Background and aims. Bacterial vaginosis (BV) is one the more frequently identified genital syndrome among childbearing aged women. The basic condition that generates this condition is a modification in the vaginal microbiota. The aim of this paper is to briefly review the current status of the art of BV and to report the results of a pilot study performed with an innovative PCR based technique.
vaginae has been identified in 20 samples by the molecular approach and Lactobacillus spp. was identified in 19 samples (by culture) and in 32 by PCR. The overall diagnosis of BV was made in 9 patients by standard techniques and in 7 by applying the molecular approach. (Cohen\u2019s kappa test: 0,84). The findings of this study clearly demonstrate that the joint use of the routine cul- ture-based techniques with the multiplex PCR methods amplifies by far the sensitivity of the overall diagnostic workflow of BV

Congestr\uec, Francesco

Dalmo, Barbara

Dirani, Giorgio

Farabegoli, Patrizia

Pedna, Maria Federica

Sambri, Vittorio

Zannoli, Silvia

Background and aims. Bacterial vaginosis (BV) is one the more frequently identified genital syndrome among childbearing aged women. The basic condition that generates this condition is a modification in the vaginal microbiota. The aim of this paper is to briefly review the current status of the art of BV and to report the results of a pilot study performed with an innovative PCR based technique.
vaginae has been identified in 20 samples by the molecular approach and Lactobacillus spp. was identified in 19 samples (by culture) and in 32 by PCR. The overall diagnosis of BV was made in 9 patients by standard techniques and in 7 by applying the molecular approach. (Cohen’s kappa test: 0,84). The findings of this study clearly demonstrate that the joint use of the routine cul- ture-based techniques with the multiplex PCR methods amplifies by far the sensitivity of the overall diagnostic workflow of BV

Congestrì, Francesco

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

Summary Background and aims. Bacterial vaginosis (BV) is one themore frequently identified genital syndrome among childbearingaged women. The basic condition that generates this condition isa modification in the vaginal microbiota. The aim of this paper isto briefly review the current status of the art of BV and to reportthe results of a pilot study performed with an innovative PCRbased technique.Materials and Methods. 36 samples of vaginal fluid routinelysubmitted for the diagnosis of BV to the Unit of Microbiology –GRHL were comparatively evaluated by standard techniques andwith the HP-Vaginiti e Vaginosi NLM kit that simultaneouslydetects in a quantitative way specific DNA from Candida (albi-cans, glabrata; krusei, tropicalis), Gardnerella vaginalis,Lactobacillus spp. and Atopobium vaginae.Results and conclusions. Candida spp. has been identified in8 samples with culture and in 15 with the molecular test. 29 G.vaginalis were found by PCR whereas only in 7 samples a specificprescription for this microbe was present (of which 4 positive). A.vaginae has been identified in 20 samples by the molecularapproach and Lactobacillus spp. was identified in 19 samples (byculture) and in 32 by PCR. The overall diagnosis of BV was madein 9 patients by standard techniques and in 7 by applying themolecular approach. (Cohen’s kappa test: 0,84). The findings ofthis study clearly demonstrate that the joint use of the routine cul-ture-based techniques with the multiplex PCR methods amplifiesby far the sensitivity of the overall diagnostic workflow of BV.IntroductionBacterial vaginosis (BV) is among the most frequent microbi-ological syndromes in the population of women in their childbear-ing phase (1). The basic condition that is found among womensuffering from BV is a substantial change in their vaginal micro-biota: the commonly predominant Lactobacillus spp. gives way toa more complex and less characterized flora composed by severaldifferent bacterial species, including Gardnerella spp., Prevotellaspp. Atopobium spp., Peptostreptococcus spp., Mobiluncus spp.,Sneathia spp., Leptotrichia spp. and Mycoplasma spp. (14). Thisuncomfortable condition has been identified back in the 50s of thelast century and since the first reporting as bacterial vaginitis itwas considered as a member of the sexually transmissible diseases(STD) (4, 5).Nowadays, BV is generally considered as a form of dysbiosisand it is linked, even if BV is mostly an asymptomatic condition,to increased risk of adverse pregnancy outcome, pelvic inflamma-tory disease (PID) and to an amplified possibility to acquire otherSTD, such as Chlamydia trachomatis, Herpes virus, Trichomonasvaginalis and Neisseria gonorrhoeae (6). BV has also been associ-ated with an increased risk of HIV transmission and acquisition (2).Despite this long lasting knowledge of the syndromic pictureof BV, the precise reason for its onset and the large difference inits prevalence among female population in diverse geographicalareas is still one the most challenging questions in the field ofwomen health.BV is usually clinically diagnosed based on the identificationof three out of the four Amsel criteria (1): non inflammatory andhomogeneous vaginal discharge; microscopic identification ofclue cells; pH over 4.5 in the vaginal fluid and a positive whiff test(fish odor after the addition of 10% potassium hydroxide to thevaginal secretions). The application of another score system basedon the microscopic semi quantitation of the presence ofLactobacillus in a gram stained vaginal smear, the so calledNugent criteria (11), has been proven more problematic under thediagnostic point of view, since a large proportion of women thatshowed a positive Nugent score are totally asymptomatic, withlarge differences among diverse ethnic groups. One possibleCorrespondence: Vittorio Sambri, Unit of Microbiology, Centro ServiziAUSL della Romagna, Piazza della Liberazione 60, 47522 Pievesestina(FC), Italy. Tel.: +39.0547.394906.E-mail: vittorio.sambri@auslromagna.itKey words: Bacterial vaginosis, Gardenerella vaginalis, Lactobacillusspp., Atopobium vaginae, Molecular Diagnosis.Contributions: VS conceived the study and wrote the manuscript; GDand SZ performed the analytical step and checked the manuscript; PFsupervised the study and BD supervised the molecular analytical steps.MFP and FC made the microbiologic tests.Conflict of interest: the authors declare no potential conflict of interest.Received for publication: 15 February 2018.Accepted for publication: 15 February 2018.©Copyright G. Dirani et al., 2017Licensee PAGEPress, ItalyMicrobiologia Medica 2017; 32:7347doi:10.4081/mm.2017.7347This article is distributed under the terms of the Creative CommonsAttribution Noncommercial License (by-nc 4.0) which permits any non-commercial use, distribution, and reproduction in any medium, provid-ed the original author(s) and source are credited.Bacterial vaginosis: epidemiologic, clinical and diagnostic updatesGiorgio Dirani,1 Silvia Zannoli,1 Maria Federica Pedna,1 Francesco Congestrì,1 Patrizia Farabegoli,1Barbara Dalmo,1 Vittorio Sambri1,21Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina (FC), 2DIMES, University of Bologna,Bologna, Italy[page 180]                                                        [Microbiologia Medica 2017; 32:7347]                                                                              Microbiologia Medica 2017; volume 32:7347Non-commercial use onlyexplanation of this rather imprecise score system could be found inthe difficulty to correctly identify diverse bacteria based on theirmicroscopic characteristics, as shown in a recent study bySrinivasan and co-workers (16).Etiology and epidemiologyMost of the improvements achieved in the last 10 years in thefield of BV have been made possible by the use of culture-indepen-dent molecular techniques. In particular, the approach based on theuse of pyrosequencing and the subsequent phylogenetic analysishas shown that the vaginal microbiota (VMB) is by far more com-plicated and includes a large variety of different fungal and bacte-rial species. As a demonstration of the power held by the molecularapproach to the investigation of BV, it is of note than in 2005, threenovel and not previously identified bacteria were found in vaginalfluids of women with BV by using PCR and FISH. These novelmicrobes, identified as BVAB1 to BVAB3 showed a 16S rRNAgene sequence not related to any other: nowadays the BVABs arephylogenetically located into the phylum Clostridium and onlyBVAB3 has been recently grown in synthetic media in vitro.BVAB3 has been officially named as Mageeibacillus indolicus andit is an obligate anaerobe (14). In addition, this more powerfulapproach to the study of the VMB has shown that the microbialcommunity in the reproductive female tract is largely varyingdepending on reproductive age and sexual activity (6).A comprehensive evaluation of the diffusion and potential clin-ical differences of BV among diverse women population is stilllacking in literature. One of the most complete set of data has beenreviewed and published in 2013 by Kenyon and co-workers (5). Atfirst, it must be clarified that: BV is significantly associated withsexual contact with new and multiple male and female partnersand that decreasing the number of unprotected sexual intercoursemay reduce incident and recurrent infection. Other relevant riskfactors for the development of BV include: smoking, low socio-economic status, douching and recent use of antibiotic (14).Overall, the collection of epidemiologic data about BV is frequent-ly hampered by different issues such as the diverse criteria for theselection of the population and the correlated samples and thegroup of women included in the surveillance. Even among differ-ent population living in a defined geographical zone BV showedassorted prevalence ratios: in the USA, BV had highest prevalenceamong African American women in respect to Caucasian andAsian, being the prevalence of BV among Hispanic women inter-mediate (5). Moving forward to an analysis of prevalence based ondifferent Countries, the area showing the highest prevalence of BVin the Sub-Saharan African region included southern and easternparts. Moving into the Latina America (including the Caribbean)the epidemiology of BV showed intermediate level of prevalence,with the only exception of selected small groups in Jamaica andPeru with rates above 40%. As far as Western European epidemi-ology is concerned, the overall prevalence of BV is quite low: theCountries showing the higher (about 20%) prevalence are Turkey,Poland and Norway. The data about BV are quite scare and scat-tered from the Middle East and North African Countries: the high-est values (about 50%) are reported in small nomadic populationsliving in these areas. Overall the epidemiologic relevance of BV islow in North America (with the only exception of AfricanAmericans and aboriginal small population in Canada) as it is forwomen living in Asia and Australia/New Zealand.DiagnosisThe microscopic evaluation of a vaginal smear stained accord-ing to the Gram technique is by far the most widely used methodfor the laboratory diagnosis of BV. The above reported score sys-tem of Nugent could be used to evaluate and interpret the micro-scopic observation. When Nugent criteria are applied a score of 0to 3 corresponds to the presence of a normal vaginal flora whereasa score above 7 (up to 10) is correspondent to a defined BV. A pos-sible variant to the Nugent score system is the Ison score that islikely more effective in identifying the intermediate BV conditions(10) based on a more comprehensive clinical and laboratory eval-uation is the set of criteria proposed by Amsel, above detailed. In the last 24 months several reports described and validatedthe possible use of different molecular based approaches for thelaboratory diagnosis of B. (3, 7-9, 12, 13, 15). Most of the dataconfirm that a molecular based diagnosis could improve the over-all sensitivity and specificity of the laboratory diagnosis of BV.Some points still need to be elucidated: in principle since a totallyaccepted definition of the microbial etiology of BV is still fluctu-ating, additional assays that detect with alternative molecularmethodologies a panel of microbes putatively related to the devel-opment of BV are required.Here we report about the use of a multiplex PCR based test(HP-Vaginitis & Vaginosis kit - AU27117, Nuclear Laser Medicine- NLM, Settala, Italy) that identifies and quantifies the presence ofspecific DNA fragments from Candida (albicans, glabrata; krusei,tropicalis), G. vaginalis, A. vaginae e Lactobacillus spp. in vaginalfluid specimens. Materials and Methods A total of 36 vaginal fluid samples, obtained from women withage ranging from 19 to 47 years, routinely evaluated at the Unit ofMicrobiology of the Great Romagna Hub laboratory were includedin the study. For all of the patients the clinical picture suggested apossible diagnosis of BV according to the Amsel criteria. The stan-dard diagnostic workflow was performed and included the micro-scopic evaluation of the vaginal smear after Gram staining associ-ated with culture based techniques for the identification of G.vagi-nalis and Candida spp.. The HP-Vaginiti e Vaginosi (AU27117 -NLM) kit is based on two different steps: the first is a multiplextarget amplification that basically could enrich the targets contentof the sample while the second step is based on a nested reactionthat further amplifies and identifies the germs. As controls a humanderived DNA and an artificial sequence, that is used as referencefor the quantitation of the different targets thus allowing the auto-mated calculation of the final score, (SPIKE) are included so thatthe complete cycle of the reaction could be checked and moni-tored. The final and overall evaluation of the results is achieved bythe software MT-PCR Analysis that provides the user with an auto-mated score correlated with the different expression of the vaginalflora from intermediate alteration to BV. ResultsThe results obtained are summarized in Table 1. In detail,Candida spp. has been identified in 8 samples with culture and in15 with the molecular test (Cohen’s kappa test: 0,57). 29 G. vagi-nalis were found by PCR whereas only in 7 samples a specific pre-scription for this microbe was present (of which 4 positive). A.vaginae is not routinely identified but this bacterium has beenidentified in 20 samples by the molecular approach. Finally,Lactobacillus spp. was identified in 19 samples (by culture) and in32 by PCR (Cohen’s kappa test: 0,25). The overall diagnosis of BV                                      [Microbiologia Medica 2017; 32:7347]                                                      [page 181]                                                                                                                                 ArticleNo-cmercial use only[page 182]                                                        [Microbiologia Medica 2017; 32:7347]                                      was made in 9 patients by standard techniques and in 7 by applyingthe molecular approach. (Cohen’s kappa test: 0,84).Discussion and ConclusionsDespite the still wide use of culture and microscopic methodsfor the laboratory diagnosis of BV, the implementation of a molec-ular approach has become more and more common in the lastyears. Among the most important factors that prompted the enlarg-ing use of PCR based techniques for the diagnosis of BV, the firstplace is due to the technical improvement that nowadays allowsthe multiplex detection of several different genetic targets withinone reaction, thus perfectly fitting the syndromic character of BV.An additional relevant issue that contributed to the wider use ofPCR based techniques is the reduction of cost of these methodsstrictly linked with the availability of automated instrumentationthat consequently allows the simple and rapid processing of largenumber of samples.One should also not forget that in recent years a more preciseclinical and epidemiological definition of BV has been made avail-able thus enlarging the request for a more focused diagnostic work-flow. The findings of this study, even if obtained with quite a lim-ited set of patients clearly demonstrate that the joint use of the rou-tine culture based techniques with the multiplex PCR methodsamplifies by far the sensitivity of the overall diagnostic workflowof BV: in particular by using the HP-Vaginiti e Vaginosi NLM kitan extended number of samples positive for all the detectablegerms. In addition this technique has also allowed the identifica-tion of A.vaginae that is likely playing a relevant role in the patho-genesis of the condition of unbalanced microbial flora behind BV.In particular this molecular assay allows an accurate and specificmeasurement of marker bacterial specie with a comparative quan-tification of these microbes. This feature allows the calculation ofa ratio, similar to the one used for the Nugent score. Additional cal-culation of the human cell number and estimation of the ratiobetween human cells and anaerobic bacteria is used as a molecularsurrogate for the microscopic clue cells detection.References1. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis.Diagnostic criteria and microbial and epidemiologic associa-tions. Am J Med 1983;74:14-22. 2. Cohen CR, Lingappa JR, Baeten JM, et al. Bacterial vaginosisassociated with increased risk of female-to-male HIV-1 trans-mission: a prospective cohort analysis among African couples.PLoS Med 2012;9:e1001251.3. Dols JA, Molenaar D, van der Helm JJ, et al. Molecular assess-ment of bacterial vaginosis by Lactobacillus abundance andspecies diversity. BMC Infect Dis 2016;16:180. 4. Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis: anewly defined specific infection previously classified non-spe-cific vaginitis Am J Obstet Gynecol 1955;69:962-76.5. Kenyon C, Colebunders R, Crucitti T. The global epidemiolo-gy of bacterial vaginosis: a systematic review. Am J ObstetGynecol 2013; 209:505-23.6. Kenyon CR, Osbak K. Recent progress in understanding theepidemiology of bacterial vaginosis. Curr Opin ObstetGynecol 2014;26:448-54. 7. Hilbert DW, Smith WL, Chadwick SG, et al. Development andvalidation of a highly accurate quantitative real-time PCRassay for diagnosis of bacterial vaginosis. J Clin Microbiol2016;54:1017-24. 8. Hilbert DW, Smith WL, Paulish-Miller TE, et al. Utilization ofmolecular methods to identify prognostic markers for recurrentbacterial vaginosis. Diagn Microbiol Infect Dis 2016;86:231-42. 9. Machado A, Cerca N. Multiplex peptide nucleic acid fluores-cence in situ hybridization (PNA-FISH) for diagnosis of bac-terial vaginosis. Methods Mol Biol 2017;1616:209-19. 10. Mashburn J. Vaginal infections update. Midw Wom Health2012;57:629-34.11. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosingbacterial vaginosis is improved by a standardized method ofgram stain interpretation. J Clin Microbiol 1999;29:297-301. 12. Pandya S, Ravi K, Srinivas V, et al. Comparison of culture-dependent and culture-independent molecular methods forcharacterisation of vaginal microflora. J Med Microbiol2016;66:149-53. 13. Plummer EL, Garland SM, Bradshaw CS, et al. Moleculardiagnosis of bacterial vaginosis: Does adjustment for total bac-terial load or human cellular content improve diagnostic per-formance? J Microbiol Methods 2017;133:66-8. 14. Onderdonk AB, Delaney ML, Fichorova RN. The humanmicrobiome during bacterial vaginosis. Clin Microbiol Rev2016;29:223-38. 15. Rumyantseva T, Shipitsyna E, Guschin A, et al. Evaluation andsubsequent optimizations of the quantitative AmpliSensFlorocenosis/Bacterial vaginosis-FRT multiplex real-timePCR assay for diagnosis of bacterial vaginosis. APMIS2016;124:1099-108.16. Srinivasan S, Morgan MT, Liu C, et al. More than meets theeye: associations of vaginal bacteria with gram stain morpho-types using molecular phylogenetic analysis. PLoS One2013;8:e78633.                                ArticleTable 1. Comparative summary of the results obtained by standard culture based methods and the HP-Vaginiti e Vaginosi NLM kit.Microbe                                                  Culture/microscopy, n. positive samples        HP-Vaginiti e Vaginosi NLM kit, n. positive samplesCandida (albicans, glabrata; krusei, tropicalis)                                     8                                                                                                           15Gardnerella vaginalis                                                                                     4                                                                                                           29Atopobium vaginae                                                                                       ND                                                                                                         20Lactobacillus spp.                                                                                          19                                                                                                          32Non-commercial use only

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Bacterial vaginosis: epidemiologic, clinical and diagnostic updates

Background and aims. Bacterial vaginosis (BV) is one the more frequently identified genital syndrome among childbearing aged women. The basic condition that generates this condition is a modification in the vaginal microbiota. The aim of this paper is to briefly review the current status of the art of BV and to report the results of a pilot study performed with an innovative PCR based technique. 
Materials and Methods. 36 samples of vaginal fluid routinely submitted for the diagnosis of BV to the Unit of Microbiology – GRHL were comparatively evaluated by standard techniques and with the HP-Vaginiti e Vaginosi NLM kit that simultaneously detects in a quantitative way specific DNA from Candida (albicans, glabrata; krusei, tropicalis), Gardnerella vaginalis, Lactobacillus spp. and Atopobium vaginae. 
Results and conclusions. Candida spp. has been identified in 8 samples with culture and in 15 with the molecular test. 29 G. vaginalis were found by PCR whereas only in 7 samples a specific prescription for this microbe was present (of which 4 positive). A. vaginae has been identified in 20 samples by the molecular approach and Lactobacillus spp. was identified in 19 samples (by culture) and in 32 by PCR. The overall diagnosis of BV was made in 9 patients by standard techniques and in 7 by applying the molecular approach. (Cohen’s kappa test: 0,84). The findings of this study clearly demonstrate that the joint use of the routine culture- based techniques with the multiplex PCR methods amplifies by far the sensitivity of the overall diagnostic workflow of BV

Bacterial vaginosis: epidemiologic, clinical and diagnostic updates

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