Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2597-2599
CORRESPONDENCE
Prediction of Human Herpesvirus 6 Infection After Allogeneic Bone
Marrow Transplantation
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LETTER |
To the Editor:Human herpesvirus 6 (HHV-6) is
a recently discovered member of the human herpesvirus
family.1 Although primary infection with variant B HHV-6
causes exanthem subitum,2 the clinical features of variant
A HHV-6 infection remains unclear. The virus probably latently infects
the body after the primary infection and then reactivates in an
immunosuppressed state like other human herpesviruses. HHV-6 has
recently been recognized as an opportunistic pathogen in transplant
recipients.3-7 It has been shown that HHV-6 might be
associated with fever and skin rash resembling acute graft-versus-host
disease (GVHD),3 interstitial pneumonitis,4
encephalitis,6 and bone marrow suppression7 after bone marrow transplantation (BMT). Since infection with the virus
after BMT could be fatal,6 it is important to prevent the
infection. Therefore, if we are able to predict HHV-6 infection after
BMT, it should prove invaluable in helping to prevent the virus
infection.
There are two likely sources for HHV-6 infection after BMT: one is
reactivation from the recipient body and another is infection via the
donor marrow from a seropositive donor. Therefore, virus genome
latently infected in peripheral blood mononuclear cells (PBMCs) of
donors and recipients could be an important source of the virus
infection after transplantation. The aim of this study is to determine
whether the presence of HHV-6 genome in PBMCs before BMT is a valuable
predictor of virus infection after BMT. We also analyzed whether HHV-6
antibody titers of donors and recipients at the time of transplantation
were associated with virus infection.
Thirty recipients (20 male and 10 female), who received allogeneic BMT
at the Children's Medical Center of the Japanese Red Cross Nagoya
First Hospital, and their donors, were employed in this study. All
guardians of these patients consented to be in this study. Patient
characteristics relating to age, sex, and underlying disease are
summarized in Table 1. The median age of
these recipients was 5.9 years old (ranging from 1 year to 15 years
old) at the time of transplantation. EDTA peripheral blood samples were
collected from donor and recipient pairs at the time of
transplantation. In addition, EDTA blood samples were collected at 2 weeks before transplantation and biweekly after transplantation until 2 months after transplantation from recipients. We attempted to isolate
HHV-6 from PBMCs and measure antibody titers to HHV-6 by indirect
immunofluorescence assay. We also analyze for the presence of HHV-6 DNA
in PBMCs obtained from the donor at the time of BMT and from the
recipient at 2 weeks before BMT.
Five hundred nanograms of DNA extracted from PBMCs obtained from
recipients approximately 2 weeks before transplantation and from donors
at the time of transplantation was used for nested polymerase chain
reaction (PCR) amplification. Nested PCR was performed for
amplification of HHV-6 DNA by using two primer sets (A/C, HS6AE/HS6AF)
as previously described.8 The PCR resulted in the
amplification of a 751-bp DNA fragment encoding a putative large
tegument protein gene. The type of HHV-6 was determined by the presence
of an HindIII site in each second PCR product. The
sensitivity of the PCR assay was determined with the use of serial
dilutions of the plasmid, pSTY-05 (kindly provided by Dr K. Yamanishi,
Department of Bacteriology, Osaka University, Osaka, Japan). As shown
in Fig 1, we could routinely detect 100 copies of the virus genome. To ensure the accuracy of each PCR assay, dilutions containing 100 copies and 10 copies of the plasmid were coamplified with each of the samples in the following analyses. Statistical analyses were performed by using Fisher's exact test and
Student's t-test.
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| Fig 1.
Representative second PCR products from serial dilution
of plasmid (pSTY-05) for determining the sensitivity of our PCR assay.
Numbers on the top of the panel indicate the copy number of the pSTY-05
plasmid.
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If HHV-6 was isolated from PBMCs or a significant increase of HHV-6
antibody was observed, we defined this as a virus infection. HHV-6
infection was confirmed in 17 (57%) of the 30 recipients. The virus
was isolated from 10 (33%) of the 30 recipients between 7 and 29 days
after transplantation. Seven additional recipients had a significant
increase in their HHV-6 antibody titer during the observation period.
All of the donors and the recipients were seropositive to HHV-6 at the
time of BMT. Seven of the 10 isolates were analyzed to determine the
variant of the virus. All 7 isolates were variant B HHV-6 on the basis
of restriction fragment length polymorphism analysis of the second PCR
products (data not shown).
HHV-6 DNA was detected in 10 (33%) of the 30 donors at the time of
BMT, and in 15 (50%) of the 30 recipients before BMT. Details of the
presence of the virus genome in PBMC and the results of virological
examinations after transplantation are shown in Table 2. HHV-6 infection occurred in 3 of 4 donor-positive/recipient-negative patients, in 7 of 9 donor-negative/recipient-positive patients, in all of the
donor-positive/recipient-positive patients, and in 1 of 11 donor-negative/recipient-negative patients. Moreover, all of the second
PCR products detected in this study were digested with
HindIII indicating variant B HHV-6 (data not shown). Details relating to the presence of the virus genome and the antibody titers of
the donor and the recipient at risk for HHV-6 infection after
allogeneic BMT are shown in Table 3. If
HHV-6 genome was detected in a donor's or recipient's PBMCs, we
defined this as a positive for the virus genome. Sixteen (84%) of the
19 patients who were positive for the virus genome before BMT were
infected with the virus after transplantation. However, only 1 (9%) of the 11 patients who were negative had HHV-6 infection. Thus, the presence of the virus genome in the donor or the recipient before transplantation was a good predictor of HHV-6 infection after allogeneic BMT (P < .001). The geometric mean titers
(GMT) (log10) of HHV-6 antibody in donors with HHV-6 infection (1.3)
and donors without infection (1.4) were not significantly different.
Moreover, GMT of the antibody to HHV-6 in recipients with infection
(1.5) and without infection (1.5) were not significantly different.
As we expected, the presence of HHV-6 DNA in a recipient's or donor's
PBMCs before transplantation correlated with the virus infection after
allogeneic BMT. Although many studies have been done to elucidate the
occurrence, time course, and clinical features of HHV-6 infection after
BMT, this is the first demonstration that the presence of the virus
genome before transplantation is predictive of virus infection after
allogeneic BMT. It is likely that infection with HHV-6 after BMT is
affected by a number of factors, such as the degree of
immunosuppression, HLA mismatch between donor and recipient, and the
occurrence of acute GVHD. We have to evaluate other factors on
prediction of the virus infection after allogeneic BMT in the future. A
large number of prospective studies is needed to perform multivariate
analysis.
Although HHV-6 DNA was not detected in 20 donors and 15 recipients, all
of them were seropositive to HHV-6 at the time of BMT. This indicates
that the amount of latently infected HHV-6 genome was below the level
of detection in our PCR assay. Hence, the sensitivity of the PCR assay
is a critical issue for this study. Therefore, all of the PCR assays
were performed carefully to eliminate contamination as previously
described. The detection limit of our nested PCR was 100 copies of
HHV-6 genome. We used standard solutions containing 10 copies and 100 copies of the plasmid pSTY-05 as negative and positive controls,
respectively, to maintain the accuracy of the PCR. Five hundred
nanograms of viral DNA was used for each PCR analysis in this study and
a good correlation was found between the results of the PCR and the
virus infection. Some of the DNA samples which gave positive PCR
results in this assay using 500 ng gave negative results when only 50 ng of DNA was used (data not shown). Therefore, it is likely that the
quantity of DNA used for PCR is another important factor to consider
for obtaining good results. We collected PBMC from the recipients 2 weeks before BMT to analyze for the presence of the virus genome. This
is because the recipients generally had a severe bone marrow
suppression at the time of BMT, and it might be impossible to obtain
sufficient PBMCs at that time.
As we expected, all of the donors and the recipients had HHV-6 antibody
at the time of BMT. It is impossible to predict virus infection on the
basis of the sero-status of the donor or the recipient, as suggested by
studies on cytomegalovirus infection. Moreover, no clear correlation
between antibody titers of either the recipients or the donors and the
virus infection was shown. Although Willborn et al9 used a
PCR assay for determining HHV-6 infection after BMT, they reported a
similar finding to what we have reported here. This indicates that
antibody titers of the donor or the recipient do not have a predictive
value for HHV-6 infection.
In this study, we demonstrated that the presence of the virus genome in
a donor's or recipient's PBMCs is a good predictor of HHV-6 infection
after allogeneic BMT. Prediction of the clinical features caused by the
virus infection will be an important issue for the future study.
Moreover, measurement of the amount of the virus genome in blood
products transfused to the recipient will be another important problem
to resolve in future studies. Prospective study consisting of a large
number of cases is now proceeding to resolve such issue.
Tetsushi Yoshikawa
Kyoko Suzuki
Masaru Ihira
Hiroshi Furukawa
Sadao Suga
Yoshizo Asano
Department of Pediatrics
Fujita Health University
School of Medicine
Toyoake, Aichi, Japan
Seiji Kojima
Koji Kato
Takaharu Matsuyama
Division of
Hematology-Oncology
Children's Medical Center
Japanese Red Cross
Nagoya First Hospital
Nagoya, Aichi, Japan
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ACKNOWLEDGMENT |
Supported in part by grants from Fujita Health University and a
Grant-in-Aid for Scientific Research, The Ministry of Education, Science and Culture, Japan. We thank Michael Conlon, PhD, for revision
of the language.
| |
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