NEW YORK (GenomeWeb) – A team led by researchers from the University of Bologna has published a study of a new assay they call VaginArray, a phylogenetic microarray-based tool that allows the qualitative and quantitative analysis of vaginal bacterial communities.
According to the results, published earlier this month in the journal Antimicrobial Agents and Chemotherapy, the array method also shows promise as a tool to assess the response of vaginosis patients to treatment by measuring changes in their microbiota.
Vaginosis, the most prevalent lower genital tract infection in reproductive-age women, is essentially a disruption of the normal microbial balance of the vagina and is characterized by reduced numbers of the protective Lactobacilli and greater abundance of anaerobic bacteria.
Standard treatment for bacterial vaginosis relies on antibiotics such as clindamycin and metronidazole, but relapse is common and poorly understood, according to the study's authors.
Rifaximin, a broad spectrum antibiotic, is a more recently proposed treatment, and using the VaginArray, the researchers were able to show in their study that this newer treatment indeed appears to reverse the microbial changes associated with the disorder.
According to Beatrice Vitali, corresponding author on the study, her group's approach for VaginArray — using a method called ligase detection reaction-universal microarray (LDR-UA) — is a continuation of sorts of previous work by some team members to develop the LDR-UA method itself and to test it initially as a tool for the phylogenetic analysis of other aspects of the human microbiome.
The method is based on the use of a so-called universal array, in which a set of oligonucleotides called ZipCodes, which are unrelated to any specific DNA sequence under study, are spotted in an array. By designing a set of probes linked to complementary ZipCodes (cZipCodes) and then using the ligase detection reaction, the resulting cZipCode-bearing products bind to the microarray where the corresponding ZipCode sequence has been spotted.
While several molecular methods have been developed for culture-independent investigation of the vaginal microbiome using various PCR techniques — for example, Beckton Dickinson has said it plans to release a vaginitis/vaginosis assay for its sample-to-answer BD Max platform in 2016 — Vitali said that most involve relatively high costs and/or long analysis times.
According to Vitali, the LDR-UA approach has a clear cost-effectiveness advantage over multiplex PCR techniques, at least when the number of targets exceeds five or six.
"LDR-UA doesn't require the optimization of PCR conditions on a number of different targets, since it is based on the molecular discrimination of the species according to their 16S rRNA gene. [All] target species can be amplified together using 'universal' PCR primers specifically designed to address conserved regions of the 16S," Vitali explained in an email.
"Other non-LDR-based array methods have indeed the possibility to detect more bacterial targets than the LDR-UA platform, due to the extreme miniaturization that can be reached with modern array production technologies," she wrote.
However, LDR-UA, because of its incorporation of a ligase-based enzymatic reaction, is very specific in correctly detecting even closely related bacterial species, for example "a single nucleotide difference between one species and another is enough for the LDR to discriminate among the two," Vitali said.
A specific LDR product is formed "only when there is a perfect identity between the probe pairs and the target," she explained.
In the adaptation of this approach to the vaginal microbiome, the Bologna researchers initially used reference sequences to develop a database of 16S rRNA gene sequences of selected bacterial groups of particular interest for the vaginal microbiome, and then used this to inform their probe design for LDR-UA.
According to the authors, because a central aim of the study was to characterize the Lactobacillus population at the level of most representative single species or groups, they included six probes targeting Lactobacillus species.
Also, in order to obtain a more complete view of the vaginal ecosystem in the context of vaginosis, the team included a number probes for species associated with the altered bacterial populations typical of bacterial vaginosis.
The team initially tested the method in a set of experiments to define the assay's sensitivity and specificity, as well as its limit of detection. The researchers then went on to study a set of 55 samples from 22 European post-menopausal, non-pregnant women from the treatment arm of a clinical study assessing the efficacy of rifaximin vaginal tablets as at treatment for bacterial vaginosis.
The researchers compared their VaginArray-based identification of target bacteria to Nugent score and Amsel's criteria, two clinical strategies used in the diagnosis of bacterial vaginosis.
They found that the array-based identification of the main Lactobacillus species associated with normal vaginal health was inversely correlated with both Nugent score and Amesel's criteria, while the detection of the major BV-related bacteria was directly correlated with both clinical BV diagnosis measures.
The team also investigated changes in patients' microbiota in response to rifaximin, seeing significant changes in several species. Variations revealed a remarkable drop in the numbers of almost all the BV-associated bacteria post-treatment, the authors reported, suggesting that the tool could be an accurate way to assess whether patients are responding to treatment with this, or other investigational drugs.
According to the study authors, the results also indicate that VaginArray could potentially be useful in a diagnostic context.
"For our group the VaginArray is primarily a tool for research studies on the vaginal ecology," Vitali wrote, although "clinical diagnosis could be a future hypothesis."
"As the probes have been published as well as the method of development of the tool, it is likely that other researchers can use a similar approach in their studies or use the same probes on other platforms," she said.