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Tuesday, January 14, 2003: TOPIC V: HORMONAL INFLUENCE ON CHANGES IN GENITAL TRACT VIROLOGICAL/IMMUNOLOGICAL PARAMETERS AND IMPLICATIONS FOR TRANSMISSION

Overview on Source of the Virus, Viral Measurement and Variablility

Coombs, Robert W

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JAIDS Journal of Acquired Immune Deficiency Syndromes: March 2005 - Volume 38 - Issue - p S31-S32
doi: 10.1097/01.qai.0000167038.50558.69

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.

Support

This study was supported by HD 40540-02, cooperative agreement AI-27664 and the University of Washington Center for AIDS Research AI-27757.

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