Download Quantification of HIV in semen: correlation with antiviral

Quantification of HIV in semen: correlation with
antiviral treatment and immune status
Pietro L. Vernazza*§, Bruce L. Gilliam†, John Dyer†, Susan A. Fiscus‡,
Joseph J. Eron‡, Andreas C. Frank§ and Myron S. Cohen†
Objective: This study examined the concentration of HIV in semen and the effects of
biological factors on HIV excretion.
Methods: Semen samples from 101 men at different stages of the disease were
evaluated by quantitative HIV culture and HIV RNA detection. Blood plasma
samples were available from 56 patients. The effects of CD4 and CD8 count, blood
plasma RNA levels, treatment status and clinical staging on the shedding of HIV
were evaluated.
Results: HIV RNA levels in semen correlated with quantitative HIV culture of
seminal cells and a strong association of positive seminal cell culture with high RNA
levels was observed. CD4 count and antiviral treatment were inversely correlated
with the concentration of HIV in semen. Blood plasma HIV RNA values were
correlated with HIV RNA levels in semen, although some patients had highly
discrepant results.
Conclusions: The strong correlation between seminal cell culture and concentration
of HIV RNA in seminal plasma suggested that HIV detected in seminal plasma was
released by productively infected cells in the male genital tract. The study showed
that the concentration of HIV in semen, which was likely to be correlated with HIV
infectivity, was a function of the immune status of the HIV-infected individual. The
results suggested that antiviral therapy may have reduced the concentration of HIV
in semen.
AIDS 1997, 11:987–993
Keywords: Semen, viral load, HIV RNA, infectivity, culture
Introduction
HIV-1 is primarily a sexually transmitted disease (STD)
that affects more than 21 million people worldwide [1].
Our current knowledge regarding the efficiency of
HIV-1 sexual transmission is limited. The spread of
HIV in a population is described by the basic reproductive rate Ro (i.e., the average number of secondary cases
infected by one index case) [2]. Ro is a function of the
efficiency (β) and duration (D) of the transmissibility
and the number of sexual partners (c), expressed by the
formula Ro = β • c • D. Partner and observational studies have indicated large variability in the sexual transmissibility of HIV [3–7]. Transmissibility of HIV
depends on the type of sexual exposure, the presence of
other cofactors in the recipient (e.g., vaginal drying
agents, mucosal disruption, resistance to infection),
and the infectivity of the donor [8]. Several biological
From the *Institute for Clinical Microbiology and Immunology, Kantonsspital, St Gallen, Switzerland, the Departments of
†
Medicine, and ‡Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,
USA and the §Department of Medicine, Kantonsspital, St Gallen, Switzerland.
Sponsorship: This study was supported by grants from the Swiss National Science Foundation and the Swiss Federal Office of
Public Health (32-38818.93 and 316.93.7159), The Australian Society for Infectious Diseases, the Cooperative Agreement
SU01AI25868 from NIAID, General Clinical Research Centers program NIH RR00046 and grant numbers UOAI31496 and
NIDDK RO149381.
Requests for reprints to: Pietro L. Vernazza, Kantonsspital, 9007 St Gallen, Switzerland.
Date of receipt: 2 August 1996; accepted: 16 March 1997.
© Rapid Science Publishers ISSN 0269-9370
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AIDS 1997, Vol 11 No 8
factors have the potential to modulate HIV infectivity.
The status of the donor immune system, local mucosal
factors, and the phenotypic characteristics of the viral
quasispecies are all likely to influence the concentration
of virus in genital secretions.
The minimum quantity of HIV required for sexual
transmission of the virus to a susceptible partner is not
known. A better understanding of the biological factors
influencing the shedding of HIV is critical for the
design of novel pharmacological and immunotherapeutic approaches that would help to reduce the infectivity
of individuals and limit the further spread of the epidemic. A critical inoculum is possibly required for
transmission of HIV by any route of exposure. For
example, higher concentration of HIV in blood is associated with increased risk of HIV transmission by transfusion, vertical transmission and after needle-stick
exposure [9,10].
In order to understand how biological factors influence
transmission, accurate quantification of HIV in semen
may be important. HIV has been recovered ex vivo by
culture of the cellular component of semen in 8–54%
of infected individuals [11–14]. However, culture techniques used in these studies were not quantitative and
HIV culture may not have been sensitive enough to
detect the viral inoculum required to support efficient
transmission. More recently, molecular amplification
techniques have been used to detect HIV DNA and
RNA in semen, but only in small studies [15–20].
Quantitative amplification techniques should allow
better definition of the range of concentration of HIV
in semen and how biological factors influence the concentration.
In the present study, quantitative HIV-1 culture and
quantitative HIV RNA amplification were used to
examine HIV-1 shedding in semen of 101 HIVseropositive men and to investigate the effect of stage
of disease, blood viral burden, CD4+ cell depletion,
and antiretroviral therapy on the concentration of virus
excreted. HIV culture of seminal cells and RNA amplification in cell-free seminal fluid were also compared to
determine the correlation between the two compartments.
(Switzerland). The study was approved by the local ethical committees and all patients gave informed consent for
participation in the study. Clinical staging was performed
according to the revised Centers for Disease Control and
Prevention classification system [21]. Participants had to
be free of signs or symptoms of STD. In addition, all
Swiss patients were screened and found to be negative
for asymptomatic chlamydia infection by polymerase
chain reaction. Patients were classified according to their
antiretroviral treatment status: treatment-naive, previous
antiretroviral treatment and active treatment. However,
treatment status had to be stable for at least 12 weeks
prior to sampling. Patients in the active treatment group
received either one or two nucleoside analogues but no
protease inhibitors. CD4 cell counts of blood lymphocytes were determined by flow cytometry using standardized methods on samples obtained within 3 months
of the date of semen collection. Results were expressed
as absolute values and as the percentage of total lymphocytes. Blood plasma samples were taken by venipuncture
in a cell-preparation Vacutainer tube (CPT; Becton
Dickinson, Franklin Lakes, New Jersey, USA) using citrate as an anticoagulant. Semen samples were obtained
by masturbation and collected in a sterile container and a
virus transport medium (RPMI plus 1000 IU/ml penicillin plus 1 mg/ml streptomycin) was added to the sample. Semen samples were processed in the laboratory
within 6 h of ejaculation.
Processing of semen samples
After liquefaction of the ejaculates, seminal cells were
separated from the seminal fluid by centrifugation in a
15 ml conical tube for 10 min at 800–1000 g. The
seminal plasma was removed, aliquotted and stored
frozen at −75°C. One aliquot of each Swiss sample was
shipped frozen in dry ice to the US site for RNA
detection. One-half of the seminal cells was used for
quantitative HIV culture.
Methods
Semen microculture assay
HIV culture of seminal cells was performed immediately after cell separation following a quantitative adaptation of a method previously described in 24-well
plates [14]. Quantification was performed by six duplicate three- or fivefold dilutions of seminal cells. The
equivalent of one-sixth of the cells from one ejaculate
was used for the first dilution. Quantitative results were
obtained by reference to the number and pattern of
serially diluted wells that were positive for p24 antigen,
using a published algorithm [22] and results were
expressed as infectious units per ejaculate (IUPE).
Study population, clinical staging, semen and
blood samples
The study population consisted of HIV-1-positive men
attending one of two collaborating HIV clinics at the
University of North Carolina at Chapel Hill (North
Carolina, USA) and at the Kantonsspital St Gallen
Quantitative detection of HIV RNA in seminal
plasma and blood plasma
A commercially available technique for the quantification of HIV RNA in cell-free seminal plasma was used
[nucleic acid sequence-based amplification (NASBA)]
following previously described methods [18,23]. A total
HIV in semen Vernazza et al.
of 100 µl of the sample were used per assay. The lower
limit of detection of the assay with this input volume is
1000 RNA copies/ml. The dilution factor of the
semen samples with the known amount of transport
medium was taken into account for the final calculation
of the HIV RNA concentration in seminal plasma.
Statistical analysis
The χ2 test was used for comparisons of distribution
ratios, and Mann–Whitney U-test for comparisons
between groups. Correlation of non-parametric variables was examined using Spearman’s rank correlation
tests. Multiple logistic regression was performed to
define the relative contribution of different factors on
the detection of HIV in seminal cells (by culture) and
in seminal plasma (HIV RNA). The factors included in
the multiple regression model were blood plasma RNA
concentration, CD4 and CD8 counts, treatment status
(currently treated: ‘yes/no’), and clinical stage (symptomatic versus asymptomatic). In addition, multiple linear
regression was performed to define the correlation of
these parameters with the quantitative HIV RNA
results in seminal plasma.
Results
Demographics
Semen samples from 101 consecutive patients were
collected at the two study sites. Paired semen and blood
samples were available from 56 patients. Clinical staging, CD4 count, status of antiviral treatment, and
demographic information are summarized in Table 1.
The median age of the study population was 34.0 years.
Fifty-eight per cent of the men were asymptomatic and
44% had a CD4 count below 200 × 106/l (or 14% of
lymphocytes). Fifty-three patients (52%) had never
been treated with an antiretroviral agent at the time of
semen donation. Twenty-nine patients were currently
taking antiretroviral agents and 19 had stopped treatment more than 3 weeks prior to semen donation. The
study populations from the two study sites were comparable in terms of disease stage and CD4 count. The
Swiss population had a significantly higher proportion
of men never treated with an antiretroviral drug (χ2
test, P = 0.0005) and a higher proportion of men
infected by needle sharing.
Quantitative aspects of HIV in semen: culture
and RNA detection
Of the 101 men studied, semiquantitative seminal cell
culture was successfully performed in samples taken from
98 (three cultures had fungal contamination). Infectious
HIV was recovered from 29 samples (30%). The infectious titre in positive semen cultures ranged from 2 to
3800 IUPE. Cell-free HIV RNA was detectable in 63
(62%) out of 101 seminal plasma samples. One patient
Table 1. Patient characteristics at the two study sites.
Study centre
US
Risk groups
Homosexual
51
Heterosexual
5
Injecting drug use
1
All patients
57
Median age (years)
33.5
Absolute CD4 count (×106/l)
>500
9
200–500
23
<200
25
Median (×106/l)
270
Relative CD4 count (% lymphocytes)
>27
7
15–27
25
0–14
25
Mean (%)
16
Peripheral blood RNA level (copies/ml)
(n = 56)
<5000
9
5000–30000
3
30000–100000
4
>100000
9
Clinical staging (CDC)
Stage A
27
Stage B
18
Stage C
12
Previous antiretroviral treatment
at study entry*
Never
21 (11)
Current
17 (9)
Discontinued
19 (5)
Mean processing time of
samples (h)
2.5
Swiss
Total
18
14
12
44
35.0
69
19
13
101
34.0
6
18
20
300
15
41
45
280
4
21
19
17
11
46
44
17
6
10
7
8
15
13
11
16
32
4
8
59
22
20
32 (25)
12 (6)
0 (0)
53 (36)
29 (15)
19 (5)
3.6
3.0
*Number of patients for whom a blood sample was available is
shown in parenthesis. Data are expressed as numbers of patients,
unless otherwise indicated.
with a past history of candida oesophagitis and a CD4
count of 10 × 106/l had the highest level of HIV RNA
in semen [3.15 × 108 (or 8.5 log10) copies/ml] and also
the highest titre of infectious virus by culture (3800
IUPE). No clinical findings suggestive of urethritis were
present on examination. The RNA values of the
remaining samples ranged from non-detectable to
9 × 106 (6.95 log10) copies/ml [median value of all samples, 5100 (3.71 log10) copies/ml]. HIV RNA levels
above 10 000 copies/ml were detectable in 44% of the
patients. Recovery rate of HIV RNA from the Swiss
patients was lower than that from the US patients (70
versus 52%; P = 0.066). This lower recovery rate was
restricted to samples from individuals with higher CD4
counts (above 14% of lymphocytes or 200 × 106/l).
Spearman’s rank correlation testing between quantitative measurements of HIV in seminal cells and cell-free
seminal plasma revealed a correlation coefficient of 0.60
(P < 0.001). In fact, in the 29 patients with a positive
HIV culture from seminal cells, only one had no
detectable HIV RNA in the seminal plasma and only
three had an HIV RNA level in semen below 10 000
copies/ml (P < 0.000001, χ 2 test; Table 2). The
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AIDS 1997, Vol 11 No 8
Table 2. Detection of HIV in semen by nucleic acid sequencebased amplification and cell culture.
(a)
HIV RNA detection in seminal plasma
No. tested
HIV culture in seminal cells
Positive
29
Negative
69
No. (%) positive
No. (%) >10000
copies/ml
28 (97)*
32 (46)
26 (90)*
18 (26)
*P < 0.001.
median HIV RNA level in seminal plasma for samples
with positive seminal cell culture was 1.2 × 105 (5.1
log10) copies/ml as opposed to 2.5 × 103 (3.4 log10) for
samples with negative culture (Fig. 1).
(b)
HIV in semen as a function of blood plasma
HIV RNA level
Seven out of 56 patients (13%) had no detectable HIV
RNA in the blood, although one of these patients had
detectable HIV RNA in semen [6500 (3.81 log 10 )
copies/ml]. HIV RNA was detectable in blood as well
as semen from 29 patients (P = 0.03, χ2 test). HIV
blood plasma level was highly predictive of detection of
HIV RNA in semen by multiple logistic regression
[odds ratio (OR), 3.07; 95% confidence interval (CI),
1.44–6.53; P = 0.003].
Spearman’s rank correlation of HIV RNA concentrations in semen and blood revealed a correlation coefficient of 0.56 (P < 0.001; Fig. 1). By multiple linear
regression analysis, blood plasma RNA levels correlated
well with the concentration of HIV RNA in semen
(P = 0.0008). Despite the correlation between HIV
RNA results in semen and blood, in some patients the
difference in HIV RNA load far exceeded the variability of the assay. When the 28 patients with positive
RNA values in both compartments were compared,
only 12 patients had semen and blood RNA levels
within 1 log 10. The HIV RNA level in semen was
more than 10-fold (1.0 log10) lower than blood in nine
subjects and 10-fold higher than blood in seven subjects. In addition, eight patients had no detectable HIV
RNA in semen despite HIV RNA concentration in
blood plasma above 30 000 copies/ml (4.5 log10).
All patients with positive semen cultures had positive
HIV RNA values in blood plasma. The median HIV
RNA value in blood was 4.91 log10 copies/ml in the
12 patients with positive culture compared with 4.25
log10 copies/ml in the 42 patients with negative culture
result. No correlation was observed between the level
of blood plasma RNA and culture positivity
(Spearman’s rank’s correlation and multiple logistic
regression).
HIV in semen as a function of blood CD4 and
CD8 count
Patients with low blood CD4 counts were more likely
Fig. 1. Correlation of seminal plasma HIV RNA concentration with (a) quantitative seminal cell culture and (b) blood
plasma HIV RNA concentration for patients with detectable
HIV in semen by (a) culture or (b) RNA detection. The box
plots indicate median and interquartile range of HIV RNA
concentration in samples with negative or positive for (a)
HIV culture or (b) HIV RNA in semen. (v), HIV RNA
detectable in (a) semen or (b) blood. (O), HIV RNA not
detectable.
to have detectable HIV RNA in the seminal plasma
(Table 3). On multiple logistic regression, CD4 count
was independently and inversely predictive of RNA
detection in semen (OR, 0.78; 95% CI, 0.63–0.93;
P = 0.01). CD4 counts were significantly correlated
with the amount of HIV RNA in semen (rs = 0.23,
P = 0.019, Spearman’s rank correlation). The correlation remained significant in a multiple linear regression
analysis that included CD4 and CD8 count, blood
plasma RNA level, treatment status and clinical stage of
disease (P = 0.04).
An increased recovery rate of HIV by culture of seminal cells was observed at lower CD4 counts (Table 3).
HIV was detected by culture in 33% of patients with
CD4 counts below 200 × 106/l compared with 14% in
patients with CD4 counts above 500 × 106/l. By multiple logistic regression, absolute CD4 count was inde-
HIV in semen Vernazza et al.
Table 3. Results of HIV seminal cell culture and HIV RNA detection in semen and clinical stage of disease.
HIV RNA†
Cell culture*
CD4 count (×106/l)
0–199
200–500
>500
CD4 count (% of lymphocytes)
0–14
15–27
>27
CDC stage
A
B
C
Median HIV RNA†
log10 copies/ml
Total
No. (%) positive
P‡
Total
No. (%) positive
P‡
43
40
14
14 (33)
14 (37)
1 (7)
NS
45
41
15
32 (71)
25 (61)
6 (40)
0.05
3.71
4.03
3.24
41
46
11
16 (39)
13 (28)
0 (0)
< 0.002
44
46
11
34 (77)
24 (52)
5 (45)
< 0.01
4.15
3.47
3.47
59
21
18
16 (27)
7 (33)
6 (33)
NS
59
22
20
33 (56)
15 (68)
15 (75)
NS
3.61
3.62
3.90
*HIV culture of seminal cells. †HIV RNA detection in seminal plasma. ‡χ2 test for trends. NS, Not significant.
Table 4. Results of HIV seminal cell culture and HIV RNA detection in semen and antiviral treatment.
Treatment status
Never treated
Previously treated
Currently treated
Median HIV RNA
(copies/ml)
Seminal plasma RNA†
Mean blood
CD4 count
(×106/l)
Blood
Semen
Total
No. (%) positive
Total
No. (%) positive
380
190
180
4.45
4.92
4.30
3.68
4.47
3.48
53
19
29
32 (60)
16 (84)
15 (51)
52
19
27
15 (29)
7 (37)
7 (26)
Seminal cell culture*
*HIV culture of seminal cells: differences not significant (χ2 test). †HIV RNA detection in seminal plasma: P = 0.02, previously treated versus
currently treated; P = 0.03, treated versus not treated (χ2 test).
pendently and inversely predictive of a positive HIV
culture result (OR, 0.45; 95% CI, 0.23–0.87;
P = 0.02). Similarly, the multiple logistic model identified CD8 counts as a predictor of positive HIV culture,
although the association did not quite reach statistical
significance (OR, 1.18; 95% CI, 0.99–1.39; P = 0.06).
Spearman’s rank correlation revealed a significant
inverse correlation between IUPE and CD4 percentage
(r = 0.25, P = 0.015).
HIV in semen as a function of clinical stage
Asymptomatic patients were less likely to have
detectable HIV RNA in semen than patients with
advanced stages of disease, although the difference was
not statistically significant (Table 3). Stage of disease
was not an independent predictor of HIV RNA detection by multiple logistic regression. The median value
of HIV RNA in semen was slightly but insignificantly
higher in patients with an AIDS-defining diagnosis
(3.9 versus 3.6; P = 0.31, Mann–Whitney U-test).
Results of HIV culture of seminal cells were independent of the clinical stage of disease, although isolation
of HIV from semen was somewhat less likely in asymptomatic patients.
HIV in semen as a function of antiviral
treatment
Drug-naive patients had higher levels of CD4 counts
and lower levels of HIV RNA in blood plasma than
patients currently or previously treated (Table 4)
reflecting the CD4 dependence of the indication for
antiviral treatment. When patients with and without
current antiviral treatment were compared, detection of
HIV RNA in semen was significantly lower in the
treated group of patients (Table 4; P = 0.03). Current
antiviral treatment was an independent inverse predictor of HIV RNA detection by multiple logistic regression, but this association did not reach the 5% level of
significance (OR, 0.38; 95% CI, 0.14–1.03;
P = 0.054). Patients currently receiving antiviral treatment had lower HIV RNA levels in semen than
patients without antiviral treatment, and antiviral treatment was inversely but not significantly correlated with
the level of HIV RNA in semen by multiple linear
regression (P = 0.07). Patients on current antiretroviral
treatment had a trend towards lower detection rates of
HIV by seminal cell culture, although the difference
did not reach statistical significance. Similarly, multiple
logistic regression analysis did not show a statistically
significant association between treatment status and culture results (OR, 0.38; 95% CI, 0.12–1.22; P = 0.1).
Discussion
HIV is primarily an STD which is spread with greater
efficiency by men than women [8]. Semen is a likely
and important, if not critical, source of virus [24].
Transmission of infectious disease requires a critical
inoculum, which is integral to the efficiency of trans-
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AIDS 1997, Vol 11 No 8
mission. Therefore, the amount of HIV in semen will
probably be one determinant of the infectivity of an
individual.
The current study was undertaken to determine the
concentration of HIV in semen from a large group of
men and to determine what factors influence this concentration. Earlier studies were limited by the small
number of subjects studied or by assays available for
detection of HIV [11–15]. Semen samples were collected from 101 men. The results demonstrated a
strong correlation of seminal cell culture results with
the level of HIV RNA in seminal plasma. These results
expanded on our earlier studies [18] and supported the
conclusion that HIV detected in seminal plasma is
released by productively infected cells in the male genital tract. This conclusion was consistent with the observation that at steady-state, HIV RNA levels in blood
plasma appeared to be directly proportional to the
number of productively infected cells contributing to
that pool [25]. Our observation that the chance of
detecting a positive culture was 59% when copies of
HIV RNA in seminal plasma exceeded 10 000
copies/ml compared with 6% when fewer copies were
detected was also consistent with the hypothesis that
HIV RNA in seminal plasma was representative of the
number of infected cells in the genital tract releasing
virus into semen. Ex vivo culture was likely to be positive only when there was a specific, relatively large
number of these infected cells shed into the semen.
A correlation was also observed between the concentration of HIV in blood and seminal plasma. Although
this correlation may have been a result of direct communication between these compartments, differences in
phenotype and genotypes that have been observed
between HIV from blood and semen in individuals
argued against this conclusion [14, 26]. An alternative
hypothesis was that HIV-infected men that had poor
control of systemic HIV-1 replication manifested by
high HIV-1 blood plasma RNA also had poor control
of genital tract HIV replication reflected by higher levels of seminal plasma HIV RNA. Exceptions to this
correlation were likely to occur as manifest by our subject who had no detectable RNA in his blood plasma
but detectable RNA in semen. Individuals such as this
one, even if they were relatively uncommon, may have
had an important role in the continuation of the HIV
epidemic if they were destined for long disease-free
survival as would be predicted by their low blood
plasma HIV RNA [27].
The concentration of HIV in the semen was also
related to the blood CD4 count and the individual’s
stage of disease. These results must be considered in the
light of a large number of epidemiological studies
designed to better understand and predict HIV transmission. In particular, existing data have suggested that
patients with late-stage disease or low CD4 count represented the greatest risk for transmission [6,28–30].
These results were consistent with the increased likelihood of finding a positive semen culture in patients
with lower CD4 counts and the increased concentration of HIV RNA in seminal plasma noted in our
study. Krieger and coworkers [12] failed to detect a
correlation between blood CD4 count and HIV in
semen but reported that positive culture was correlated
with peripheral CD8 counts. The effect of CD8 count
was also noted in our study but did not reach statistical
significance. This difference might have been explained
by different culture techniques and size of groups
examined. In addition, the fact that CD4 and CD8
counts were related variables (reflecting a proportion of
the total number of lymphocytes) reduced the power of
the multiple logistic regression analysis to identify both
factors as independent predictors. In addition, in a
more recent study, Krieger and coworkers [31] had
used amplification techniques and noted a correlation
of HIV RNA levels in semen with the stage of disease,
CD4 count, and positive HIV culture.
Given our observation of a correlation between blood
plasma RNA and seminal plasma RNA, future epidemiological studies of transmission that collect blood
RNA data may find that this measure is a stronger predictor of transmission than disease stage or absolute
CD4 count. Mathematical models of HIV transmission
have predicted that patients with primary disease (who
have very high blood RNA levels) may be most
important for the continuation of the HIV epidemic
[32]. Although consistent with our observations, our
study did not include patients with primary HIV infection. However, specimens have been recently obtained
from three patients with primary disease whose semen
and blood viral burden were studied before therapy.
None of these patients had concentrations of HIV in
their semen outside the range of the entire group in
this study despite having high levels of blood RNA
[33]. Other factors, such as antiretroviral therapy, may
also be important in determining the concentration of
HIV in semen. Musicco et al. [34] reported a 50%
reduction of the risk of transmission in men treated
with zidovudine.
Although our study was not designed to address this
question, it was observed that patients who were currently on therapy had significantly lower HIV RNA in
seminal plasma (approximately 10-fold lower) than
patients who had discontinued therapy at least 3 weeks
prior to the study. In addition, active antiretroviral
treatment was an independent predictor of a negative
culture. In an unpublished prospective trial of a nucleoside combined with a non-nucleoside agent or placebo,
a median 1.4 log10 decline (25-fold) was observed in
seminal plasma RNA after 8 weeks on therapy in 11
patients [35].
HIV in semen Vernazza et al.
The effects of vaccines, antiretroviral therapy and
immune modulating therapy on the concentration of
HIV in semen are of considerable importance. Data
collected in this study, including the demonstration that
HIV-1 RNA could be quantified in seminal plasma in a
majority of individuals, has set the stage for development of interventions specifically designed to reduce
the concentration of HIV in semen below the level of
efficient transmission.
14.
15.
16.
17.
18.
Acknowledgement
Special thanks to A. Tschopp for performing the statistical analysis and D. Feldman for editing the manuscript.
19.
20.
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