The main goal of this study was to compare the diversity of the lower genital tract microbiota in HIV+ and HIV- women and is, to our knowledge, the first study to compare microbial diversity in these groups using culture-independent methods. Analysis of the sequences obtained by pyrosequencing showed that there was a trend (p=0.07) towards higher microbial diversity in HIV+BV+ when the number of taxa found at ≥1% of the microbiota in this group was compared with HIV-BV+. Additionally, three different taxa, Propionibacterineae, Anaerococcus and Citrobacter were found only in HIV+ women and this difference was statistically significant. While the total number of taxa found in HIV+BV+ women was higher than in HIV-BV+ women, this difference was not significant. Taken together, sequence comparisons showed higher microbial diversity in HIV+BV+ women compared to HIV-BV+. Sequence analysis did not show any differences between HIV+BV- women and HIV-BV- women. In contrast to the sequence analysis, LH-PCR did not distinguish any significant differences in microbial diversity between HIV+ and HIV- women.
Several studies have found that HIV-infected women with BV have higher levels of HIV in genital secretions [7
] and levels of specific bacteria have been associated with HIV levels [7
]. It is possible that changes in the diversity of microbiota associated with HIV infection could in turn be associated with changes in HIV expression in the genital tract. Changes in microbial diversity could also affect either severity or recurrance of BV which could in turn affect pregnancy in HIV-infected women.
While we found a trend towards higher microbial diversity in HIV+ women, an underlying cause for this association is not yet known. Since HIV infection suppresses immunity, it is possible that reduced immunity could play a role in the higher diversity. Candida vaginitis is more frequent and gynecologic diseases are more severe in HIV infected women suggesting that immunity in the lower genital tract that normally restricts growth of microbes is reduced in HIV-infected women [23
]. An influence of HIV on the incidence of BV was not previously noted [23
Another goal of this study was to determine the utility of molecular techniques, LH-PCR and pyrosequencing, for analyzing female genital microbiota. Our data confirmed numerous previous studies [1
] showing that the microbiota in BV+ women is much more diverse than in BV- women with lactobacilli constituting most of the bacteria in the latter. Thus, both pyrosequencing and LH-PCR can differentiate these clinically important conditions, while only pyrosequencing was capable of showing the more subtle differences between HIV+ and HIV-. LH-PCR has the additional limitation of not providing identification of the organisms. Pyrosequencing confirmed that BV microbiota can be highly variable between women. Thus, although some bacteria (e.g. Prevotella, Megasphaera, Gardnerella and Lachnospira) were found in essentially all BV+ women at ≥1% of microbiota, there were 11 taxa found in only one of the BV+ women at these levels and 8 taxa found in 2 women ().
While LH-PCR has been used to analyze diversity of intestinal microbiota [32
], there are no reports for genital microbiota. Recently, pyrosequencing was used to analyze genital microbiota in six samples pooled from HIV-negative pregnant women [33
]. As in our study, high levels of lactobacilli, Prevotella
, Bifidobacterium and Veillonella
were found although it was not clear how many individuals were pooled in samples. The gynecologic health of the women was not defined in contrast to our study and the read lengths of the amplified products averaged 100 bp; less than half the size of the median reads in our study (249). Longer read lengths and use of 16S rRNA Variable Region 1 and 2 in our study likely results in higher accuracy of identification.
A possible limitation of our study is a potential bias introduced during PCR amplification so that the proportion of reads may not represent the actual proportions of organisms in the women. However, both methods showed high levels of diversity in individuals with BV compared to no BV providing confirmation of utility. In conclusion, this study provides evidence that HIV infection is associated with increased diversity of the lower genital tract microbiota which could have pathogenic consequences for HIV sexual transmission.