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Analysis of the Vaginal Microbiome by Next-Generation Sequencing and Evaluation of its Performance as a Clinical Diagnostic Tool in Vaginitis

View Article: PubMed Central - PubMed

ABSTRACT

Background: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis.

Methods: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis.

Results: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%).

Conclusions: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.

No MeSH data available.


Shannon diversity indices and the number of taxa (more than 5%) according to the Nugent score group. (A) The Shannon diversity indices according to the Nugent score group are shown in the form of Tukey's boxplots. The Shannon diversity index, including both bacteria and fungi, was calculated for each sample. In total, 67 Shannon diversity indices were classified into three groups according to the Nugent score of the specimen. (B) The total number of taxa (more than 5%) according to the Nugent score group are shown in the form of Tukey's boxplots.
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Figure 2: Shannon diversity indices and the number of taxa (more than 5%) according to the Nugent score group. (A) The Shannon diversity indices according to the Nugent score group are shown in the form of Tukey's boxplots. The Shannon diversity index, including both bacteria and fungi, was calculated for each sample. In total, 67 Shannon diversity indices were classified into three groups according to the Nugent score of the specimen. (B) The total number of taxa (more than 5%) according to the Nugent score group are shown in the form of Tukey's boxplots.

Mentions: The Shannon diversity index including the 16S rRNA and ITS showed significant associations with the groups based on the Nugent scores (P=0.0037, Fig. 2A). With Bonferroni correction, there was a significant association between the normal Nugent score group and the vaginitis Nugent score group (P=0.033). The number of taxa representing more than 5% of the reads significantly differed among the Nugent score groups (Fig. 2B, P=0.0163). With Bonferroni correction, there was a significant association between the normal Nugent score group and the vaginitis Nugent score group (P=0.004). A one-way ANOVA showed that the proportions of Lactobacillus spp. differed significantly among the Nugent score groups (P<0.0001), and the proportions did not increase in accordance with the Nugent score group. The mean proportions of Lactobacillus spp. in the normal, intermediate, and vaginitis groups were 0.827, 0.262, and 0.426, respectively.


Analysis of the Vaginal Microbiome by Next-Generation Sequencing and Evaluation of its Performance as a Clinical Diagnostic Tool in Vaginitis
Shannon diversity indices and the number of taxa (more than 5%) according to the Nugent score group. (A) The Shannon diversity indices according to the Nugent score group are shown in the form of Tukey's boxplots. The Shannon diversity index, including both bacteria and fungi, was calculated for each sample. In total, 67 Shannon diversity indices were classified into three groups according to the Nugent score of the specimen. (B) The total number of taxa (more than 5%) according to the Nugent score group are shown in the form of Tukey's boxplots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940487&req=5

Figure 2: Shannon diversity indices and the number of taxa (more than 5%) according to the Nugent score group. (A) The Shannon diversity indices according to the Nugent score group are shown in the form of Tukey's boxplots. The Shannon diversity index, including both bacteria and fungi, was calculated for each sample. In total, 67 Shannon diversity indices were classified into three groups according to the Nugent score of the specimen. (B) The total number of taxa (more than 5%) according to the Nugent score group are shown in the form of Tukey's boxplots.
Mentions: The Shannon diversity index including the 16S rRNA and ITS showed significant associations with the groups based on the Nugent scores (P=0.0037, Fig. 2A). With Bonferroni correction, there was a significant association between the normal Nugent score group and the vaginitis Nugent score group (P=0.033). The number of taxa representing more than 5% of the reads significantly differed among the Nugent score groups (Fig. 2B, P=0.0163). With Bonferroni correction, there was a significant association between the normal Nugent score group and the vaginitis Nugent score group (P=0.004). A one-way ANOVA showed that the proportions of Lactobacillus spp. differed significantly among the Nugent score groups (P<0.0001), and the proportions did not increase in accordance with the Nugent score group. The mean proportions of Lactobacillus spp. in the normal, intermediate, and vaginitis groups were 0.827, 0.262, and 0.426, respectively.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis.

Methods: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis.

Results: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%).

Conclusions: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.

No MeSH data available.