Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators

Abstract

Background: Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods.

Methods: Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth.

Results: Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status.

Conclusions: Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.

Figure 1. Sample collection algorithm.

Sample collection proceeded from the introitus to the cervix. A Dacron swab was rolled 360° along the vaginal lateral wall. A flocked swab was rolled 360° along the opposite vaginal lateral wall. After vaginal swabs, endocervical swabs were taken with first a Dacron swab and then a flocked swab inserted into the cervical os and turned 360°. Finally, the women were randomized to have a CVL collected with 10 ml of saline, Normosol-R, or tap water. Samples were processed as described in the Methods section.

Figure 2. Protein levels in female genital tract secretions collected by swabs and cervicovaginal lavages (CVL).

Female genital tract secretions were collected by Dacron swabs (DS) and flocked swabs (FS) from the vagina and the endocervix (cervix) and by CVL using Normosol-R, saline, or water. Protein levels were determined using the Bradford assay. Data are presented as box and whisker plots where the median is the horizontal line through the vertical box which represents the 25–75^th percentiles. Values within the 10–90^th percentiles are represented by the error bars. Outliers are shown by filled circles. Data were log-transformed and significant changes were determined using linear mixed model and discussed in the results section.

Figure 3. Levels of mediators in female genital tract secretions collected by swabs and cervicovaginal lavages (CVL).

Female genital tract secretions were collected by Dacron swabs (DS) and flocked swabs (FS) from the vagina and the endocervix (cervix) and by CVL using Normosol-R, saline, or water. (A) Cytokine levels and IL-8 were measured by luminex technology. (B) Antimicrobial protein levels were measured by commercially available ELISA. β-defensins were measured only in CVL due to limited sample volume from the swabs. Data are presented as box and whisker plots where the median is the horizontal line through the vertical box which represents the 25–75^th percentiles. Values within the 10–90^th percentiles are represented by the error bars. Outliers are shown by filled circles. Data were log-transformed and significant changes were determined using linear mixed model and discussed in the results section.

Figure 4. Levels of secretory leukocyte protease inhibitor (SLPI) in female genital tract secretions collected by swabs and cervicovaginal lavages (CVL) in women without or with bacterial vaginosis (BV).

Female genital tract secretions were collected by Dacron swabs (DS) and flocked swabs (FS) from the vagina and the endocervix (cervix) and by CVL using Normosol-R, saline, or water. Women with BV (n = 24) had a Nugent score of ≥7. Data are presented as box and whisker plots where the median is the horizontal line through the vertical box which represents the 25–75^th percentiles. Values within the 10–90^th percentiles are represented by the error bars. Data were log-transformed and significant changes were determined using linear mixed model and discussed in the results section.

Figure 5. Levels of antimicrobial activity in female genital tract secretions collected by cervicovaginal lavages (CVL) and swabs.

Female genital tract secretions were collected by Dacron swabs (DS) and flocked swabs (FS) from the vagina and the endocervix (cervix) and by CVL using Normosol-R, saline, or water. Anti-E. coli activity was calculated as the % inhibition of bacterial growth compared to an untreated control. Anti-HIV-1 activity was calculated as the % inhibition of infection as compared to an untreated control. CVL/water was not tested (nt) because water would lyse the pathogens or cells providing non-reportable results. Negative values reflect increased E. coli growth or enhanced HIV-1 infection. Data are presented as box and whisker plots where the median is the horizontal line through the vertical box which represents the 25–75^th percentiles. Values within the 10–90^th percentiles are represented by the error bars. Outliers are shown by filled circles. Significant changes were determined using linear mixed model and discussed in the results section.