283
Detection of Human Herpesvirus 8 DNA Sequences before the Appearance of
Kaposi's Sarcoma in Human Immunodeficiency Virus (HIV)-Positive Subjects
with a Known Date of HIV Seroconversion
J. J. Lefrere, M. c. Meyohas, M. Mariotti, J. L. Meynard,
M. Thauvin, and J. Frottier
Institut National de Transfusion Sanguine and Service des Maladies
Infectieuses, Hopital Saint-Antoine, Paris, France
The presence of human herpesvirus 8 (HHV-8) DNA sequences was sought by polymerase chain
reaction (PCR) in peripheral blood mononuclear cells of 4 groups: 6 human immunodeficiency virus
(HIV)-infected persons with well-defined dates of seroconversion, during the period between the
diagnosis of HIV infection and the appearance of Kaposi's sarcoma (KS); 45 HIV -positive persons
with no symptoms of HIV infection; 11 AIDS patients with KS; and 14 AIDS patients without KS.
HHV-8 DNA PCR was positive in 3 of the 6 patients during HIV infection preceding the appearance
of KS and in all but 1 of 11 AIDS patients with KS. HHV-8 DNA PCR was negative in all but 1
of the 45 HIV -positive persons with no symptoms of infection and in all but 1 AIDS patient without
KS. These results indicate that HHV-8 DNA may be detected several years before the occurrence
of KS in HIV-infected subjects.
The presence of DNA sequences of a herpes-like virus was
detected in the vast majority of biopsy specimens of AIDSassociated Kaposi's sarcoma (KS) lesions [1] and in peripheral
blood mononuclear cells (PBMC) from human immunodeficiency virus type 1 (HIV-1) - infected patients with KS lesions
[2]. The same viral sequences were also found in KS samples
from HIV-negative subjects, such as elderly persons of Mediterranean, Eastern European, or African ethnic origin, and immunosuppressed organ transplant recipients [3 -9]. They were
also detected in B cell lymphomas of the abdominal cavity
[10] and in multicentric Castleman's disease (benign mediastinal lymph node hyperplasias) [11]. Although these sequences
were not found in some KS cases or in KS-derived cell lines
[12], the causal role of this viral agent is strongly suspected in
the pathogenesis of KS. This virus is presumed to belong to
the subfamily of Herpesvirinae and has been provisionally
termed KS-associated herpesvirus [13], but its formal designation will probably be human herpesvirus 8 (HHV-8).
Recently, HHV-8 DNA has been found in PBMC of HIVinfected persons several months before the emergence of KS
[14], but no sequential detection ofHHV-8 DNA has been done
during the AIDS incubation period in patients who eventually
develop KS. To investigate this issue, we used polymerase
chain reaction (PCR) with PBMC of HIV-infected persons who
had a well-defined date of HIV seroconversion to detect HHV8 DNA during the clinically silent period between HIV infection and the appearance ofKS. This study was possible because
lymphocytes were collected, then frozen, from persons with a
Received 8 January 1996; revised 21 March 1996.
Financial support: Paris American AIDS Committee.
Reprints or correspondence: Dr. Jean-Jacques Lefrere, Institut National de
Transfusion Sanguine, 53 Blvd. Diderot, 75012 Paris, France.
The Journal of Infectious Diseases 1996; 174:283-7
© 1996 by The University of Chicago. All rights reserved.
0022-1899/96/7402-0006$01.00
known date of HIV seroconversion who were prospectively
followed through annual visits. Furthermore, HHV-8 DNA sequences were sought in the PBMC of HIV-positive subjects at
various stages of immunodeficiency with no symptoms of HIV
infection, of AIDS patients with or without KS, and of sex
partners of KS patients included in the longitudinal study.
Patients and Methods
HIV-positive subjects: KS patients and partners of KS patients.
Six patients who had developed biopsy-proven KS lesions between
1994 and 1995 were studied. They were drawn from a cohort of
HIV-I-seropositive subjects followed in the same Paris hospital.
All 6 had been diagnosed between 1985 and 1989 through systematic screening for anti-HIV antibody in blood donations. The diagnosis of HIV infection was confirmed by Western blot. Five patients were white men and had been infected through homosexual
contacts. The sixth was a black man and had been infected through
heterosexual contacts. The mean age of the 6 patients was 31 years
(range, 24-41) at the time of diagnosis of HIV infection. All 6
had a well-defined date of HIV seroconversion (negative ELISA
in the 6 months preceding the first positive ELISA). The time from
seroconversion to enrollment was <3 months in each case. At each
visit, the patients underwent a physical examination and provided
specimens for laboratory evaluation.
No patient had had evidence of an acute opportunistic event
before the diagnosis ofKS, which was in all 6 patients the indicator
of CDC stage C (1993 definition). The mean duration of stage A
was 6 years (range, 5-8). CDC stage, CD4 cell count, and initial
year of antiviral therapy are indicated in table 1 for each year of
follow-up for the 6 patients. Mean CD4 cell counts at the beginning
of follow-up and the appearance of KS in the 6 patients were 421/
mrrr' (200-596) and 130/mm 3 (50~ 194), respectively. Longitudinal study was possible because serum and lymphocyte samples
had been obtained (and frozen) annually for the whole cohort since
the first visit following diagnosis of HIV infection. An additional
lymphocyte sample had been collected from 1 of these 6 subjects
2 years before HIV seroconversion (at that time, he participated
no
Lefrere et al.
284
1996; 174 (August)
Table 1. Results of HHV-8 DNA detection and other biologic parameters in the 6 HIV-positive patients with a known date of HIV infection
who later developed KS.
Patient
no.
2
3
4
5
6
Years of
follow-up
CDC
stage
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
A
A
A
A
A
A
A
A
C
C
1987
1988
1989
1990
1991
1992
1993
1994
A
A
A
A
A
A
A
C
1987 t
1989
1990
1991
1992
1993
1994
1995
A
A
A
A
A
A
C
1988
1989
1990
1991
1992
1994t
A
A
A
A
C
C
1988
1989
1990
1991
1992
1993
1994
A
A
A
A
A
A
C
1989
1990
1991
1992
1993
1994
A
A
A
C
C
C
First KS
diagnosis
+
+
+
+
+
+
+
Antiviral
therapy*
CD4
cells/mm"
p24 antigen
(pg/mL)
Anti-p24
antibody
HIV-I RNA level
(viral copies/mL)
HHV-8
DNA
+
+
+
+
+
+
+
+
+
+
5500
52,800
146,000
114,000
364,000
81,000
325,000
268,000
122,000
255,000
+
+
+
+
+
200
300
280
310
290
250
350
236
171
89
+
+
+
596
520
710
600
540
230
208
120
+
+
+
+
+
+
22,000
10,300
26,900
46,900
51,600
112,500
27,800
435,000
+
+
+
+
+
+
+
+
+
+
+
ND
430
230
340
258
215
220
120
ND
15,800
10,500
10,000
8100
38,200
32,800
21,100
+
+
+
+
+
+
+
+
+
+
+
390
570
470
280
150
50
+
+
480
560
640
600
445
370
194
+
+
430
310
380
191
93
125
37
72
58
+
+
+
+
+
+
+
DR
+
+
+
+
+
+
+
+
2400
45,000
ND
42,000
34,100
90,000
+
+
+
+
10,200
17,800
15,000
127,400
220,000
200,700
420,000
+
+
+
+
+
+
+
+
+
+
4000
48,800
54,600
390,000
143,000
350,000
+
+
+
+
235
275
460
89
19
79
100
NOTE. DR, discrepant results on duplicate samples; third test was positive; ND, not done (sample was used up).
* Zidovudine, didanosine, or both.
t Samples were collected 2 years before HIV seroconversion (see text).
t No 1993 sample was available.
in a study of persons at risk for HIV [15] and was negative by
peR for HIV DNA).
Partners of 2 of these 6 KS patients (patients 4 and 5, table 1)
were also HIV-infected and followed by our center. Patient 4's partner
did not have any symptoms of HN infection in 1995; patient 5's
partner died of AIDS without having KS. Frozen lymphocyte samples
obtained annually during the follow-up of these 2 patients (8 years
for patient 4's partner, 3 years for patient 5's partner) were available.
Fresh lymphocyte samples from 11 AIDS patients with KS were
studied.
JID 1996; 174 (August)
HHV-8 in HIV-Infected Persons
AIDS patients without KS. Fresh lymphocyte samples of 14
AIDS patients without KS were studied.
HIV-infected subjects with no symptoms ofHIV infection. The
presence of HHV -8 DNA was also sought in fresh PBMC from
45 asymptomatic HIV-positive subjects followed in our outpatient
clinic who were, at time of sampling (1995), at various stages of
immunodeficiency as determined by CD4 cell count; the 3 groups
(15 patients each) had >400,200-400, and <200 cells/mrrr'. For
these 45 subjects, a lymphocyte sample had been obtained (and
frozen) annually since the beginning of their follow-up. All samples collected before a fresh sample in 1995 that was positive for
HHV-8 DNA were assayed retrospectively for HHV-8 DNA by
PCR. All 45 persons were white (40 men, 5 women) and their
average age was 35 years (range, 21- 57). The risk factors for HIV
infection were homosexuality in 32, intravenous drug use in 3,
heterosexual intercourse with an HIV -infected partner in 6, and
unknown in 4.
HIV-negative subjects. Fresh lymphocyte samples from 20
healthy seronegative subjects at low risk of HIV infection (used
as negative controls) were studied.
HHV-8 DNA PtlR procedure. The lymphocyte samples studied were duplicated and coded for blind analysis. Genomic DNA
was extracted from cryopreserved PBMC suspension by digestion
with proteinase K and extraction with phenol chloroform and precipitated with ethanol. Samples were amplified by PCR using a
specific primer pair (KS l/KS2) to amplify a sequence of 233 bp
designated KS330 m that specifically hybridize to the internal
probe KSS [1]. The PCR conditions were as follows: 92°C for 1
min (1 cycle); 92°C for 45 s, 58°C for 1 min, 72°C for 1 min (40
cycles); 72°C for 7 min (1 cycle). Each 50-j.lL reaction contained
0.32 p,g of genomic DNA, 50 pmol of each primer, 1.25 U of Taq
polymerase, 100 p,M each dNTP, 10 mMTRIS-HCl, 1 mMMgCh,
and 50 mM KCl (pH = 8.3).
Amplification products were visualized (after electrophoresis)
on 1.5% agarose gels containing ethidium bromide and were scored
for the presence or absence of the 233-bp fragments, which were
transferred to a nylon membrane and subjected to Southern blot
hybridization with the 25-bp internal oligonucleotide (3' -tailing
with digoxigenin-l1-dUTP/dATP [DIG]). Detection of DIG-labeled nucleic acids required a chemiluminescent substrate (Lumigen CSPD; Boehringer, Mannheim, Germany). Chemiluminescence detection was a three-step process. First, membranes were
treated with blocking reagent to prevent nonspecific attraction of
antibody to the membrane. Second, the membranes were incubated
with a dilution of anti-digoxigenin Fab fragments conjugated to
alkaline phosphatase. Third, membranes carrying the hybridized
probe and bound antibody conjugate reacted with the substrate and
were exposed to x-ray film (15 min) to record chemiluminescent
signal.
Other assays. Follow-up serum samples that had been routinely collected and stored at - 80°C were subjected to other assays.
The number of HIV-l RNA copies in serum was determined
through nucleic acid sequence-based amplification (NASBA; Organon Teknika, Boxtel, Netherlands) according to a previously
described method [16]. Briefly, this assay is based on the simultaneous activity of three isothermal enzymes (avian myeloblastosis
virus reverse transcriptase, T7 RNA polymerase, and RNase H),
each acting continuously on their appropriate substrates. The isolated nucleic acid is amplified by interaction of these enzymes
together with a primer set selected in the gag region of the HIV
285
sequence. This cyclic phase is performed under isothermal conditions at 41°C. In this assay, many copies are generated from each
RNA target that reenters the reaction, resulting in exponential
amplification.
Quantification of HIY -1 RNA was based on coamplification of
HIV-l RNA with an internal standard Q-RNA dilution series, thus
ensuring equal efficiency of amplification. Three distinguishable
Q-RNAs (Qa, Qb, and Qc) were mixed with the wild type sample
in different amounts (10 4 Qa, 103 Qb, and 102 Qc molecules) and
coamplified with the wild type RNA in one tube. Using electrochemiluminescence-labeled oligonucleotides, the wild type, Qa,
Qb, and Qc amplification products were separately detected with
a semiautomated electrochemiluminescence detection instrument
(NASBA QR system; Organon, Teknika), and the ratio of the
signals was determined. The amount of initial wild type RNA was
calculated from the ratio of wild type signal to Qa, Qb, and Qc
signals. Results are expressed as number of viral copies (YC) per
milliliter of plasma.
Concentrations of serum p24 antigen were determined through
monoclonal immunoassay (Abbott Laboratories, Abbott Park, IL)
according to the manufacturer's instructions. The specificity of
each positive result was confirmed by a neutralization assay (Abbott). Results are expressed as picograms per milliliter. Concentrations of serum anti-p24 antibody were determined with HIY p24
antibody (rDNA; Abbott) through end-point dilution according to
the manufacturer's instructions.
Results
Table 1 details the results obtained for the 6 patients who
developed KS and for whom lymphocyte samples collected
since HIV seroconversion were analyzed for HHV-8 DNA. A
duplicate sample that gave discrepant results (patient 1, table
1) was positive on a third testing. HHV-8 DNA PCR was
positive in 3 cases (patients 1-3) for all studied samples (i.e.,
during the entire period of HIV infection preceding the appearance of KS). The patient who had a frozen lymphocyte sample
from 2 years before HIY infection was among these 3 patients
(no. 3); this sample was positive for HHV-8 DNA. In patient
4, HHV-8 DNA PCR was negative on all samples collected
over the study period. In patients 5 and 6, initial samples gave
negative PCR results, but subsequent samples (after 3 and 2
years of HIV infection in patients 5 and 6, respectively) were
positive. Serum concentrations of p24 antigen and anti-p24
antibody and the number of serum HIV-l RNA copies were
detailed for the 6 patients for each annual visit. The mean
serum HIV RNA levels in the first sample and at the appearance
of KS were 9983 VC/mL (range, 2400-22,000) and 239,683
YC/mL (range, 21,100-435,000), respectively.
Patient 4's partner was negative for HHV-8 DNA in lymphocyte samples collected during the first 3 years of follow-up but
positive in later samples (at the last visit, this patient had a
low CD4 cell count of 102/mm3 but did not have KS). Patient
5's partner was positive for HHV-8 DNA in the 3 available
sequential lymphocyte samples. The results of HIV serology
and HHV-8 DNA PCR are detailed in table 2 for patients 4
and 5 and their partners.
Lefrere et al.
286
Table 2. Results of HIV serology and HHV -8 DNA peR for patients 4 and 5 and their partners.
Patient 4
Patient 4' s partner
Patient 5
Patient 5' s partner
HIV serology
HHV-8 DNA peR
+
+
+
+
- then +
- then +
+
Among the 11 AIDS patients with KS, HHV-8 DNA was
detected in all but 1. PCR negativity was confirmed for this I
patient with 2 lymphocyte samples collected later.
Among the 14 AIDS patients without KS, HHV-8 DNA
PCR was negative in all but 1. peR positivity was confirmed
for this patient with a lymphocyte sample collected later (the
amplification signals observed with these 2 samples were the
strongest among the samples PCR-positive for HHV-8 DNA
in this study).
Mean serum HIV RNA levels in the 45 patients grouped
according to CD4 cell count (>400, 200-400, and <200/
mm') were 19,100 (range, 800-92,000),74,600 (range, 4000202,000), and 153,000 (range, 8500-750,600) VC/mL, respectively. No HHV-8 DNA was found in subjects with >400 or
<200 CD4 cells/mrrr'. Among the 15 subjects with 200-400
CD4 cells/mrrr', 1 was positive for HHV-8 DNA. This individual was a homosexual man who had irregularly protected contacts with multiple partners. He had a well-defined date of
HIV seroconversion (negative ELISA 8 months before the first
positive HIV ELISA and seroconversion on the first HIV-l
Western blot). Frozen lymphocyte samples collected each year
since his first year of HIV infection were assayed with HHV8 DNA PCR; the first 3 samples were negative and the following 2 were positive. At last testing (1995), the HIV infection
was CDC stage A and physical examination detected no cutaneous KS. The patient's CD4 cell counts at baseline and the
most recent visits were 340 and 387/mm3 , respectively. His
serum HIV RNA levels at baseline and most recent visits were
22,500 and 24,500 VC/mL, respectively. At the end of the
observation period, the patient had not received any antiviral
therapy.
No HHV-8 DNA was detected in the 20 healthy low-risk
HIV-negative persons used as negative controls.
Discussion
Our results indicate that HHV-8 DNA may be detected well
before the occurrence of KS in Hl V-infected persons, even
>8 years for certain patients in our study. These data were
established through follow-up of HIV-infected subjects with a
known date of seroconversion. HHV-8 infection being clinically silent raises the problem of systematically screening for
it in the still asymptomatic HIV-infected population. Indeed,
HIV-seropositive subjects positive for HHV-8 DNA appear at
JID 1996; 174 (August)
high risk of developing KS and could benefit from surveillance
for early diagnosis of KS lesions. Furthermore, if a negative
PCR result does not formally exclude a diagnosis of KS (2 of
our KS patients were negative for HHV -8 DNA), a positive
PCR result could contribute to the diagnosis of internal KS
(e.g., gastrointestinal) when it is suspected from clinical or
radiologic signs or when biopsy is impossible or inconclusive.
As expected, the results of our study indicate that HHV-8
may infect persons already carrying HIV and that HIV infection
may occur in persons previously positive for HHV-8. The detection of HHV-8 DNA in samples collected during the first year
of HIV infection in 3 patients in our longitudinal study suggests
that they either were simultaneously infected by both viruses or
had HHV-8 before HIV infection. HHV -8 DNA was detected
2 years before HIV infection in I of these 3 patients (no. 3).
Cases of KS have been reported in HIV-negative homosexual
subjects [17], and recently HHV-8 sequences were detected in
HIV-negative homosexual subjects with KS [18]. To our knowledge, patient 3 represents the first reported case of KS linked to
HHV -8 infection in an individual who was HIV seronegative,
then seropositive. Furthermore, our study is the first to detect
HHV-8 DNA in the partner of an HHV-8-infected KS patient.
Due to differences in immune status, HHV -8 infection is
much more frequently responsible for KS in HIV-positive than
HIV -negative homosexual subjects. As the epidemiology of
some forms of KS is consistent with sexual transmission of
HHV-8, the risk of developing HHV-8 -linked KS constitutes
by itself a new reason for encouraging HIV-positive patients
to protect their sex partners. However, the declining incidence
of AIDS-associated KS as the first manifestation of AIDS observed during the last decade [19,20] could reflect a decreasing
trend in exposure to the causative agent of KS as a result of
altered sexual behavior of homosexual men. This latter phenomenon could also explain the relatively low (3.3%) frequency of HHV-8 infection in the asymptomatic HIV -infected
subjects of our study.
Since HHV-8 is a transmissible agent, it must be present in
at least a small proportion of the general population. Until now,
published studies have not detected HHV-8 in healthy blood
donors [14], but the number of subjects studied is very low.
Large studies based on serologic assays specific for HHV-8
will be necessary to detennine the precise frequency of HHV 8 in the general population. Nevertheless, the fact that some
HIV -seronegative persons carry the KS agent without symptoms [6] raises the hypothesis of transmission of HHV-8
through transfusion. It is reassuring, however, that reported
cases of AIDS-associated KS are not frequently associated with
transfusions [21], and epidemiologic studies have demonstrated
that the KS agent is more frequently transmitted through sexual
contact than through intravenous drug use or transfusion [22].
If HHV -8 is highly cell-associated, it could lose its efficacy
for transfusional transmission with increasing blood product
age, which has been demonstrated for human T cell lymphotropic virus type I (HTLV-I), the pathogen of adult T cell
leukemia and of HTLV-I - associated myelopathy [23, 24].
JID 1996; 174 (August)
HHV-8 in HIV-Infected Persons
The inability to detect HHV-8 DNA in 2 KS patients in our
study could be linked to sequence polymorphism at the PCR
priming site. In reported KS cases of various etiologies, HHV8 sequences were highly conserved, but a certain variation was
noted according to the form of KS [5]. The detection of HHV8 DNA in the partner of patient 4 in our study was in sharp
contrast to the fact that patient 4 had negative HHV-8 DNA
PCR results over his entire follow-up period. However, patient
4's partner could have received HHV-8 from another partner.
Further studies, based on the quantitative determination of
HHV-8 DNA copies in PBMC of HIV-infected subjects with
no symptoms of HIV infection, are necessary to establish
whether a progressive increase in HHV-8 DNA load in PBMC
may constitute a predictor of development of KS lesions. Furthermore, if a direct role for HHV-8 is definitively demonstrated in the pathogenesis of KS, the possibility of detecting
HHV -8 before the appearance of KS lesions may allow preventive therapy through antiviral drugs. Clinical trials will be necessary to establish whether preventive therapy against KS in
HIV infection can improve the overall survival of asymptomatic HHV -8 carriers.
Acknowledgments
We thank L. Joubert for determination of p24 antigen and anti24 antibody levels and F. Roudot-Thoraval for her helpful comment on the manuscript.
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