Between November 2007 and July 2009, 52 men enrolled (). Participants were older than the 43 men who attended clinic during the study period but did not enroll (median age 31.3 vs. 28.9 years, p=0.02). Participants were less likely to be single, although this difference was borderline (69.2% vs. 86.0%, p=0.06). There were no differences in education, sexual orientation, ethnicity, or religion between groups.
Participant Characteristics (N = 52)
Participants attended quarterly visits until July 31, 2009, completion of five visits, or enrolment in a sub-study of men initiating ART. Forty-seven men had visits contributing to the present analysis; the other five participated only in the sub-study. Participants attended a median of 4 visits (inter-quartile range [IQR], 2 – 5 visits). Overall, 167 of 208 (80.3%) expected visits were completed, and 29 men (61.7%) missed no visits. Nine participants (19.1%) were lost to follow-up and two (4.3%) withdrew due to relocation outside the clinic area.
Adherence and acceptability
Complete ACASI information was available for 128 of 167 full visits (76.6%) and for 111 of 136 semen collections (81.6%), and was missing in the remaining cases due to technical failures or non-completion of the ACASI questionnaire by participants. Reported adherence to the prescribed abstinence period was 79.5% at quarterly post-PMF/U collections and 96.4% at semen collections 1 week later. Median acceptability for post-PMF/U was 10 (IQR 10 – 10, range 0 – 10), with reservations reported at 8.4% of collections. For semen collections, median acceptability was 10 (IQR 10 – 10, range 0 – 10), with reservations reported at 6.4% of collections. There was no difference in acceptability by collection method (p=0.8). Median discomfort reported with post-PMF/U collection was 0 (IQR 0 – 0, range 0 – 9), with at least some discomfort reported at 20.5% of collections. For semen collections, median discomfort was 0 (IQR 0 – 0, range 0 – 7), with some discomfort reported at 3.7% of collections. The level of discomfort reported at paired visits was higher for post-PMF/U than for semen collection (p=0.003).
All men with successful collections stated that they would be willing to provide post-PMF/U again. Reasons documented for 46 post-PMF/U refusals (75.4% of unsuccessful collections) included discomfort (12), pain (6), embarrassment (6), and other (22). Difficulty producing semen was reported at two collections (1.8%), and all men who submitted semen said they would do so again. Reasons documented for 13 missed semen collections (41.9% of unsuccessful collections) included being too busy (2), forgetting the appointment (2), having penile pain (1), being unable to ejaculate (1), and other (7).
Successful post-PMF/U collections occurred at 106 of 167 visits (63.5%), with EPS obtained during 62 collections (58.5%). At 4 visits, EPS was obtained but the participant was subsequently unable to provide post-PMF/U, possibly due to dehydration. Semen was collected after 136 of 167 visits (81.4%), a higher success rate than for post-PMF/U (p<0.001). Most semen samples were provided in clinic (85.3%) using sterile urine containers (67.0%). Both specimens were collected at least once from 35 men (74.5%), only semen from 8 men (17.0%) and only post-PMF/U from 3 men (6.4%). One man (2.1%) provided neither specimen. Semen processing took approximately 3 hours, and post-PMF/U processing <1 hour on average.
In univariate analysis, MSM and transactional sex workers were more likely to have successful post-PMF/U collections (). Success was less likely with the female clinician and for repeat visits. A multivariate GEE model showed that female clinician and repeat visits were associated with lower odds of successful post-PMF/U collection. In contrast, unsuccessful semen collection was associated with having a CD4 count ≤200 cells/μL (OR 0.35, 95% CI 0.14 – 0.88, p=0.03).
Factors Associated with Successful Post-Prostate Massage Fluid/Urine (Post-PMF/U) Sample Collection
HIV-1 RNA levels were compared for post-PMF/U and semen samples from 29 men who had provided samples through June 8, 2008 (). Of eight participants for whom paired samples were unavailable, six provided only post-PMF/U, one provided only semen, and one was unable to provide either sample. Ten participants were taking ART, including nine who received a standard first-line regimen of stavudine, lamivudine, and efavirenz and one who received a second-line regimen of tenofovir, abacavir, and boosted lopinavir. Additional sample sets were collected at repeat visits from 19 men, for a total of 48 paired sample sets.
Characteristics of 29 Men Providing Paired Genitourinary and Blood Samples
HIV-1 RNA Detection
At initial visits, HIV-1 RNA was detected in post-PMF/U alone from two men (7%), semen alone from five men (17%), and both specimens from eight men (28%). In contrast, HIV-1 RNA was detected in blood only from six men (21%), semen only from no man (0%), and both specimens from thirteen men (45%). HIV-1 RNA was undetectable in post-PMF/U and semen in all ten men taking ART, but was detectable in three blood samples (30%). HIV-1 RNA detection in post-PMF/U was associated with HIV-1 RNA in semen (p=0.02).
Stability of HIV-1 RNA Detection
Concordance of test results at 19 repeat visits was 78.9% for post-PMF/U (κ = 0.519, p = 0.02), and 89.5% for both blood and semen (κ = 0.774, p = 0.001). For the nine men taking ART who had repeat visits, concordance was 100% for both semen and post-PMF/U, and 90% for blood (κ = 0.727, p = 0.02).
Post-PMF/U sensitivity for predicting seminal HIV-1 was 61.5%, with 87.5% specificity (joint 95% CI, [28.3% – 88.3%] × [58.0% – 98.9%]). The sensitivity of blood for predicting seminal HIV-1 was 100%, with 62.5% specificity (joint 95% CI, [71.4% – 100%] × [32.3% – 86.9%]). The full dataset of 48 sample sets was explored for factors associated with post-PMF/U sensitivity and specificity, including disease stage (i.e., HIV-1 RNA level in blood, CD4 cell count, ART status) and collection technique (i.e., time since last ejaculation, whether EPS was obtained, volume of post-PMF/U collected, and clinician obtaining the sample). Relative post-PMF/U sensitivity increased by 1.35 (95% CI, 1.04-1.75) for each log10 increase in blood HIV-1 RNA level. No other factor was found to influence the sensitivity or specificity of HIV-1 detection in post-PMF/U.
HIV-1 RNA Quantitation
At the initial visit, 10 of 29 men (34.5%) had detectable post-PMF/U RNA with a median level of 222.5 copies/mL (range, 30–21,221 copies/mL). Thirteen men (44.8%) had detectable semen RNA with a median level of 3,901 copies/mL (range, 150–121,633 copies/mL). presents log10 HIV-1 RNA levels for the 48 sample sets. Correlations between semen and post-PMF/U RNA level (ρ = 0.657, p <0.001), post-PMF/U and blood RNA level (ρ = 0.502, p = 0.006), and semen and blood RNA level (ρ = 0.792, p <0.001) were all highly significant.
Comparison of Log10-Transformed HIV-1 RNA Levels in (A) Blood and Urine, (B) Blood and Semen, and (C) Urine and Semen
Stability of HIV-1 RNA level
Among 19 participants with repeat visits, the standard deviation of differences in log10 HIV-1 RNA were 1.00 for post-PMF/U, 0.88 for semen, and 1.29 for blood when restricted to paired samples in which HIV-1 RNA was detected in both samples. When restricted to paired samples in which HIV-1 RNA was detected in either sample, the standard deviations of the differences in log10 HIV-1 RNA were 1.05 for post-PMF/U, 0.99 for semen, and 1.25 for blood. Results were similar (1.05 for post-PMF/U, 0.99 for semen, and 1.36 for blood) when restricted to men not taking ART.