Full text of DOI:10.1111/j.1348-0421.2002.tb02754.x

Microbiol. Immunol., 46(10), 701―705, 2002
Monitoring of Active HHV-6 Infection in Bone
Marrow Transplant Recipients by Real Time PCR;
Comparison to Detection of Viral DNA in Plasma
by Qualitative PCR
Masaru Ihira1, Tetsushi Yoshikawa*, 2, Kyoko Suzuki1, Masahiro Ohashi1, Sadao Suga1,
Keizo Horibe3, Naoko Tanaka4, Hiroshi Kimura4, Seiji Kojima4, Koji Kato5,
Takaharu Matsuyama5, Yukihiro Nishiyama2, and Yoshizo Asano1
1Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 470
ogy, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Aichi 466
Japan, 3Department of Pediatrics, 4Developmental Pediatrics, Nagoya University School of Medicine, Nagoya, Aichi 466
―
1192, Japan, 2Laboratory of Virol-
―
8550,
8550,
―
Japan, and 5Division of Hematology-Oncology, Children’s Medical Center, the Japanese Red Cross Nagoya First Hospital,
Nagoya, Aichi 453―0046, Japan
Received April 8, 2002; in revised form, July 19, 2002. Accepted July 22, 2002
Abstract: Twelve (46%) of the 26 patients had human herpesvirus 6 (HHV-6) viremia after bone marrow
transplant (BMT). All isolates were recovered from the samples obtained at 2 weeks after BMT. The sen-
sitivity and the specificity of detection of viral DNA in plasma by qualitative polymerase chain reaction
(PCR) for monitoring active virus replication were 92% and 97% respectively. Moreover, the positive (85%)
and negative (99%) predictive values were also high. The patients with HHV-6 viremia showed a clear peak
in HHV-6 DNA in peripheral blood mononuclear cells (PBMCs) at 2 weeks after BMT, which was measured
by real time PCR. The virus DNA level in PBMCs between the two groups (patients with viremia and
patients without viremia) was statistically different at 2 weeks after BMT (P＝0.033). In patients with HHV-
6 viremia, mean HHV-6 DNA copy number was higher in the samples collected at 2 weeks after BMT than
the samples collected at any other time period.
Key words: HHV-6, Real time PCR, Plasma PCR, Bone marrow transplantation
Human herpesvirus 6 (HHV-6) was originally isolat-
ed from peripheral blood mononuclear cells (PBMCs) of
patients with lymphoproliferative disorders or acquired
immunodeficiency syndrome (12). On the basis of
genomic DNA sequences, cell tropism, and reactivity to
several monoclonal antibodies, two distinct variants
(variant A and variant B) have been described (18).
Although primary HHV-6 variant B infection occurs
early in childhood (20) and causes exanthem subitum (1,
19), clinical features of HHV-6 variant A infection
remain unclear. After primary infection, HHV-6 latent-
ly infects PBMCs (7, 15) or several organs including kid-
neys, and salivary glands throughout life. Moreover,
the virus can reactivate in patients with immunosup-
pression, such as bone marrow transplant (BMT) recip-
ients. Recent studies have suggested that HHV-6 reac-
tivation in BMT recipients could cause several manifes-
tations including fever, skin rash (2, 21, 24), acute graft
versus host disease (17), encephalitis (5), interstitial
pneumonitis (3), and bone marrow suppression (4).
Therefore, monitoring of active HHV-6 infection is
important to ensure the health of immunocompromised
patients. Polymerase chain reaction (PCR) assay is a
commonly used method for monitoring active viral
infection. However, this sensitive assay may detect the
genome of the latent virus in PBMCs (false positive). To
remedy the problem, it is necessary to measure the quan-
tity of the virus DNA in PBMCs. Recently, it has been
shown that a real time PCR is useful for analysis of
copy numbers of viral DNA (6, 16) because of high
accuracy and reproducibility. Meanwhile, it has been
*Address correspondence to Dr. Tetsushi Yoshikawa, Labora-
tory of Virology, Research Institute for Disease Mechanism and
Control, Nagoya University School of Medicine, Nagoya, Aichi
Abbreviations: BMT, bone marrow transplantation; HHV-6,
human herpesvirus 6; PBMC, peripheral blood mononuclear
cell; PCR, polymerase chain reaction.
466
―8550, Japan. Fax:＋81―52―744―2452. E-mail: tetsushi@
med.nagoya-u.ac.jp
701

702
M. IHIRA ET AL
reported that detection of viral genome in serum or plas-
ma by qualitative PCR (plasma PCR) is another good
indicator of active HHV-6 infection (14). However,
there has been no report examining an association
between the results of these two methods and virus iso-
lation, which is considered to be the gold standard for
detecting active virus replication. In this paper, the
quantity of HHV-6 DNA in PBMCs was measured by
using a real time PCR in BMT recipients, and the kinet-
ics of viral DNA between the patients with HHV-6
viremia and those without viremia were compared. The
reliability of plasma PCR for monitoring active viral
infection in BMT patients was also assessed.
analysis, and plasma samples were used for qualitative
PCR assay. DNA was extracted from 200 µl of plasma
by using a QIAamp Blood Kit (QIAGEN, Chatsworth,
Calif., U.S.A.), eluted in 100 µl of distilled water and
stored at －70 C until analysis. Ten µl of eluted DNA
was used for PCR assay. Nested PCR was carried out for
amplification of HHV-6 DNA using two primer sets
(A/C, H6AE/H6AF) as described previously (15).
Aliquots of TE buffer were processed as a negative con-
trol, and appropriate precautions were taken during sam-
ple preparation and performance of the PCR to avoid
cross contamination. The sensitivity of the nested PCR
assay was 10 copies/reaction.
Twenty-six patients who received BMT at Nagoya
University Hospital or the Japanese Red Cross Nagoya
First Hospital were enrolled in this study. Thirteen
patients were male and 13 were female. Twenty-five of
the 26 patients underwent allogeneic BMT and the
remaining one underwent autologous BMT. Eighteen
of the 25 patients received marrow from HLA-matched
siblings and the remaining 7 received marrow from
HLA-matched unrelated donors. Transplantations were
performed for severe aplastic anemia (n＝4), acute lym-
phoblastic leukemia (n＝5), acute myelogenous leukemia
(n＝9), myelodysplastic syndrome (n＝2), neuroblas-
toma (n＝2), acute undifferentiated leukemia (n＝1),
chronic myelogenous leukemia (n＝1), juvenile chronic
myelogenous leukemia (n＝1), and Niemann-Pick Dis-
ease (n＝1). Details of the conditioning regimen and
graft versus host disease prophylaxis are described else-
where (10). In brief, patients with hematological malig-
nancy were conditioned with melphalan (180 mg/m2)
plus busulfan (16 mg/kg) or total body irradiation (12
Gy) with high dose chemotherapy. Patients with severe
aplastic anemia were conditioned with cyclophosphamide
(50 mg/kg) and anti-thymocyte globulin (ATG; Lym-
phoglobulin, 15 mg/kg). Graft versus host disease pro-
phylaxis consisted of methotrexate with or without
cyclosporine. Patients received intravenous γ-globulin
preparations weekly during the first 3 months as pro-
phylaxis for cytomegalovirus infection. Oral acyclovir at
doses of 300 to 1,000 mg was given daily.
DNA was extracted from PBMCs by using a QIAamp
Blood Kit (QIAGEN), eluted in 100 µl of distilled water.
DNA concentration was measured by spectrophotometer.
Ten µl of extracted DNA was used for real time PCR
assay, and it was performed with an ABI Prism 7700
Sequence Detection System (PE Applied Biosystems,
Foster City, Calif., U.S.A.) according to the guidelines
supplied. The primers and probe setting for HHV-6 have
been described elsewhere (16). The PCR reaction was
performed using a TaqMan PCR Kit (PE Applied Biosys-
tems). A standard graph was constructed using the CT
values obtained from serially diluted pSTY05 plasmid
containing the U31 gene of HHV-6. The CT values from
the clinical samples were plotted on the standard curve,
the copy number was calculated automatically using
Sequence Detector v1.6 (PE Applied Biosystems), a
software package for data analysis. This assay detects
both variants of HHV-6. The limit of quantification
was 10 copies/reaction.
Student’s t-test was used to compare the mean copy
numbers of viral DNAs. The association between iso-
lation of HHV-6 and presence of the virus DNA in plas-
ma was assessed by the chi-square test.
Twelve (46%) of the 26 patients had HHV-6 viremia
after BMT. All isolates were recovered from the samples
obtained at 2 weeks after BMT. Since OHV-3 (variant B
specific monoclonal antibody) was used to confirm the
virus isolation, all isolates were considered to be HHV-
6 variant B. Thirteen (14%) of the 91 plasma samples
collected from the patients throughout the study were
positive for the HHV-6 DNA (Table 1). An associa-
tion between the virus isolation and detection of viral
DNA in plasma was evaluated. The sensitivity and the
specificity of the assay for monitoring active virus repli-
cation was 92% and 97% respectively. Moreover, the
positive (85%) and negative (99%) predictive values
were also high.
Samples were obtained after the project was thor-
oughly explained to the guardians of the patients. EDTA-
treated peripheral blood was collected from the patients
at the time of BMT and at 2 weeks, 1 month, and 2
months after BMT. Since the main focus of this study
was to evaluate the reliability of the two assays for mon-
itoring active HHV-6 infection, samples were not col-
lected from donors in this study.
The procedures for isolation and identification of
HHV-6 have been described elsewhere (1). PBMCs
were used for the virus isolation and real time PCR
The kinetics of HHV-6 DNA copy numbers in PBMCs
are shown in Fig. 1. We compared the HHV-6 DNA
copy numbers in the patients with HHV-6 viremia and

703
NOTES
Table 1. Association between virus isolation and presence of viral DNA in plasma
Category of the sample
HHV-6 DNA in plasma
Positive Negative
Positive of HHV-6 isolation
Negative of HHV-6 isolation
11
2
1
77
* Sensitivity: 92%, specificity: 97%, positive predictive value: 85%, negative pre-
dictive value: 99%.
those in the patients without viremia. In the patients
without HHV-6 viremia, mean copy numbers of HHV-6
DNA were less than 10 copies/µg DNA. Moreover, no
remarkable peak in copy numbers of viral DNA was
demonstrated during the observation period. In contrast,
the patients with HHV-6 viremia showed a clear peak in
HHV-6 DNA copy numbers at 2 weeks after BMT.
Throughout the observation period, the amount of HHV-
6 DNA was higher in the patients with HHV-6 viremia as
compared with those without viremia. The difference in
viral DNA levels between the two groups was statistically
significant at 2 weeks after BMT (at the same time as the
virus isolation) (P＝0.033).
Changes in copy numbers of viral DNA at each time
period were also analyzed in patients with and without
HHV-6 viremia (Fig. 1). In patients with HHV-6 viremia,
mean HHV-6 DNA copy number at 2 weeks after BMT
was significantly higher than at any other time period.
Moreover, the HHV-6 DNA copy number at 2 weeks
after BMT appeared to have reached a maximum, as
levels were greater than those at day 0 and 1 month
after BMT in each patient (Fig. 1A). In contrast to the
patients, mean copy numbers of HHV-6 DNA in each
time period were stable in the patients without HHV-6
viremia (Fig. 1B).
In order to elucidate the reliability of the newly devel-
oped real time PCR assay as a predictor of active viral
infection, which was determined by virus isolation, we
postulated that a value of at least 10 copies/µg of viral
DNA would be an indicator for active virus infection. All
12 samples from which the virus was isolated contained
more than 10 copies/µg of viral DNA. Sixty of the 79
samples from which the virus was not isolated were
negative (less than 10 copies/µg of DNA), and more
than 10 copies/µg of viral DNA was detected from the
remaining 19 samples. Therefore, the sensitivity and
the specificity of this assay were 100% and 74% respec-
tively, and the positive predictive value was 35% while
the negative predictive value was 100%.
Fig. 1. Comparison of HHV-6 DNA levels in patients with (A) and
without HHV-6 viremia (B) at different periods of time. All 12
isolates were recovered at 2 weeks after bone marrow trans-
plant. Open circle indicates the sample without the virus isolation
and closed circle indicates the sample with the virus isolation. n:
___
number of samples,
: mean copy numbers of HHV-6 DNA.
*Mean copy numbers in patients with HHV-6 viremia was sig-
nificantly higher than those in patients without viremia at 2
weeks after bone marrow transplantation (P＝0.033). No sta-
tistical difference in copy numbers of viral DNA was observed in
other time periods.
Due to evidence that HHV-6 can latently infect
PBMCs (7, 15), detection of the viral DNA in PBMCs
obtained from BMT recipients is meaningless for diag-
nosis of active infection of the virus. To solve the prob-
lem, it has been postulated that a quantitative analysis of
viral DNA in PBMCs or plasma PCR is suitable for
evaluation of active HHV-6 infection. Recently, the
establishment and basic evaluation of real time PCR for
HHV-6 has been reported (9). Nitsche et al. (11) have
shown that HHV-6 variant A DNA is detected frequent-

704
M. IHIRA ET AL
ly in plasma, but not PBMCs, in BMT recipients on the
basis of real time PCR analysis. However, evaluation of
these methods for monitoring active HHV-6 infection in
BMT recipients has not been carried out. In this study,
we used the isolation of the virus from PBMCs as an
indicator of active viral infection after BMT, and evalu-
ated the reliability of quantitative analysis of HHV-6
DNA in PBMCs by real time PCR and plasma PCR for
monitoring active HHV-6 infection after BMT.
between active viral replication and latent infection of the
virus by real time PCR. As shown in Fig. 1, some
PBMC samples from which the virus was not isolated
contained larger amounts of viral DNA than the samples
from which the virus was isolated. It is likely that this is
the reason why the specificity and the positive predictive
value were low. Ljungman et al. (8) have also demon-
strated a wide variation in HHV-6 DNA levels present in
PBMC samples. In order to solve the problem, mea-
surement of copy numbers of viral DNA in plasma sam-
ples by real time PCR should be carried out in future
studies.
Plasma PCR had high sensitivity and high specificity
for detecting active viral infection after BMT. This
result is consistent with the result demonstrated by Sec-
chiero et al. (14). Recently, we have demonstrated that
plasma PCR is useful for monitoring active HHV-6
infection after liver transplantation (23). This method has
the advantage of being more readily available than real
time PCR in most laboratories. These findings suggest
that plasma PCR could be a useful tool for the detection
of active viral infection. However, it is difficult to mon-
itor the kinetics of viral activities over time. Monitoring
of HHV-6 DNA copy numbers in plasma by real time
PCR is a possible new strategy to solve the problem.
DNA concentrations extracted from the 2 false positive
(positive for plasma PCR and negative virus isolation)
PBMC samples was low. Therefore, it is possible that
sufficient numbers of PBMCs for virus isolation were not
collected. Meanwhile, a false negative (negative for
plasma PCR and positive virus isolation) sample con-
tained high copy number of viral DNA (639 copies/µg
DNA) in PBMCs. It is difficult to explain why plasma
PCR became negative. The frequency of false nega-
tive result should be re-evaluated in future large case
analysis.
The authors gratefully acknowledge Takeda Chemical Indus-
tries, Ltd., Osaka, Japan, which kindly supplied recombinant
human interleukin-2.
This work was supported by Japan Society for the Promotion
of Science; Grant number: JSPS-RFTF97L00703 and Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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