
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Introduction |
Human herpesvirus-8 (HHV-8) primary infection and
reactivation are associated with the development of Kaposi sarcoma,
primary effusion lymphoma, Castleman disease, and plasmacytic
proliferation in patients who undergo solid organ
transplantation.1-9 In contrast, in patients who undergo
bone marrow (BM) and peripheral blood stem cell (PBSC) transplantation,
Kaposi sarcoma is the exception.10 Only one study has so
far been conducted to investigate HHV-8 seroprevalence in patients
after allogeneic BM transplantation. It did not find any association
between the presence of antibodies to HHV-8 latent nuclear antigen
before or after transplantation, to chronic graft-versus-host disease,
or to overall BM transplantation survival.11
In this report we used a combination of polymerase chain reaction
(PCR)-based assays, with degenerate and specific primers, and several
serologic assays to describe a temporal association between HHV-8
reactivation and the development of fever, cutaneous rash, and
hepatitis in an Italian patient who received autologous PBSC infusions.
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Study design |
In September 1996, a 35-year-old man was found to have
B-cell non-Hodgkin lymphoma of Burkitt-like type, involving the jejunum and the mesenteric, laterocervical, and supraclavicular nodes. He
underwent excision of multiple parts of the jejunum and received a
first treatment with chemotherapy according to the BFM-LNH81 protocol
(cyclophosphamide, prednisone, methotrexate, aracytine, teniposide,
doxorubicin), achieving complete clinical remission. His PBSC
were mobilized with cyclophosphamide and granulocyte colony-stimulating
factor (G-CSF). Then he underwent autologous PBSC transplantation in
July 1997. The conditioning regimen consisted of BEAM chemotherapy
(carmustine, etoposide, aracytine, and melphalan). A dose of
4.5 × 106/kg unmanipulated CD34+ cells was
reinfused, and the patient's neutrophil count became greater than
0.5 × 109/L in 10 days, and his platelet count became
20 × 109/L in 12 days. Prophylactic treatment with
ciprofloxacin, itraconazole, acyclovir, and immunoglobulin was
administered. Seventeen days after PBSC reinfusion, the patient had
intermittent fever higher than 38°C, asthenia, and increased levels
of the liver enzymes aspartate aminotransferases (AST; 145 IU/L) and
alanine aminotransferases (ALT; 255 IU/L). Two days later (day 19) the
patient contracted a cutaneous maculopapular rash, which regressed in 4 days, and diarrhea, which lasted for 3 days. Liver enzyme levels
reached a peak (AST, 263 IU/L; ALT, 367 IU/L) and then progressively
decreased but maintained elevated, with variations, for 6 months
(Figure 1). Chest radiograms were
negative, and ultrasonographic examination of the abdomen only revealed
homogeneous enlargement of the liver. The patient has remained
asymptomatic and in complete remission, and neither Kaposi sarcoma nor
lymphoproliferative disease has been diagnosed in the posttransplant
period, after a follow-up of approximately 2 years.
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| Figure 1.
Clinical history and virologic study.
Kinetics of AST and ALT, clinical signs, HHV-8 DNA (copies/mL), and
anti-HHV-8 antibodies (Ab) in the patient, after autologous peripheral
blood stem cell transplantation (APBSCT).
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Results and discussion |
Routine blood and urine cultures and serologies for common
bacterial, fungal, and viral infections were negative. Nuclei acid extraction was performed in serial cell-free serum samples, as described.12 Herpesvirus (HSV-1, HSV-2, VZV, EBV, HHV-6,
HHV-7, CMV), adenovirus, polyomavirus (JC, BK) and hepatitis (A, B, C, G) virus DNA and RNA were not detected in the sera by PCR assays, using
described protocols.12,13 Using PCR with degenerate
primers targeting the highly conserved DNA polymerase gene of
herpesviruses,14 amplification products of the predicted
size of 236 base pairs were detected in the sera collected at days 12, 21, and 23 after transplantation, immediately before and concomitant
with the clinical symptoms. The resultant PCR fragments were sequenced,
and the alignment of the nucleotide and amino acid sequences revealed that the DNA sequences identified in the sera were 100% identical to
the prototype HHV-8 DNA polymerase gene sequence (Figure
2).14 The presence of the
HHV-8 genome was further confirmed by PCR amplification of specific orf
26 and K1 sequences of HHV-8 in the same serum
samples.15-17 Nested PCR for the K1 gene detected as few
as 50 HHV-8 copies, determined by serial dilutions of the HHV-8-positive BCBL-1 cell line. HHV-8 DNA was not detectable in the
serum collected before transplantation. It was first detected by nested
PCR (10-100 copies/mL, as determined semiquantitatively by 10-fold
serial dilutions of patient DNA) in the serum collected at day 12 after
transplantation (day +12), before the onset of fever and the increase
of aminotransferase levels (Figure 1). HHV-8 viremia persisted
throughout the duration of the clinical symptoms (Figure 1).
Semiquantitative PCR analysis also showed that the viral load increased
progressively to 1000 to 10 000 copies/mL at day +21, to be detectable
even by 1-step PCR. Then it decreased to 10 to 100 copies/mL at
day + 23 and was no longer detectable after the regression of
clinical symptoms (Figure 1). HHV-8 sequences were also detected by
nested PCR (orf 26 and K1) in the DNA extracted from the
Ficoll-separated PBMC collected on days +21 and +23. Phylogenetic
analysis and sequence analysis of the 2 highly variable regions of the
K1 gene from this patient showed that the infecting strain belonged to
clade C, known to be common in Italy.17 This patient had
antibodies to HHV-8 that were detectable by immunofluorescence assay
for lytic antigens before transplantation (day 
60) and in all but one
serum sample collected after transplantation. Interestingly, no
antibodies were detectable in the serum sample (at day +21) with the
highest viral load. The patient had no detectable antibodies to the
latent nuclear antigen or to recombinant capsid protein encoded by orf 6518-20 or recombinant membrane protein encoded by
K8.1.21
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| Figure 2.
HHV-8 DNA polymerase sequence identified by degenerate
PCR assay.
Alignment of amino acid translations of sequences from the herpesvirus
DNA polymerase gene obtained from the patient's sera, with analogous
regions from the DNA polymerase sequences of other
herpesviruses14: human herpesvirus-8 (HHV-8), human
herpesvirus-7 (HHV-7), human herpesvirus-6 (HHV-6), human
cytomegalovirus (HCMV), Epstein-Barr virus (EBV), varicella-zoster
virus (VZV), herpes simplex virus type 2 (HSV-2), and herpes simplex
virus type 1 (HSV-1). Dots indicate identity with the first sequence in
the alignment.
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Thus far, HHV-8 infection has been involved in the development of
neoplastic diseases in patients after transplantation.1-9 Our study shows that active HHV-8 infection may be associated with
nonmalignant pathologic events in PBSC recipients, at least in our
geographic area (lower Po Valley, northern Italy), where HHV-8
seroprevalence in the blood donor population is approximately 13%.22,23 The detection of anti-HHV-8 antibodies before
transplantation suggests that HHV-8 viremia is likely to be caused by
viral reactivation rather than by primary infection with HHV-8. The
fact that HHV-8 viremia was detectable in serial cell-free serum
samples collected immediately before or concomitant with the clinical
symptoms, but undetectable in the serial sera collected after the
regression of the symptoms and before transplantation, support a causal
association between HHV-8 infection and the disease in this patient.
HHV-8 reactivation occurred early (within the first 30 days) after
transplantation, was transient, and was associated with an acute but
self-limited clinical syndrome characterized by fever, hepatitis, and
cutaneous rashsymptoms that have been noted in the context of other
human herpesvirus infections. HHV-8 reactivation in this patient could not be prevented by prophylactic treatment with acyclovir, consistent with the reported in vitro resistance of HHV-8 to this and to the most
common antivirals.24,25 Finally, the identification of a
serologic response to HHV-8 with only the lytic immunofluorescence assay suggests that using a single antibody assay may underestimate the
true prevalence of HHV-8 infection in HIV-negative patients without
Kaposi sarcoma and that at least some patients with "lytic immunofluorescence assay only" reactivity are genuinely infected with
HHV-8. Thus, PCR appears useful for the early identification and
monitoring of HHV-8 viremia and its associated complications in
patients who undergo BM and PBSC transplantation.
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Footnotes |
Submitted March 6, 2000; accepted May 15, 2000.
Supported by a grant from Associazione Italiana per la Ricerca sul
Cancro (M.L.) and a grant from National Health Service Biomedical Research and Development Fund (RDO/22/09) (T.F.S), and from
Ministero Universita-Ricerca Scientifica e Tecnologica (MURST) (G.T.).
M.L., P.B., and T.F.S. contributed equally to this study.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Giuseppe Torelli, Department of Medical Sciences,
Section of Hematology, Policlinico, Via del Pozzo 71, 41100, Modena,
Italy; e-mail: gtorelli{at}unimo.it.
| |
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Abstract
Introduction
