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Vaginal Microflora Associated with Bacterial Vaginosis in Nonpregnant Women: Reliability of Sialidase Detection

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Vaginal Microflora Associated with Bacterial Vaginosis in Nonpregnant Women: Reliability of Sialidase Detection Infect Dis Obstet Gynecol 2001;9:17–22 Vaginal microflora associated with bacterial vaginosis in nonpregnant women: reliability of sialidase detection Jorgelina Smayevsky, Liliana Fernández Canigia, Alejandra Lanza and Hebe Bianchini Laboratorio de Microbiología, Centro de Educación Médica e Investigaciones Clínicas Dr. Norberto Quirno CEMIC, Buenos Aires, Argentina Objective: Todeterminethe prevalenceof Gardnerellavaginalis, anaerobicbacteria and Mycoplasmahominis in vaginal specimensof women with andwithout bacterial vaginosis(BV) aswell asto determine the sensitivityand specificity of the direct sialidase assay of vaginal fluid as a rapid test for diagnosing this syndrome. Methods: Vaginal cultureswere obtainedfrom 109 nonpregnantwomen (mean age33 ± 7.1 years),47 of them with clinical signsof BV(BV+) and62 of them without BV(BV ).Inaddition,we determinedthe vaginal sialidase activity in both groups, which may serve as a feature of this syndrome. Results: Anaerobic bacteria wereisolated in91% and18% of the BV+and BV groups,respectively ( p < 0.001). Peptostreptococcus spp., Prevotella bivia and Porphyromonas spp.were stronglyassociated with BV. P. bivia and Prevotella spp.represented 44% of all the anaerobesisolated inthe BV+group. All the isolated P. bivia strains presentedsialidase activity. G.vaginalis and M. hominis wereisolated in76% and42% of the BV+and 1% and0% of the BV women, respectively( p < 0.001). Mobiluncus morphotypeswere observedin 34% ofthe BV+and 0% of BV women. Sensitivity,specificity, positive predictive value and negative predictive value of sialidase activity were 81%, 94%, 90% and 86%, respectively. Conclusions: Ourdata demonstratea strongassociation between G.vaginalis , M. hominis, and P. bivia and BV. Sialidase activity and Gram stain of vaginal fluid represent accurate methods for diagnosing BV. Key words: V AGINOSIS; ANAEROBES; DIAGNOSIS Bacterial vaginosis, previously knownas non- Bacterial vaginosis represents asynergistic specific vaginitis, Haemophilusvaginalis vaginitis, polymicrobialinfection, characterized by anover- Corynebacteriumvaginale vaginitis, Gardnerella growthof bacterial species usually foundin vaginalis vaginitis andanaerobic vaginosis, is an thevagina. Thelactobacilli-dominated flora is abnormalcondition of the vaginal ecosystem replacedby amixed flora, consistingof Gardnerella causedby overgrowthof bothaerobic and anaero- vaginalis;anaerobes suchas Bacteroides spp., bicvaginal bacteria flora 1–4.Itis themost common Prevotella spp. and Mobiluncus spp.; and Mycoplasma vaginal disorderin women of reproductiveage and hominis10. is responsible forapproximately one-third of all Several bacterial enzymes, includingsialidases, cases ofvulvovaginitis. Itis nowregarded as arisk have beenimplicated as virulence factorsin factorfor complications of pregnancy, including pregnancycomplications such as prematurityand chorioamnionitis and prematurity 5–9. chorioamnionitis.Sialidases, formerly known Correspondenceto: JorgelinaSmayevsky, PhD, Camargo581, Piso 4, BuenosAires 1414, Argentina. E-mail: [email protected] Clinical study 17 Bacterial vaginosis and sialidase detection Smayevsky et al. as neurominidases, are enzymes thatcleave transportmedium, andinto mycoplasma transport a-ketosidiclinkages betweenthe glycosyl residues medium. Anothervaginal swabwas collectedto ofglycoproteins,glycolipids and sialic acids 11. The performa sialidase activity assay. Endocervical purposeof this studywas todetermine the swabspecimens were takenfor screening for prevalence of Gardnerellavaginalis , anaerobic Neisseria gonorrhoeae and Chlamydia trachomatis. bacteria and Mycoplasmahominis in vaginal Vaginal swabs were placedonto chocolate agar, specimens fromwomen with and without bacterial humanblood agar, modifiedThayer-Martin vaginosis, as well astodetermine thesensitivity and medium andFeimberg medium, as described specificity ofthe direct sialidase assay onvaginal elsewhere13. G.vaginalis culturesrecovered from fluid as a rapid test for diagnosing this syndrome. thethird and fourth streak zoneson anagar plate were considered significant. Brucella bloodagar +vitamin K(1 mg/ml) + MATERIALS AND METHODS hemin (5 mg/ml) andbrucella blood agar + Intotal 109 women (mean age 33 ± 7.1 years) K vitamin (1 mg/ml) +hemin (5 mg/ml) + were studied.Forty-seven womenwith BV were amikacin (50 mg/ml) were incubatedfor 7 days at includedin the BVgroup.Sixty-two women from 35°Cinan anaerobic chamber 14.Allother plates thesame hospital,free ofBV, were includedas were incubatedat 37 °C in 5% CO2 for 96 h. controls.All women were nonpregnant,and none Feimberg medium was incubatedin air for96 h. was menstruatingat thetime ofexamination. Mobiluncus culture was not performed. Womenwere excludedfrom the studywhen they Ureaplasmaurealyticum and Mycoplasmahominis had used antibiotics 3 days before the study. were cultivated inurea broth,arginine brothand BVwas definedby thepresence ofvaginal pH A7Sheppardagar andincubated for 5 days at 35 °C > 4.5, fishyodor-positive in presence of10% in 5% CO2 atmosphere. U. urealyticum cultures KOH(positive whifftest), andpresence ofclue were consideredas significant whenthe colony cells. Controls(women without BV) were defined count was higher than 10 4 ccu/ml15. bythe absence ofall these three clinical criteria. Aerobicand facultative anaerobicisolates Homogeneousvaginal dischargewas notconsid- were identifiedby conventional methods 13,16. ered. Specimens were takenfrom the posterior Anaerobicbacteria growingin the third and vaginal fornixusing a sterile, nonlubricated fourthstreaks were identifiedby performingbio- speculum. Odorwas tested byimmersing aswab chemical tests 14,17.Sialidase activity ofanaerobic containingthe vaginal fluidin 10% KOH and bacteria was measured byafilter-paper spottest 18. smelling theodor. The pH of vaginal secretions Selective medium for lactobacilli was not used. was measured withpH paper(Spezialindikator pH Specimens in2SP medium were storedfrozen at 4.0–7.0; Merck). Asecondvaginal swabwas 70°Cforup to 5 days. Aliquotsof 250 ml were collectedin saline solutionand examined micro- inoculatedonto McCoy cells monolayers growing scopicallyfor bacterial morphologictypes, clue onglass coverslips inshell vials bystandard cells, whitecells, trichomonadsand yeasts bywet methods19.Thetissue cultureswere incubatedat mount (´ 400). 35°C in a 5% CO2 atmosphere. After72 h the Gram stain was performedusing a thirdswab cultureswere stained withJones’ s iodinestain andevaluated forbacterial morphologictypes andexamined microscopicallyfor chlamydial usingNugent’ s score ( ´ 1000)12. Women were inclusions. categorizedas BV-positive (scores 7–10) and Sialidase activity was qualitatively determined BV-negative (score <7) usingthe Gram stain bya filter-paper spottest usinga stocksolution scoringsystem forvaginal smears. Having more of 2¢-(4-methylumbelliferyl) a-D-N-acetyl-neur- than10 polymorphonuclearcells perfield ( ´ 1000) aminic acidin buffer acetate. Priorto use, it was was consideredas asignificant inflammatory dilutedand filter paperstrips were saturatedwith reaction. this solution.Paper strips were theninoculated Threeother vaginal swabs were placedinto witha spotof vaginal fluidand incubated for Cary Blair transportmedium, intoanaerobic 15 min at 37°C. Thetest was interpretedby 18 INFECTIOUSDISEASES IN OBSTETRICS AND GYNECOLOGY Bacterial vaginosis and sialidase detection Smayevsky et al. Table 1 Prevalance of microorganisms in the vaginal fluid of 109 women with and without bacterial vaginosis (BV) BV+ BV– (n = 47) (n = 62) Microorganisms isolated* Number Percentage Number Percentage p value** Anaerobic Gram-negative rods Prevotella bivia 22 47 1 1 <0.001 P. intermedia 03 06 1 1 NS P. disiens 01 02 0 0 NS Prevotella spp. 05 10 0 0 NS Porphyromonas asacharolytica 17 36 1 1 <0.050 Porphyromonas spp. 03 06 1 1 NS Bacteroides spp. 03 06 0 0 NS Anaerobic Gram-positive rods Mobiluncus morphotypes 16 34 0 0 <0.010 Anaerobic Gram-positive cocci Peptostreptococcus spp. 23 49 8 13 <0.001 Facultative anaerobic bacteria Gardneralla vaginalis 36 76 1 1 <0.001 Streptococcus agalactiae 02 04 5 8 NS Mycoplasmas Mycoplasma hominis 20 42 0 0 <0.001 Ureaplasma urealyticum 10 21 9 14 <0.050 Yeasts Candida albicans 02 04 7 11 NS *T. vaginalis , N. gonorrhoeae and C. trachomatis have not been isolated; **the c2 tests and the Fisher’s exact test were used examining thestrips undera long-wavelength Table 2 Numberof isolates invaginal samples of109 (365 nm) lamp. Afluorescentblue spot was women with and without bacterial vaginosis (BV) 11 indicative of sialidase activity . Women Women 2 The c tests, Fisher’s exact test andMann– with BV without BV Whitneytest were usedfor comparative analysis of Isolates (n = 47) (n = 62) p value* thedata obtained, and significance was assigned at Total number 163 34 p < 0.05. Mean ± SD per specimen 3.47 ± 1.20 0.55 ± 0.71 <0.001 Values are reported as mean ± standard deviation (SD); *the RESULTS Mann–Whitney test was used Theprevalence ofmicroorganisms isolated inthe vaginal fluidin women with and without BV is shownin Table 1. Neither Neisseria gonorrhoeae nor Chlamydia trachomatis was isolated. Anaerobic associated withBV ( p < 0.001). P. bivia and bacteria were isolated in91% and 18% of the Prevotella spp.represented 44%of all ofthe womenwith and without BV, respectively anaerobes isolated in the BV+ group. (p < 0.001). G.vaginalis and M. hominis were isolated in76% Themean numberof isolates perspecimen from and42% of thewomen with BV andin 1.0% and womenwith
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