Patent Application: US-31271107-A

Abstract:
the present invention relates to a method for in vitro diagnosis and follow - up of vaginal bacterial flora status in relation to the presence of bacterial vaginosis , and for monitoring its treatment where applicable , wherein the presence of bacterial vaginosis or failure of on - going treatment is determined if the concentrations of specific sequences present in a single copy in the dna of the bacteria atopobium vaginae and gardnerella vaginalis in the dna extracted from a vaginal discharge specimen from a patient are such that at least one of the following two conditions a ) and b ) are met : a ) the concentration ca of said dna fragment of atopobium vaginae is greater than or equal to 10 8 copies / ml , and b ) the concentration cg of said dna fragment of gardnerella vaginalis is greater than or equal to 10 9 copies / ml .

Description:
the pregnant women whose pregnancies were followed were recruited at the la conception hospital in marseille . an informed consent was the pre - condition for inclusion . the specimens were taken from the posterior vaginal fornix with an unlubricated sterile speculum without antiseptic . four samples were taken from each woman : two samples by a cotton swab placed in a dry tube ( copan innovation ®, brescia , italy ) and two samples by cytobrush ( scrinet ® 5 . 5 mm , c . c . d . international , paris , france ). one standard cotton swab was used fresh for bacterial culturing . a second swab was placed in a specific transport medium ( r1 urea - arginine lyo 2 , biomerieux sa , marcy l &# 39 ; etoile , france ) to look for genital mycoplasmas ( m . hominis and m . urealyticum ). one cytobrush was used for smearing on a slide and gram staining . a second cytobrush to extract the dna for quantification by molecular amplification was carried in 500 μl of mem transport medium ( minimum essential medium , invitrogen life technologies , carlsbad , calif ., usa ). it was frozen at − 80 ° c . from the time it arrived in the laboratory to the time it was used . the trichomonas vaginalis investigation was done by examination while fresh between the slide and cover slip under the optical microscope ( 10 × objective ). after smearing the specimen onto a glass slide and drying , gram staining was comprised of : staining with oxalate crystal violet ( 1 minute ), staining with lugol &# 39 ; s solution ( 1 minute ), decoloration with alcohol / acetone and coloration with safranin in solution ( biomerieux ). each step was followed by rinsing in water . gram staining enables the nugent score to be established by a semi - quantitative evaluation of three bacterial morphotypes ( table 1 ). according to the score , three vaginal flora are identified : normal flora ( nf ) for a score of 0 to 3 , intermediate flora ( if ) for a score of 4 to 6 , and bv for a score of over 7 . the vaginal specimens were seeded onto three culture media : columbia anc agar plus 5 % sheep &# 39 ; s blood ( biomérieux ), chocolate poly vitex pvx agar ( biomerieux ), choc vcat agar ( biomérieux ) incubated at 37 ° c . for 48 hours . to detect mycoplasmas , the specimens were seeded onto a specific kit ( urea - arginine lyo 2 , biomérieux ) then incubated at 37 ° c . and inoculated into anaerobic culture media ( biomerieux ) for 48 hours . dna extraction was done with the qiamp ® dna mini kit ( qiagen ®, courtaboeuf , france ). the protocol was modified as follows : incubation for 12 hours at 56 ° c . of 200 μl of sample per 200 μl of lysis buffer and 20 μl of proteinase k . the lysate was treated according to the manufacturer &# 39 ; s recommendations . the dna was eluted in 100 μl of elution buffer then stored at − 20 ° c . analysis of the data in the literature and the sequences deposited at the genbank site gave information on the sequences available for each of the target microorganisms . the specificity of the probes and fragments of target sequences of each chosen microorganism were tested for specificity at the nbci website . the inventors chose the targets from all the potential vaginosis agents , including agents whose role was uncertain . surprisingly , the most significant target atopobium vaginae was not considered to be an essential agent . the selected targets were located on the gene sequence coding for the 60 kda chaperone protein ( cpn60 ) for g . vaginalis and m . curtisii , on that of 16s rna for m . mulieris and a . vaginae , the fts y sequence for m . hominis , the urease sequence for u . urealyticum , and the topoisomerase iii gene for c . albicans . for the lactobacillus sp ., target , a sequence common to : lactobacillus crispatus , lactobacillus jensenii , lactobacillus gasseri , lactobacillus iners and lactobacillus acidophilus was chosen ; it is located on the gene coding for the elongation factor tu ( tuf ). a sequence located in exon 12 of the human albumin gene was chosen to attest for the presence and quantity of dna in the test specimen . for each microorganism studied ( lactobacillus sp ., g . vaginalis , m . curtisii , m . mulieris , u . urealyticum , m . hominis , a . vaginae , c . albicans ) and human albumin , a probe , and a sense plus anti - sense primer pair were chosen from the target sequences defined above using the primer 3 ® program . the primers and probes are listed below . each primer and probe were analyzed at the nbci website to ensure their specificity in silico . in the listing below , the “ sense primer ” and “ probe ” sequences are written in the 5 ′→ 3 ′ direction , and the “ anti - sense primer ” sequences are written in the reverse complementary direction 5 ′→ 3 ′. molecular target and nucleotide sequence of the primer pair and probe for each microorganism studied . in order to perform real - time dual recognition pcrs ( duplex pcrs ), recognition pairs of microorganisms were chosen arbitrarily : one of the probes was labeled fam and the other , vic . four pcr pairs were defined in this way . lactobacillus sp . ( fam ) and g . vaginalis ( vic ) m . curtisii ( fam ) and m . mulieris ( vic ) u . urealyticum ( vic ) and human albumin ( fam ) a . vaginae ( vic ) and c . albicans ( fam ) mycoplasma hominis ( fam ) were to be quantified alone . the primers were synthesized by eurogentec ® ( seraing , belgium ), and the probes , by applied biosystems ( warrington , cheshire , uk ). to test the specificity of the primers and probes , the dna extracted from the reference bacteria strains ( l . acidophilus cip 104464 , a . vaginae cip 106431 , g . vaginalis cip 103660 , m . curtisii subsp . holmesii atcc 35242 , m . mulieris atcc 35239 , c . albicans umip 1180 . 79 , u . urealyticum cip 103755 and m . hominis cip 103715 ) was used in three dilutions ( 1 : 10 , 1 : 100 , and 1 : 1000 ) for developing real - time pcrs ( determination of relative primer and probe quantities , specificities , cross reactivities ). each strain was tested with the primers and the specific probe , but also with the probes and primers of the 7 other microorganisms and those of human albumin . the ct ( cycle threshold ) values were measured . the ct is the intersection point between the baseline of the reaction and the logarithmic curve representing the amplification . this ct value corresponds to the number of amplification cycles necessary for amplification to begin . it is related to the concentration of the nucleic product to be quantified : the higher the concentration , the lower the ct . the quantities of primers and probes necessary for obtaining optimal amplification with single and double fluorescence are defined by testing the amplification reaction on a mixture of the bacterial dna from the 8 reference strains under equimolar conditions , as well as with pure strains , for a final concentration of 1 : 10 in both cases . the experimental conditions used were those for which the ct values were identical to those obtained with single fluorescence . the genomic dna samples from 40 different bacterial strains ( appendix ) excluding the 8 reference bacterial strains were tested in a 1 : 10 dilution . care was taken with all these tests to introduce , with each amplification reaction , negative controls containing no dna or positive controls ( mixing of the dna from the 8 reference strains ). specificity was tested with the four pcr pairs in real time as well as mycoplasma hominis . the preparation method described in fr 2 882 063 was used . the nucleotide sequence of the quantification hybrid fragment was obtained by aligning the pcr target sequences in real time for the 8 microorganisms as well as for those of human albumin . we obtained a hybrid fragment with 931 base pairs , described below . boldfaced sequences = sequences of sense and anti - sense primers underlined sequences = specific probe sequences black sequences = intercalary sequences sequences in parentheses = sequences of xhol restriction site the hybrid nucleotide fragment sequence was divided into the following 6 consecutive oligonucleotide fragments . ( 1 ) sense oligonucleotide 1 , 178 nucleotides : m . curtisii and m . mulieris sequences ( 2 ) anti - sense oligonucleotide 2 , 183 nucleotides : g . vaginalis and lactobacillus sp . ( 5 ) sense oligonucleotide 5 , 152 nucleotides : a . vaginae and c . albicans to ensure continuity of adjacent oligonucleotides , 10 additional nucleotides at the 3 ′ end on the upstream oligonucleotide were added to the 5 ′ end of the downstream oligonucleotide . the sizes of the 6 oligonucleotide sequences ranged from 155 to 172 nucleotides . the consecutive oligonucleotides were alternatively in forward and reverse sequence . thus , oligonucleotides 1 , 3 , and 5 were used in the forward sequencing form while oligonucleotides 2 , 4 , and 6 were used in the reverse sequencing form . sense and anti - sense “ construction ” primers ( below ), whose sequences corresponded to those of the 20 nucleotides at 5 ′ and 3 ′ of the quantification hybrid fragment , were to be synthesized . construction primers for recombinant plasmid with the fragment of 939 base pairs the double - stranded nucleotide fragment was constructed by an amplification reaction thanks to the complementarity of the ends of two adjacent oligonucleotides . two successive pcrs were necessary . this allowed hybridization of the oligonucleotides by their ends and partial elongation when this is compatible with activity of the taq polymerase . the 6 oligonucleotides were introduced under equimolar conditions ( 0 . 2 mmol ), with polymerase buffer 1 × mgcl 2 ( 1 . 5 mmol ), dntp ( 0 . 2 mmol ), 0 . 2 μl of taq roche ( roche ®), 5 iu / μl , in a reaction volume of the amplification program was : 95 ° c . 2 min , followed by 40 cycles comprising 94 ° c . 30 sec ( denaturation ), 37 ° c . 1 min ( hybridization ), 72 ° c . 1 min 30 sec ( elongation ). this allows a pcr fragment containing the expected oligonucleotide groups end - to - end to be obtained ( fig5 ). one μl of amplification product from the first pcr was added to the following reaction mixture : polymerase buffer hotstar ( qiagen ®) 1 × mgcl 2 ( 1 . 5 mmol ), dntp ( 0 . 2 mmol ), 0 . 2 μl of hotstar ( qiagen ®) with 5 iu / μl , and 0 . 2 mmol of sense and anti - sense construction primers . the pcr program was : 95 ° c . 15 min , followed by 95 ° c . 30 sec , 58 ° c . 45 sec , 72 ° c . 2 min 40 cycles , 72 ° c . 5 min . the pcr fragment obtained was analyzed on 1 . 5 % b et agarose gel in tbe buffer 0 . 5 ×. if the size of the fragment was the expected size , it was purified using the qiaquick ® pcr purification kit 250 pcr qiakit ( qiagen ®). two μl of the fragment obtained previously were introduced into a ligation reaction mixture containing 5 μl of ligase buffer , 1 μl of ligase , and 1 μl of linearized , dephosphorylated plasmid pgem ( kit pgem ®- t easy vector system 2 promega ®, madison , wis ., usa ). the final volume was 10 μl . the ligation product was incubated at 15 ° c . overnight . seven μl of the ligation product were mixed with 50 μl of competent cells ( escherichia coli jm 109 ) kept in ice for 20 minutes then incubated for 1 minute at 42 ° c . after addition of 950 μl of lb broth ( usb ®, cleveland , ohio , usa ), and incubation for 1 h 30 min at 37 ° c ., 500 and 250 μl of this culture medium were smeared on 2 petri dishes containing lb agar ( usb ®) with 100 μg / ml of ampicillin . the dishes were incubated overnight at 37 ° c . the recombinant colonies were deposited both in 50 μl of sterile distilled water and on an lb agar ampicillin petri dish . the recombinant e . coli colonies were sampled for pcr analysis : 5 μl of bacterial suspension in distilled water , the pair of m13 primers ( 10 pm / μl ), and the previously described pcr reaction medium . the pcr program was identical to that used for the second construction step . the pcr products obtained were analyzed in 1 . 5 % gel agarose in tbe buffer 0 . 5 ×. the fragments of the expected size were purified using the qiaquick ® pcr purification kit 250 qiakit ( qiagen ®). they were then sequenced using big dye ® terminator v1 . 1 cycle sequencing kit ( applied biosystems ®, warrington , uk ), 3 . 2 pmol / μl of sense and anti - sense primers chromatographed on the abi prim 3100 sequencer ( applied biosystems ®). the sequence obtained was compared to the sequence of the expected fragment using the auto - assembler and sequence navigator ( applied biosystems ®) programs . one of the recombinant clones , conforming to the expected sequence , was to be produced in large amounts in 100 ml of lb broth ampicillin and purified according to the high speed plasmid midi kit ( qiagen ®) protocol . the purified plasmid was then stored at − 20 ° c . the selected recombinant strain was frozen at − 80 ° c . its concentration was determined by measuring the optical density at 260 nm . for example , let 0 . 38 be for the initial solution . 1 unit od corresponds to 50 μl / ml : 0 . 38 × 50 = 19 μg / ml = 19 × 10 − 6 g / ml of plasmid in the initial solution . plasmid size = size of pgem ®- t easy + fragment size = 3015 + 931 = 4334 bp , or 8668 bases . the molar mass of one nucleotide is 330 da ( g / mol ). the molar mass of the plasmid will be 8668 × 330 = 2 . 860 × 10 6 g / mol . the plasmid concentration will be in mol / ml , or 19 × 10 − 6 / 2 . 860 × 10 6 = 6 . 64 × 10 − 12 mol / ml . multiplied by avogadro &# 39 ; s number , we obtained the number of plasmid copies per 1 mml of solution , namely : 6 . 64 × 10 − 12 × 6 . 023 . 10 23 = 40 × 10 11 copies / ml or 2 × 10 10 copies / 5 μl . a first dilution of the initial 1 : 2500 plasmid solution enabled the concentration to be adjusted to the first step of the plasmid range , or 10 7 copies / 5 μl of plasmid solution . a dilution cascade in steps of 10 enabled successive steps of the range to be created ( of 10 7 copies to 1 copy per 5 μl of solution ). the quantification reactions were done by real - time pcr on dna extracts from vaginal specimens . the following were placed on each reaction plate : 4 negative controls ( ntc ), the calibration plasmid range ( 10 7 to 1 copy par well ), and 24 test specimens , pure and diluted to 1 : 10 and 1 : 100 , to look for inhibitors . the negative controls and the points on the plasmid range were tested in duplicate . for amplification and quantification of the 8 microorganisms and human albumin , 4 pcr plates were run with double fluorescence and one with single fluorescence . for preparation of the reaction mixture , the kit quantitect probe pcr kit ( qiagen ®) containing the 2 × reaction mixture combining the taq polymerase and taq polymerase buffer ( hotstar ), dntp , and dutp was used . to this reaction mixture were added the sense and anti - sense probes necessary for single or double fluorescence pcr according to the experimental conditions reported and described in appendix 6 , with the test specimens diluted or undiluted ; the points on the plasmid range were 5 μl and 0 . 25 μl of udg ( uracil dna glycosylase , 100 units , sigma - aldrich , lyon , france ) was added for a final reaction volume of 25 μl . the pcr reactions were run on the stratagene ® mx 3000p ( la jolla , calif .). the amplification program was the following : 2 minutes at 50 ° c ., 15 minutes at 95 ° c ., followed by 45 pcr cycles consisting of denaturation at 95 ° c . for 30 seconds then the hybridization and elongation phase at 60 ° c . for 1 minute . for each vaginal specimen and each microorganism , in order to ensure comparability of the specimens , the bacterial load was defined as follows . the quantification in number of dna copies from each microorganism per 5 μl of dna extract was reported as the number of bacteria per 1 ml of initial specimen . the dna elution volume ( 100 μl ), the volume of the transport medium per initial specimen ( 200 μl ), and the fact that the quantified gene is a single gene must be taken into account . the value obtained for each microorganism in numbers of copies per 5 μl of dna extract was multiplied by 10 2 to obtain a concentration in number of bacteria per ml of specimen , then an elution volume of 100 μl of dna extracted from 200 μl of sample was created . the statistical analysis of the real - time pcr bacterial quantification data employed the wilcoxon test and the mann - whitney test for one degree of significance p & lt ; 0 . 05 . for the statistical analysis , all the quantification values were taken into account , including those below the positivity threshold . for quantification values equal to zero , the lowest concentration of the total specimens analyzed was attributed to them . the wilcoxon statistical test was applied to describe the distribution of each microorganism within each group of flora . the mann - whitney test was applied to compare the quantification of each of the 8 microorganisms in the bv and nf group . in order to investigate the molecular criteria for bv , each bacterial quantification threshold ( de 10 3 / ml to 10 9 / ml ) was studied for sensitivity , specificity , and positive and negative predictive value , calculated according to the identification by the nf and bv thresholds previously defined by the nugent score . the quantification thresholds , taken in isolation or combined , having the best sensitivity and specificity were used as molecular criteria for identification of bv . from june 2005 to april 2006 , 204 pregnant women aged 18 to 49 ( average age 28 . 9 ± 6 . 2 ) were included . the ethnic origins were north africa ( 43 %), europe ( 42 %), south africa ( 14 %), and other origins ( 1 %). one vaginal specimen was taken from each woman . seventy - two percent of the specimens were taken in the third trimester , 20 % in the second , and 8 % in the first trimester of pregnancy . twenty - one women received bacteriological follow - up , with 2 to 4 specimens being taken . hence , a total of 231 specimens was analyzed . from the 231 vaginal specimens from 204 women , the nugent &# 39 ; s score identified : 167 nf , 44 if , and 20 bv . the frequency of vaginal flora abnormalities in the first vaginal specimen for each of the 204 women was 71 % for nf ( 145 cases ), 19 % for if ( 39 cases ), and 10 % for bv ( 20 cases ). half of the women with bv were symptomatic . the most frequently observed symptom was abundant vaginal discharge . culturing enabled g . vaginalis , c . albicans , m . hominis , u . urealyticum , and streptococcus agalatiae to be isolated ( table 1 ). none of the vaginal specimens examined exhibited t . vaginalis on direct examination . when the single and double fluorescence pcrs were developed , no cross reactions were done , with no competition . similar ct value results were obtained with single and double fluorescence using the pure bacterial strains and the plasmid range . the optimum quantities of primers and probes necessary for obtaining optimum amplification are presented below . quantity of probe and sense and anti - sense primers necessary for the amplification reaction of 5 μl of dna extract for each microorganism and human albumin . bacterial quantification for each specimen was provided by the plasma dilution range . for each amplification reaction , the 8 points on the plasmid range , from 10 7 copies to 1 copy per 5 μl , were tested . the 10 7 copies range point was identified early for a ct of about 17 . for the 1 copy range point , detection was later with a ct at 37 . for all the points on the range , the amplification reaction was linear . the graphic representation is a straight line with a slope of − 3 . 2 to − 3 . 5 . this linearity was confirmed by testing the pure strains diluted to 1 : 10 , 1 : 100 , and 1 : 1000 . a positivity threshold was defined above 10 copies ( or 10 3 bacteria / ml ), whatever the type of microorganism considered ( table 2 ). statistically , amplification of the “ 1 copy ” point occurs in 75 % while it is 100 % for the “ 10 copies ” point . within each amplification plate , each specimen was tested in undiluted solution , and one - tenth and one - hundredth dilutions . reproducibly , detection of the amplification product followed the dilution step , since a one - tenth dilution corresponds to an increase of 3 cts . only the pure solution was considered for calculating the bacterial load . for each specimen , we tested human albumin by real - time pcr . the results obtained were homogenous for the set of 231 samples . the ct values were distributed between 19 and 22 ( or 10 5 to 10 6 copies of albumin dna per 5 μl of dna extracted ). no specimen was excluded from the analysis . the median concentrations of a . vaginae and g . vaginalis were 1 . 1 × 10 9 / ml and 1 . 2 × 10 9 / ml respectively , and there was no statistically significant difference with the wilcoxon test ( p = 0 . 3755 ). these two bacteria were in high concentrations ( fig1 a and 3 ) relative to the other microorganisms whose median concentrations were statistically lower ( p = 0 . 0001 ). the median concentration of lactobacillus sp . was significantly lower ( p & lt ; 0 . 0001 ) for bacterial vaginoses ( median concentration 3 × 10 3 / ml ) than for the nf ( median concentration 5 . 7 × 10 7 / ml ). on the other hand , the median concentrations of a . vaginae , g . vaginalis , m . curtisii and m . hominis were significantly higher ( p & lt ; 0 . 0037 ) for bacterial vaginoses ( median concentrations respectively 1 × 10 9 / ml ; 1 . 2 × 10 9 / ml ; 5 × 10 3 / ml and 5 . 5 × 10 2 / ml ) than for the nf . for the median concentrations of u . urealyticum and c . albicans , there was no statistically significant difference between the bv and the nf . finally , m . mulieris was not identified ( positivity threshold ≧ 10 3 / ml ) in any of the bvs or any of the nf . the analysis by quantification threshold for a . vaginae and g . vaginalis taken in isolation has the best criteria for sensitivity , specificity , and positive and negative predictive values for molecular identification of bv and nf defined by the nugent score ( table 4 ). the combination of quantification of a . vaginae ≧ 10 8 / ml and / or quantification of g . vaginalis ≧ 10 9 / ml has a sensitivity of 95 %, specificity of 99 %, positive predictive value ( ppv ) of 95 %, and negative predictive value ( npv ) of 99 %. application to if of the nugent score ( fig2 ) of the molecular criteria for bv identification previously defined by quantification of a . vaginae ≧ 10 8 / ml and / or quantification of g . vaginalis ≧ 10 9 / ml , allowed 25 flora to be characterized ( 57 %) that had a bv profile ( fig3 ) and 19 flora ( 43 %) with an nf profile ( fig4 ). eight women with bv or if by the nugent score were followed ( table 6 ). molecular quantification showed a relapse of a bv not identified by the nugent score for subject 1 . it confirmed disappearance of bv for subjects 7 and 8 . it characterized as bv the if in the nugent score persisting over a month later for subjects 2 and 5 . finally , it confirmed the normal nature of the vaginal flora followed in subject 3 . the distribution of vaginal flora into nf ( 71 %), bv ( 10 %) and if ( 19 %) according to the nugent score conforms to those reported in the literature in france [ goffinet f , ejogr 2003 ], europe [ guise j m , amjpm 2001 ], and the united states [ delaney m l , og 2001 ]. the original feature of the present invention is establishing a rational tool for vaginal flora identification by combining the specific real - time pcr detection technique with relative quantification by a calibration plasmid . the most surprising result was for a . vaginae . this bacterium was identified for the first time in 1999 by 16s rna amplification and sequencing from a vaginal specimen from a healthy subject [ rodriguez j m , ijsb 1999 ]. in 2003 , a . vaginae was isolated by culturing a specimen from a tuboovarian abscess , suggesting that the bacterium played a pathogenic role [ geissdorfer w , jcm 2003 ]. in 2004 , an approach combining 16s rrna with cloning techniques revealed the bacterium in specimens obtained perioperatively from patients with salpingitis [ hebb j k , jid 2004 ]. according to the present invention , this bacterium is frequently identified in 19 out of 20 bv ( 95 %) and 115 out of 167 nf ( 69 %). the most discriminating criterion for bv and nf diagnosis is an a . vaginae concentration ≧ 10 8 / ml with a sensitivity of 90 % ( 18 cases out of 20 bv ) and specificity of 99 % ( 1 case out of 167 nf ). since 2004 , four studies based on various molecular techniques have shown a possible association between a . vaginae and bv , but none of them used rigorous quantification criteria . first of all , a pcr approach targeting 16s rrna followed by gel migration showed a . vaginae in only 12 bv out of 20 ( 60 %) and 2 nf out of 24 ( 8 . 3 %) [ ferris m j , bmcid 2004 ]. by pcr specifically targeting the 16s rrna gene of a . vaginae , the dna of the bacterium was found in 7 out of 9 bv cases ( 77 . 8 %) and 22 nf out of 112 ( 19 . 6 %) [ verhelst r , bmcm 2004 ]. applying the same technique , a . vaginae dna was shown in 19 out of 22 bv ( 86 . 4 %) and 59 nf out of 403 ( 14 . 7 %) [ verhelst r , bmcm 2005 ]. finally , by amplification , cloning , and sequencing techniques , a . vaginae dna was observed in 26 bv out of 27 ( 96 %) and 9 nf out of 46 ( 19 . 5 %) [ fredricks d n , nej m 2005 ]. none of these studies quantified the a . vaginae dna , which is essential in bacterial vaginosis where the bacterial concentration is an important factor in the diagnosis . a . vaginae is missing from the nugent score despite its leading role in vaginal flora abnormalities . its identification by morphological criteria is ill - suited . its variable morphology in the form of a small gram - positive coccobacillus ( 0 . 6 - 0 . 9 μm ) sometimes grouped in pairs or short chains camouflages it when it contacts other bacteria , rendering it undetectable [ verhelst r , bmcm 2004 ]. its resemblance to lactobacillus sp . and to streptococci is hence a source of identification error [ rodriguez j m , ijsb 1999 ]. the description of an association between a . vaginae and g . vaginalis in bv is recent , sparsely documented , and of limited diagnostic scope in the absence of quantification . this association is 87 . 8 % ( 8 out of 9 bv ) [ verhelst r , bmcm 2004 ] and 90 % ( 20 out of 22 bv ) [ zariffard m r , fems 2002 ] by specific pcr that targets the 16s rrna gene of a . vaginae and g . vaginalis . a recent publication notes the presence of a . vaginae in bacterial vaginoses by a semi - quantitative method [ bradshaw c s , jid 2006 ]. according to the present invention , this combination of a . vaginae and g . vaginalis is 90 % ( 18 out of 20 bv ) but consideration of the quantification of a . vaginae ≧ 10 8 / ml and / or of g . vaginalis ≧ 10 9 / ml with a sensitivity of 95 % ( 19 out of 20 bv ) offers the best criteria for specificity ( 99 %), ppv ( 95 %) and npv ( 99 %) ever to have been achieved for identification of bv . hence , an a . vaginae concentration of ≧ 10 8 / ml and / or a g . vaginalis concentration of ≧ 10 9 / ml is used for molecular diagnosis of bv . the results according to the present invention suggest that the quantification of g . vaginalis is less discriminating than that of a . vaginae . for lactobacillus sp ., their diminution in bv shown objectively by the nugent score is confirmed by these results : if we consider a lactobacillus sp . concentration less than or equal to 10 7 / ml , the sensitivity for diagnosing bv is 100 % and the specificity , 44 %. moreover , if we consider a lactobacillus sp . concentration greater than or equal to 10 8 / ml , we observe 100 % specificity in demonstrating normal vaginal flora . for mobiluncus spp ., despite the position afforded them by the nugent score , no pcr is positive for m . mulieris . only m . curtisii is associated with bv . however , its utility as a possible molecular diagnostic criterion remains minor . genital mycoplasmas are not taken into account by the nugent score . they are however identifiable by culturing or molecular biology . a study culturing 445 bv and 2729 nf identified m . hominis as being significantly associated with bv with a prevalence of 29 % ( 129 bv ) and u . urealyticum as not being associated with bv despite a prevalence of 56 % ( 253 bv ) [ thorsen p , ajog 1998 ]. a study on 203 bv and 203 nf targeting m . hominis by real - time pcr suggested involvement of m . hominis in bv [ zariffard m r , fems 2002 ]. however , an identical study on a smaller cohort ( 5 bv and 16 nf ) did not demonstrate this link [ sha b e , jcm 2005 ]. according to the present invention , only m . hominis correlates with bv , but this correlation is insufficient for proposing this microorganism as a diagnostic criterion for bv . the results according to the present invention do not show a link between bv and c . albicans as shown by the data in the literature [ thorsen p , ajog 1998 ]. this is interesting because while genital carriage of candida albicans does not increase the risk of prematurity [ cotch m f , ajog 1998 ], a recent study [ kiss h , bmj 2004 ] showed a reduction in prematurity rate by treatment of c . albicans genital portage . the originality of the present invention is that for the first time for the first time it provides a diagnostic tool for bv based on quantification of a . vaginae and g . vaginalis . the criteria in current use combine a concentration of a . vaginae ≧ 10 8 / ml and / or a g . vaginalis concentration of ≧ 10 9 / ml . this diagnostic test has a sensitivity of 95 %, a specificity of 99 %, a ppv of 95 %, and a npv of 99 %. several methods of reading the pcr results , particularly those in which the ratio and products of the bacterial concentrations are calculated , have been tested so that the most pertinent method could be identified . one of the most disturbing applications of the molecular biology tool according to the present invention is the one performed on the if in the nugent score . we know that the if , identified solely by the nugent score , constitutes a non - negligible fraction ( 8 % to 22 %) of all the flora identified by the latter [ guerra b , ejogrb 2006 ; goffinet f , ejogrb 2003 ; delaney m l , og 2001 ; libman m d , dmid 2006 ; larsson p g , sti 2004 ] and that it is associated with many uncertainties . indeed , its interpretation requires caution because we do not know the microbiological reality to which this intermediate flora corresponds . some authors believe it to be a transitional flora between nf and bv [ ison c a , sti 2002 ; guerra b , ejogrb 2006 ; goffinet f , ejogrb 2003 ; ugwumadu a , lancet 2003 ; carey j c , nejm 2000 ]. others consider it to be a heterogenous group including nf and bv [ ison c a , sti 2002 ; libman m d , dmid 2006 ; larsson p g , sti 2004 ]. attempts to characterize the if in nf or bv are reported in the literature . by applying amsel &# 39 ; s criteria to 13 if , 12 if were reclassified as nf and 1 as bv [ icon c a , sti 2002 ]. using kopeloff &# 39 ; s stain to establish the nugent score , 69 of the 232 if ( 30 %) were reclassified as nf or bv [ libman m d , dmid 2006 ]. in this stain , fuchsin replaces the safranin in gram &# 39 ; s stain for better identification of gram - negative bacteria . finally , the standardization of the nugent score criteria as a function of the surface of the optical field of the microscope used enabled 458 out of 1176 if ( 39 %) to be reclassified as nf or bv [ larsson p g , sti 2004 ]. the application to if of the molecular criteria according to the present invention associating an a . vaginae concentration of ≧ 10 8 / ml and / or a g . vaginalis concentration of ≧ 10 9 / ml with bv enables this if to be rationally characterized . thus , 24 if ( 57 %) have a profile similar to that of nf . these results thus suggest that the if are heterogenous in nature , and that the nugent score is so insensitive that it cannot diagnose over half the instances of bv . thus , these data confirm the limitations of the nugent score . the etiology of bv remains quite mysterious , but for the first time we have an objective quantification tool providing a rational approach to the diagnosis of bv . the singular nature of the present invention is showing both the crucial role of a . vaginae in bv and its utilization by quantification as a main diagnostic criterion for bv . it offers better understanding of the therapeutic problem posed by the relative resistance of a . vaginae to metronidazole , which could in part account for the frequency of relapses after treatment [ anaes 2001 ; ferris m j , bmc id 2004 ; secor a m , cnp 1997 ; geissdorfer w , jcm 2003 ; de backer e , bmc id 2006 ]. the molecular criteria used to diagnose bv combine an a . vaginae concentration ≧ 10 8 / ml and / or a g . vaginalis concentration ≧ 10 9 / ml . this molecular tool enables bv to be diagnosed and some if to be characterized as bv . we can also envisage our molecular tool as being a bv diagnostic tool and a follow - up method for evaluating therapeutic management of bv during pregnancy . anaes . 2001 . prévention anténatale du risque infectieux bactérien anténatal . recommandations pour les professionnels de santé . 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