Quantification of normal vaginal constituents using a new wet preparation technique.

OBJECTIVE: This study aimed to evaluate a new method for preparing vaginal wet preparations to enable quantification of cells and lactobacilli. The current nonstandardized technique allows for a variable amount of vaginal fluid collected, diluted by a variable amount of saline/KOH, and no quantification of constituents. MATERIALS: The vaginal fluids from 100 randomly selected women without vulvovaginitis symptoms presenting to the author's practice at Mayo Clinic underwent analysis by the quantification technique. Women were excluded if they were younger than 18 years, had antibiotics within the past 2 months, currently on their period, had placed anything in the vagina for the past 24 hours, used Depo-Provera, or were lactating. METHODS: All the wet preparations were made by mixing the natural vaginal fluids with 3 mL of sterile normal saline. Spinal diluting fluid was added to the saline preparation. The saline and KOH mixtures were injected into separate wells of KOVA Glasstic Grid Slide and analyzed with a phase-contrast microscope at 40× and 60×. The concentration of leukocytes, lactobacilli, and squamous cells and the degree of maturation of the majority (>50%) of squamous cells were assessed, and it was determined whether there was excessive non-lactobacilli bacteria (EB) as evident by clumps of bacteria in the background fluid and speckling on the squamous cells. RESULTS: The 3 most common patterns to occur were as follows: First, 51% (95% confidence interval [CI] = 41%-60%) of the total specimens had abundant lactobacilli, no leukocytes, more than 50% fully maturated squamous cells, and no EB. Second, 22% (95% CI = 14%-32%) of the total specimens had low lactobacilli counts, no leukocytes, more than 50% undermaturated squamous cells, and no EB. Third, 12% (95% CI = 6%-20%) of the total specimens had abundant lactobacilli, leukocytes, more than 50% fully maturated squamous cells, and no EB. CONCLUSIONS: It is imperative to be able to objectively quantify normal vaginal secretion constituents so that (1) the abnormal patterns can be demarcated and (2) treatment targets of what constitutes healthy vaginal conditions can be provided.

Expansion of altered vaginal flora states in vaginitis to include a spectrum of microflora.

OBJECTIVE: To define the existence of 2 patterns of altered vaginal flora in symptomatic women identified on wet preparations that are not in the current vaginitis classification system. STUDY DESIGN: Testing of vaginal secretions from gynecologic patients at Mayo Clinic, Scottsdale, Arizona, who presented with vulvo-vaginal symptoms by vaginal pH, whiff testing, and saline and potassium hydroxide wet preparations. Over 14 years, approximately 5,000 samples were analyzed. Wet preparations were analyzed under low- (x 100) and high-power (x 400) phase-contrast microscopy. RESULTS: The first pattern has mixed bacteria with few or no lactobacilli, increased leukocytes and elevated pH. This pattern has a spectrum of severity, with the severe form meeting the diagnostic definition of desquamative inflammatory vaginitis. The mild-to-moderate form, termed inflammatory vaginitis, falls outside established diagnostic categories. The second pattern also has a spectrum of severity. The mild-to-moderate form, termed noninflammatory vaginosis, has mixed bacteria speckling squamous cells (but not obscuring edges), few or no lactobacilli, no leukocyte response and mildly elevated pH. This form differs from the severe form, which meets Amsel's criteria for bacterial vaginosis. CONCLUSION: The current classification system requires revision because it oversimplifies and ignores the full spectrum of altered vaginal microflora.

Activated lactoferrin's ability to inhibit Candida growth and block yeast adhesion to the vaginal epithelial monolayer.

OBJECTIVE: To study in vitro growth-inhibitory effects of activated lactoferrin (ALF) against vaginal isolates of Candida species and to measure the ability of ALF to block interactions of Candida albicans and Candida glabrata to the vaginal epithelial (VE) monolayer. STUDY DESIGN: In vitro effects of ALF on growth of C albicans and C glabrata in Sabouraud dextrose (SD) broth were measured as change in broth turbidity by microscale optical density assay. ALF was tested at 5 and 2.5 mg/mL concentrations against 105 yeast cell inoculum at 370 degrees C for 96 hours and compared with native lactoferrin and control (growth in broth without ALF). VE cells were isolated from human vaginal tissue biopsies to establish a functional monolayer for yeast interaction studies. ALF effects on Candida interactions with the VE monolayer were tested using 3H-thymidine-labeled yeast. Prophylactic (treatment prior to yeast inoculation onto VE) and therapeutic (treatment to detach VE-adherent yeast) potential of ALF (5 mg/mL) was evaluated against vaginal isolates of C albicans strain NTRL809A and C glabrata strain NTRL131G. RESULTS: Growth of Candida species indicated that a 105 yeast inoculum in SD broth proliferated to a stationary growth equilibrium (approximately 10(9) yeast cell density) in 18 hours (approximately 2 hours of generation time). ALF (5 mg/mL) elicited >96 hours of total stasis (100% growth inhibition) and was significantly effective against both Candida species (p < 0.0001). At 2.5 mg/mL dilution, ALF sustained total stasis activity to an average of 18 hours and 24 hours for C albicans (n = 5) and C glabrata (n = 5), respectively. Interaction studies indicated avid binding of C albicans (70 - 140 x 10(3) yeast) and C glabrata (50 - 75 x 10(3) yeast) per square centimeter of VE monolayer. ALF-treated VE showed significant blockade (p < 0.05) of yeast adhesion by 33% and 58% with C albicans and C glabrata, respectively. ALF treatment of yeast-VE complexes resulted in significant detachment (p < 0.05) of C albicans and C glabrata, by 58% and 51%, respectively. CONCLUSION: ALF is a natural fungistatic agent with potent yeast adhesion-blocking and detachment properties and is effective against the vaginal pathogens C albicans and C glabrata.

Activated lactoferrin and fluconazole synergism against Candida albicans and Candida glabrata vaginal isolates.

OBJECTIVE: To evaluate the fungistatic activity of activated lactoferrin (ALF), fluconazole (FCN) individually and in combination against Candida vaginal isolates as well as to measure the time to recovery from the fungistatic effects after exposure in vitro to threshold minimal inhibitory concentrations (MIC). STUDY DESIGN: Fungistasis patterns for ALF (2.5 mg/mL) and FCN (0.25 mg/mL) were tested at threshold MIC against vaginal isolates of C albicans (n = 5) and C glabrata (n = 5) grown in Sabouraud's dextrose broth against 10(5) yeast inoculum at 37 degrees C for 48 hours by microscale optical density (OD) assay according to the following criteria: "Total stasis" indicates that an agent elicited no change or a change in turbidity <0.1 OD unit for >48 hours (complete growth inhibition), "stasis recovery" (SR) is the time point at which turbidity of a previous stasis system shows an upward growth trend for >0.1 OD unit (recovery from growth inhibition), and "partial stasis" (PS) is proliferation after stasis recovery, measured as a percentage relative to growth control at any time (incomplete growth inhibition). RESULTS: For ALF (2.5 mg/mL), the mean SR time was 15.6 +/- 2 hours for C albicans (n = 5) and 27.5 +/- 2 hours for C glabrata (n = 5). The SR patterns for FCN were strain dependent and showed a wide range of deviation for both Candida species; accordingly, the values were 15.8 +/- 9 hours for C albicans and 25.5 +/- 12 hours for C glabrata. After 48 hours exposure to C albicans, ALF and FCN elicited a mean PS of 27.5 +/- 2% and 24.8 +/- 7%, respectively. The PS values at 48 hours showed a marked variation between C glabrata isolates, 29.1 +/- 24% for ALF and 21.5 +/- 38% for FCN. However, a combination of ALF and FCN at their threshold MIC showed significant drug synergism, causing total stasis of both species of Candida isolates. Thus, no SR for any Candida isolate was detected at or beyond 48 hours. Conversely, native lactoferrin failed to demonstrate such potent synergism with FCN against either Candida species. CONCLUSION: The combination of ALF and FCN at the threshold MIC elicited potent synergism, leading to total fungistasis of C albicans and C glabrata vaginal pathogens. ALF is a new class of fungistatic agent with a mode of action distinct from that of azoles.