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American Journal of Epidemiology
Copyright O 1999 by The Johns Hopkins University School of Hygiene and Public Health
All rights reserved
Vol. 150, No. 6
Printed In USA.
Risk Factors for HIV-1 Shedding in Semen
Carl E. Speck, u Robert W. Coombs,34 Laura A. Koutsky,1 Judith Zeh,5 Susan O. Ross,6 Thomas M. Hooton,4
Ann C. Collier,4 Lawrence Corey,34 Anne Cent,3 Joan Dragavon,3 Willa Lee,3 Eric J. Johnson,3 Reigran R.
Sampoleo,3 and John N. Krieger*
Semen is the body fluid most commonly associated with sexual transmission of human immunodeficiency
virus type-1 (HIV-1). Because the male genitourinary tract is distinct immunologically from blood, compartmentdependent factors may determine HIV-1 shedding in semen. To identify these factors, the authors obtained 411
semen and blood specimens from 149 men seen up to three times. Seminal plasma was assayed for HIV-1 RNA
and semen was cocultured for HIV-1 and cytomegalovirus (CMV), which may up-regulate HIV-1 replication. The
best multivariate model for predicting a positive semen HIV-1 coculture included two local urogenital factors,
increased seminal polymorphonuclear cell count (odds ratio (OR) = 12.6 for each log10 increase/mL, 95%
confidence interval (Cl) 12.2, 134.5) and a positive CMV coculture (OR = 3.0, 95% Cl 1.2, 7.7). The best
multivariate model for predicting semen HIV-1 RNA included two systemic host factors, CD4+ cell counts
<200/|xliter (OR = 3.0, 95 percent Cl 1.3, 6.9) and nucleoside antiretroviral therapy (monotherapy: OR = 0.5, 95%
Cl 0.3, 1.0; combination therapy: OR = 0.4, 95% Cl 0.2, 0.9), and a positive CMV coculture (OR = 1.7, 95% Cl
1.0, 3.0). Thus, both systemic and local genitourinary tract factors influence the risk of semen HIV-1 shedding.
These findings suggest that measures of systemic virus burden alone may not predict semen infectivity reliably.
Am J Epidemiol 1999; 150:622-31.
acquired immunodeficiency syndrome; coculture; cytomegalovirus; HIV-1; polymerase chain reaction; RNA, viral;
semen
Compelling epidemiologic data suggest that direct
contact with semen is the major route of human
immunodeficiency virus type-1 (HTV-1) transmission
from seropositive men to their sex partners (1-16).
However, little is known about the factors that influ-
ence HTV-1 shedding in semen or the biology of HTV1 within the male genital tract. Although HTV-1 nucleic
acid (DNA and RNA) is detected in most semen specimens from infected men, infectious virus is cocultured from fewer than half of specimens (17-24). In
addition, factors that influence the infectivity of semen
may differ from those that influence the infectivity of
blood, lymphatic tissue, or the central nervous system
because the male reproductive tract is a distinct
immunologic compartment (24-27).
Most of our work has used semen HTV-1 coculture as
the outcome measure. While somewhat insensitive, a
positive semen HTV-1 coculture indicates the presence
of infectious virus. The results of our earlier studies
suggest that shedding of cultivable HIV-1 in semen is
intermittent (20, 28); is not statistically associated with
clinical stage of HIV-1 disease or CD4+ T-lymphocyte
count (CD4+ count) (18, 29); is not reduced by zidovudine monotherapy (18, 20); but is associated with low
peripheral blood CD8+ T-lymphocyte counts (CD8+
counts) (20). Taken together, these findings suggest that
systemic host factors alone are poor predictors of
semen infectivity as assessed by HIV-1 coculture.
Reports from other researchers who have used semen
HTV-1 coculture as the outcome have varied. In 1992,
Anderson et al. (30) reported that seminal HTV-1 cocul-
Received for publication May 2, 1997, and accepted for publication February 1, 1999.
Abbreviations: AIDS, acquired immune deficiency syndrome; Cl,
confidence interval; CMV, cytomegalovirus; GEE, generalized estimating equation; HIV-1, human immunodeficiency virus type-1; OR,
odds ratio; PBMC, peripheral blood mononuclear cell; RT-PCR,
reverse transcription polymerase chain reaction; PMN, polymorphonuclear; RNA, ribonuclefc acid; SD, standard deviation.
1
Department of Epidemiology, University of Washington, Seattle,
WA.
2
Department of Research and Evaluation, Southern California
Permanente Medical Group, Pasadena, CA.
3
Department of Laboratory Medicine, University of Washington,
Seattle, WA.
4
Department of Medicine, University of Washington, Seattle, WA.
5
Department of Statistics, University of Washington, Seattle, WA.
6
Department of Urology, University of Washington, Seattle, WA.
Reprint requests to Dr. John N. Krieger, Department of Urology,
University of Washington, Box 356510, Seattle, WA 98195.
Written informed consent was obtained from subjects in accordance with guidelines of the University of Washington and the US
Department of Health and Human Services.
Presented in part in the HIV in Semen: Research Issues workshop, NIH, Bethesda, MD, February 1-2,1996 (Abstract p. 23), and
at the Eleventh International Conference on AIDS, Vancouver, British
Columbia, July 7-12, 1996 (Abstract We.B.3383).
622
Risk Factors for HIV-1 Shedding in Semen
ture positivity was associated with low peripheral blood
CD4+ counts and with seminal inflammation. These
authors also reported that zidovudine monotherapy significantly reduced HTV-1 shedding in semen. However,
in 1993, Hamed et al. (19) reported no association
between detection of HTV-1 RNA in semen and clinical
stage of HIV-1 infection, CD4+ count, or long-term
zidovudine monotherapy. In 1994, Vernazza et al. (23)
reported higher semen HTV-1 coculture positivity rates
among symptomatic men and among men with CD4+
cell counts <100/nliter of peripheral blood. However,
positive peripheral blood plasma HTV-1 coculture did
not predict positive semen HTV-1 coculture.
Cytomegalovirus (CMV), a sexually transmitted
virus, is shed in semen. In 1994, Detels et al. (31)
reported that after they controlled for peripheral blood
CD4+ cell count, HTV-1 seropositive men who persistently shed CMV in their semen were more likely to
develop acquired immune deficiency syndrome
(AIDS) than men whose semen was either intermittently positive or persistently negative. In a subsequent
analysis, the same group of researchers (32) reported
that men who shed multiple strains of CMV in their
semen were more likely to progress to AIDS than men
who either shed the same strain of CMV or who did
not shed CMV. Krieger et al. (33) reported that CMV
shedding was associated with HTV-1 shedding in
semen, but that this association was attenuated when
they controlled for other factors.
We used a prospective, repeated measures design, to
reassess local urogenital and systemic host factors as
determinants of HTV-1 shedding in semen. The local
urogenital factors that we examined included seminal
leukocyte levels and concurrent seminal CMV shedding, while the systemic host factors included lymphocyte subset counts, HTV-1 level in peripheral blood,
clinical status, and antiretroviral therapy use. Because
there is no perfect measure of semen HTV-1 infectivity,
we used two outcome measures, coculture of semen
and detection of HTV-1 RNA by reverse transcription
polymerase chain reaction (RT-PCR) amplification of
seminal plasma. Coculture techniques are much less
sensitive than RT-PCR amplification, but a positive
coculture indicates the presence of infectious virus.
However, in defining the "infectious transmissible
unit," the relation between the detection of semen viral
RNA and semen infectivity can only be inferred.
MATERIALS AND METHODS
Subjects
Subjects were recruited using an advertisement in a
local gay newspaper (the Seattle Gay News), and
through the Harborview Medical Center (Madison)
Am J Epidemiol Vol. 150, No. 6, 1999
623
HTV/AIDS Clinic, the University of Washington AIDS
Clinical Trials Unit, Center for AIDS Research, and
Virology Research Clinic. At the clinic sites, potential
subjects were recruited by a study nurse who briefly
described the study. Interested men were then introduced to the study manager, who provided details and
obtained informed consent. Subjects recruited through
the newspaper advertisement called a confidential telephone number to speak with the study manager.
Study eligibility. HIV-1 seropositive men, aged >18
years, who had a local address and telephone number
and were physically capable of providing three semen
specimens at 4-week intervals were eligible for the
study. There were no exclusions based on disease
stage, antiretroviral therapy status, or CD4+ cell count.
Specimen collection. At each visit, blood was
drawn, a clinical questionnaire was administered, and
a semen specimen was scheduled for the following
week. Semen specimens were collected by masturbation into sterile containers after 2 days' abstinence
from ejaculation and immediately sent to the
University of Washington Male Fertility Laboratory.
Laboratory methods
Semen processing, cell-staining, and analysis.
Semen specimens were generally processed within 2
hours of collection (mean, 72 minutes; standard deviation (SD), 40; range, 20-210). Each specimen was
diluted 1:1 with culture medium and the seminal
plasma and seminal cell-pellet were separated by centrifugation at 2,940 g for 2-4 minutes (18). An aliquot
of seminal plasma was stored at -70°C for HTV-1 RNA
analysis. If sufficient specimen was available, each
fraction was cocultured separately for HIV-1 and for
CMV. For each semen analysis, a thin smear was
stained using a modified Bryan-Leishman stain and
examined for sperm and leukocyte morphology
according to a World Health Organization protocol
(34, 35). This method allows discrimination of leukocytes from immature sperm forms. Leukocyte types
were enumerated per 100 sperm and the concentrations
(millions/mL) of total leukocytes and each leukocyte
subset (polymorphonuclear (PMN) cells, lymphocytes,
and monocytes) were calculated by multiplying the
leukocytes per 100 sperm times the sperm concentration and dividing by 100. We found excellent agreement between this staining and evaluation of the same
samples by monoclonal antibody IILe-1 (panleukocyte)-stained slides (36). Weekly quality control was
provided by cryopreserved aliquots of semen or by
semen samples from normal donors.
Detection of cell-free HIV-1 genomic RNA in seminal
plasma. A reverse transcriptase-based polymerase
chain reaction (RT-PCR) amplification method was
624
Speck et al.
used to assay HTV-1 ribonucleic acid (RNA) in the
cell-free seminal plasma (Amplicor HTV Monitor,
Roche Molecular Systems, Branchburg, New Jersey).
Prior to amplification, seminal plasma was pretreated
using a silica gel extraction method to remove inhibition of the internal assay standard (24, 37). The manufacturer's internal quantitative standard was used for
each assay, and negative controls were routinely run to
ensure that results fell within the range of acceptance
for truly negative assays. Results were reported as positive or negative. The lower limit of detection for this
assay was 400 copies of HTV-1 RNA/mL of diluted
seminal plasma (38, 39). We did not use bDNA to
assay semen specimens due to insufficient volume.
However, we did compare the RT-PCR to bDNA
assays using HTV-1 spiked pooled HTV-1 seronegative
seminal plasma provided by the National Institute of
Allergy and Infectious Disease (NIAID) Division of
AEDS-sponsored Virology Quality Assurance
Laboratory (VQAL). The relation between the two
assays is described by the following equation: RT-PCR
log10 copy number/mL = 0.987 (bDNAlog10 copy number/mL) + 0.716, Pearson correlation coefficient, r2 =
0.986 (24). Thus, the semen HTV-1 RNA levels are
approximately fivefold greater when measured by the
RT-PCR assay than by the bDNA assay.
Viral cultures. HTV-1 was cocultured from blood
and semen using standard methods (24,40). To coculture for CMV, seminal cell pellets and plasma were
diluted with RPMI 1640 medium containing heatinactivated (30 minutes, 56°C) fetal calf serum (16
percent), penicillin G (100 units/mL), streptomycin
(100 (Xg/liter), and additional L-glutamine (0.03
mg/mL), then stored at 4°C for <6 hours (41, 42).
Human fetal tonsil fibroblast cell monolayers were
inoculated with 0.2 mL of the diluted specimen and
with 1:10 and 1:100 dilutions, then examined weekly
for 5 weeks. CMV isolates were identified by their
characteristic cytopathic effects, then confirmed by
indirect immunoperoxidase staining and by failure to
grow in HeLa-M cells (41^3).
Quantitation of HIV-1 RNA in peripheral blood
plasma. Using the Quantiplex branched chain DNA
assay (Chiron Corporation, Emeryville, California),
ACD-anti-coagulated blood plasma was assayed without
pretreatment (44). The manufacturer's standards were
run with each assay with results reported as HTV-1 RNA
copies/mL of blood plasma. The lower limit of HTV-1
RNA detection was 10,000 copies/mL of blood plasma
(44). The University of Washington Retro virology
Laboratory participates in the VQAL proficiency testing
program for HTV-1 RNA quantitation (39, 45).
T-lymphocyte subset analyses. Counts of total
lymphocytes and of lymphocyte subsets were done in
the University of Washington's Hematopathology
Laboratory using a standard, quality assured flow
cytometric technique (46). Blood cells were fractionated by Ficoll-Hypaque density gradient centrifugation
(40). The mononuclear cell-enriched layer was then
incubated with OKT-3, OKT-4, and OKT-8 fluorescein-conjugated monoclonal antibodies. Reactions
were read on an EPICS flow cytometer (Beckman
Coulter, Inc., Fullerton, California) and the proportions of each subset were calculated. The absolute
number of lymphocytes was determined on a simultaneous complete blood count and the absolute numbers
of each T-cell subset were calculated. This procedure
was run daily.
Statistical analyses
Continuous variables were transformed on a logarithmic base-10 scale. The SAS package (SAS
Institute, Inc., Cary, North Carolina) was then used to
perform generalized estimating equation (GEE) analyses to determine the best univariate and multivariate
predictors for each outcome measure (47). The GEE
approach was chosen because ordinary logistic regression techniques assume that all data points are independent, and thus fail to account for the intra-subject
correlation inherent in longitudinal data sets such as
that produced in this study. When intra-subject correlation is not accounted for, incorrect indications of statistically significant effects may result. Usually, GEE
approaches attenuate this problem by calculating
wider confidence intervals. Although risk estimates
generated by ordinary and GEE logistic regression
analyses are nearly identical, the GEE approach is
almost always more conservative and confidence
intervals are wider.
In the univariate and multivariate models, we performed both "unrestricted" and "restricted" analyses.
The restricted analyses included only observations for
which results were available for HTV-1 and CMV
coculture and seminal plasma HIV-1 RNA PCR
assays. We conducted these restricted analyses to
determine whether odds ratios differed between data
sets with complete information for all variables and
data sets with either some outcome or CMV coculture
data missing or both missing. This facilitated assessment of potential bias caused by differential testing
rates of semen specimens by HTV-1 coculture, CMV
coculture, and HIV-1 RNA assays. In all, four analyses
were run for each potential predictor variable for each
of the two study outcomes (univariate unrestricted;
univariate restricted; multivariate unrestricted; and
multivariate restricted).
When we constructed the multivariate models, we
evaluated all factors associated with HTV-1 shedding in
Am J Epidemiol
Vol. 150, No. 6, 1999
Risk Factors for HIV-1 Shedding in Semen
the univariate models, as well as factors that have been
consistently identified in the literature to influence
detection of HTV-1 in semen, such as antiretroviral therapy. For each model, we selected the factor most
strongly associated with HTV-1 shedding in semen, and
then placed other variables into the model one at a time.
If the variable was statistically significant in the multivariate model, it was retained. In all multivariate modeling, we used categorical (as opposed to continuous)
CD4+ cell count because the relation between CD4+
cell count and HIV-1 shedding in semen was non-linear.
RESULTS
Overview
A total of 161 men were enrolled between April 4,
1994 and August 18, 1995. Of these 161 men, 12 were
lost to follow-up prior to providing their first semen
specimen. Of the remaining 149 men, 13 (8.7 percent)
completed only one visit; 8 (5.4 percent) completed
exactly two visits; and 128 (85.9 percent) completed
all three visits. The 149 study participants provided
411 semen specimens overall. The mean CD4+ count
for subjects who completed <3 visits was 277 (SD,
281), while that for subjects who completed all 3 visits was 375 (SD, 241) (t = -1.66, p = 0.10). The mean
log10 copy number of HTV-1 RNA/mL of blood plasma
for subjects who completed <3 visits was 4.68 (SD,
0.68); the mean for subjects who completed 3 visits
was 4.38 (SD, 0.56) (t = 1.43, p = 0.15).
At study entry, about 25 percent of the subjects had
clinical AIDS (Centers for Disease Control Stage C);
about 30 percent had CD4+ counts <200/(xliter of
peripheral blood and just under 40 percent of subjects
took one or more nucleoside antiretroviral medications. Average semen volume was 1.84 mL (range,
0.05-6.5 mL). These and other baseline subject characteristics are summarized in table 1.
HIV-1 isolation from semen by coculture. HTV-1
was isolated from 34 (12.4 percent) of 274 specimens
cocultured for HTV-1. Of the 34 culture positives, 28
(82.3 percent) were positive only in the cellular fraction of the specimen; 4 (11.8 percent) were positive in
both fractions of the specimen, and 2 (5.9 percent)
were positive in the plasma fraction only. Of 53 men
cocultured for HTV-1 at all 3 visits, 8 (15.1 percent)
were positive at visit 1, 9 (17.0 percent) had >1 positive coculture at visit 2, and 12 (22.6 percent) had >1
positive coculture after visit 3.
CMV isolation from semen by coculture. Of 320
specimens evaluated for CMV, 126 (39.4 percent)
were positive. Of these 126 positive specimens, 94
(74.6 percent) were positive in both the cellular and
plasma fractions of the specimen. Of the 69 men
Am J Epidemiol
Vol. 150, No. 6, 1999
625
TABLE 1. Sociodemographlc and Immunologic characteristics
of 149 participants In study of risk factors for HIV-1* shedding
In semen, Seattle, Washington State, 1994-1995
Characteristic
Age (years)
20-29
30-39
40-49
50-59
Race
Caucaslon
Hispanic
African American
Other
Current employment status
Full time
Part time
Not working
Recruitment procedure
Harborview Madison Clink;
Seattle Gay News advertisement
Other AIDS»-related programs
Physician referral
Another enrolled patient
HIV-1 risk factors
Male-male sex
Injection drug use
Health care/occupational exposure
Female-male sex
Blood transfusion
Multiple risk factors
Unknown
Baseline CD4 count):
£500 (Class 1)
200-499 (Class 2)
<2OO(C4ass3)
Baseline clinical stage of HIV-1 disease
Asymptomatic (Class A)
Mild constitutional symptoms (Class B)
Clinical AIDS
Baseline CD4 count and clinical stage
of HIV-1 disease
A-1
A-2
A-3
B-1
B-2
B-3
C-1
C-2
C-3
Baseline nucleoside antiretroviral
therapy status
Combination tnerapy§
Monotherapyfl
No antiretroviral therapy
No.
%t
27
73
44
5
18.1
49.0
29.5
3.4
121
12
12
4
81.2
8.1
8.1
2.7
44
16
89
29.5
10.7
59.7
54
75
8
6
6
362
50.3
5.4
4.0
4.0
121
4
3
2
1
17
1
812
2.7
2.0
1.3
0.1
11.4
0.1
47
58
44
31.6
38.9
29.5
41
71
37
27.5
47.7
24.8
20
18
3
15
34
22
0
12
25
13.4
12.1
2.0
10.1
22.8
14.8
0.0
8.1
16.8
17
38
94
11.4
25.5
63.1
• HIV-1, human Immunodeficiency virus type-1; AIDS, acquired Immunodeficiency syndrome.
t Percent was calculated based on the 149 men who completed at least
one visit
t Per mm 1 of peripheral blood.
§ Use of 22 licensed nucleoside analog drugs.
H Use of exactly one licensed nucleoside analog drug.
cocultured three times for CMV, 25 (36.2 percent)
were positive at visit 1, 30 (43.5 percent) had been
positive at least once at visit 2, and 33 (47.8 percent)
had been positive at least once after visit 3.
HIV-1 RNA detection in seminal plasma by RTPCR. Of 350 specimens evaluated for HTV-1 RNA,
210 (60.0 percent) were positive. Among 91 men
assayed at all three time points, 56 (61.5 percent) were
positive at visit 1, 65 (71.4 percent) were positive after
626
Speck et al.
visit 2, and 71 (78.0 percent) were positive at least
once after visit 3.
HIV-1 and CMV isolation from semen by
coculture. A total of 236 specimens were of sufficient volume to be cocultured for both HTV-1 and
CMV. Of those 236 specimens, 18 (7.6 percent) were
positive for both HTV-1 and CMV; 69 (29.2 percent)
were CMV positive but HTV-1 negative; 15 (6.4 percent) were HTV-1 positive but CMV negative; and 134
(56.8 percent) were negative for both viruses.
HIV-1 in semen by RNA RT-PCR and coculture. Of
274 specimens cocultured for HTV-1, 244 (89.1 percent) were also assayed for HTV-1 RNA. Twenty-five
(89.3 percent) of 28 HTV-1 culture positive samples
were positive for HTV-1 RNA. Of 216 HTV-1 culture
negative samples, 121 (56.0 percent) were positive for
HTV-1 RNA.
HIV-1 in semen by RNA RT-PCR, HIV-1 coculture,
and CMV coculture (table 2). Of 210 semen specimens with results for all three assays, 127 (60.5 percent)
were HTV-1 RNA positive, 27 (12.9 percent) were HTV1 coculture positive, and 75 (35.7 percent) were CMV
coculture positive. Eighteen (24.0 percent) of 75 CMV
coculture positive specimens were HTV-1 negative by
both coculture and RT-PCR and 16 (21.3 percent) were
positive for HTV-1 by both coculture and RT-PCR. In
contrast, only 8 (5.9 percent) of the 135 CMV coculture
negative specimens were positive for HTV-1 by both
coculture and RT-PCR.
Detection of HIV-1 in peripheral blood plasma by
RNA RT-PCR and coculture. Among 398 peripheral
blood specimens assayed for HIV-1 RNA, 300 (75.4
percent) were positive, with a median of 39,400 HIV1 RNA copies/mL (range, 10,400-1,600,000). Of 400
peripheral blood specimens cocultured for HFV-1,
362 (90.5 percent) were positive, with a median of
28.3 infectious units/10* cells (range, 0.13-5,608).
Seminal leukocyte counts. Seminal lymphocyte,
PMN cell, and monocyte cell levels were assessed per
TABLE 2. HIV-1 and CMV Isolation in semen by coculture and
HIV-1 RNA detection In semen by RNA RT-PCR amplification
among 149 men, Seattle, Washington State, 1994-1995*
Results ot
HIV-1 Isolation
RNA RT-PCR,
cocutture
No.
of
specimens
CMV coculture-posltive
No.
Negative, negative
Negative, positive
Positive, negative
Positive, positive
80
3
103
24
18
0
41
22.5
0.0
39.8
16
66.7
Total CMV-posWve
210
75
35.7
* Abbreviations: HIV-1, human immunodeficiency virus type-1;
CMV, cytomegaiovirus; RNA, ribonudeic add; RT-PCR, reverse
transcription polymerase chain reaction.
100 sperm in 285 (69.3 percent) of the 411 specimens.
Of those 285 specimens, 44 (15.4 percent) had one or
more seminal lymphocytes (range, 0-12); 77 (27.0
percent) had one or more PMN cells (range, 0-22);
and 3 (1.1 percent) had one or more monocytes (range,
0-2). Because few specimens contained monocytes,
we did not evaluate this variable as a potential predictor of HTV-1 in semen.
HIV-1 coculture-positivity in relation to systemic
and local host factors (tables 3 and 4). We analyzed
the relation between detection of HTV-1 in semen by
coculture and a variety of systemic host factors and
local urogenital tract factors. Systemic host factors
included: peripheral blood CD4+ and CD8+ counts,
clinical stage of HTV-1 infection, antiretroviral therapy use, the presence and levels of HTV-1 RNA in
peripheral blood plasma, and quantitative and qualitative HTV-1 peripheral blood mononuclear cell culture result. Local urogenital tract factors included:
concurrent seminal CMV shedding, seminal PMN
and lymphocyte cell counts per mL of semen, and
semen volume.
Predictors of positive semen HIV-1 cocultures:
unlvariate analyses (table 3)
Systemic host factors. None of the systemic variables evaluated was associated with semen HTV-1
coculture positivity in the univariate analyses. Odds
ratios were elevated for specimens from men with
<200 CD4+ cells/(i.liter of peripheral blood, but not
significantly so.
Local urogenital tract factors. Urogenital tract factors associated with a positive HIV-1 coculture
included log|0 seminal PMN cells/mL of semen (odds
ratio (OR) = 3.6, 95 percent confidence interval (CI)
1.1, 12.3) and semen CMV coculture positivity (OR =
2.3, 95 percent CI 1.0, 5.4). Thus, for each one-log10
increase in the number of PMN cells/mL of semen, the
odds of a positive semen HTV-1 coculture increased
by a factor of 3.6. The odds ratios for a one-log|0
increase in seminal PMN cells per 100 sperm and per
ejaculate were significantly increased (data not
shown). HTV-1 shedding rates were increased in specimens that had higher seminal lymphocyte counts/mL
of semen, but this association did not reach statistical
significance. The univariate predictors of semen HTV1 coculture outcome did not differ in the restricted
analyses (data not shown).
Predictors of positive semen HIV-1 cocultures:
multlvariate analyses (table 4)
Systemic host factors. Although not associated
with HTV-1 coculture positivity in the univariate analyAm J Epidemiol Vol. 150, No. 6, 1999
Risk Factors for HIV-1 Shedding in Semen
627
TABLE 3. Unlvarlate predictors of semen HIV-1 Isolation by coculture and detection of HIV-1 RNA In seminal plasma by RT-PCR
amplification assay estimated using GEE analysis, Seattle, Washington State, 1994-1995*
Semen HIV-1 coculture
Odds
ratio
A Systemic host factors
Peripheral Wood CD4+ count/jiLt
£500
200-499
<200
Peripheral blood CD8+ count/|iLt
51,200
800-1,199
<800
CD4:CD8 ratio (for each unit Increase)
Clinical stage of disease
Asymptomatic (Class A)
Symptoms (Class B)
AIDS (Class C)
HIV-1 PBMC coculture titer (for each log10 increase)
Blood plasma RNA
Negative
Positive
Blood plasma RNA level (for each log10 increase)
Antiretroviral therapy
No therapy
Monotherapyf
Combination therapy§
B. Local urogenital factors
Semen CMV coculture
Negative
Positive
Seminal PMN count (for each log10 increased
Seminal lymphocyte count (for each log10 increased
Seminal volume (for each 1-mL Increase in
ejaculate volume)
95% Cl
No. of
specimens/
men
Semen HIV-1 RNA RT-PCR
Odds
ratio
95% Cl
274/127
1.0
0.8
2.2
348/132
1.0
1.6
0.3, 2.4
0.8, 6.2
2.4
0.8, 2.9
1.2,4.8
274/127
1.0
1.2
2.0
0.5
348/132
1.0
0.3, 4.0
0.6, 6.7
0.1,2.2
0.7
274/127
274/127
1.1
2.4
1.1
0.3, 3.6
0.6, 9.0
0.8, 1.6
1.0
1.0
1.4
0.9, 1.1
0.8, 2.5
1.4
0.3
1.0
1.7
1.0
1.0
0.9
0.8
No. of
specimens/
men
3.3
268/127
268/127
1.5
1.0
2.3
268/127
274/127
2.0
0.9, 3.3
1.4,7.9
1.0,2.0
1.0, 5.4
1.1, 12.3
0.1, 348.7
0.7
0.1,3.1
340/132
345/131
1.5,3.6
1.3,3.3
0.5, 1.6
0.2, 1.2
276/121
2367119
1.0
2.3
3.6
5.8
348/132
350/132
350/132
1.0
0.9
0.5
0.3, 2.8
0.3, 2.1
0.4, 1.4
0.7, 2.7
0.1,0.8
237/114
237/114
1.0
1.8
1.6
5.6
1.1,3.1
0.6, 4.6
0.3, 96.7
307/124
307/124
270/126
0.9
0.4, 2.0
344/131
* Abbreviations: Cl, confidence interval; CMV, cytomegalovirus; GEE, generalized estimating equation; HIV-1, human immunodeficiency
virus type-1; PBMC, peripheral blood mononuclear cells; PMN polymorphonuclear; RNA, ribonucleic acid; RT-PCR, reverse transcription
polymerase chain reaction.
t Per mm3 of peripheral blood.
t Use of exactly one licensed nucleoside analog drug.
§ Use of £2 licensed nucleoside analog drugs.
H Per mL of semen.
sis, we assessed CD4+ count and antiretroviral therapy
as potential confounders by including them (alone and
together) in the model containing CMV shedding and
log|0 number of PMN cells/mL of semen. Neither of
the systemic host factors was statistically associated
with HTV-1 coculture positivity in these analyses, nor
did they substantially alter risk estimates for local urogenital factors. Therefore, neither of these systemic
host factor variables was included in the final model.
Local urogenital tract factors. PMN cells per mL of
semen and semen CMV coculture outcome were tested
in multivariate models because they were statistically
associated with shedding of HTV-1 in the univariate
analyses. When placed together in a model, PMN cell
Am J Epidemiol Vol. 150, No. 6, 1999
count (irrespective of the manner in which it was
assessed) and concurrent CMV shedding each independently predicted positive semen HTV-1 coculture
(ORs (95 percent CIs) 12.6 (1.2, 134.5) and 3.0 (1.2,
7.7), respectively). Results were similar in the
restricted analyses (data not shown).
Predictors of HIV-1 RNA in seminal plasma:
univariate analyses (table 3)
Systemic host factors. Systemic factors associated
with HTV-1 RNA detection in semen included: CD4+
count <200/(iliter (compared with CD4+ count = 500;
OR = 2.4, 95 percent Cl 1.2, 4.8), CD4+:CD8+ cell
ratio (for each one unit increase, OR = 0.3, 95 percent
628
Speck et al.
TABLE 4. Multivariate models for predicting semen HIV-1 Isolation (n = 207 specimens from 108 men)
and detection of HIV-1 RNA In seminal plasma by RT-PCR (n n 275 specimens from 121 men) estimated
using GEE analysis, Seattle, Washington State, 1994-1995*
Semen HIV-1 cocutture
Odds ratio
if in
model
95% Cl
A. Systemic (actors
Peripheral Wood CD4+ countf
2500
200-t99
<200
Antiretroviral therapy
No therapy
MonotherapyJ
Combination therapy§
B. Local urogenltal factors
Semen CMV coculture
Negative
Positive
Each log t0 increase in seminal PMN
cell courrtH
1.0
3.0
12.6
1.2,7.7
Semen HIV-1 RNART-PCR
Odds ratio
[fin
model
95% a
1.0
1.6
3.0
0.8, 3.3
1.3,6.9
1.0
0.5
0.4
0.3, 1.0
0.2, 0.9
1.0
1.7
1.0,3.0
1.2, 134.5
• Abbreviations: Cl, confidence interval; CMV, cytomegalovirus; GEE, generalized estimating equation; HIV-1,
human Immunodeficiency virus type-1; PMN polymorphonuclear; RNA, ribonucleic acid,
t Per mm3 of peripheral Wood.
$ Use of exactly one licensed nucleoside analog drug.
§ Use of two or more licensed nucleoside analog drugs.
H Per mL of semen.
Cl 0.1, 0.8), class "C" clinical disease (AIDS) (OR =
3.3, 95 percent Cl, 1.4, 7.9), log10 copy number of
infectious units/10* peripheral blood mononuclear
cells (OR = 1.5, 95 percent Cl 1.0, 2.0), and the presence (OR = 2.3, 95 percent Cl 1.5, 3.6) and levels (OR
= 2.0, 95 percent Cl 1.3, 3.3) of HTV-1 RNA in peripheral blood plasma. The odds ratio for clinical stage B
(mild constitutional symptoms) was elevated but not
statistically significant. Combination nucleoside antiretroviral therapy was associated with a decrease in the
odds of detecting HTV-1 RNA in seminal plasma, but
this decrease was not significant. CD8+ count was not
associated with semen HTV-1 RNA assay outcome.
Local urogenital tract factors. Of the local urogenital risk factors assessed, only semen CMV coculture
positivity was associated with detection of HTV-1
RNA in seminal plasma (OR = 1.8, 95 percent Cl 1.1,
3.1). Seminal PMN and lymphocyte cell counts/mL of
semen, per 100 sperm, and per ejaculate, and semen
volume, were not associated with detection of HTV-1
RNA in seminal plasma. Results were similar in the
restricted analyses (data not shown).
Predictors of HIV-1 RNA in seminal plasma:
multivarlate analyses (table 4)
Systemic host factors. Systemic factors statistically associated with seminal plasma HTV-1 RNA copy
number in univariate analyses were considered for
inclusion in the multivariate model. Antiretroviral
therapy was also considered to assess its effect after
adjustment for CD4+ count. Of these systemic host
factors, low CD4+ count <200/jxliter of peripheral
blood (compared with £500) increased the odds of
detection of HTV-1 RNA in seminal plasma (OR = 3.0,
95 percent Cl 1.3, 6.9). In contrast, monotherapy (OR
= 0.5, 95 percent Cl 0.3, 1.0) or combination nucleoside antiretroviral therapy (OR = 0.4, 95 percent Cl
0.2, 0.9) decreased the odds of detecting HTV-1 RNA
in seminal plasma.
Local urogenital tract factors. Semen CMV
coculture positivity, the only local urogenital host
factor associated with detection of HIV-1 RNA
in semen in the univariate analyses, was also
evaluated for inclusion in the multivariate model.
When peripheral blood CD4+ count and antiretroviral therapy status were taken into account, this
variable remained associated with detection of
HTV-1 RNA in seminal plasma (OR = 1.7, 95 percent
Cl 1.0, 3.0). The factors that comprised the best
multivariate model were therefore CD4+ count,
antiretroviral therapy use, and semen CMV
coculture result. The same factors formed the multivariate model in the restricted analyses (data not
shown).
Am J Epidemiol
Vol. 150, No. 6, 1999
Risk Factors for HIV-1 Shedding in Semen
DISCUSSION
We believe this study to be one of the largest investigations of risk factors for detection of HTV-1 in
semen, as well as one of the few studies in which rigorous epidemiologic and biostatistical methods were
incorporated into the design. Subjects were seen at 4week intervals, and semen and blood were collected at
each study visit. Subject compliance was excellent;
most men (85.9 percent) completed all three study visits. Nevertheless, we did three things to assess the
potential for bias due to differential dropout rates
among study subjects. First, we conducted restricted
analyses in which only observations with complete
data for all three semen assays (HTV-1 coculture, CMV
coculture, and HTV-1 RNA) were analyzed. Neither
risk estimates nor the risk factors that comprised the
multivariate models differed in these analyses. Second,
we compared baseline CD4+ counts and systemic viral
load values between men who completed less than
three visits and those who completed all three visits.
These baseline values did not differ significantly.
Third, we conducted univariate analyses using a GEE
approach to assess the association between number of
visits completed (<3 visits vs. 3 visits), semen HTV-1
coculture results, and semen HTV-1 RNA assay results.
Number of visits completed was not associated with
either of these outcomes (data not shown).
CMV shedding in semen, a local urogenital tract
factor, was the only variable assessed that predicted
both positive semen HTV-1 coculture and positive
HTV-1 RNA assay of seminal plasma. Increased seminal PMN cell count, another local urogenital tract factor, predicted positive semen HTV-1 coculture but did
not predict detection of HTV-1 RNA in seminal plasma.
In contrast, low CD4+ count and lack of antiretroviral
therapy, both systemic host factors, predicted detection
of HTV-1 RNA in seminal plasma, but not by semen
cocultivation.
We are not aware of prior reports that concurrent
shedding of CMV in semen predicts detection of HTV1 in semen by coculture or RT-PCR after controlling
for CD4+ counts and antiretroviral therapy. However,
the relation between CMV shedding and HTV-1 shedding in semen is biologically plausible. In vitro data
suggest that bi-directional interactions occur between
HTV-1 and CMV (48); these interactions could provide
mechanisms for the activation of T-lymphocytes,
which has been proposed as a prerequisite for efficient
HTV-1 replication (49), and is analogous to immunologic activation of retroviruses in murine models (50).
It has also been suggested that herpes group viruses,
such as CMV, may enhance HTV-1 production by
infected immune cells (51), forming a positive feedback loop among herpes viruses and HTV-1 -induced
Am J Epidemiol Vol. 150, No. 6, 1999
629
growth factors, cell proliferation, and HTV-1 replication (52). A chemokine receptor homologue (US28)
encoded by CMV could be an additional cofactor for
HTV-1 entry (53). Together, these observations suggest
that interactions between HTV-1 and CMV may play a
role in the sexual transmission of HTV-1.
Our report that seminal PMN levels predict detection of HTV-1 by coculture but not by viral RNA is
consistent with the observations of other investigators
(23, 30, 54, 55) on the importance of sexually transmitted diseases and genital tract inflammation as
potential risk factors for increased shedding of HTV-1.
These observations suggest that there may be a compartmentalization of infectious virus to the cell-associated semen pellet, while cell-free viral RNA in the
seminal plasma may arise from other cellular compartments within the male genital tract (24). In addition,
these observations suggest that efforts to treat urethritis, prostatitis, and other inflammatory conditions that
affect the male urogenital tract may limit the potential
for sexual transmission of HTV-1 (55).
Our findings are to some degree consistent with
other reports that low CD4+ count, a systemic host
factor, predicts shedding of HTV-1 in semen, in that
detection of HTV-1 RNA in seminal plasma was more
likely if specimens were obtained from men with <200
CD4+ cells/nliter of peripheral blood (19, 30).
However, the reports by others were based on coculture. This study is consistent with our previous work,
in that we did not find an association between CD4+
counts and HTV-1 shedding in semen as assessed by
coculture (29, 33). Our finding that both mono- and
combination nucleoside antiretroviral reduced shedding of HTV-1 in semen confirms the report by
Anderson et al. (30) that zidovudine use was associated with decreased rates of detection of HTV-1 in
semen.
In contrast to the local urogenital tract factors, systemic host factors were inconsistent predictors of HTV1 shedding in semen. In univariate analyses, clinical
stage of HTV-1 infection, counts of CD4+ and CD8+
cells in peripheral blood, CD4+:CD8+ cell ratio,
nucleoside antiretroviral therapy, and systemic viral
load (assessed by either peripheral blood mononuclear
cell culture or plasma HTV-1 RNA) were associated
with detection of HTV-1 RNA in the seminal plasma,
but not with cocultivation of HTV-1 from semen. In the
multivariate analyses, none of the systemic host factors approached significance as potential predictors of
HTV-1 shedding in semen as assessed by cocultivation.
CD4+ cell counts <200/^liter of peripheral blood,
mono-antiretroviral therapy, and combination antiretroviral therapy predicted detection of cell-free HTV1 RNA in the seminal plasma. These findings suggest
630
Speck et al.
that our study may have lacked sufficient power to
detect an effect of CD4+ cell count or combination
nucleoside antiretroviral therapy on isolation of cultivable HTV-1 in semen. Quantitative determinations of
HTV-1 RNA levels in semen or studies that employ
even larger sample sizes may be necessary to establish
the effect of these factors on HTV-1 shedding in semen
(24). These findings do suggest that highly active antiretroviral agents, such as protease inhibitors, that produce larger reductions in the systemic viral burden,
may be more effective in reducing seminal shedding of
HTV-1 (56). From a clinical standpoint, the important
implication is that healthy, HTV-1 seropositive patients
on antiretroviral therapy should still regard themselves
as infectious for their sexual partners.
This report has several strengths and limitations. Its
strengths include the large number of men enrolled
and specimens collected; its high patient compliance;
the use of random effects techniques in statistical
analyses to recognize and account for intra-subject
correlations; and the use of sensitive molecular techniques for detection of HTV-1 RNA in seminal plasma.
Thus, because many semen specimens were of insufficient volume to be evaluated by HTV-1 coculture,
CMV coculture, and HTV-1 RNA RT-PCR, study
power was reduced in models containing more than
two variables. Neither the univariate nor the multivariate models presented here differed in the restricted
analyses, which lessens the likelihood that systematic
bias substantially influenced our results. However, the
low study power may have prevented us from detecting significant relations between semen HTV-1 coculture positivity and some of the other risk factors
investigated. In addition, other than direct measurement of cultivable virus from semen, we can only
infer the impact of presence of viral RNA on semen
infectivity. Furthermore, the insensitivity of coculture
may also have limited study power to detect relations
between infectivity, presence of viral RNA, and our
inferences about sexual transmissibility.
In summary, our findings confirm and extend the
results of our prior reports and those other researchers
that local urogenital tract factors, such as seminal PMN
cell count and concurrent shedding of CMV in semen,
were the best predictors of HTV-l shedding in semen, hi
contrast, systemic viral load was a poor predictor of
HIV-1 shedding. CD4+ cells counts <200/|iliter of
peripheral blood and the use of nucleoside antiretroviral
therapy were associated with shedding of HTV-1 as
assessed by cell-free HTV-1 RNA assay, but not by
cocultivation. In addition to direct measurements of
HTV-1 in semen, an assessment of local urogenital tract
factors may be necessary to determine the potential for
sexual transmission of HTV-1.
ACKNOWLEDGMENTS
Supported by NIH grant nos. DK-49477, AI-27664, AI30731, and AI-27757 and the Paul G. Allen Foundation for
Medical Research. Dr. Speck was a University of
Washington NIH/NIAID STD/HIV Predoctoral trainee, supported by grant no. 05-T32-AI-07140.
The authors thank Dr. Diana Petitti for her help in assembling the final manuscript and Pam Easterling for technical
assistance in preparing the manuscript.
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