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      Comparison of the vaginal microbiota diversity of women with and without human papillomavirus infection: a cross-sectional study

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          Abstract

          Background

          The female genital tract is an important bacterial habitat of the human body, and vaginal microbiota plays a crucial role in vaginal health. The alteration of vaginal microbiota affects millions of women annually, and is associated with numerous adverse health outcomes, including human papillomavirus (HPV) infection. However, previous studies have primarily focused on the association between bacterial vaginosis and HPV infection. Little is known about the composition of vaginal microbial communities involved in HPV acquisition. The present study was performed to investigate whether HPV infection was associated with the diversity and composition of vaginal microbiota.

          Methods

          A total of 70 healthy women (32 HPV-negative and 38 HPV-positive) with normal cervical cytology were enrolled in this study. Culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis was used to measure the diversity and composition of vaginal microbiota of all subjects.

          Results

          We found significantly greater biological diversity in the vaginal microbiota of HPV-positive women ( p < 0.001). Lactobacillus, including L. gallinarum, L. iners and L. gasseri, was the predominant genus and was detected in all women. No significant difference between HPV-positive and HPV-negative women was found for the frequency of detection of L. gallinarum ( p = 0.775) or L. iners ( p = 0.717), but L. gasseri was found at a significantly higher frequency in HPV-positive women ( p = 0.005). Gardnerella vaginalis was also found at a significantly higher frequency in HPV-positive women ( p = 0.031). Dendrograms revealed that vaginal microbiota from the two groups had different profiles.

          Conclusions

          Our study is the first systematic evaluation of an association between vaginal microbiota and HPV infection, and we have demonstrated that compared with HPV-negative women, the bacterial diversity of HPV-positive women is more complex and the composition of vaginal microbiota is different.

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          Most cited references 48

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          16S ribosomal DNA amplification for phylogenetic study.

          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            Bias in template-to-product ratios in multitemplate PCR.

            Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.
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              Using ecological diversity measures with bacterial communities.

              Abstract There are many ecological diversity measures, but their suitability for use with highly diverse bacterial communities is unclear and seldom considered. We assessed a range of species richness and evenness/dominance indices, and the use of species abundance models using samples of bacteria from zinc-contaminated and control soils. Bacteria were assigned to operational taxonomic units (OTUs) using amplified ribosomal DNA restriction analysis of 236 clones from each soil. The reduced diversity apparent in the contaminated soil was reflected by the diversity indices to varying degrees. The number of clones analysed and the weighting given to rare vs. abundant OTUs are the most important considerations when selecting measures. Our preferences, arrived at using theory and practical experience, include: the log series index alpha; the Q statistic (but only if coverage is 50% or more); the Berger-Parker and Simpson's indices, although their ecological relevance may be limited; and, unexpectedly, the Shannon-Wiener and Shannon evenness indices, even though their meanings may not be clear and their values inaccurate when coverage is low. For extrapolation, the equation for the log series distribution seems the best for extrapolating from OTU accumulation curves while non-parametric methods, such as Chao 1, show promise for estimating total OTU richness. Due to a preponderance of single-occurrence OTUs, none of the five species abundance models fit the OTU abundance distribution of the control soil, but both the log and log normal models fit the less diverse contaminated soil. Species abundance models are useful, irrespective of coverage, because they address the whole distribution of a sample, aiding comparison by revealing overall trends as well as specific changes in particular abundance classes.
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                Author and article information

                Journal
                BMC Infect Dis
                BMC Infect. Dis
                BMC Infectious Diseases
                BioMed Central
                1471-2334
                2013
                10 June 2013
                : 13
                : 271
                Affiliations
                [1 ]Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Gynecologic Oncology, Peking University school of Oncology, Peking University Cancer Hospital and Institute, No 52, Fucheng Road, Haidian District, Beijing 100142, PR China
                [2 ]First Dental Clinic, Peking University School and Hospital of Stomatology, 37 Xishiku Dajie, Xicheng District, Beijing 100034, PR China
                [3 ]Department of Oral Biology, Peking University School and Hospital of Stomatology, No 22, Zhongguancun Nandajie, Haidian District, Beijing 100081, PR China
                Article
                1471-2334-13-271
                10.1186/1471-2334-13-271
                3684509
                23758857
                Copyright ©2013 Gao et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Research Article

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