A study of the susceptibility of three species of primate to vaginal colonization with Gardnerella vaginalis

In an attempt to develop an animal model of Gardnerella-associated vaginitis, several strains of Gardnerella vaginalis were inoculated into the lower genital tract of female pig-tailed macaques, tamarins and chimpanzees. G. vaginalis was not recovered from either tamarins or chimpanzees, but was recovered from each of 1O pig-tailed macaques inoculated with either of two freshly isolated Gardnerella strains, colonization persisting for 11-39 days. Examination of Gram-stained vaginal smears obtained from infected pig-tailed macaques failed to demonstrate clue cells, a feature which is pathognomonic of Gardnerella-associated vaginitis in humans. Other features characteristic of non-specific vaginitis, namely an increase in vaginal pH, and an increase in the ratio of succinate to lactate (S/L ratio) in vaginal fluid were not found. However, the physiology of the macaque vagina was found to be different from that of the human, the vaginal pH and S/L ratio of uninfected macaques both being higher than that seen in humans. The physiological differences between the macaque and human vagina may be due, in part, to a difference in their anaerobic vaginal flora. While these inter-species differences in vaginal physiology and microbiology limit the relevance of the pig-tailed macaque as a model of Gardnerella-associated vaginitis, the ease with which macaques are colonized with G. vaginalis may prove useful in studying bacterial adhesion and local immunity.     

Detection of Gardnerella vaginalis in vaginal specimens by direct immunofluorescence.

The preparation of a fluorescein-labeled Gardnerella vaginalis polyclonal antibody is described, and its usefulness is assessed for the detection of this microorganism in vaginal samples obtained from 263 women attending the gynecological department of a general hospital, 66 of whom harbored an intrauterine device. The direct immunofluorescence technique was positive for G. vaginalis in 21% of the specimens, whereas only 12.5% of the total bacteriological cultures were positive. The frequency was higher in patients harboring intrauterine devices since 34.8% exhibited positive immunofluorescence and 30.3% exhibited positive cultures.     

Longitudinal study of the biotypes of Gardnerella vaginalis.

Gardnerella vaginalis is the predominant vaginal microorganism in women with bacterial vaginosis. However, this organism is also frequently isolated from women without signs or symptoms of vaginitis. Earlier studies have not revealed whether certain biotypes of G. vaginalis are more often associated with bacterial vaginosis or are more common in women who acquire bacterial vaginosis. We used a typing scheme based on tests for beta-galactosidase, hippurate hydrolysis, and lipase, using oleate as a substrate. Of 261 strains tested, the distribution of biotypes observed was as follows: 1, 13%; 2, 9%; 3, 5%; 4, 7%; 5, 41%; 6, 15%; and 8, 10%. Biotype 7 was not observed. The distributions of biotypes from women with and without bacterial vaginosis were found to be significantly different, with the lipase-positive biotypes (biotypes 1, 2, 3, and 4) being more predominant in women with vaginosis (41 versus 23%, P = 0.003). Of 40 women with normal vaginal flora at the index visit who remained normal at follow-up, 23 (57%) acquired a new biotype of G. vaginalis. By comparison, 90% of the 30 women who developed bacterial vaginosis acquired a new biotype of G. vaginalis (P = 0.003). Women with bacterial vaginosis at the index visit who were not treated were no more likely than normal women to have a shift in G. vaginalis biotype. However, 86% of the 30 women with bacterial vaginosis who were treated with an antibiotic at the index visit acquired a different biotype (P = 0.04 compared with the value for untreated women) regardless of treatment success. A trend toward the acquisition of a new biotype was observed among women who had contact with a new sexual partner (81 versus 65%, P = 0.15). These data demonstrate that the lipase-positive isolates of G. vaginalis are associated with bacterial vaginosis. Women who acquire bacterial vaginosis are more likely to have a shift in biotype than women who had normal flora at he follow-up, suggesting that the G. vaginalis isolates recovered from women who develop bacterial vaginosis represent newly acquired strains rather than overgrowth of previously colonizing biotypes.     

Biotypes of Gardnerella vaginalis.

A simple and reproducible scheme for identifying biotypes of Gardnerella vaginalis has been developed, based on reactions for lipase, hippurate hydrolysis, and beta-galactosidase. Among a total of 359 strains tested, eight biotypes were observed, the most common ones being types 1 (beta-galactosidase positive, lipase positive, hippurate positive), 2 (beta-galactosidase negative, lipase positive, hippurate positive), and 5 (beta-galactosidase negative, lipase negative, hippurate positive). The distribution in biotypes was similar among isolates from Antwerp, Seattle, and Nairobi. There were no differences in biotypes between strains isolated from patients with and without bacterial vaginosis (nonspecific vaginitis). Up to 14% of women with bacterial vaginosis harbored at least two different biotypes of G. vaginalis in the vagina. G. vaginalis strains isolated before and after treatment for bacterial vaginosis belonged to identical biotypes when the time interval between two specimens was less than 1 week. Similarly, G. vaginalis isolates from the vaginas of women with bacterial vaginosis and from the urethras of their male sex partners belonged to identical biotypes when strains were isolated within the same 24-h period from both partners (P less than 0.005).     

Humoral circulatory immune response to Gardnerella vaginalis.

Strain-specific circulating immunoglobulin G and/or M was detected by enzyme-linked immunosorbent assay and immunofluorescence test by using Formol-treated suspensions of Gardnerella vaginalis from 28 women with overt vaginitis but only three symptom-free subjects among 43 otherwise healthy women found to be colonized by G. vaginalis. Analogous but less stringent strain specificity patterns were elicited by immunization of BALB/c mice.     

Identification of Gardnerella vaginalis with the API 20 Strep system.

A total of 137 strains of Gardnerella vaginalis were examined by the API 20 Strep system. The system was shown to be reliable when the tests were compared with standard identification methods, and very little confusion occurred with streptococcal profiles; consequently, G. vaginalis has been included in the API 20 Strep data base.     

Prevalence of Gardnerella vaginalis in the urinary tract.

Midstream urine samples from 106 patients presenting to the Casualty Department of The Royal Melbourne Hospital with frequency or dysuria were cultured for Gardnerella vaginalis and conventional uropathogens. Urine samples collected via an open-end catheter from 70 healthy pregnant women were examined similarly. Midstream urine and other samples, including the seminal fluids and swabs of the mouths, throats, rectums, and vaginas of 33 healthy subjects, were cultured for G. vaginalis. Another 15 female patients with proven G. vaginalis bacteriuria were given a bladder washout localization test to determine the site of infection in the urinary tract. G. vaginalis in counts greater than 10(3) CFU/ml was recovered from the midstream urine of 27 of 106 patients (25%), 7 of whom also harbored conventional pathogens in counts greater than 10(4) CFU/ml. Another 11 patients with cultures negative for G. vaginalis yielded greater than 10(4) CFU of conventional pathogens per ml. G. vaginalis was cultured (greater than 10(3) CFU/ml) from catheter samples of 19 of 70 healthy pregnant women (27%), 6 of whom also harbored greater than 10(3) CFU of conventional uropathogens per ml. Two women yielded growths of conventional pathogens only. Midstream urine samples from 13 of 13 healthy males were free of G. vaginalis, whereas 5 of 20 healthy nonpregnant females yielded greater than 10(3) CFU of G. vaginalis per ml from midstream urine samples. G. vaginalis was recovered from 4 of 12 semen samples and from urethral samples from four of seven males and four of eight females. All four culture-positive females also harbored G. vaginalis in their vaginas. There was no evidence of oral or rectal carriage of G. vaginalis in 15 healthy subjects. Localization studies with 15 female patients having underlying renal disease showed that 11 patients harbored G. vaginalis in their kidneys. The result suggest that colonization or infection of the bladder and upper urinary tract by G. vaginalis is very largely a phenomenon of females, with the highest frequency in pregnant women. The prevalence of G. vaginalis in the urinary tracts of healthy females is similar to that of symptomatic subjects. However, G. vaginalis in counts greater than 10(5) CFU/ml is more likely to be associated with urinary tract symptoms. In males, this bacterial species infects the genital tract rather than the urinary tract.     

Evaluation of the Affirm Ambient Temperature Transport System for the detection and identification of Trichomonas vaginalis, Gardnerella vaginalis, and Candida species from vaginal fluid specimens

The objective of this study was to measure the performance of the Affirm Ambient Temperature Transport System (ATTS) over time and to estimate the length of time the system can preserve a vaginal specimen containing the three common organisms causing vaginitis: Trichomonas vaginalis, Candida species, and Gardnerella vaginalis (one of the causative agents of bacterial vaginosis). Women with symptoms of vaginitis presenting to one of three clinical centers were evaluated over a 4- to 8-week period. Four simultaneously obtained swabs were collected and tested by the Affirm VPIII assay at time zero with and without a preservative reagent, at 24 h with reagent, and at either 48 or 72 h with reagent. For each of the three organisms, Trichomonas, Gardnerella, and Candida, positivity at each time point was evaluated and compared to that at reference time zero with and without the ATTS. A total of 940 specimens were obtained from the three clinical sites. Eight hundred three were positive for one or more of the three organisms. Gardnerella had the highest overall positive rate (62%), followed by Candida with 18% and Trichomonas at 9%. The percent sensitivity versus control for Trichomonas ranged from 100% at time zero with and without reagent to 91% by 72 h. Gardnerella and Candida sensitivity remained at 100% for each time period. The Affirm VPIII ATTS system performed within 10% of the control swab (no transport reagent) at all four time points (0, 24, 48, and 72 h) for Trichomonas, Gardnerella, and Candida.     

Evaluation of the enhanced rapid identification method for Gardnerella vaginalis.

The enhanced rapid identification method (RIM; Austin Biological Laboratories), a micromethod for the identification of Gardnerella vaginalis, is based on starch and raffinose fermentation and hippurate hydrolysis. We tested 105 clinical isolates of G. vaginalis with both the RIM and standard biochemical tests. The RIM agreed with the standard biochemical methods for 96 (91.4%) of the strains; nine isolates which were hippurate hydrolysis positive by standard biochemical tests were hippurate hydrolysis negative in the RIM. RIM may serve as a useful adjunct to Gram stain and colony morphology for the identification of G. vaginalis.     

Evaluation of affirm VP Microbial Identification Test for Gardnerella vaginalis and Trichomonas vaginalis.

A commercial system (Affirm VP Microbial Identification Test; MicroProbe Corp.) for detection of vaginal pathogens was evaluated with 176 consecutive women attending a sexually transmitted disease clinic for genital complaints. Vaginal swab specimens were used for culture of Gardnerella vaginalis and Trichomonas vaginalis, preparation of a vaginal smear for Gram stain interpretation, and wet mount evaluation. An additional swab was used to evaluate the 30-min nonisotopic oligonucleotide probe test. The automated probe system detected G. vaginalis in 69 (95%) of 73 women having > 5 x 10(5) CFU of G. vaginalis per ml by culture, and 20 (43%) of 47 specimens with < or = 5 x 10(5) CFU of G. vaginalis per ml. There were three false positives and four false negatives for the Affirm VP test compared with > 5 x 10(5) CFU of G. vaginalis per ml. The probe system detected G. vaginalis in 57 (90%) of 63 vaginal specimens from women having clue cells on wet mount examination, and in only 3 (3%) of 113 women without clue cells, suggesting that the Affirm probe for G. vaginalis could be used as a surrogate for wet mount examination for clue cells. The T. vaginalis probe was positive for 12 of 12 specimens positive by wet mount and 12 of 15 specimens positive by culture. There were no false positives and three false negatives for the Affirm VP test compared with culture and/or wet mount for T. vaginalis. The Affirm VP Microbial Identification System is a rapid, objective, and automated test for the detection of T. vaginalis and clinically significant levels of G. vaginalis that is comparable to wet mount examination for clue cells and is superior to wet mount examination for the detection of trichomonads.     

Symptomatic candidiasis: Using self sampled vaginal smears to establish the presence of Candida, lactobacilli, and Gardnerella vaginalis

In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis ("clue cells") and neutrophils.All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle.Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus-predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida.     

Involvement of Gardnerella vaginalis in urinary tract infections in men.

Fifteen male patients from whose urine samples Gardnerella vaginalis was isolated (clinical incidence of 0.1%) were evaluated for clinical signs and symptoms of urinary tract infection and modality of acquisition of the organism. Ten of 15 (67%) patients were symptomatic or had signs of inflammation as manifested by an increased number of urinary neutrophils. One patient had two bouts of infection caused by this organism which required two courses of antibiotic therapy. Colonies of diphtheroidlike organisms found in urine cultures should not be ignored as insignificant but should be further investigated to determine whether G. vaginalis is present.     

Heterogeneity in restriction patterns of Gardnerella vaginalis isolates from individuals with bacterial vaginosis.

This study was undertaken to resolve the genetic make up of Gardnerella vaginalis present in bacterial vaginosis (BV). DNA from several G. vaginalis isolates from within and between individual BV patients were compared by BamHI, ClaI and EcoRI restriction endonuclease analysis (REA) followed by a restriction fragment length polymorphism (RFLP) study, utilizing a 5.7-kb BamHI G. vaginalis ATCC14018 DNA probe. Four G. vaginalis isolates from one patient (GVP-062) were composed of 3 different biotypes (biotypes 3, 5 and 8), and while the REA mirrored the biotype, in RFLP studies at least 3 isolates had DNA fragments in common. All of the isolates from 2 other patients (GVP-063 and GVP-072) represented a single biotype (biotype 2), but under REA and in RFLP studies, the isolates GVP-063 differed from GVP-072. An opposite case existed with the isolates GVP-072 (biotype 2) and GVP-065 (biotype 5), which appeared similar under REA and in RFLP studies. Finally, reisolates after 8 weeks (GVP-080) from a BV patient (isolates GVP-065) representing the same biotype (biotype 5) differed under REA and in RFLP studies. Thus, lacking any unique DNA fingerprint, G. vaginalis occurring in BV represents a (genetically) mixed population.     

Genetic susceptibility to vaginal candidiasis.

To enable future studies on host resistance factors and therapy, inbred and outbred mouse strains were tested for susceptibility to vaginal candidiasis. Groups of mice were given 0.5 mg estradiol 3 days before and 4 days after intravaginal challenge with a suspension of Candida albicans. On day 1 after challenge, a swab was used to quantitate infection in all groups and to assure equivalent infection levels. On day 6, this was repeated and the experiment was terminated. BALB/c, the reference strain in repeated experiments, was susceptible, showing persistent infection with levels of cfu at day 6 falling within a range between a twofold decrease and a fourfold increase in relation to day 1 levels. CD-1 outbred mice were markedly resistant, with day 6 cfu levels showing a 74- to 87-fold decrease with respect to day 1 levels, whereas other outbred strains (CF-1, SW, ICR) were susceptible. A BALB/c substrain (ByJ) was also susceptible. With exception of CBA/J, which showed modest resistance, all inbred strains were similarly susceptible, including DBA/2, AKR/J, C3H/HeN, A/J and C57BL/6. The differences between CD-1 and BALB/c mice were also seen with a second C. albicans isolate. Our results show susceptibility to vaginal candidiasis is independent of the major histocompatibility locus H2 haplotype and any effect ascribable to use of particular commercial mouse suppliers. Differences among mouse strains in susceptibility to C. albicans, as seen in previous studies involving nonvaginal challenge routes, are not reflected in this vaginal candidiasis model; in general, such resistance patterns appear specific to the route of challenge administration. The resistance seen in mouse strain CD-1 is of particular interest in that CD-1 is known to be resistant to endocrine disruption by estrogen. Our results suggest this estrogen insensitivity may have broad-ranging effects on processes other than gametogenesis, including vaginal susceptibility to candidiasis.     

Comparison of culture and microscopy in the diagnosis of Gardnerella vaginalis infection.

A comparison was made between human blood agar containing amphotericin B, nalidixic acid and either gentamicin or colistin for the isolation of Gardnerella vaginalis from cases of non-specific vaginitis seen in a clinic for sexually transmitted diseases. The medium containing gentamicin was more inhibitory for non-Gardnerella species, but not sufficiently inhibitory to allow direct plating in the clinic without spreading for single colonies. The diffuse beta haemolysis produced by G vaginalis on human, but not on horse blood agar, proved very useful in differentiating it from other vaginal organisms and was not affected by the antibiotics used. This characteristic, together with Gram stain morphology, oxidase and catalase, provides a simple, reliable methods of identifying G vaginalis. Sixty women with symptoms of vaginitis, in whom no other pathogen was isolated, were examined by culture and microscopy. Gardnerella vaginalis was grown from 45 whereas only 31 had positive microscopy (clue cells or Gram-variable bacilli). There was no significant difference between the rate of isolation of G vaginalis in the group with positive microscopy (25/31) and that with negative microscopy (20/31).     

Use of a sodium polyanetholesulfonate disk for the identification of Gardnerella vaginalis.

Several methods have been previously suggested for the presumptive identification of Gardnerella vaginalis in clinical laboratories, but none is entirely satisfactory. We previously found that sodium polyanetholesulfonate (SPS) inhibits G. vaginalis in blood culture media. We compared susceptibility to an SPS-containing paper disk with beta-hemolysis on human blood agar, hippurate hydrolysis, and inhibition by alpha-hemolytic streptococci for identification of 62 previously confirmed G. vaginalis strains. All strains were positive by SPS disk and alpha-hemolytic streptococcus inhibition, 78% were positive by beta-hemolysis, and 81% were positive by hippurate hydrolysis. Although positive reactions occurred with SPS disk and alpha-hemolytic streptococcus tests for 5 and 9 of 84 other bacteria tested, respectively, none of these bacteria were positive for both tests. We conclude that a combination of SPS disk susceptibility and alpha-hemolytic streptococcus inhibition provides excellent identification of G. vaginalis when performed by the methods suggested.