Quantification of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp. in bacterial vaginosis.

INTRODUCTION: The aim of the study was to investigate prevalence of bacteria most frequently associated with bacterial vaginosis using Amsel's criteria as well as to quantify these bacteria by real-time PCR and to explore the difference in their quantity between healthy and bacterial vaginosis samples. METHODOLOGY: For classification of vaginal discharge samples Amsel's criteria have been used. To detect and quantify Gardnerella vaginalis Atopobium vaginae, Lactobacillus spp. and total vaginal microbiome, real-time PCR has been applied. RESULTS: According to results of our study Amsel's criteria matched well with real-time PCR diversification of healthy women and women with BV. Nevertheless, real-time PCR has been more sensitive in diagnosis of bacterial vaginosis. DNA quantification of bacteria demonstrated that mutual abundance of G.vaginalis and A. vaginae was good bacterial vaginosis marker . On the contrary, Lactobacillus spp. was present in high amount in both healthy and bacterial vaginosis samples, but ratio of investigated bacteria was different between them. In fact, G. vaginalis and A. vaginae comprised only 0.1% of total microbiome in healthy, whereas Lactobacillus spp. took 99.3% of it. Nonetheless, in bacterial vaginosis, G. vaginalis and A. vaginae made up 34.4% of total microbiome, while Lactobacillus spp. was 21.6%. CONCLUSIONS: According to the results of our study real-time PCR analysis was more sensitive in diagnosis of bacterial vaginosis than Amsel's method, as well as it represented fine tool in making a difference between microbial entities in healthy and bacterial vaginosis samples.

A novel microscopic method for analyzing Gram-stained vaginal smears in the diagnosis of disorders of vaginal microflora.

BACKGROUND/AIM: The Nugent's score is still the gold standard in the great majority of studies dealing with the assessment of vaginal flora and the diagnosis of bacterial vaginosis (BV). The aim of this study was to show that the analysis of Gram-stained vaginal samples under microscope at the magnification of x200 (a novel microscopic method--NMM), as a fast and simple tool, easily applicable in everyday practice, better reflects complexity of vaginal microflora than the Nugent's methodology (x1000). METHODS: Gram-stained vaginal smears from 394 asymptomatic pregnant women (24-28 week of pregnancy) were classified according to the Nugent's microscopic criteria (immersion, magnification x1000). The smears were then reexamined under immersion but at magnification x200. All samples were classified into 6 groups according to semiquanititative assessment of numbers (cellularity) and the ratio of rod (length < 1.5 microm) and small bacterial (< 1.5 microm) forms: hypercellular (normal full--NF), moderately cellular (normal mid-NM), hypocellular (normal empty--NE), bacterial vaginosis full (BVF), bacterial vaginosis mid (BVM), and bacterial vaginosis empty (BVE). Also yeasts, coccae, bifido and lepto bacterial forms as well polymorphonuclear (PMN) leukocytes were identified. RESULTS: According to the Nugent's scoring, BV was found in 78, intermediate findings in 63, and yeasts in 48 patients. By our criteria BV was confirmed in 88 patients (37 BVF, 24 BVM, and 27 BVN). Generally, both tools proved to be highly concordant for the diagnosis of BV (Lin's concordance correlation coefficient = 0.9852). In 40% of the women mixed flora was found: yeasts in 126 (32%), coccae in 145 (37%), bifido forms in 32 (8%) and lepto forms in 20 (5%). Almost a half of BV patients had also yeasts (39/88). Elevated PMN numbers were found in 102 (33%) patients with normal and in 36 (41%) women with BV. CONCLUSION: The newly described methodology is simpler to apply and much better reflects diversity of vaginal microflora. In this way it may be more valuable to molecular biologists and their attempts based on quantitative polymerase chain reaction (PCR) to define formulas for molecular diagnosis of bacterial vaginosis.

Value of bacterial culture of vaginal swabs in diagnosis of vaginal infections.

BACKGROUND/AIM: Vaginal and cervical swab culture is still very common procedure in our country's everyday practice whereas simple and rapid diagnostic methods have been very rarely used. The aim of this study was to show that the employment of simple and rapid diagnostic tools [vaginal fluid wet mount microscopy (VFWMM), vaginal pH and potassium hydroxide (KOH) test] offers better assessment of vaginal environment than standard microbiologic culture commonly used in Serbia. METHODS: This prospective study included 505 asymptomatic pregnant women undergoing VFWMM, test with 10% KOH, determination of vaginal pH and standard culture of cervicovaginal swabs. Combining findings from the procedures was used to make diagnoses of bacterial vaginosis (BV) and vaginitis. In addition, the number of polymorphonuclear leukocytes (PMN) was determined in each sample and analyzed along with other findings. Infections with Candida albicans and Trichomonas vaginalis were confirmed or excluded by microscopic examination. RESULTS: In 36 (6%) patients cervicovaginal swab cultures retrieved several aerobes and facultative anaerobes, whereas in 52 (11%) women Candida albicans was isolated. Based on VFWMM findings and clinical criteria 96 (19%) women had BV, 19 (4%) vaginitis, and 72 (14%) candidiasis. Of 115 women with BV and vaginitis, pH 4.5 was found in 5, and of 390 with normal findings 83 (21%) had vaginal pH 4.5. Elevated numbers of PMN were found in 154 (30%) women--in 83 (54%) of them VFWMM was normal. Specificity and sensitivity of KOH test and vaginal pH determination in defining pathological vaginal flora were 95% and 81%, and 79% and 91%, respectively. CONCLUSION: Cervicovaginal swab culture is expensive but almost non-informative test in clinical practice. The use of simpler and rapid methods as vaginal fluid wet mount microscopy, KOH test and vaginal pH offers better results in diagnosis, and probably in the treatment and prevention of sequels of vaginal infections.

[Primary ovarian malignant melanoma].

BACKGROUND: Primary ovarian malignant melanoma is extremely rare. It usually appears in the wall of a dermoid cyst or is associated with another teratomatous component. Metastatic primary malignant melanoma to ovary from a primary melanoma elsewhere is well known and has been often reported especially in autopsy studies. CASE REPORT: We presented a case of primary ovarian malignant melanoma in a 45-year old woman, with no evidence of extraovarian primary melanoma nor teratomatous component. The tumor was unilateral, macroscopically on section presented as solid mass, dark brown to black color. Microscopically, tumor cells showed positive immunohistochemical reaction for HMB-45, melan-A and S-100 protein, and negative immunoreactivity for estrogen and progesteron receptors. CONCLUSION: Differentiate metastatic melanoma from rare primary ovarian malignant melanoma, in some of cases may be a histopathological diagnostic problem. Histopathological diagnosis of primary ovarian malignant melanoma should be confirmed by immunohistochemical analyses and detailed clinical search for an occult primary tumor.

[Reliability of the CINtec p16INK4a immunocytochemical test in screening cervical precancerous lesions].

BACKGROUND/AIM: Overexpression of p16INK4a has been found to be linked with genomic integration of high-risk human papillomavirus (HPV) and the developement of precancerous cervical intraepithelial lesions. The aim of this study was to examine is there a higher positive level of correlation between grade of histological dysplasia and p16INK4a level of expression in cervical smear, compared to results of Papanicolaou test. We also examined the correlation between HPV type, p16INK4a expression and Papanicolau test results. METHODS: A total of 48 women with precanceorous cervical lesions and HPV cervicitis and 10 healthy women were enrolled in the study. Papanicolaou test, CINtec p16INK4a cytological immunohistochemical test, polymerase chain reaction (PCR) HPV 16, 18, 31, 33 analysis and histopathology of the lesion were performed in all the patients. RESULTS: Comparing the results of Papanicoulaou test and the grade of histological dysplasia, low-grade squamous intraepithelial lesion (LSIL) was confirmed in 38%, and high-grade squamous intraepithelial lesion (HSIL) in 69.2% of the patients (p > 0.05). Significant positive correlation was found between p16 overexpression and grade of histological dysplasia (p = 0.000). Overexpression p16 was found in 70% of LSIL and 94.4% of HSIL. Positive correlation was found between p16 overexpression and grade of dysplasia in Papanicolaou test (p = 0.011). In 38% of LSIL and 15% of HSIL cases p16 was not expressed. The most frequently found HPV type in PCR analysis was HPV16. Analysing the results of p16 test according to HPV status and Papanicolaou test rather heterogenous results were obtained. CONCLUSION: In the patients with precancerous cervical lesions a higher level of correlation was found between the grade of histological dysplasia and p16INK4a level of expression in the cervical smear, compared to the results of Papanicolaou test.

Cervical fluid cytokines in pregnant women: Relation to vaginal wet mount findings and polymorphonuclear leukocyte counts.

OBJECTIVE: The purpose of the study was to measure and compare concentrations of IFN-gamma, IL-8, IL-10 and IL-12 in cervical fluids of pregnant women with normal and pathologic microscopic findings of vaginal wet mount. STUDY DESIGN: Vaginal samples were obtained from 100 pregnant women (22-32 weeks of gestation) without signs and symptoms of preterm labour (PTL), selected on the basis of the microscopic examination of vaginal wet mounts. Cytokine (IFN-gamma, IL-8, IL-10 and IL-12) concentrations were measured in cervical fluids by an enzyme-linked immunosorbent assay. RESULTS: The cytokines were found in cervical fluids of the majority of pregnant women, with IFN-gamma and IL-8 detected in all samples. Concentrations of IL-8 were significantly higher in pregnant women with pathologic findings on vaginal wet mount, including elevated polymorphonuclear (PMN) cell counts. CONCLUSION: A significant correlation between microscopic findings and cervical fluid IL-8 concentrations may make vaginal wet mount microscopy a useful tool for identification of patients with cervicovaginal inflammation, which is considered a risk factor for spontaneous preterm delivery.