Randomized Cross-Sectional Study to Compare HIV-1 Specific Antibody and Cytokine Concentrations in Female Genital Secretions Obtained by Menstrual Cup and Cervicovaginal Lavage

Abstract

IntroductionOptimizing methods for genital specimen collection to accurately characterize mucosal immune responses is a priority for the HIV prevention field. The menstrual cup (MC) has been proposed as an alternative to other methods including cervicovaginal lavage (CVL), but no study has yet formally compared these two methods.MethodsForty HIV-infected, antiretroviral therapy-naïve women from the CAPRISA 002 acute HIV infection cohort study were randomized to have genital fluid collected using the MC with subsequent CVL, or by CVL alone. Qualitative data, which assessed levels of comfort and acceptability of MC using a 5-point Likert scale, was collected. Luminex multiplex assays were used to measure HIV-specific IgG against multiple gene products and 48 cytokines.ResultsThe majority (94%) of participants indicated that insertion, wearing and removal of the MC was comfortable. Nineteen MCs with 18 matching, subsequent CVLs and 20 randomized CVLs were available for analysis. Mucosal IgG responses against four HIV-antigens were detected in 99% of MCs compared to only 80% of randomized CVLs (p = 0.029). Higher specific antibody activity and total antibodies were observed in MCs compared to CVL (all p<0.001). In MCs, 42/48 (88%) cytokines were in the detectable range in all participants compared to 27/48 (54%) in CVL (p<0.001). Concentrations of 22/41 cytokines (53.7%) were significantly higher in fluid collected by MC. Both total IgG (r = 0.63; p = 0.005) and cytokine concentrations (r = 0.90; p<0.001) correlated strongly between MC and corresponding post-MC CVL.ConclusionsMC sampling improves the detection of mucosal cytokines and antibodies, particularly those present at low concentrations. MC may therefore represent an ideal tool to assess immunological parameters in genital secretions, without interfering with concurrent collection of conventional CVL samples.

Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women

“…Because antibody concentrations were generally low in CVLs, we next tested IgG antibodies freshly collected from these women using cervical Softcups to improve IgG recovery [38] and allow for more neutralization assays to be performed (Supplementary Table 1). This included two CAPRISA 002 women (CAP8 and CAP248) who were receiving ART at the time of Softcup sampling, as well as three additional women from the CAPRISA 004 cohort who were ART-naive.…”

“…Antibody recovery was significantly improved through the use of the cervical Softcup [64, 65], which provided more consistent data and higher IgG recovery from genital fluids. The Softcup collects undiluted samples that are processed immediately [38], and thus are not exposed to freeze-thaw cycles, which may reduce the concentration of IgG antibodies in CVL [66]. Furthermore, other studies have shown that the endocervix, which is more specifically sampled by Softcup, has more concentrated antibodies than the vagina [60] which is a wash-out mainly of the vaginal vault.…”

“…In addition, cervical secretions were collected using a Softcup® Menstrual cup (EuroFemPro, Netherlands) and processed as previously described with modifications [37]. For this, the Softcup was inserted into the vagina for a minimum of 1 hour by a clinician, placed into a 50 ml tube and centrifuged [38]. The fluid phase was collected into an eppendorf tube and the volume measured.…”

Comparison of sampling methods to measure HIV RNA viral load in female genital tract secretions

“…11,12 Yet, relatively few studies have tested the validity and reproducibility of these methods to collect samples for genital HIV VL detection. 8,11,12 Volumes recovered in CVLs can be highly variable relative to the amount of fluid instilled, 13 which can affect the volume of cell-free and cell-associated virus collected, prompting transition to swab enrichment of lavage specimens. 12 CVL provides an advantage over ECS in that increased surface area is sampled.…”

Accurate measurement of female genital tract fluid dilution in cervicovaginal lavage samples

“…Vaginal proinflammatory cytokines and other markers of immune activation are routinely measured in CVL [4-9]. More recently, a multiplexed assay to analyze antimicrobial peptides in CVL has been reported [10].…”