Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence<i>in situ</i>hybridization method

Machado, Antonio Liles Tavares; Castro, Joana; Cereija, T.B.; Almeida, Carina; Cerca, Nuno

doi:10.7717/peerj.780

Cited by 25 publications

(16 citation statements)

References 47 publications

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“…Gard162 is the first PNA FISH probe designed specifically for G . vaginalis and has extensively been tested by Machado et al on a variety of cultured bacterial strains and clinical samples [ 30 , 31 ]. Using this probe, we obtained clear hybridization for all G .…”

Section: Discussionmentioning

confidence: 99%

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Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

et al. 2015

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Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

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Section: Discussionmentioning

confidence: 99%

“…For the detection of G . vaginalis , a previously described probe, Gard162 [ 30 , 31 ], was used. The broad-range bacterial probe, BacUni-1, previously designed [ 32 ] as a modified version of the broad-range eubacterial DNA probe [ 33 ], was used as a positive control.…”

Section: Methodsmentioning

confidence: 99%

Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

et al. 2015

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“…Then based on the criteria for BV assessment developed by Nugent et al 30 , participants with the Gram stain score of ≥7 were finally confirmed as BV ( Supplementary Table S3 ). We also probed the samples with a novel PNA-FISH probe against G. vaginalis 31 .…”

Section: Methodsmentioning

confidence: 99%

Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Castro

Alves

Sousa

et al. 2015

Sci Rep

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Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development.

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“…However, the great diversity of vaginal microbiota in women with BV affects its potential to develop preterm birth and other negative outcomes in pregnancy ( Hyman et al, 2014 ; Nelson et al, 2015 ) and it becomes BV treatment in pregnant women a challenging task ( Haahr et al, 2016 ). Despite of its high impact, few epidemiological studies on BV have been conducted in Portugal ( Guerreiro, Gigante & Teles, 1998 ; Silva et al, 2014 ; Machado et al, 2015 ). Specifically, there is no information related to the prevalence of BV among Portuguese pregnant women.…”

Section: Introductionmentioning

confidence: 99%

Prevalence of bacterial vaginosis in Portuguese pregnant women and vaginal colonization byGardnerella vaginalis

Machado

Castro

Martinez-de-Oliveira

et al. 2017

PeerJ

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BackgroundWe aimed to determine the prevalence of vaginal colonization by Gardnerella vaginalis and of bacterial vaginosis (BV) in Portuguese pregnant women, and to identify risk factors for BV and G. vaginalis colonization in pregnancy.MethodsA cross-sectional study was conducted among pregnant women aged ≥ 18 years who were attending in two public hospitals of the Northwest region of Portugal. Epidemiological data was collected by anonymous questionnaire. BV was diagnosed by Nugent criteria and G. vaginalis presence was identified by polymerase chain reaction. Crude associations between the study variables and BV or G. vaginalis colonization were quantified by odds ratios (ORs) and their 95% confidence intervals (CIs).ResultsThe prevalences of BV and of G. vaginalis colonization among Portuguese pregnant women were 3.88% and 67.48%, respectively. Previous preterm delivery and colonization by G. vaginalis were factors with very high OR, but only statistically significant for a 90% CI. Conversely, higher rates of G. vaginalis colonization were found in women with basic educational level (OR = 2.77, 95% CI [1.33–5.78]), during the second trimester of pregnancy (OR = 6.12, 95% CI [1.80–20.85]) and with BV flora (OR = 8.73, 95% CI [0.50–153.60]).DiscussionDespite the lower number of women with BV, prevalence ratios and association with risk factors were similar to recent European studies. However, the percentage of healthy women colonized by G. vaginalis was significantly higher than many previous studies, confirming that G. vaginalis colonization does not always lead to BV development.

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Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescencein situhybridization method

Cited by 25 publications

References 47 publications

Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Prevalence of bacterial vaginosis in Portuguese pregnant women and vaginal colonization byGardnerella vaginalis

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