Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

Increased vaginal pH in Ugandan women: what does it indicate?

Abnormal vaginal flora (AVF), indicative of bacterial vaginosis (BV) and/or aerobic vaginitis (AV), amongst other abnormalities, is a risk factor for multiple complications in pregnant as well as non-pregnant women. Screening for such conditions could help prevent these complications. Can self-testing for increased vaginal pH reliably detect BV and other high-risk microflora types, and is this more accurate than performing Gram stain-based Nugent score when screening for high-risk microflora? A total of 344 women presenting at different outpatient clinics in Mulago Hospital and Mbuikwe Outpatient clinics in Kampala, Uganda, were asked to test themselves by introducing a gloved finger into the vagina and smearing it on a microscopy slide, on which a pH strip was attached. Self-assessed categories of normal (pH 3.6-4.4), intermediate (4.5-4.7) or high pH (>4.7) were compared with demographic and with centralised microscopic data, both in air-dried rehydrated wet mounts (Femicare), as well as in Gram-stained specimens (Nugent). AVF was present in 38 %, BV in 25 % and AV in 11 % of patients. High pH and AVF is correlated with human immunodeficiency virus (HIV), infertility, frequent sex, but not vaginal douching. Screening for raised pH detects 90 % of AVF cases, but would require testing over half of the population. As AV and non-infectious conditions are frequent in women with AVF and high pH, Nugent score alone is an insufficient technique to screen women for a high-risk vaginal microflora, especially in infertile and HIV-infected women.

Diagnosis of aerobic vaginitis by quantitative real-time PCR.

PURPOSE: To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. METHODS: Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. RESULTS: Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p < 0.0001). Streptococcus spp. dominated in the majority of AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. CONCLUSIONS: PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

Multiple human papillomavirus infections and HIV seropositivity as risk factors for abnormal cervical cytology among female sex workers in Nairobi.

We estimated type-specific prevalence of human papillomavirus (HPV) and examined risk factors for abnormal cervical cytology among 296 female sex workers from Nairobi, Kenya. Over half (54%) were infected with a high-risk (HR) HPV type, of which HPV16 and 52 were the most common types. HIV-1 prevalence was 23% and HIV-1 sero-positivity was associated with high-grade cervical lesions, particularly among women with CD4 count less than 500 cells/mm(3) (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 1.7-24.9). Among women who had normal cytology at the time of entry into the study, the risk of having an abnormal Pap smear within one year was significantly elevated for women with multiple HPV types at study entry (adjusted odds ratio [aOR] = 6.0; 95% CI: 2.3-15.7) and with a subset of HR HPV types (aOR = 4.2; 95% CI: 1.6-11.2). Detection of multiple concurrent HPV infections may be a useful marker to identify women at risk of developing precancerous lesions in populations of high HPV prevalence.

Sialidase activity in aerobic vaginitis is equal to levels during bacterial vaginosis.

OBJECTIVE: To evaluate levels of proinflammatory cytokines and sialidase activity in aerobic vaginitis (AV) in relation to normal vaginal flora and bacterial vaginosis (BV). STUDY DESIGN: In this cross-sectional study, a total of 682 consecutive non-pregnant women attending the gynecology service were assessed and 408 women were included. Vaginal rinsing samples were collected from 223 women with microscopic finding of BV (n=98), aerobic vaginitis (n=25) and normal flora (n=100). Samples were tested for interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, and sialidase activity. RESULTS: Compared to women with normal flora, vaginal levels of IL-1ß were highly increased in both BV and AV (p<0.0001). Significantly higher vaginal IL-6 was detected in AV (p<0.0001) but not in BV, in relation to normal flora. Women with AV also presented increased IL-8 levels (p<0.001), while those with BV presented levels similar to normal flora. Sialidase was increased in BV and AV compared with the normal group (p<0.0001) but no difference in sialidase activity was observed between BV and AV. CONCLUSION: A more intense inflammatory host response occurs for AV than for BV when compared with normal flora. Furthermore, the increased sialidase activity in AV and BV indicates that both abnormal vaginal flora types can be harmful to the maintenance of a healthy vaginal environment.

Acceptance of self-testing for increased vaginal pH in different subsets of Ugandan women.

We assessed the acceptance of self-testing for vaginal pH in 344 Ugandan women in different clinical settings. Women tested themselves by insertion of a gloved finger into the vagina to test vaginal pH and provide a smear on a glass slide. None of the tested women found the test very difficult: 8% found it somewhat difficult, 16% rather easy and 76% very easy to do. Of the 20% who found it difficult to read the test result, more women were attending a family planning clinic or had a higher diploma (P = 0.001). Pregnant women were least likely to understand of the meaning of the test, while those visiting family planning clinics had the opposite experience. HIV-infected women were most motivated to accept: 95% would be happy to use the test more often if requested, and another 3.5% felt they might be better motivated to do repeat testing after extra explanation. Self-sampling of vaginal pH is well accepted by Ugandan women. Our new method also allows diagnostic work-up by formal microscopy. Before commencing large-scale population screening, unexpected reactions of different subpopulations should be taken into account.

Effect of lyophilized lactobacilli and 0.03 mg estriol (Gynoflor®) on vaginitis and vaginosis with disrupted vaginal microflora: a multicenter, randomized, single-blind, active-controlled pilot study.

OBJECTIVES: To evaluate the efficacy of lyophilized lactobacilli in combination with 0.03 mg estriol when compared to metronidazole in the treatment of bacterial vaginal infections. SETTING: Multicenter, randomized, single-blind, active-controlled pilot study in 3 independent gynecological practices in Belgium. METHODS: Forty-six, 18- to 50-year-old premenopausal women with a disrupted vaginal flora due to a bacterial vaginal infection (bacterial vaginosis, aerobic vaginitis) were included, provided that fresh phase-contrast microscopy of the vaginal fluid showed lactobacillary flora grade 2B or 3. Patients were given a blinded box with either 12 vaginal tablets of Gynoflor® (study medication) or 6 vaginal suppositories containing 500 mg metronidazole (control medication). Eight efficacy variables were studied to assess the status of the vaginal flora at entry, 3-7 days (control 1), 4-6 (control 2) weeks and 4 months after the end of therapy. RESULTS: At control 1, the combined variables equally improved in the lactobacilli group as in the metronidazole group. At control 2, the lactobacillus preparation showed slightly inferior results when compared to metronidazole. At 4 months, this analysis could not be performed due to low numbers, but analysis of recurrence rate and extra medication needed was not different between both groups. CONCLUSION: Lyophilized lactobacilli in combination with low-dose estriol are equivalent to metronidazole in the short-term treatment of bacterial vaginal infections, but have less effect after 1 month. Further studies are required to evaluate the long-term efficacy of lactobacilli when applied repeatedly.

Variability in diagnosis of clue cells, lactobacillary grading and white blood cells in vaginal wet smears with conventional bright light and phase contrast microscopy.

OBJECTIVE: Study the reproducibility of wet smear interpretation of clue cells, lactobacillary grades and leukocyte dominance with conventional bright light and phase contrast microscopy. STUDY DESIGN: Sets of vaginal specimens were taken from unselected consecutive women attending an outpatient gynaecology clinic. Air-dried vaginal fluid on a microscope slide was rehydrated with isotonic saline before examination by six independent international investigators. Some investigators initially used a conventional bright light microscope, followed by phase contrast technique. RESULTS: Using phase contrast microscopy, an excellent inter-observer agreement was obtained among all investigators for clue cells detection (Kappa values from 0.69 to 0.94) and lactobacillary grades (Kappa 0.73-0.93). When conventional light microscopes were used, poor agreement was obtained for these criteria (Kappa index 0.37-0.72 and 0.80, respectively), but switching to phase contrast microscopy by the same investigators, improved Kappa to 0.83-0.85 and 0.88, respectively. The inter-observer agreement for estimation of the leukocyte/epithelial cell ratio (Kappa index 0.17-0.67) was poor, irrespective of the type of microscopy applied. Intra-observer agreement of clue cell detection and lactobacillary grading was also found to be excellent if phase contrast microscopy was used (Kappa 0.87-0.93), and poor with conventional bright light microscopy (Kappa 0.45-0.66). CONCLUSION: Clue cells and the lactobacillary grades are reliably identified by phase contrast microscopy in wet smears, with excellent intra- and inter-observer reproducibility agreement, and better than when simple bright light microscopy was used. Evaluation of leukocyte grading, on the other hand, was inconsistent among the different microscopists, irrespective of the type of microscope used. We propose to grade the leukocytes in a different way than searching for leukocyte dominance over epithelial cells, namely by counting them per high power field and per epithelial cell.

Predictive value for preterm birth of abnormal vaginal flora, bacterial vaginosis and aerobic vaginitis during the first trimester of pregnancy.

INTRODUCTION: Abnormal vaginal flora (AVF) before 14 gestational weeks is a risk factor for preterm birth (PTB). The presence of aerobic microorganisms and an inflammatory response in the vagina may also be important risk factors. AIM: The primary aim of the study was to investigate the differential influences of AVF, full and partial bacterial vaginosis, and aerobic vaginitis in the first trimester on PTB rate. The secondary aim was to elucidate why treatment with metronidazole has not been found to be beneficial in previous studies. SETTING: Unselected women with low-risk pregnancies attending the prenatal unit of the Heilig Hart General Hospital in Tienen, Belgium, were included in the study. MATERIALS AND METHODS: At the first prenatal visit, 1026 women were invited to undergo sampling of the vaginal fluid for wet mount microscopy and culture, of whom 759 were fully evaluable. Abnormal vaginal flora (AVF; disappearance of lactobacilli), bacterial vaginosis (BV), aerobic vaginitis (AV), increased inflammation (more than ten leucocytes per epithelial cell) and vaginal colonisation with Candida (CV) were scored according to standardised definitions. Partial BV was defined as patchy streaks of BV flora or sporadic clue cells mixed with other flora, and full BV as a granular anaerobic-type flora or more than 20% clue cells. Vaginal fluid was cultured for aerobic bacteria, Mycoplasma hominis and Ureaplasma urealyticum. Outcome was recorded as miscarriage

Quantitative variations in the vaginal bacterial population associated with asymptomatic infections: a real-time polymerase chain reaction study.

The real-time polymerase chain reaction (PCR) quantification of several vaginal bacterial groups in healthy women and patients developing asymptomatic bacterial vaginosis (BV) and candidiasis (CA) was performed. Statistical analysis revealed that the BV condition is characterised by a great variability among subjects and that it is associated with a significant increase of Prevotella, Atopobium, Veillonella and Gardnerella vaginalis, and a drop in Lactobacillus. On the contrary, the vaginal microflora of healthy women and patients developing CA was found to be homogeneous and stable over time.

Mannose-binding lectin gene polymorphism and resistance to therapy in women with recurrent vulvovaginal candidiasis.

PRECIS: Women with recurrent vulvovaginal candidiasis (RVC) due to a polymorphism in codon 54 of the MBL2 gene respond better to fluconazole maintenance therapy than do women with other underlying causes. OBJECTIVE: To explain differences in response rates to maintenance therapy with fluconazole in women suffering from RVC by evaluating associations with a polymorphism in the gene coding for mannose-binding lectin (MBL). DESIGN: Follow-up study, neted case-control group. SETTING: Women attending vulvoginitis clinic for RVC. POPULATION: Women participating in a multicentric study in Belgium with a degressive dose of fluconazole for RVC (the ReCiDiF trial) were divided into good responders, intermediate responders and nonresponders according to the number of relapses they experienced during therapy. From 109 of these women with adequate follow-up data, vaginal lavage with 2 ml of saline were performed at the moment of a proven acute attack at inclusion in the study, before maintenance treatment was started. A buccal swab was obtained from 55 age-matched women without a history of Candida infections, serving as a control group. METHODS: Extracted DNA from buccal or vaginal cells was tested for codon 54 MBL2 gene polymorphism by polymerase chain reaction and endonuclease digestion. MAIN OUTCOME MEASURES: Frequency of MBL2 condon 54 allele B in women with optimal or poor response to maintenance therapy in composition with controls. Results Women (n = 109) suffering from RVC were more likely to carry the variant MBL2 codon 54 allele B than control women (20 versus 6.6%, OR 3.4 [95% CI 1.3-8.2], P = 0.01). B alleles were present in 25% of the 36 women not suffering from any recurrence during the maintenance therapy with decreasing doses of fluconazole (OR 4.9 [95% CI 1.9-12.5], P = 0.0007 versus controls), in 20% of the 43 women with sporadic recurrences (OR 3.6 [95% CI 1.4-9.2], P = 0.007 versus controls) and in 15% of the 30 women who had to interrupt the treatment regimen due to frequent relapses (P = 0.097 versus controls). CONCLUSIONS: The MBL2 codon 54 gene polymorphism is more frequent in Belgian women suffering from RVC than in controls. The presence of the B allele is associated with a superior response to fluconazole maintenance therapy as compared with RVC patients without this polymorphism. We conclude that RVC due to deficient MBL production is more easily helped with antifungal medication than is RVC due to some other mechanism.

Ultrasonographic features of the endometrium and the ovaries in women on etonogestrel implant.

OBJECTIVE: To evaluate the ultrasound features of the endometrium and ovaries in women on etonogestrel implant, and to correlate these features with the bleeding pattern. METHODS: Observational study including 188 consecutive women presenting for follow-up transvaginal ultrasound examination after insertion of an etonogestrel implant contraceptive device. Thirty women had more than one follow-up examination. The bleeding pattern was considered abnormal if, in the last 3 months, there were more than five episodes of vaginal bleeding, or there was prolonged bleeding exceeding 14 consecutive days. RESULTS: At first follow-up examination, the mean age was 29.7 years and 47% of women had an abnormal bleeding pattern. Most bleeding episodes were of less intensity than menses. The mean endometrial thickness (ET) on ultrasound was 2.9 mm (standard deviation, 2.0). Ovarian follicle growth exceeding 5 mm was observed in 60% of the cases. Ovulation was demonstrated in one woman. Univariate analysis showed a positive association (P < 0.01) between ET, bleeding pattern, and bleeding intensity. Follicle growth was positively associated (P < 0.01) with ET, bleeding pattern, and interval between insertion and examination. Multivariate analysis showed that the ET was on average 1.25 mm greater in women with abnormal bleeding (P = 0.0001). The odds of finding follicle growth were 2.8 times higher (95% confidence interval, 1.2-6.2) in women presenting with a three-layer type of endometrial morphology. There was no association between the other patients' characteristics and the bleeding pattern. CONCLUSIONS: Abnormal uterine bleeding in women on etonogestrel implant was associated with follicle growth and a thicker, three-layer type of endometrium, suggesting incomplete ovarian inhibition and estrogen stimulation of the endometrium.

Relationship of bacterial vaginosis and mycoplasmas to the risk of spontaneous abortion.

OBJECTIVE: This study was undertaken to investigate a possible link between first-trimester diagnosis of bacterial vaginosis and cessation of pregnancy at < or =20 weeks' gestation. STUDY DESIGN: Women (n = 228) who received routine prenatal care in Flanders, Belgium, during the first trimester (14 weeks' gestation) and had a living singleton fetus were examined for microbiologic flora of the vagina. Bacterial vaginosis was assessed either clinically (Amsel et al criteria), microscopically (clue cells), or by culture of bacterial vaginosis-associated bacteria. Data were analyzed univariately (relative risk) and multivariately. RESULTS: The presence of bacterial vaginosis at the first prenatal visit was strongly associated with subsequent early pregnancy loss (relative risk, 5.4; 95% confidence interval, 2.5-11). After multivariate analysis bacterial vaginosis, Mycoplasma hominis, and Ureaplasma urealyticum but not other microorganisms remained associated with an increased risk of miscarriage. CONCLUSION: Bacterial vaginosis and mycoplasmas may play causative roles in spontaneous abortion and early pregnancy loss.

Wet mount microscopy reflects functional vaginal lactobacillary flora better than Gram stain.

AIM: The status of vaginal lacto-bacillary flora, an indicator of possible genital infection and pregnancy complications, can be assessed on wet mount or Gram stained specimens. The former is quick, the latter more routine. The accuracy of the two preparative techniques to detect normal vaginal lacto-bacillary microflora was compared for 646 patients. The effect of delay in transport medium before Gram staining was also investigated. METHODS: Patients presented with infectious vaginitis or for a routine prenatal visit. After placement of a speculum, duplicate smears were taken from the upper vaginal vault and examined fresh or after Gram staining. Lacto-bacillary grades from both methods were compared with lactate concentration in vaginal rinses. In a subgroup of 238 patients, Gram staining was performed both on fresh smears and those that had been transported in Stuart's growth medium. RESULTS: Higher lacto-bacillary grades (more disrupted flora) were diagnosed 2.9 times more frequently on Gram stained specimens than on wet mounts (p < 0.0001), a difference even more pronounced after transport in Stuart's medium (relative risk, 4.2; p < 0.0001). Lacto-bacillary grades assessed on wet mounts correlated better with vaginal lactate concentration than those assessed on Gram stains. CONCLUSIONS: Easier recognition of lacto-bacillary morphotypes on wet mounts than on Gram stains might result from the loss of lactobacilli by the process of fixation or Gram staining. Wet mount microscopy of vaginal smears for assessment of lacto-bacillary grades, rather than Gram staining, is strongly recommended.

Treatment of sexually transmitted bacterial diseases in pregnant women.

Testing for and treating sexually transmitted diseases (STDs) in pregnant women deserves special attention. Treatment possibilities are limited because of potential risks for the developing fetus, and because effects can differ in pregnant compared with non-pregnant women, re-infection may be missed because of the intrinsic delicacy of contact-tracing during pregnancy and because pregnant women are more reluctant to take the prescribed medication in its full dose, if at all. However, the devastating effects of some of these genital infections far outweigh any potential adverse effects of treatment. Although active syphilis has become a rarity in most Western countries, it is still prevalent in South America, Africa and South-East Asia. Benzathine benzylpenicillin (2.4 million units once or, safer, twice 7 days apart) is the treatment of choice, although patients with syphilis of longer standing require 3 weekly injections as well as extensive investigation into whether there has been any damage due to tertiary syphilis. Despite declining rates of gonorrhea, the relative rate of penicillinase-producing strains is increasing, especially in South-East Asia. The recommended treatment is intramuscular ceftriaxone (125 or 250 mg) or oral cefixime 400 mg. Despite good safety records after accidental use, fluoroquinolones are contraindicated during pregnancy. An alternative to a fluoroquinolone in pregnant women with combined gonorrhea and chlamydial infection is oral azithromycin 1 or 2 g. Azithromycin as a single 1 g dose is also preferable to a 7 day course of erythromycin 500 mg 4 times a day for patients with chlamydial infection. Eradication of Haemophilus ducreyi in patients with chancroid can also be achieved with these regimens or intramuscular ceftriaxone 250 mg. Trichomonas vaginalis, which is often seen as a co-infection, has been linked to an increased risk of preterm birth. Patients infected with this parasite should therefore received metronidazole 500 mg twice daily for 7 days as earlier fears of teratogenesis in humans have not been confirmed by recent data. Bacterial vaginosis is also associated with preterm delivery in certain risk groups, such as women with a history of preterm birth or of low maternal weight. Such an association is yet to be convincingly proven in other women. The current advice is to treat only women diagnosed with bacterial vaginosis who also present other risk factors for preterm delivery. The treatment of choice is oral metronidazole 1 g/day for 5 days. The possible reduction of preterm birth by vaginally applied metronidazole or clindamycin is still under investigation. In general, both test of cure and re-testing after several weeks are advisable in most pregnant patients with STDs, because partner notification and treatment are likely to be less efficient than outside pregnancy and the impact of inadequately treated or recurrent disease is greater because of the added risk to the fetus. Every diagnosis of an STD warrants a full screen for concomitant genital disease. Most ulcerative genital infections, as well as abnormal vaginal flora and bacterial vaginosis, increase the sexual transmission efficiency of HIV, necessitating even more stringent screening for and treating of STD during pregnancy.

Pathogenesis of abnormal vaginal bacterial flora.

OBJECTIVE: This study was undertaken to determine the relationships between microscopy findings on wet mounts, such as lactobacillary grade or vaginal leukocytosis, and results of vaginal culture, lactate and succinate content of the vagina, and levels of selected cytokines. STUDY DESIGN: In a population of 631 unselected women seeking treatment at an obstetrics and gynecology outpatient clinic, vaginal fluid was obtained by wooden Ayre spatula for wet mounting and pH measurement, by high vaginal swab for culture, and by standardized vaginal rinsing with 2 mL 0.9% sodium chloride solution for measurements of lactate, succinate, interleukin 1beta, interleukin 8, leukemia inhibitory factor, and interleukin 1 receptor antagonist concentrations. Lactate and succinate levels were measured by gas-liquid chromatography and the cytokine concentrations were measured by specific immunoassays. Both univariate analysis (Student t test, Welch test, chi(2) test, and Fisher exact test) and multivariate regression analysis (Cox analysis) were used. RESULTS: Increasing disturbance of the lactobacillary flora (lactobacillary grades I, IIa, IIb, and III) was highly correlated with the presence of Gardnerella vaginalis, Trichomonas vaginalis, enterococci, group B streptococci, and Escherichia coli. Vaginal pH and interleukin 8 and interleukin 1beta concentrations increased linearly with increasing lactobacillary grade, whereas lactate concentrations and the presence of epithelial cell lysis decreased. A similar pattern of associations with increasing leukocyte count was clear, but in addition there was an increase in leukemia inhibitory factor concentration. Multivariate analysis of vaginal leukocytosis, lactobacillary grades, and the presence of positive vaginal culture results showed that interleukin 1beta concentration was most closely related to the lactobacillary grade, leukemia inhibitory factor concentration was most closely related to the lactobacillary grade and positive culture results, interleukin 8 concentration was most closely related to positive culture results, and interleukin 1 receptor antagonist concentration was most closely related to vaginal leukocytosis and positive culture results. The concentration ratio of interleukin 1beta to interleukin 1 receptor antagonist remained stable, except when vaginal leukocytosis increased. In its most severe form, with >10 leukocytes per epithelial cell present, a decompensation of the vaginal flora with a collapse in interleukin 1beta and interleukin 1 receptor antagonist concentrations was seen, but there was a concurrent sharp increase in leukemia inhibitory factor concentration. This pattern was completely different from the course of the cytokine concentrations associated with a lactobacillary grade increase. CONCLUSION: Both disturbed lactobacillary grade and the presence of increasing vaginal leukocytosis were correlated with lactobacillary substrate (lactate) concentration, pH, and the concentrations of a variety of cytokines. There was a remarkably linear increase in these cytokines as either leukocytosis or lactobacillary grade became more severe. In circumstances in which leukocytosis was extreme, however, interleukin 1beta was no longer produced but leukemia inhibitory factor concentrations increased. We speculate that in extreme inflammation the body tries to limit the damage that can be done by exaggerated cytokine production.