Various factors determine the infectiousness of HIV-1 in semen, including the viral phenotype , whether the virus is cell associated or cell free  and the chemokine receptor status of the recipient . The quantity of virus in ejaculate, as reflected by seminal plasma viral load (SVL) is probably the most important, a fact supported by both epidemiological and biological evidence [4–12].
Previous studies have shown, in general, a good correlation between HIV-1 RNA load in seminal and blood plasma in antiretroviral drug-naive and experienced patients [9,10,11,13,14]. SVL is generally lower than blood plasma viral load (BVL), but most comparisons report a minority of patients where seminal values are higher. This may, in part, reflect independent replication of HIV-1 in the genital compartment  and the differential penetration of antiretroviral drugs into it [16,17]. In addition, the presence and quantity of HIV-1 proviral DNA in semen cells is correlated with both SVL and BVL and HIV-1 DNA has been found in semen from patients with undetectable SVL and BVL [9,14,18].
Other work has also shown that, in those not taking antiretroviral therapy, SVL and proviral HIV-1 DNA in semen and genital secretions may increase considerably (up to 20-fold) during episodes of gonococcal, non-gonococcal and chlamydial urethritis and fall following treatment for the urethritis [19–23]. There is also evidence that gonococcal infection is more likely to raise SVL than non-gonococcal urethritis because of the more pronounced inflammatory effect of the former . In vitro studies have demonstrated enhanced replication of HIV-1 in the presence of chlamydia  and also increased expression of chemokine co-receptors for HIV-1 in the presence of bacterial lipopolysacharide, Treponema pallidum  and other infectious agents [26,27].
However the effects of urethritis in patients taking combination antiretroviral therapy have not been explored. We have conducted a study in two centres to investigate whether urethritis leads to an increase in SVL and HIV-1 proviral DNA in semen cells in patients being treated with combination antiretroviral therapy.
Subjects and samples
Gay men attending as outpatients and who had been receiving combination antiretroviral therapy for at least 3 months were eligible for the study. Exclusion criteria were any previous episode of urethritis or intercurrent illness in the month prior to study. Patients with symptomatic urethritis (PWU) and those attending for a sexually transmitted infection screen in whom no diagnosis was found (controls) were recruited prospectively from the Mortimer Market Centre between November 1998 and March 2000. The patients with symptomatic urethritis attending the Department of Sexual Medicine (Birmingham Heartlands Hospital) were recruited between February 1999 and February 2000.
Written informed consent was obtained from all patients and a urethral smear and culture for diagnosing gonorrhoea, a swab for diagnosing chlamydial infection using the ligase chain reaction and blood for viral load were taken on their first visit. Patients then provided a semen sample by masturbation. Most patients provided semen samples prior to voiding urine. This study was approved by the ethics committees of Camden & Islington Community Health Services and Birmingham Heartlands NHS Trusts. There were no conflicts of interest.
Those patients diagnosed with urethritis, whether gonococcal or non-gonococcal, were treated with appropriate antibiotics. All patients attended 2 weeks later for repeat smears, gonococcal culture, blood and semen samples. Most patients also provided samples 1 week after the first visit. At all visits sexual histories were taken to exclude further exposure to sexually transmitted infections.
Semen and blood samples were centrifuged within 2 h of collection and the plasma and cellular components stored at −70°C. RNA was extracted from blood and seminal plasma by a silica gel capture method previously observed to remove inhibitors of the polymerase chain reaction (PCR) successfully  and was measured using an in-house, internally calibrated reverse transcribed PCR assay (RT-QPCR, Department of Virology, University College London) or NASBA (nucleic acid sequence base assay; Nuclisens HIV-1 QT, Organon Teknika, Durham, North Carolina, USA; used at PHLS, Birmingham) as previously described . For the in-house RNA assay, the level of detection was 500 copies/ml for blood plasma and 1000 copies/ml for seminal plasma; NASBA had a level of detection of 400 copies/ml for both blood and seminal plasma. A good correlation was previously documented between these two assays (data not shown).
To assay proviral DNA in the cellular component of semen, stored cell samples were extracted using a modified protocol with the QIAamp DNA mini Kit (Qiagen Ltd, Crawley, West Sussex, UK) . Provirus DNA loads were assayed as described previously . The assay was performed once only for each sample measured.
Eligible patients that were offered participation in the study at the Mortimer Market Centre were compared with those that were not; among those offered, those who agreed to participate were compared with those who declined. These comparisons were performed separately among PWU and controls with respect to age and stage of disease. In the study population, PWU were compared with controls with respect to the most recent CD4 cell count prior to first visit, and CD4 cell count and BVL prior to ever starting combination antiretroviral therapy. For comparisons of age, the t-test was used and for CD4 cell counts and viral loads the Mann–Whitney test was used. The chi-square test for trend was used for HIV disease stage. The chi-square test was also used to test for an association between the detection of HIV RNA in semen and blood.
Of 84 eligible patients at the Mortimer Market Centre, 47 (60%) were offered entry into the study and of these, 36 (77%) agreed to take part. A comparison within control and PWU groups between those offered and those not offered entry into the study showed there was no significant difference in age (P = 0.154 and P = 0.550, respectively) or stage of HIV infection (P = 0.345 and P = 0.799, respectively). In those offered entry into the study, there was no difference between those recruited or not in the age of controls and PWU (P = 0.117 and P = 0.295, respectively) or in the stage of HIV infection among controls (P = 0.829). PWU, however, appeared to be more likely to decline study participation if they had more advanced HIV disease stage (P = 0.043). (data not shown).
Samples from 16 control patients and 24 PWU (10 with gonorrhoea, six with chlamydial infection and eight with non-gonococcal urethritis) were collected. Baseline characteristics of all patients, and differences between PWU and controls are shown in Table 1.
The majority of patients had undetectable BVL a few months prior to the study. Both the median nadir CD4 cell count before antiretroviral therapy was started and current CD4 cell count just prior to the study were similar between PWU and controls. All participants were taking at least three antiretroviral drugs (Tables 2 and 3).
In 13 of 16 controls and 18 of 24 PWU on antiretroviral therapy, HIV RNA was undetectable in semen at visit 1. In general, among all patients, there was a strong association between detectability of HIV-1 RNA in seminal plasma and detectability in blood plasma at visit 1 (P < 0.0005 and P = 0.06 for PWU and controls, respectively;Table 4). In those with detectable BVL at visit 1, 0/5 PWU compared with 4/7 controls had undetectable SVL at visit 1. However, median BVL was higher in the PWU compared with controls at visit 1 (69 000 versus 1052 copies/ml).
Among controls, consistent with earlier studies in patients on combination antiretroviral therapy, when virus was undetectable in blood it was undetectable in seminal plasma, and low BVL (ranging from 600 to 11 000 copies/ml) was associated with low or undetectable SVL. Also in line with earlier studies, some controls with undetectable BVL and SVL had detectable proviral DNA in semen despite most of them being on suppressive therapy for more than 1 year (Table 2). Time on antiretroviral therapy ranged from 2 to 42 months for controls.
Among PWU with undetectable BVL prior to recruitment, BVL and SVL remained undetectable at visit 1 and subsequent visits in 5/5 episodes of chlamydial urethritis, 6/7 episodes of non-gonococcal urethritis and 4/5 episodes of gonococcal urethritis. Two PWU (patients 23 and 24 with gonococcal urethritis and non-gonococcal urethritis, respectively) with undetectable BVL just prior to the first study visit, had low to moderate SVL (5928 and 1512 copies/ml, respectively), which became undetectable by visit 2 following treatment for urethritis. The reduction in SVL in patient 24, however, may be accounted for by assay variability. (Table 3).
Of seven PWU with detectable BVL prior to recruitment, three (one with gonococcal urethritis, one with chlamydial urethritis and one with non-gonococcal urethritis) had undetectable BVL and SVL at visit 1. These results were maintained at subsequent visits apart from patient 4, who had low levels of detectable RNA in blood at visits 2 and 3. (In patient 5, because of difficulties with diluting the sample, the limit of detection in semen at visit 1 was 10 000 copies/ml). Four PWU (patients 19–22, all with gonococcal urethritis ) had poor control of BVL prior to presentation with their urethritis (and at visit 1) and all of them had high SVL. In one of these (patient 22), in particular, the SVL was higher than the BVL but was reduced 20-fold following treatment for gonorrhoea (Table 3). Time on antiretroviral therapy for PWU ranged from 7 to 51 months.
In patients 19–21, (with gonococcal urethritis), HIV-1 RNA was detectable in seminal plasma at lower concentration than in blood and these loads did not change significantly following treatment for gonorrhoea. Two of these patients (20 and 21) voided urine prior to providing semen samples and the first catch of urine from patient 20 was retained at visit 1 and follow-up. It was not possible to amplify the amount of HIV-1 RNA in cell-free urine from this patient because of inhibitors. However, HIV-1 proviral DNA was found in urinary cells at visit 1 but was not detected at follow-up (data not shown). Patient 21 was the only one in this group that had detectable HIV-1 proviral DNA in semen at the first visit and this remained detectable following treatment.
In 12 PWU with undetectable SVL at visit 1, semen samples were available for measuring proviral HIV-1 DNA both at the first visit and following treatment for urethritis. In 10 of these, provirus was undetectable during urethritis; it remained undetectable in eight following treatment. In two PWU, provirus was detectable during urethritis; following treatment it remained detectable in one patient but was undetectable in the other.
In PWU patients 23 and 24, in whom SVL was reduced following treatment for urethritis, HIV-1 provirus was not detected in semen at visit 1, a finding not inconsistent with their respective low BVL and SVL . Unfortunately, samples were not available for measuring HIV-1 proviral DNA in patient 22.
In 18 of 24 PWU and 13 of 16 controls, SVL was undetectable and, where tested, HIV-1 proviral DNA was also largely undetectable. In general, there was a strong association between detectability of HIV-1 RNA in seminal plasma and detectability in blood plasma in all patients, confirming previous findings in patients taking antiretroviral therapy who did not have urethritis [11,13,14]. The present study, therefore, provides important evidence that antiretroviral therapy may not only reduce sexual transmission of HIV-1 by reducing viral load in semen but may also reduce the potential facilitating effects of sexually transmitted infections on HIV-1 transmission.
SVL was detectable in 3 of 16 controls (at low levels) and in 6 of 24 PWU. In three PWU, SVL was reduced following treatment for urethritis. In one PWU, with poorly controlled BVL, SVL was reduced 20-fold following treatment for gonorrhoea. These observations suggest that, at least in some patients, antiretroviral therapy may not always control the potentiating effects of urethritis on HIV replication in the genital tract.
There was a suggestion that, among those with detectable BVL, SVL was more likely to be detected in PWU compared with controls, but we believe this is explained by the higher BVL in PWU.
This study also confirms that HIV-1 provirus may still be detectable in semen in patients who have undetectable levels of HIV-1 RNA in blood and seminal plasma for more than 1 year [9,14,18].
In the three PWU (patients 19–21) in whom antiretroviral therapy had clearly failed virologically prior to their acquiring urethritis, SVL was lower than BVL and did not change significantly following urethritis treatment. These results may seem surprising given that urethritis might have been expected to raise SVL above BVL in the presence of failing therapy. There are several possible explanations for this observation. First, even though BVL and SVL were high in these patients, it is possible that antiretroviral therapy may still have been sufficiently effective to contain the potentiating effects of urethritis on SVL. Second, it is possible that the viral load in the semen of these patients was already too high for there to be a potentiating effect of urethritis on replication. Third, patients were not asked to void urine before ejaculating in a previous study showing large increases in SVL during urethritis (; Dr Irving Hoffman, AIDSCAP Malawi Research Group, personal communication). Previous studies on patients with vasectomies have demonstrated that the vast majority of HIV-1 in semen arises distal to the vas deferens [32,33], but the source of increased SVL following urethritis has not been established. HIV-1 contained in the urethral discharge may be an important part of this source. In patients 20 and 21, urine was voided just prior to producing semen samples and HIV-1 provirus was found in first voided urinary cells of patient 20. Therefore, these patients may have had increased amounts of HIV-1 RNA in the urethra that was washed away by voiding urine before semen collection. However, patient 19 voided urine after semen production and still had no increase in SVL. Other explanations for rises in SVL not being seen in these patients include decreased replicative fitness of HIV-1 owing to the presence of drug-resistant mutations prior to acquiring urethritis or drug resistance acquired during urethritis.
Patients such as these may have increased infectiousness for drug-resistant strains of HIV-1 and contribute to the prevalence of sexually transmitted drug-resistant HIV-1 [34–37].
Our study has shown that antiretroviral therapy may be important in limiting the effect of sexually transmitted infections on the transmission of HIV-1. For this potential benefit to be fully realised, viral load in semen as well as in peripheral blood plasma may need to be optimally suppressed. Where this is not the case, urethritis may still increase the risk of HIV-1 transmission of both wild-type and drug-resistant strains.
The authors would like to thank the reception, nursing and medical staff at The Mortimer Market Centre and Birmingham Heartlands Hospital, and Caroline Dann for assisting with typing and tabulation.
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