Ann Lab Med.  2016 Sep;36(5):441-449. 10.3343/alm.2016.36.5.441.

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

• Hong KH
• Hong SK
• Cho SI
• Ra E
• Han KH
• Kang SB
• Kim EC
• Park SS
• Seong MW

• KMID: 2373574
• DOI: http://doi.org/10.3343/alm.2016.36.5.441

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.

Keyword

Vaginal microbiome; NGS; Vaginitis

MeSH Terms

Area Under Curve
Bacteria/*genetics/isolation & purification
Bacterial Proteins/genetics
Candida/*genetics/isolation & purification
Female
Fungal Proteins/genetics
Gardnerella vaginalis/genetics/isolation & purification
High-Throughput Nucleotide Sequencing
Humans
*Microbiota
RNA, Ribosomal, 16S/chemistry/genetics/metabolism
ROC Curve
Sequence Analysis, DNA
Trichomonas vaginalis/genetics/isolation & purification
Vagina/*microbiology
Vaginitis/*diagnosis/microbiology
Bacterial Proteins
Fungal Proteins
RNA, Ribosomal, 16S

Figure

• 

  Fig. 1 Composition of the microbiomes in the 89 specimens. Each line shows the taxonomy at the order level, and each column shows a single specimen. The colored bar below the tree indicates the Nugent score group of the 67 specimens with the Gram stain results. Green, normal group; Yellow, intermediate group; Red, vaginitis group; Gray, samples without Nugent score data.

• 

  Fig. 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.

• 

  Fig. 3 ROC curves of the 11 predictors of diversity and three vaginitis criteria. The ROC curves of the 11 predictors are shown according to (A) vaginitis criterion 1, (B) vaginitis criterion 2, and (C) vaginitis criterion 3.*P<0.05.(1) Vaginitis criteria Vaginitis criterion 1: vaginitis when the Nugent score≥4.Vaginitis criterion 2: vaginitis when the Nugent score≥7.Vaginitis criterion 3: vaginitis otherwise the culture results are either normal flora or Lactobacillus spp. (2) Predictors The specimen indicated vaginitis when the fraction of that taxon in total reads, including both the 16S rRNA and ITS genes, was more than zero: (a), 0.1% (b), 1% (c), and 5% (d).The specimen indicated vaginitis when the fraction of that taxon in total reads, including only the 16S rRNA gene, was more than zero: (e), 0.1% (f), 1% (g), and 5% (h).(i): The fraction of Lactobacillus spp. in the specimen.(j)-(k): Shannon diversity index of the specimen, when the index was calculated from both the 16S rRNA gene and the ITS gene (j) or was calculated from the 16S rRNA gene only (k). Abbreviations: AUC, area under the curve; ITS, internal transcribed spacer; rRNA, ribosomal RNA.

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