Although HIV can be recovered from the vagina of women who have had a total hysterectomy, the majority of genital virus probably arises from the cervix and possibly the upper genital tract1–3. The proximity to the vaginal lumen of cervical-stroma lymphocytes, which compose the genital-associated lymphoid tissue, may contribute both to the shedding of HIV from the genital tract of HIV-infected women and to the susceptibility to mucosal transmission of HIV infection in HIV-uninfected women. Moreover, there are important changes that occur in the histology of the lower female reproductive tract over the course of a woman's reproductive life that influence the choice of sampling methods used to evaluate HIV shedding4.
Unlike the measurement of virological and immunological parameters in peripheral blood, which are easily standardized, measurements of the same parameters in the female genital tract are complex. Sampling methods for the female genital tract traditionally involved cervical vaginal lavage with sterile saline or swabs of the cervix. More recently, sampling methods have utilized direct aspirates of cervical mucous5, cytobrush6–8, tampons9, swabs10,11 and Sno-strip® wicks8,11. The advantage of the wick is that it allows for the localization and collection of a defined volume of genital fluid from a specific anatomical location such as the vaginal fornix, endocervix or exocervix with little or no disruption of the epithelial surface. The moistened Weck-Cel® sponge has been utilized for sampling mucosal antibodies12,13. Like the Sno-strip®, the Weck-Cel® sponge is an ophthalmic fluid collection device that has been adapted for the collection of cervical secretions.
All sampling methods offer advantages and disadvantages. Lavage samples have the benefit of volume of sample and can be readily fractionated into cellular and cell-free components. Moreover, the lavage method can be performed easily in the clinical setting and yields a volume for virological or immunological analysis 400 times greater than that of either wick or sponge collection methods. The disadvantage of the lavage method is that the levels of virus and antibody will be diluted, and standardization of the volume is problematical. Volume standardization can be performed with the addition of 10 mM lithium to the washing buffer14,15. Although this procedure has been tested on spiked specimens, the method has not been routinely applied to clinical samples. Swabs collect both cellular and cell-free material, as do direct aspirates. In contrast, a cytobrush primarily collects cell-associated material (epithelial cells and inflammatory cells present on the mucosal surface), whereas the wick and the sponge primarily collect cell-free material. Direct aspirates have problems in volume standardization, similar to those for lavage.
The variability of the results obtained is a function of the sampling method, assay used and biological variation in the compartment sampled16. Variability in female genital tract measurement parameters has only been determined for the HIV-1-RNA level. Variability is least for wicks and greatest for lavage samples8. Variability is similar between the wick and cytobrush, and within-subject variability approximates ± 1.1 log10 RNA copies/ml (11-fold). This variation is greater than that for blood plasma HIV-1 RNA, which is two to sevenfold8,17,18. The wick is an optimum method for collecting a known volume of sample; however, the sample volume is quite small (∼8 μl per Sno-strip® wick), which limits the use of the specimen to one assay.
Because more than one sampling method may be used in complex studies, the order of sampling becomes important. Depending upon the assay used, sampling by one method may actually enhance sampling by another method19. In general, the approach to sampling has been to proceed from the least intrusive to the most intrusive. The detection and quantification of virus is greatest in the cervix and least in the vagina17.
This study was supported by HD 40540-02, cooperative agreement AI-27664 and the University of Washington Center for AIDS Research AI-27757.
1. Nuovo GJ, Forde A, MacConnell P, Fahrenwald R. In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. Am J Pathol 1993; 143:40–48.
2. Hocini H, Becquart P, Bouhlal H, et al
. Active and selective transcytosis of cell-free human immunodeficiency virus through a tight polarized monolayer of human endometrial cells. J Virol 2001; 75:5370–5374.
3. Farrar DJ, Cu Uvin S, Caliendo AM, et al
. Detection of HIV-1 RNA in vaginal secretions of HIV-1-seropositive women who have undergone hysterectomy. AIDS 1997; 11:1296–1297.
4. Coombs RW, Reichelderfer PS, Landay AL. Recent observations on human immunodeficiency virus (HIV) type-1 infection in the genital tract of men and women. AIDS 2003; 17:455–480.
5. Rasheed S. Infectivity and dynamics of HIV type 1 replication in the blood and reproductive tract of HIV type 1-infected women. AIDS Res Hum Retroviruses 1998; 14(Suppl. 1):S105–S118.
6. Iversen AK, Larsen AR, Jensen T, et al
. Distinct determinants of human immunodeficiency virus type 1 RNA and DNA loads in vaginal and cervical secretions. J Infect Dis 1998; 177:1214–1220.
7. Hart CE, Lennox JL, Pratt-Palmore M, et al
. Correlation of human immunodeficiency virus type 1 RNA levels in blood and the female genital tract. J Infect Dis 1999; 179:871–882.
8. Coombs RW, Wright DJ, Reichelderfer PS, et al
. Variation of human immunodeficiency virus type 1 viral RNA levels in the female genital tract: implications for applying measurements to individual women. J Infect Dis 2001; 184:1187–1191.
9. Webber MP, Schoenbaum EE, Farzadegan H, et al
. Tampons as a self-administered collection method for the detection and quantification of genital HIV-1. AIDS 2001; 15:1417–1420.
10. Mostad SB. Prevalence and correlates of HIV type 1 shedding in the female genital tract. AIDS Res Hum Retroviruses 1998; 14(Suppl. 1):S11–S15.
11. John GC, Sheppard H, Mbori-Ngacha D, et al
. Comparison of techniques for HIV-1 RNA detection and quantitation in cervicovaginal secretions. J Acquir Immune Defic Syndr 2001; 26:170–175.
12. Kozlowski PA, Lynch RM, Patterson RR, et al
. Modified wick method using Weck-Cel sponges for collection of human rectal secretions and analysis of mucosal HIV antibody. J Acquir Immune Defic Syndr 2000; 24:297–309.
13. Rohan LC, Edwards RP, Kelly LA, et al
. Optimization of the weck-Cel collection method for quantitation of cytokines in mucosal secretions. Clin Diagn Lab Immunol 2000; 7:45–48.
14. Belec L, Meillet D, Levy M, et al
. Dilution assessment of cervicovaginal secretions obtained by vaginal washing for immunological assays. Clin Diagn Lab Immunol 1995; 2:57–61.
15. Mohamed AS, Becquart P, Hocini H, et al
. Dilution assessment of cervicovaginal secretions collected by vaginal washing to evaluate mucosal shedding of free human immunodeficiency virus. Clin Diagn Lab Immunol 1997; 4:624–626.
16. Brambilla D, Reichelderfer PS, Bremer JW, et al
. The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements. The Women Infant Transmission Study Clinics. Virology Quality Assurance Program. AIDS 1999; 13:2269–2279.
17. Reichelderfer PS, Coombs RW, Wright DJ, et al
. Effect of menstrual cycle on HIV-1 levels in the peripheral blood and genital tract. WHS 001 Study Team. AIDS 2000; 14:2101–2107.
18. Villanueva JM, Ellerbrock TV, Lennox JL, et al
. The menstrual cycle does not affect human immunodeficiency virus type 1 levels in vaginal secretions. J Infect Dis 2002; 185:170–177.
19. Baron P, Bremer J, Wasserman SS, et al
. Detection and quantitation of human immunodeficiency virus type 1 in the female genital tract. The Division of AIDS Treatment Research Initiative 009 Study Group. J Clin Microbiol 2000; 38:3822–3824.