title: 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.
authors.affiliation: Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America. cdezzutti@mwri.magee.edu
authors.forename: Charlene S
authors.initials: CS
authors.lastname: Dezzutti
authors.forename: Craig W
authors.initials: CW
authors.lastname: Hendrix
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: Marrazzo
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Pan
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Nicolette
authors.initials: N
authors.lastname: Louissaint
authors.forename: Sabah
authors.initials: S
authors.lastname: Kalyoussef
authors.forename: N Merna
authors.initials: NM
authors.lastname: Torres
authors.forename: Florian
authors.initials: F
authors.lastname: Hladik
authors.forename: Urvi
authors.initials: U
authors.lastname: Parikh
authors.forename: John
authors.initials: J
authors.lastname: Mellors
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Hillier
authors.forename: Betsy C
authors.initials: BC
authors.lastname: Herold
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D015415:Biomarkers / Q000032:analysis*; D002584:Cervix Uteri / Q000378:metabolism / Q000382:microbiology; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D005260:Female; D005836:Genitalia, Female / Q000378:metabolism*; D015658:HIV Infections / Q000175:diagnosis / Q000276:immunology / Q000517:prevention & control; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis / Q000276:immunology; D009092:Mucous Membrane / Q000378:metabolism*; D012307:Risk Factors; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000032:analysis / Q000276:immunology; D012995:Solubility; D013048:Specimen Handling / Q000379:methods*; D007507:Therapeutic Irrigation / Q000379:methods; D014621:Vagina / Q000378:metabolism / Q000382:microbiology; D014626:Vaginal Smears / Q000379:methods; D016585:Vaginosis, Bacterial / Q000276:immunology / Q000382:microbiology; D055815:Young Adult; D023084:alpha-Defensins / Q000032:analysis / Q000276:immunology
chemical_list: D015415:Biomarkers; D016207:Cytokines; D016209:Interleukin-8; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D023084:alpha-Defensins; C406413:human neutrophil peptide 1; C406416:human neutrophil peptide 2; C406427:human neutrophil peptide 3