Background and ObjectiveBacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.Materials and MethodsVaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3–V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated.ResultsRelative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor.ConclusionsQuantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.

Diagnosing bacterial vaginosis (BV) has long been based on the clinical criteria of Amsel et al., whereby three of four defined criteria must be satisfied. Though there are other criteria and scoring methods which function well in comparison (i.e. Nugent scoring), it is not certain that they will always identify the same category of patients. Point-of-care methods based on various combinations of microbial products, presence of RNA, or more complex laboratory instrumentations such as sensor arrays, have also been introduced for the diagnosis of BV. No method for diagnosing BV can at present be regarded as the best. It could be that--based partly on tacit knowledge on the part of the clinical investigators scoring in the clinic--various scoring systems have been chosen to fit a particular BV-related problem in a particular population. In this review we critically examine these pertinent issues influencing clinical scoring and laboratory diagnostics of BV.

The laboratory diagnosis of sexually transmitted infections in many Eastern European countries remains suboptimal.The main objective of the present evidence-based guidelines is to provide comprehensive information regarding the laboratory diagnosis of infections caused by Trichomonas vaginalis in East European countries. In particular, the present guidelines recommend: (i) to encourage examination of the wet mounts of vaginal exudates, instead of stained smears, at all clinical settings; (ii) nucleic acid amplification tests (NAATs) or culture could be employed if no trichomonads are detected on microscopic examination of the wet preparation and there is a strong indication of infection and (iii) the use of NAATs is encouraged in screening, using non-invasive specimens, or high volume testing situations. In the absence of internationally recognized commercial NAAT systems, tests developed in-house should be validated using obtainable international standards and quality assured strictly. Individual East European countries may be required to make minor national adjustments to these guidelines as a result of lack of accessibility to some reagents or equipment, or laws in a specific country.

interest,4 as it has been isolated almost exclusively from women with BV.5 6 In contrast, the first organism associated with BV, Gardnerella vaginalis, has been isolated from about 50% of clinically healthy women.78 The position of Mobi-

The presence of a fishy odor emanating from women who present with a malodorous vaginal discharge is well known. The odor is due to bacterial reduction of trimethylamine oxide to trimethylamine (TMA) in vaginal secretion. The release of TMA from specimens of vaginal fluid following the addition of alkali is often used in making a clinical diagnosis of bacterial vaginosis (BV). We now report a sensitive gas chromatographic method for analysis and quantification of TMA in vaginal fluid in which weighed samples were used. In addition, a proper diagnosis of BV was obtained using Gram-stained smears of the vaginal fluid according to the method of Nugent et al. (R. P. Nugent et al., J Clin Microbiol 1991;29:297-301). We also diagnosed BV according to Hallén et al. (A. Hallén et al. Genitourin Med 1987;63:386-9). TMA was present in all women with a Nugent score between 7 and 10 and in almost all women diagnosed with BV according to the method of Hallén et al. TMA was not found or was only found in very low concentrations in vaginal fluid from women with Nugent scores of 0 to 3. TMA was also found in four women with a negative sniff test. It seems that high levels of TMA in samples of vaginal fluid are typical for BV regardless of the scoring method used for diagnosis. However, low levels of TMA, <5 microg/g vaginal fluid, do not always correlate with BV.

There were significant shortcomings in STI testing availability and practices. Among all participating laboratories diagnosing STIs, only a minority (n = 16, 28%) offered testing for the full minimum range of relevant STIs in Estonia, i.e., Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. In addition, because testing methods used were not properly selected, confirmation of several infections in accordance with evidence-based requirements was not possible, which has an impact both on STI diagnostic quality and surveillance.

The present guidelines aim to provide comprehensive and precise information regarding the laboratory diagnosis of the sexually transmitted infection (STI) syphilis in East European countries. These recommendations contain important information for laboratory staff working with STIs and/or STI-related issues. Individual East European countries may be required to make minor national adjustments to these guidelines as a result of lack of accessibility to some reagents or equipment, or laws in a specific country.

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