Prospective pilot study on the relationship between seminal HIV-1 shedding and genital schistosomiasis in men receiving antiretroviral therapy along Lake Malawi

Male genital schistosomiasis (MGS) is hypothesized to increase seminal shedding of HIV-1. This prospective pilot study assessed seminal HIV-1 RNA shedding in men on long-term ART with and without a diagnosis of MGS. Study visits occurred at 0, 1, 3, 6 and 12 months. MGS was diagnosed by egg positivity on semen microscopy or PCR of seminal sediment. After optimization of the HIV-RNA assay, we examined 72 paired plasma and semen samples collected from 31 men (15 with and 16 without MGS) over 12 months. HIV-1 RNA was detected in 7/72 (9.7%) seminal samples and 25/72 (34.7%) plasma samples. When comparing sample pairs, 5/72 (6.9%) showed HIV-1 RNA detection only in the seminal sample. Overall, 3/31 (9.7%) participants, all with MGS, had detectable HIV-1 RNA in semen while plasma HIV-1 RNA was undetectable (< 22 copies/mL), with seminal levels ranging up to 400 copies/mL. Two participants showing HIV-1 RNA in seminal fluid from the MGS-negative group also had concomitant HIV-1 RNA detection in plasma. The findings suggest that MGS can be associated with low-level HIV-1 RNA shedding despite virologically suppressive ART. Further studies are warranted to confirm these observations and assess its implications.

who attended HIV outpatient services along Lake Malawi between October 2017 and December 2018 (Fig. 1).Mid-morning urine, semen and whole blood in EDTA were to be collected at each planned study visit (0, 1, 3, 6 and 12 months).The sampling methodology was reported previously 23 and is described in Supplementary Information.Seminal samples were collected in a clear plastic bag following two days of abstinence from sexual activity.In total, 74 participants enrolled, of which 23 did not submit blood or urine samples for schistosomiasis testing.Of the remaining 51, 33 tested negative for schistosomiasis, of whom 16 provided blood, urine, and semen.This cohort of 16 was deemed the 'MGS-negative group' .Of the 18 participants that tested positive for schistosomiasis, 15 provided blood, urine, and semen.This cohort of 15 was deemed the 'MGS-positive group' .The disposition of the whole study population is shown in Supplementary Fig. 1.Given the high prevalence of schistosomiasis in the region 22 , participants received PZQ at all study visits regardless of a diagnosis of MGS.
Laboratory procedures.Samples were processed within 3 hours of collection.Whole blood samples were centrifuged at 3000 xg for 5 minutes to separate plasma for storage at −80°C.Seminal samples were allowed to liquefy at ambient temperature and examined under microscopy for the presence of Schistosoma eggs, followed by centrifugation at 3000 xg for 5 minutes to separate supernatant seminal fluid and sediments.Seminal fluid was stored at −80°C.Seminal sediment samples were re-dissolved in saline and 2-3 drops were placed on a glass slide for microscopy to detect Schistosoma eggs.Leftover seminal sediments were preserved in ethanol and shipped to the Elisabeth-TweeSteden Ziekenhuis (ETZ) Hospital in Tilburg, the Netherlands, for Schistosoma DNA detection by in-house real-time PCR as previously described 23 .The parasitological procedure was previously detailed 23 and is described in Supplementary Information.Urine samples were tested for Schistosoma circulating cathodic antigen (CCA) using the point-of care parasite CCA test (POC-CCA) (Rapid Medical Diagnostics, South Africa), followed by urine filtration and microscopy for Schistosoma eggs.Cryopreserved seminal fluid and plasma samples were shipped on dry ice to the United Kingdom for HIV-1 RNA testing.
Schistosomiasis diagnosis and definition of MGS.Schistosoma positivity was defined by visual evidence of eggs by microscopy of filtrated urine, semen, or seminal sediment; PCR detection in seminal sediment, urine or stool; or positive POC-CCA test in urine.Male genital schistosomiasis (MGS) was defined as Schistosoma positivity in semen.
HIV-1 RNA testing.HIV-1 RNA was detected in plasma and seminal fluid using the Cepheid Xpert assays 25 .The assays perform qualitative (HIV-1 Qual assay) and quantitative (HIV-1 Viral Load assay) detection of HIV-1 RNA in plasma 26 .With 1 mL input, the HIV-1 Qual assay reports qualitative HIV-1 RNA detection with a lower limit of detection (LLOD) of 278 copies/mL; the manufacturer describes 25% detection rate at 60 copies/ mL 27,28 .With 1mL input, the HIV-1 Viral Load assay has a lower limit of quantification (LLOQ) of 40 copies/ mL and a LLOD of 22 copies/mL 26 .To detect HIV-1 RNA in seminal fluid, the assays were first validated using seminal fluid samples collected from two HIV negative donors and spiked with known amounts of HIV-1 RNA, as detailed in Supplementary Information.The assay LLOD for seminal fluid ranged from 55 to 220 copies/mL according to the dilution applied.HIV-1 RNA results were reported as either a quantified level or as qualitative targeted detected.

Results
Study population and sampling.A total of 31 participants established on ART provided at least one paired semen and blood sample over the 12 months of the study.Their baseline characteristics are summarised in Table 1.At study entry, participants had received ART for a median of 7.5 years (IQR 1.9-13.1).Most were receiving coformulated tenofovir disoproxil fumarate/lamivudine/efavirenz (TDF/3TC/EFV).A total of 72 paired samples were collected, with a median of 2 paired samples per participant (range 1-5), including 30 samples at study entry (baseline), and 10, 12, 9 and 11 samples at 1, 3, 6 and 12 months.Eleven participants donated only one set of paired samples.Overall, based on the date of the last paired samples, the median duration of Figure 1.Schematic map of study area showing health facilities along the shores of Lake Malawi (https:// www.cia.gov/ libra ry/ publi catio ns/ the-world-factb ook/ attac hments/ locat or-maps/ MI-locat or-map.gif and https:// www.cia.gov/ libra ry/ publi catio ns/ the-world-factb ook/ attac hments/ maps/ MI-map.gif) 24 .
www.nature.com/scientificreports/follow-up was 10.2 months (IQR 4.0-14.2).All except 6 participants (one with MGS and 5 without) had received PZQ in the 12 months prior to recruitment.None showed symptoms suggestive of sexually transmitted infections (STIs) and no samples were collected to investigate asymptomatic STIs.

MGS status.
Details of parasitology testing are summarised in Table 2.At baseline, 8 participants tested MGS positive by semen microscopy, 4 tested positive only by real-time PCR of seminal sediment, and 20 tested negative by all tests.Among the 20 participants with a negative baseline test, 3 had a positive test during followup (1 by real-time PCR only; 1 by PCR and POC-CCA test and 1 by urine filtration, PCR and POC-CCA test) yielding a total of 15 participants who were classed as MGS positive (A01-A15), whereas 16 were classed as MGS negative (A16-A31).There were no significant differences when comparing the baseline characteristics of the two groups (Table 1).During the study period, 42 paired samples were provided by the MGS positive participants while 30 samples were provided by MGS negative participants.

Patterns of HIV-1 RNA detection by MGS status.
When comparing the 72 paired plasma and seminal samples, HIV-1 RNA detection (with or without quantification) was concordant in 44/72 (61.1%) samples (Table 4).The remaining 28/72 (38.9%) sample pairs were discordant; most had HIV-1 RNA detected in plasma only.In the MGS-negative group, 2/16 participants had detectable HIV-1 RNA in 2 seminal samples, and in both cases the paired plasma showed high HIV-1 RNA levels.In contrast, in the MGS-positive group, 3/15 participants had detectable HIV-1 RNA in 5 seminal samples, and in all cases the paired plasma showed undetectable HIV-1 RNA.The characteristics of the 3 participants are detailed in the Table 5.They had been established on ART long-term, ranging between 8 and 12 years at study entry.Overall, 4 of the 5 seminal samples with detectable HIV-1 RNA had concomitant Schistosoma test positivity.

Discussion
Across sub-Saharan Africa, evidence of the impact of MGS on HIV-1 shedding in semen is limited despite the significant epidemiological overlap between HIV-1 infection and endemic schistosomiasis 29 .We sought to assess prospectively over 12 months, the rates and kinetics of seminal HIV-1 RNA shedding in heterosexual men living with HIV-1 and established on first-line NNRTI-based ART in Malawi, stratifying the data according to a diagnosis of MGS.In our pilot study, we observed low-level HIV-1 RNA shedding in seminal fluid of men with MGS despite fully suppressed plasma HIV-1 RNA, whereas in those without an MGS diagnosis seminal HIV-1 RNA shedding was always concomitant with high-level viremia.Our numbers are limited.In total, we observed HIV-1 RNA detection in 5 seminal samples from 3 of 15 participants in the MGS group.Larger studies are needed to confirm these interesting observations.Urogenital schistosomiasis is thought to be similar to STIs such as HSV-2 in facilitating HIV-1 transmission; this is due to presence of mucosal lesions and local recruitment of HIV-susceptible cells through egg-induced inflammation 3 .Collectively, this is expected to increase the risk of HIV acquisition and propagation by increasing viral replication.Whilst data reporting the impact of urogenital schistosomiasis on HIV-1 shedding in semen are scarce, one study showed a decrease in seminal HIV-1 RNA levels following treatment with PZQ during a follow-up period of 10 weeks 12 .
Virologically effective ART reduces the risk of transmitting HIV sexually via both heterosexual and homosexual routes 14,15,30 .One question remains as to the relevance of the reported delay in HIV-1 RNA suppression  www.nature.com/scientificreports/ in seminal fluid relative to plasma in the early phase of ART.In our study, participants entered the analysis while already established on ART with a median ART duration of 7.5 years (IQR 1.9-13.0).In this population established on long-term ART, 5/31 (16.1%) participants had at least one episode of seminal HIV-1 RNA detection over 12 months of follow-up.Two of the participants were negative for Schistosoma and had concomitant high HIV-1 RNA detection in plasma.Among participants positive for Schistosoma, 3 were established on ART with TDF/3TC/EFV for 8-12 years and had fully suppressed, undetectable HIV-1 RNA in plasma, yet they showed detectable HIV-1 RNA in a total of 5 seminal samples.It is important to highlight that the levels of seminal HIV-1 RNA in these 5 samples were low and never exceeded an estimated 400 copies/mL, despite 4 of the 5 samples coinciding with a positive Schistosoma test.The implications in terms of risk of HIV-1 transmission are uncertain.
Table 3. HIV-1 RNA testing results (in copies/mL) in plasma and seminal fluid by MGS status. a a With plasma, HIV-1 RNA results were either quantifiable ( ≥ 40 copies/ml), detectable qualitatively (D, estimated level 22-39 copies/ml), or undetectable (UD, < 22 copies/ml); with seminal fluid, the lower limit of detection varied according to the sample dilution and is specified by sample as follows (the estimated HIV-1 RNA levels are given in parenthesis): UD 1a = HIV-1 RNA undetectable (< 110 copies/ml); UD 2b = HIV-1 RNA undetectable (< 55 copies/ml); UD 3c = HIV-1 RNA undetectable (< 220 copies/ml); D = HIV-1 RNA detected (viral load in copies/ ml).Shadowing legend Orange color: HIV-1 RNA detectable in semen and undetectable in plasma.Blue color: HIV-1 RNA detectable in plasma and undetectable in semen.Green color: HIV-1 RNA detectable in plasma and semen.www.nature.com/scientificreports/As a pilot study, we acknowledge several limitations.The study population was small and participants did not provide samples at all scheduled time points.We had small seminal sample volumes which constrained our ability to use a high input volume in the HIV-1 RNA tests.Due to lack of samples, we did not attempt to perform HIV drug resistance testing when HIV-1 RNA was detected and we did not investigate the concomitant occurrence of clinically unrecognised STIs.

Conclusion
Taken together, our study found that 3/15 (20%) men with HIV-1 and MGS who were established on NNRTIbased first-line ART for at least 8 years had detectable HIV-1 RNA in 5 seminal samples while plasma HIV-1 RNA was fully suppressed.Seminal HIV-1 RNA shedding coincided with Schistosoma detection in 4 of the 5 samples.However, HIV-1 RNA was detected in seminal fluid at low levels, raising doubts about significance in terms of risk of transmission, particularly if shedding is sporadic or intermittent.We recommend that future studies should aim to (1) evaluate the definitive role of MGS in enhancing HIV-1 RNA shedding in semen during ART; (2) investigate the infectiousness and drug resistance profile of seminal HIV-1 RNA; and (3) determine how the introduction of dolutegravir-based ART across Africa may impact on the findings especially as reports have started to observe treatment failure across populations 31,32 . https://doi.org/10.1038/s41598-023-40756-8