Rapid diagnosis of bacterial vaginosis by terminal restriction fragment length polymorphism profiling
Abstract number: o169
Thies F., König W., König B.
Bacterial vaginosis (BV) is an ill-defined disorder of the vaginal microflora associated with impaired health conditions of women and, possibly, of fetuses and newborns. As cultivation is of limited value, the diagnosis of BV is currently based on microscopical techniques (e.g., Nugent's Gram stain criteria). Molecular identification methods are supposed to provide more detailed insight into the bacterial composition of the disturbed vaginal flora. However, most techniques described so far, as sequencing of 16S rDNA clone libraries, are time-consuming and not suitable for routine purposes. Therefore, we adapted a culture-independent method for bacterial community analysis, called terminal restriction fragment length polymorphism (T-RFLP), to the needs of a routine microbiology laboratory.
Vaginal swabs were collected from 50 patients with BV and, as control, from 25 healthy subjects. Disease status was assessed using Nugent's criteria. We analysed the bacterial community directly from the swab specimens by means of the T-RFLP method. T-RFLP analysis is based on the restriction endonuclease digestion of fluorescently end-labelled PCR amplificates (derived from the 16S rRNA gene). For the identification of bacteria from T-RFLP raw data, we developed an in-house computer software.
Using T-RFLP, analysis of the vaginal microflora could be accomplished within 12 hours. In each BV sample, we found, on average, 6 to 8 typical fingerprints corresponding to definite bacterial species. Atopobium vaginae (95 % of all BV samples) and Gardnerella vaginalis (60 %) proved to be the predominant species, followed by Lactobacillus iners (50 %), Prevotella sp. (30 %) and Peptoniphilus sp. (15%). At least 3 different, and so far uncharacterized, bacteria from the Clostridiales order could be regularly detected, with one of them (a presumed Megasphaera sp.) at a frequency of 70 %. In healthy controls, only lactobacilli were detected.
Molecular tools are currently favoured for the analysis of the vaginal microflora. As such a tool, T-RFLP proved to be very promising for the rapid and detailed analysis of the bacterial communities inhabiting the vagina. This technique may be especially helpful in the context of clinical studies, when collecting reliable and extensive microbiological data may be crucial for the interpretation of study results.
|Session name:||XXIst ISTH Congress|
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