Haemophilia (2003), 9, 657–659
LETTER TO THE EDITOR
Prevalence of Kaposi’s sarcoma-associated herpesvirus
infection in haemophilic patients
G. THEODOSSIADES,* L. ARVANITAKIS, V. TSEVRENIS,* E. NOMIKOU,* A. ZOGRAFIDIS,
D. BOURBOULIAà and I. KONTOPOULOU-GRIVA*
*First Regional Transfusion and Haemophilia Centre, Hippokration Hospital, Molecular Virology Laboratory,
Hellenic Pasteur Institute, Athens, Greece; and àWolfson Institute for Biomedical Research, University College
London, London, UK
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a
gamma herpesvirus, the sequence of which was
identified by representative differential amplification
in the lesion of HIV-infected patients with Kaposi’s
sarcoma (KS) in 1994 [1]. KSHV has also been found
in classic Mediterranean KS and other tumours such
as multicentric Castleman’s disease and primary
effusion lymphoma [2].
Little is known about the precise way in which
KSHV is transmitted. It is known that KSHV
transmission occurs during sex in homosexual men
[3]. In classic and endemic KS countries, KSHV
seroprevalence is high in adults [4,5], but also occurs
in children because of close contact within infected
members of the families [6–8]. Although the presence
of viral genome in peripheral blood mononuclear
cells and CD19+ B cells has been demonstrated
[9,10], various studies have shown that blood-borne
transmission of KSHV in multitransfused patients
receiving white blood cell-reduced components, if it
exists, is rare [11,12].
Epidemiological studies, even before the discovery
of KSHV, have confirmed that HIV-positive patients
with haemophilia had a low incidence of KS
compared with HIV-positive homosexual or bisexual
men. It was concluded that the presumed infectious
agent causing KS was not normally transmissible
parenterally [13]. After KSHV identification, studies
have failed to reach a definite conclusion whether the
virus could be transmitted by coagulation factor
concentrates. Two studies have reported a lower
Correspondence: George Theodossiades, MD, First Regional
Transfusion and Haemophilia Centre, Hippokration Hospital,
Vassilissis Sophias 114, Athens 11527, Greece.
Tel.: + 302 10777 4833; fax: + 302 10770 2959;
e-mail: gdtheo1@otenet.gr
Accepted after revision 10 June 2003
 2003 Blackwell Publishing Ltd
KSHV seroprevalence in patients with haemophilia
than in blood donors [14,15], while another study
has shown a significantly higher prevalence of KSHV
in HIV-infected and non-HIV-infected haemophilic
patients than in controls [16].
In view of this, we investigated the seroprevalence
of KSHV in patients with haemophilia by serological
methods (anti-KSHV antibodies) and detection of
KSHV DNA in blood.
We tested blood samples from 50 patients (39 with
haemophilia A, three with haemophilia B and eight
with von Willebrand’s disease), of whom 25 were
HIV- and 44 HCV-infected. All HIV-infected patients
except two, and 16 of the 25 non-HIV-infected
patients with haemophilia suffered from severe dis-
ease. All the haemophilic patients have received
non-virus-inactivated factor VIII concentrates before
routine viral inactivation techniques were implicated,
i.e when the risk for viral contamination of the
concentrates was high. We also tested samples from
50 age- and sex-matched controls (HIV- and HCV-
negative) obtained from the volunteer blood donors.
Indirect immunofluorescence assay (IFA) was used
to detect antibodies against the latent nuclear antigen
(LANA) encoded by open reading frame 73 (ORF73).
Briefly, the BCP-1 cell line was used [17,18]. The cells
were washed twice in phosphate-buffered saline (PBS),
fixed in 4% paraformaldehyde and permeabilized
with 0.2% Triton-X-100. Cells were resuspended in
PBS, spotted and dried on glass slides. Human sera
were diluted (1:100) in PBS with 3% foetal bovine
serum (FBS). The diluted sera (1:100) were added to
the fixed BCP-1 cells and incubated for 45 min at
22
C. Slides were then washed in PBS with 3% FBS
and fluorescein isothiocyanate-(FITC)-labelled rabbit
anti-human IgG Ab (DAKO, High Wycombe, UK)
diluted (1:60) in PBS and 3% FBS was added. After
20 min of incubation at 22
C, the slides were washed
657
658 THEODOSSIADES et al.
in PBS only and screened under ultraviolet microsco-
py. All positive sera were scored by IFA as +, ++ or +++
according to fluorescence signal intensity and diluted
two-fold to determine the antibody titre [19]. As a
negative control cell line, the KSHV-negative and
EBV-negative Ramos cells were used. No nuclear
staining is observed in these cells.
Genomic DNA was extracted from whole blood
(Wizard System; Promega, Madison, Wisconsin,
USA) as per the manufacturer’s instructions. PCR
conditions and sequences of oligonucleotides used
for the amplification and hybridization of both the
KSHV KS330233 Bam fragment and its flanking
sequences were as originally reported [1]. All oligo-
nucleotides used in these studies as PCR primers or
probes were obtained commercially (MWG Biotech
AG, Edersberg, Germany). Primers for amplification
of the 233 bp KS330 fragment were as follows: sense
(+) primer (20mer): 5¢-AGC-CGA-AAG-GAT-TCC
ACC-AT-3¢; antisense ()) primer (20mer): 5¢-TCC
GTG-TTG-TCT-ACG-TCC-AG-3¢. The probe used
for Southern hybridization (25mer) was 5¢-CTG
CAG-CAG-CTG-TTG-GTG-TAC-CAC-A-3¢. PCR
was performed on 100–200 ng genomic DNA. For
DNA from the KSHV-positive cell line, BC-3 was
used as the positive control, while DNA for the
KSHV-negative cell line, BJAB was used as the
negative control. Amplified products were electro-
phoretically separated on 1.2–1.5% agarose gels,
and the identity of the 233 bp fragment was
confirmed by Southern hybridization.
Antibody reactivity to KSHV by IFA was detected
in two (one HIV-infected) of 50 haemophilic patients
(4%) and exactly the same prevalence was observed
in the control group. The anti-KSHV antibody titre
was higher in the patients with haemophilia
(1/51 200, 1/12 800) compared with the controls
(1/100, 1/400). KSHV DNA could not be amplified
in any sample either from the patients or the controls.
KSHV infection can be studied with genomic
amplification by direct or nested PCR on peripheral
blood mononuclear cells and by antibody detection
assays to latent or lytic antigens. Although antibody
detection is more sensitive than PCR, not all PCR
positive samples are seropositive [15]. We thus used
the combined approach to screen our cohort of
haemophilia patients for KSHV infection.
KSHV transmission by blood components is still
debated and raises public health issues. By using the
immunofluorescence assay for antibodies to a latent
antigen, the prevalence of KSHV infection in hae-
mophilic patients was found at 4% (two of 50)
similar to that of the age- and sex-matched blood
donors. These results support the previous studies
Haemophilia (2003), 9, 657–659
that reported no transmission of KSHV by factor VIII
concentrates [14,15]. As our patients had received
factor VIII concentrates before routine viral inacti-
vation of the concentrates, it is suggested that KSHV
could be inactivated during factor VIII purification.
KSHV expresses different antigens when in the
latent state (latent antigens) than in a stimulated
cytolytic form (lytic antigens). The KSHV nuclear
antigens are defined as latent because their synthesis is
neither enhanced by phorbol esters nor inhibited by
anti-viral drugs that inhibit the viral DNA polym-
erase. The transcription of other presumed lytic-
phase KSHV genes, such as the major capsid-protein
gene, is induced and inhibited under these conditions.
If, like EBV, KSHV is a transforming virus, latently
expressed KSHV proteins may play an important role
in KSHV-related tumorigenesis [20]. The finding of
significantly higher titre of anti-latent antibodies in
our haemophilic patients (one HIV-seropositive)
compared with that of the controls has not been
observed in earlier studies. The importance of this
finding is not yet known. None of our patients has
shown any clinical sign associated with KSHV
infection so the significance of this observation will
be defined in the future on follow-up of these patients.
The fact that we failed to detect KSHV DNA in the
peripheral blood in patients with haemophilia is
considered plausible. Previous studies have reported
the presence of KSHV DNA in peripheral blood of
patients with KS or HIV-seropositive homosexual
men or women attending STD clinics [12]. Conse-
quently, antibody detection is believed to be a more
sensitive indicator of KSHV infection.
In conclusion, our findings support the previous
studies that failed to detect KSHV transmission by
factor VIII concentrates. Nevertheless, the observa-
tion of significantly higher antibody titres in haemo-
philic patients than in the controls needs further
study to delineate the clinical relevance of KSHV
infection.
Acknowledgements
The authors would like to thank Dr Chris Boshoff
(Wolfson Institute for Biomedical Research, Univer-
sity College London, UK) for his kind support in
performing the indirect immunofluorescence assay
(IFA) for anti-KSHV antibodies.
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