There was an overall greater nucleotide divergence in protease and reverse transcriptase of women's vaginal secretions compared with blood. The median DNA pairwise distances between clinic visits were significantly higher in vaginal secretions versus blood for all women tested and for those subgroups either with or without DRM or compartmentalized DRM (P < 0.01, Table 2). These higher divergence values in vaginal secretions were not significantly different between women with or without DRM or compartmentalized DRM (P ≥ 0.6, Table 2). In contrast, blood viral divergence was greater for women who had either DRM or compartmentalized DRM detected in blood (P < 0.05). There was no significant difference between the time intervals for collecting blood and vaginal secretions samples used for this pairwise analysis (Table 2).
Our results provide evidence that compartmentalized DRM can be infrequent and transient over time in antiretroviral-experienced women. There was no effect of reverse transcriptase vs. protease mutations on this transience suggesting that the class of drug does not alter this relationship. In addition, sequence analysis revealed an apparent greater divergence of the HIV pol gene over time in vaginal secretions than in blood. Thus, viral subpopulations in the genital tract are reflective of those in the blood over time, which supports the concept of an ongoing migration of infected cell populations from the blood to the genital tract [15–17]. This lack of independent evolution also supports the continuous exchange of viral genomes from the blood to the female genital tract and gives the appearance of greater genetic divergence in the genital tract.
In contrast to our longitudinal analysis, there are previous cross-sectional analyses that have reported more and less extensive occurrences of compartmentalized DRM between the blood and genital tract of infected women [4,6,9,11,18]. The disparate outcomes between these studies may, in part, reflect the differences in techniques used for sampling genital virus or the types of mutations used to define compartmentalization, differences in drug regimens used by participants, and their cross-sectional nature. Across studies, the highest levels of compartmentalization reported in previous studies used swabs to collect genital virus , whereas those with some of the lowest levels of compartmentalization used cervical-vaginal lavages [6,9]. Recent observations show that different HIV-1 quasispecies are more likely found at separate locations within the genital mucosa of an infected woman , suggesting that procedures which sample a large area of the mucosa, such as cervical-vaginal lavage, may produce a more accurate representation of the genital tract quasispecies.
The similarities we observed over time in DRM between virus shed in vaginal secretions and blood occurred while the major species of pol RNA shed in vaginal secretions appeared to be diverging at a faster rate. The apparent higher divergence in vaginal secretions we observed is likely a reflection of the lack of independent proviral evolution within genital tissues of women reported previously . Although there is sufficient evidence that local cellular virus replication in the female genital tract accounts for most HIV-1 shed in vaginal secretions , the majority of those infected genital tract cells replicating virus are believed to have migrated from blood and to have undergone clonal expansion in genital tissues [15–17]. Thus, an ongoing influx and turnover of a subpopulation of virus-infected cells from blood would result in the appearance of a higher level of viral RNA divergence in vaginal secretions between clinic visits. In addition, the increase of pol divergence over time in blood, but not in vaginal secretions, in the presence of DRM or compartmentalized DRM also supports the concept that a temporally linked evolution of HIV-1 is occurring in blood but not in vaginal secretions.
Our data are limited in that more contemporary samples would be expected to show different patterns of DRM. However, the mutations we report are reflective of those most commonly seen in studies of transmitted drug resistance [1–3]. We also did not measure drug concentrations in vaginal secretions or blood. Though previous data do report differences in drug penetration to the female genital tract , the effect on compartmentalized DRM has not been shown . Given the use of bulk sequencing employed in this analysis, which generally detects variants comprising more than 20% of the viral subpopulation, we are unable to make conclusions about the presence of minority DRM, which may be present [6,21]. Nonetheless, our longitudinal data suggest that more sensitive sequencing methodologies might show even less evidence of DRM compartmentalization and a more stochastic relationship between the blood and female genital tract. Finally, in general, the women in this study had high levels of virus shed in the vaginal secretions making inadequate PCR input copies an unlikely cause of the greater divergence we saw.
In summary, we have demonstrated the infrequent and transient nature of compartmentalized DRM over time in the female genital tract. Our results indicate that the majority of HIV-1 transmitted drug resistance, which occurs from exposure to a woman's genital secretions could be predicted from DRMs in her blood. Taken together, the pol divergence and DRM compartmentalization results reported in this study provide new evidence that the genital mucosa does not support an independently evolving subpopulation of HIV-1 genomes.
C.F.K. completed the analysis and writing of the manuscript. S.T.T. began the analysis and writing of the manuscript. J.L.L. developed the study and contributed to the writing of the manuscript. T.E.S. did the blood and vaginal secretion HIV viral loads. C.E.H. developed the study, supervised collection of the virus load and sequence data, and contributed to the analysis and writing of the manuscript.
The findings and conclusions of this manuscript are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
1. Novak RM, Chen L, MacArthur RD, Baxter JD, Huppler Hullsiek K, Peng G, et al
. Prevalence of antiretroviral drug resistance mutations in chronically HIV-infected, treatment-naive patients: implications for routine resistance screening before initiation of antiretroviral therapy. Clin Infect Dis 2005; 40:468–474.
2. Weinstock HS, Zaidi I, Heneine W, Bennett D, Garcia-Lerma JG, Douglas JM Jr, et al
. The epidemiology of antiretroviral drug resistance among drug-naive HIV-1-infected persons in 10 US cities. J Infect Dis 2004; 189:2174–2180.
3. Hurt CB, McCoy SI, Kuruc J, Nelson JA, Kerkau M, Fiscus S, et al
. Transmitted antiretroviral drug resistance among acute and recent HIV infections in North Carolina from 1998 to 2007. Antivir Ther 2009; 14:673–678.
4. De Pasquale MP, Leigh Brown AJ, Uvin SC, Allega-Ingersoll J, Caliendo AM, Sutton L, et al
. Differences in HIV-1 pol sequences from female genital tract and blood during antiretroviral therapy. J Acquir Immune Defic Syndr 2003; 34:37–44.
5. Ellerbrock TV, Lennox JL, Clancy KA, Schinazi RF, Wright TC, Pratt-Palmore M, et al
. Cellular replication of human immunodeficiency virus type 1 occurs in vaginal secretions. J Infect Dis 2001; 184:28–36.
6. Kemal KS, Burger H, Mayers D, Anastos K, Foley B, Kitchen C, et al
. HIV-1 drug resistance in variants from the female genital tract and plasma. J Infect Dis 2007; 195:535–545.
7. Kemal KS, Foley B, Burger H, Anastos K, Minkoff H, Kitchen C, et al
. HIV-1 in genital tract and plasma of women: compartmentalization of viral sequences, coreceptor usage, and glycosylation. Proc Natl Acad Sci U S A 2003; 100:12972–12977.
8. Philpott S, Burger H, Tsoukas C, Foley B, Anastos K, Kitchen C, Weiser B. Human immunodeficiency virus type 1 genomic RNA sequences in the female genital tract and blood: compartmentalization and intrapatient recombination. J Virol 2005; 79:353–363.
9. Si-Mohamed A, Kazatchkine MD, Heard I, Goujon C, Prazuck T, Aymard G, et al
. Selection of drug-resistant variants in the female genital tract of human immunodeficiency virus type 1-infected women receiving antiretroviral therapy. J Infect Dis 2000; 182:112–122.
10. Sullivan ST, Mandava U, Evans-Strickfaden T, Lennox JL, Ellerbrock TV, Hart CE. Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status. J Virol 2005; 79:9799–9809.
11. Tirado G, Jove G, Kumar R, Noel RJ, Reyes E, Sepulveda G, et al
. Differential virus evolution in blood and genital tract of HIV-infected females: evidence for the involvement of drug and non-drug resistance-associated mutations. Virology 2004; 324:577–586.
12. Diem K, Nickle DC, Motoshige A, Fox A, Ross S, Mullins JI, et al
. Male genital tract compartmentalization of human immunodeficiency virus type 1 (HIV). AIDS Res Hum Retroviruses 2008; 24:561–571.
13. Hart CE, Lennox JL, Pratt-Palmore M, Wright TC, Schinazi RF, Evans-Strickfaden T, 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.
14. Rhee SY, Gonzales MJ, Kantor R, Betts BJ, Ravela J, Shafer RW. Human immunodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res 2003; 31:298–303.
15. Bull ME, Learn GH, McElhone S, Hitti J, Lockhart D, Holte S, et al
. Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus. J Virol 2009; 83:6020–6028.
16. Poss M, Rodrigo AG, Gosink JJ, Learn GH, de Vange Panteleeff D, Martin HL Jr, et al
. Evolution of envelope sequences from the genital tract and peripheral blood of women infected with clade A human immunodeficiency virus type 1. J Virol 1998; 72:8240–8251.
17. Bull M, Learn G, Genowati I, McKernan J, Hitti J, Lockhart D, et al
. Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations. PLoS One 2009; 4:e7122.
18. Frenkel LM, McKernan J, Dinh PV, Goldman D, Hitti J, Watts DH, et al
. HIV type 1 zidovudine (ZDV) resistance in blood and uterine cervical secretions of pregnant women. AIDS Res Hum Retroviruses 2006; 22:870–873.
19. Min SS, Corbett AH, Rezk N, Cu-Uvin S, Fiscus SA, Petch L, et al
. Protease inhibitor and nonnucleoside reverse transcriptase inhibitor concentrations in the genital tract of HIV-1-infected women. J Acquir Immune Defic Syndr 2004; 37:1577–1580.
20. Kwara A, Delong A, Rezk N, Hogan J, Burtwell H, Chapman S, et al
. Antiretroviral drug concentrations and HIV RNA in the genital tract of HIV-infected women receiving long-term highly active antiretroviral therapy. Clin Infect Dis 2008; 46:719–725.
21. Johnson JA, Li JF, Wei X, Lipscomb J, Irlbeck D, Craig C, et al
. Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy. PLoS Med 2008; 5:e158.