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

Introduction: Optimizing 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.

Methods: Forty 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.

Results: The 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.

Conclusions: MC 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.

Fig 1. Schema of study design.

Fig 2. HIV specific activity [Log₁₀ MFI*dilution factor ng ml^-1 (MFI/total Ig)] for gag p24- 2A, p66- 2B, gp41- 2C and gp120- 2D in MC (n = 19) and randomized CVL samples (n = 20).

Limit of detection is shown as the dotted line on the figures.

Fig 3. Comparison of total antibody (ng ml-1) in MCs (n = 19)- 3A, randomized CVL (n = 20) and matched post-MC CVLs (n = 18) [Log₁₀ MFI*dilution factor ng ml^-1 (MFI/total Ig)]- Correlation plots between total antibody in MCs and matched post MC CVLs- 3B.

Correlation plots (Fig 3C–3F) of HIV specific activity in MCs (n = 18) and matched post-MC CVLs (n = 18) [Log₁₀ MFI*dilution factor ng ml^-1 (MFI/total Ig)] for gag p24- 3C, p66- 3D, gp41- 3E and gp120- 3F.

Fig 4. Comparison of median cytokine concentrations observed in genital fluid isolated from CVL, MC and matching subsequent CVL specimens in a randomised study.

Cytokine concentrations were compared in genital fluid isolated from n = 19 MC- 4A and n = 20 randomized CVL, and n = 18 matching MC and subsequently-sampled CVL specimens- 4B. The concentrations of 48 cytokines [(regulatory ○; growth factor ✚; haematopoietic ✖; adaptive ◆; inflammatory ●)] were determined in each specimen by luminex technology and the median of each cytokine concentration was compared by Spearman rank correlation. Data are depicted on Log₁₀ scales. P-values <0.05 were considered significant.