Vaginal microbiome.

OBJECTIVE: Presentation of complex information about the vaginal microbiota from historical view to current concepts with focus on latest findings on the structure and functioning of the vaginal microbiome. DESIGN: Review article. SETTING: Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradci Králové, Charles University in Prague. METHODS: Literature review using the databases (PubMed, Web of Science, Ovid, etc.) with keywords (vaginal microbiota/ microbiom; vaginal discharge; bacterial vaginosis; vulvovaginitis; vaginal Lactobacillus). RESULTS: The vaginal microbiome is a specific compartment of the human microbiome. Unique conditions of the vagina are characterized by a few microbial species, usually lactobacilli, which are able to utilize glycogen, which is under control of estrogens. Lactobacilli and other fermentative bacteria together with vaginal epithelial cells produce lactic acid and are responsible for acidifying vaginal milieu. Lactic acid occurs in two isomeric forms, and their relative ratio is likely to give the vaginal microbiota a certain degree of stability and ability to withstand some infections. This microbiota is manifested by a low degree of diversity and by the high dynamics of changes of its composition under the influence of various exogenous and endogenous factors. Increase in diversity can be paradoxically associated with a dysbiosis such as bacterial vaginosis. Individual species of lactobacilli mainly Lactobacillus crispatus characterize the main community state types in the vagina. Apart from lactobacilli, healthy women may be colonized with a non-lactobacillary microbiota whose rate is dependent on ethnicity. CONCLUSION: The definition of vaginal microbiota cannot be only related to the presence or absence of individual microorganisms, although the incidence of some of them can be correlated with dysbiosis or eubiosis. The composition of microbiota is important, but it is only one of the basic attributes of normal vaginal microbiota, but not sufficient; that is the functional definition of vaginal microbiota in relation to its structure and dynamics, including the influence of ethnicity, physiological status of the vagina, and genetic disposition of woman.

[Contemporary technicqes in diagnostic patients with vaginal microbiota]. / Soucasné vyuzití technik v diagnostice recidivujícího vulvovaginálního dyskomfortu.

OBJECTIVE: Review of literature dealing with the specificity of the vagina from the standpoint of microbial colonization. DESIGN: Literature review. SETTING: Department of Obstetrics and Gynecology, Department of Clinical Microbiology, University Hospital and Medical Faculty in Hradec Kralove, Charles University Prague. METHODS: Analysis of the literature review and personal experience dealing with vaginal microbiota.

High frequency of Candida fabianii among clinical isolates biochemically identified as Candida pelliculosa and Candida utilis.

Clinical yeast isolates belonging to Candida pelliculosa, Candida utilis and Candida fabianii are difficult to distinguish in a routine mycology laboratory using common biochemical tests. The aims of this study were to determine the prevalence of C. pelliculosa, C. utilis and C. fabianii in clinical samples and to compare their minimum inhibitory concentrations (MICs) to systemic antifungals. Two hundred and forty-eight clinical yeast isolates obtained from eight large hospitals in the Czech Republic were included in this study. Identification was performed biochemically using ID 32C kit and by MALDI-TOF MS. MICs were determined using colorimetric broth dilution Sensititre YeastOne panels. From a total number of 248 isolates, 175 were identified as C. pelliculosa and 73 as C. utilis using the biochemical kit. In contrast, MALDI-TOF MS identified 222 isolates as C. fabianii, 20 as C. pelliculosa and 6 as C. utilis. The highest mean MICs were found in C. fabianii and, regardless of the studied species, in isolates from blood cultures and central venous catheters. MALDI-TOF MS revealed C. fabianii to be most prevalent in clinical samples as compared with the other studied species. Higher MIC values in C. fabianii support the importance of correct identification of this species.

[What is the current stage of vulvovaginal discomfort diagnostics in the Czech Republic? Pilot analysis]. / Jaký je aktuální stav diagnostiky vulvovaginálního dyskomfortuv Ceské republice? Pilotní analýza.

OBJECTIVE: To find out the level of the diagnostic effort of gynaecologists which is focused on the issue of vulvovaginal discomfort. DESIGN: Pilot questionnaire study. SETTINGS: Department of Obstetrics and Gynecology, Department of Clinical Microbiology, University Hospital and Medical Faculty Hradec Králové, Charles University, Prague, Department of Biological and Medici Sciences, FaF UK in Hradec Králové. METHODS AND RESULTS: The evaluation of selected parametres of entrance questionnaire in patients with chronic vulvovaginal discomfort (itching, burning, discharge, vulvodynia more than 4 times a year). It has been confirmed, with the questionnaires, that almost no gynaecologists are interested in the issue of this matter. CONCLUSION: After evaluating the questionnaires a diversion of patient-tailored attitude has been found out, which can lead to negative consequences, particularly, in the future. Overuse of antibacterial and antimycotic medication and blind treatment have been prevailing.

[Is diabetes mellitus a risk factor in genital yeast infections?]. / Je diabetes mellitus rizikový faktor kvasinkového posevního zánetu?

OBJECTIVE: To analyze experimental and clinical data on diabetes mellitus (DM) related to infections with focus on vaginal mycosis. To evaluate a role of DM in the epidemiology of vulvovaginal candidiasis. DESIGN: Review. SETTING: Department of Clinical Microbiology, Department of Biological and Medical Sciences, Faculty of Pharmacy, Department of Obstetrics and Gynecology, Charles University in Prague, Faculty of Medicine and University Hospital Hradec Kralove. METHODS: Review of literature data. RESULTS: DM is a risk factor for fungal infections caused by yeasts (Candida albicans), members of Mucorales fungi, some dimorphic fungi (Coccidioides) and agents of onychomycosis. DM is usually associated only with increased colonization of the anatomical sites (oral cavity, vagina), and/ or with an intensified symptomatology of infection (onychomycosis, mucormycosis). Diabetic patients with oropharyngeal and vulvovaginal candidiasis have frequently changed etiology spectrum. The patients with VVC and DM, especially of older age or with prone to relapses, have tendency to shift of the spectrum to non-albicans species, mainly C. glabrata. Treatment of VVC in diabetic patients can be complicated owing to unfavourable antifungal susceptibility profile (C. glabrata) and/ or adverse interactions between some azole antifungals and sulfonylurea-based antidiabetics. CONCLUSION: Diabetes mellitus is often cited as a risk factor, although in many cases there is a lack of reliable and clinically relevant information. This does not mean that this disease can be underestimated. On the contrary, it is necessary to get the DM as soon as possible under control and thus prevent complications when infection develops. Individual approach should be applied to the diabetic patients at risk of an infection. Yeast colonization and development of vaginal infection is complex process primarily dependent on sexual hormones, indigenous microbiota and finely tuned mechanisms of local immunity. Role of DM consists in the fact that it is one of the important co-factors that can change the setup of the vaginal environment in favour of yeasts and thus promote or facilitate the development of VVC.

[Vulvodynia and the possibility of its management]. / Nový pohled na problematiku vulvodynie.

OBJECTIVE: A study of vulvodynia and the possibility of its management. SUBJECT: Original study. SETTING: Department of Obstetrics and Gynecology, Teaching Hospital and Medical Faculty Hradec Králové, Charles University, Prague. Department of Neurology, Teaching Hospital and Medical Faculty Hradec Králové, Charles University, Prague. Outpatient psychiatric department Galenus, Hradec Králové. Department of Clinical Microbiology, Teaching Hospital and Medical Faculty Hradec Králové, Charles University, Prague. Department of Biological and Medical Sciences, Faculty of Pharmacy Hradec Králové, Charles University, Prague. METHODS: Analysis and discussion focused on our experience in a long time follow-up of patients with chronic vulvovaginal discomfort and vulvodynia. CONCLUSION: Especially the management of indolent form of vulvodynia would be taken into consideration of idiopathic origin and problematic management. Our study emphasizes the necessity of individual approach. The goal of therapy in this most problematic form is the improving of the actual quality of life. In the case of idiopathic forms of vulvodynia.

[Possibilities of diagnostics yeasts in gynecologist patients]. / Jak dáIe v diagnostice kvasinek u gynekologickch pacientek?

Problem of mycoses in gynecology is considered very frequent and yeasts are traditionally described as most common cause of vulvovaginal disorders. While acute disorders can be promptly cured in most cases, there is a group of women suffering from those repeatedly. Following article refers to a group of patients in long-term follow-up and shows realistic possibilities of diagnostics, available to out-patient gynecologist. Preliminary results of our pilot project do not confirm yeasts have to be the primary etiologic factor, nevertheless it is logical to be in search of them in these patients.

[Penetration of ceftriaxone into the cerebrospinal fluid and its relationship to inflammatory markers during bacterial meningitis]. / Prunik ceftriaxonu do likvoru a jeho v vztah k markerum zánetu v prubehu invazivní bakteriální infekce.

AIM: To evaluate the penetration of ceftriaxone into the cerebrospinal fluid (CSF) in patients with invasive bacterial infection and to define correlation between the penetration and laboratory markers of inflammation. MATERIAL AND METHODS: Levels of ceftriaxone in the serum and CSF of 17 patients with purulent meningitis were examined. Serum concentrations of ceftriaxone before and after its administration were measured in 9 patients (18 samples, 52.9 %) by microbiological assay based on the agar diffusion test. In all patients, the CSF/serum quotient for ceftriaxone was calculated and correlated with laboratory markers of inflammation (C-reactive protein, fibrinogen and neutrophils). The CSF from nine patients with positive culture for bacteria was used for a modified bactericidal test. RESULTS: Ceftriaxone levels in the serum before and after administration (31.2 mg/l -/+ SD 12.29 and 300.0 mg/l -/+ SD 125.9, respectively) were different (p = 0.000156). The decrease of ceftriaxone levels in the CSF was gradual. There was also a significant difference between the levels of inflammatory markers and CSF/serum quotient of ceftriaxone. Patients with the values higher than 0.1 had higher CRP serum levels (p = 0.00192), fibrinogen serum levels (p = 0.0178) as well as neutrophil count in the CSF (p = 0.0112). However, no inflammatory markers (or their combinations) predicted the extent of penetration of ceftriaxone into the CSF. CONCLUSION: High serum concentration of ceftriaxone causes higher penetration through the inflamed blood-brain barrier. Higher antibiotic penetration correlated with the extent of systemic inflammatory response. However, no inflammatory marker predicted the rate of ceftriaxone crossing the blood-brain barrier. Ceftriaxone penetration, with a 24-hour regimen of administration, remains reliable and efficient therapy of purulent meningitis.

Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region.

The rapid identification of fungal pathogens in clinical specimens is a prerequisite for timely onset of the most appropriate treatment. The aim of the present study was to develop a sensitive and rapid method for the species-specific identification of clinically relevant fungi. We employed fluorescent polymerase chain reaction (PCR)-fragment length analysis of the highly variable internally transcribed spacer 2 (ITS2) region to identify individual fungal species by their specific amplicon sizes. The specificity of the technique was ascertained by the detailed analysis of 96 strains derived from 60 different human-pathogenic fungal species. To achieve adequate sensitivity for species identification in patients with invasive fungal infection, who often display very low pathogen loads in peripheral blood, the ITS2 region was amplified by semi-nested PCR prior to amplicon-length analysis. Serial specimens from 26 patients with documented fungal infections were investigated. The fungal pathogens identified included different Aspergillus and Candida species, Rhizopus oryzae and Fusarium oxysporum. Fragment length analysis of the ITS2 region upon amplification by semi-nested PCR permits the sensitive identification of fungal species. The technique can be readily implemented in routine diagnostics.

Comparison of disk diffusion test and Etest for voriconazole and fluconazole susceptibility testing.

The distribution for voriconazole and fluconazole susceptibility was determined by Etest and disk diffusion test in 143 clinical isolates. The majority of the strains of Aspergillus spp., Candida krusei, C. inconspicua, C norvegensis and Saccharomyces cerevisiae displayed resistance or decreased susceptibility to fluconazole in contrast to voriconazole. The absolute categorical agreement for voriconazole and fluconazole susceptibility results by the disk method and Etest was 90.5 and 74.8 % respectively. The error rate bounding analysis showed only 0.7 % of false susceptible results ( very major error) with voriconazole, but 2.8 % with fluconazole. Fluconazole can be used as a surrogate factor to predict voriconazole susceptibility but with lower reliability for susceptible-dose dependent and resistance category, especially in Candida glabrata isolates. The results of the disk method were not substantially influenced by the composition of media (Mueller-Hinton agar vs antimycotic Sensitivity Test agar), even if with the latter the results had fewer tendencies to produce false susceptibility of C.glabrata isolates to both of the triazole drugs. Disk test as well as Etest were shown to represent suitable methods for routine evaluation of susceptibility of clinical isolates of pathogenic fungi, including aspergilli, to fluconazole and voriconazole.

[Multidisciplinary approach to the management of vulvovaginal discomfort]. / Problematika vulvovaginálního dyskomfortu z hlediska mezioborové spolupráce.

Vulvovaginal discomfort is generally the most common reason for visiting a gynaecological clinic. The acute complaints are, in most cases, solved by a gynaecologist himself and the decision procedure is based on possibilities arising from outpatient examination. If need be, the evaluation of wet preparation should be carried out. In many cases we have been confronted with discrepant diagnosis where severe difficulties are not accompanied by relevant development of inflammatory changes and thus the diagnosis gives us an impression of quandary. In this case microbiological examination is the logic solution. With regard to changing living conditions we can expect in the future, that clinical symptoms of vulvovaginitis will not be entirely clear. The overview article points out the possibilities, which a gynecologist is able to use within differential diagnosis in the outpatient department. It also emphasis the necessity of interdisciplinary co-operation, particularly in patients with chronic difficulties where erudite evaluation of fixed preparation and culture examination are beneficial.

[Recurrent vulvovaginal candidiasis--possibility of its treatment]. / Rekurentní vulvovaginální kandidóza--soucasný stav problematiky a lécebné moznosti.

OBJECTIVE: A review of recurrent vulvovaginal candidiasis and the possibility of its treatment. SUBJECT: Original study. SETTING: Department of Obstetrics and Gynaecology, Medical Faculty Hradec Králové, Charles University, Prague. Department of Clinical Microbiology, Medical Faculty Hradec Králové, Charles University, Prague. Department of Biological and Medical Sciences, Faculty of Pharmacy Hradec Králové, Charles University, Prague. Department of Clinical Imunology and Allergology, Medical Faculty Hradec Králové, Charles University, Prague. Department of Psychiatry, Medical Faculty Olomouc, Palacky University, Olomouc. METHODS: Analysis and discussion focused especially on our results and experience in a long time followup of patients with confirmed recurrent vulvovaginal candidiasis. CONCLUSION: Owing to the multifactorial character of etiopathogenesis, the management of recurrent vulvovaginal candidiasis would be taken into consideration the complexity of the disease, not only the treatment of individual episodes with antimycotics.

[Treatment of invasive candidiasis--recommendations of professional]. / Lécba invazivní kandidózy--doporucení odborných spolecností.

National working group representing clinicians (hematologists, oncologists, infection diseases and ICU specialists), microbiologists, and different special medical societies and working groups prepared evidence-based guidelines for the treatment established fungal infection--invasive candidiasis in the adult hematology and ICU patients. These guidelines updated those published in the Czech Republic in 2003-2004. Evidence criteria of the Infectious Diseases Society of America (IDSA) were used for assessing the quality of clinical trials, and EORTC/MSG Consensus Group for definitions of invasive fungal disease.

[Treatment for invasiveness aspergillosis--recommendations of professionals]. / Lécba invazivní aspergilózy--doporucení odborných spolecností.

An increasing incidence of invasive aspergillosis is observed in most immunocompromised patients, and especially patients with acute leukemia and after hematopoietic stem cell transplantation. In order to decrease the mortality due to this infection, the clinicians need to optimise their treatment choice. The objective of these guidelines is to summarize the current evidence for treatment of invasive aspergillosis. The recommendations have been developed by an expert panel following an evidence-based search of literature with regard to current recommendation of European Conference in Infections in Leukemia and Infectious Diseases Society of America.

Cryptococcosis--a review of 13 autopsy cases from a 54-year period in a large hospital.

From 1952 to 2005, 13 cases of cryptococcosis confirmed by postmortem examination were diagnosed in autopsy material from the University Hospital in Hradec Králové, the Czech Republic. Histologically, Cryptococcus was found in multiple organs (brain and spinal cord, lungs, lymph nodes, spleen, bone marrow, liver, kidneys and adrenal glands). The lungs and CNS were the organs most often involved. Only in two cases was the diagnosis of cryptococcal infection established during the patient's lifetime, in both presenting clinically as meningitis, with positive result of CSF cultivation. Data and issues of diagnostics and treatment of cryptococcosis are discussed.

Effect of subinhibitory concentration of some established and experimental antifungal compounds on the germ tube formation in Candida albicans.

The influence of subinhibitory concentrations of six established and 19 newly synthesized antifungal compounds on the dimorphic transition of three C. albicans strains was evaluated in the filamentation-inducing medium. Amphotericin B was found to produce almost complete inhibition in the germination at a concentration of 1/10 of the corresponding MIC and partial inhibition at a concentration as low as MIC/50. Flucytosine and four azole derivatives were proven ineffective. From the newly synthesized drugs, the incrustoporin derivative LNO6-22, two phenylguanidine derivatives (PG15, PG45), and four thiosalicylanilide derivatives, in particular, showed results comparable to those of amphotericin B, with a high inhibition of germ tube formation at concentrations of MIC/10. In general, concentrations of MIC/50 had no visible effect.

Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients.

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.

[Systemic immunity in patients with recurrent vulvovaginal candidiasis]. / Systémová imunita u pacientek s rekurentní vulvovaginální kandidózou.

OBJECTIVE: This study was undertaken to characterize systemic immunity in the patients with recurrent vulvovaginal candidiasis. SUBJECT: Original prospective study. SETTING: 1. Department of Biological and Medical sciences, Charles University in Prague, Faculty of Pharmacy, Hradec Kralove, 2. Department of Obstetrics and Gynaecology, Medical Faculty, Hradec Kralove, Charles University in Prague, 3. Department of Clinical Immunology and Allergology, University Hospital, Hradec Kralove, 4. Department of Clinical Microbiology, University Hospital, Hradec Kralove, Czech republic. SUBJECT AND METHOD: Basic immunological parameters in 44 patients with recurrent vulvovaginal candidiasis were investigated and analyzed in this longitudinal follow-up study. CONCLUSION: Although there is no fundamental disturbance in systemic immunity in patients with recurrent vulvovaginal candidiasis and the majority of systemic immunological parameters in the patients were within normal range, some subtle changes of a few parameters of humoral and cellular immunity in contrast to the controls were identified.

[Chlamydia infections in urogenital tract--application of direct immunofluorescence in the diagnostics of Chlamydia trachomatis in the East Bohemian Region of Czech Republic during 1997-2003]. / Chlamydiové infekce urogenitáln'iho systému-- vyuzití prímé imunofluorescence v diagnostice Chlamydia trachomatis ve východoceském regionu Ceské republiky v letech 1997-2003.

OBJECTIVE: The aim of the study was an evaluation of results of Chlamydia trachomatis antigen detection by means of direct immunofluorescence in samples from patients suspected from this infection. DESIGN: Retrospective study. SETTING: Institute of clinical microbiology, University Hospital and Medical Faculty of Charles University, Hradec Králové. METHODS: Samples from females were swabs from uterine cervix, vagina and urethra, from male urethral swabs; moreover conjunctival swabs, bronchoalveolar irrigation, lower respiratory tract aspirates and sputum. Samples were fixed and "dyed" with monoclonal specific antibody labeled by fluorescent dye in the lab with a commercial diagnostic kit and they were evaluated microscopically after adding a drop of glycerol and covering with a top slide. RESULTS: Within a group of 6126 samples from patients suspected from Chlamydia infection we have found positivity in 14.4%. Of this subgroup 14.1% in samples from genitourinary tract of females and 15.2% in males, 14.1% from conjunctival swabs and 3.7% from lower respiratory tract. According to an age differentiation, positive samples in individual life-decades from genitourinary tract were 0-13-14.4-13.9-13.9 and in females over 60 13.9%. In males the corresponding walues were 0-21.4-15.1-16-13.4 and those over 60 years 16%. So, the highest positivity in our region has been detected in samples from young men aged 20-30, where it is nearly two-fold higher comparison to age-matched females. CONCLUSION: Direct detection of Chlamydia trachomatis antigen with immunofluorescence method still remains a reliable diagnostic tool. But for a control or confirmation of disputable results it is inconditionally necessary to have at least one more method available, e.g. PCR.

3-Phenyl-5-acyloxymethyl-2H,5H-furan-2-ones: synthesis and biological activity of a novel group of potential antifungal drugs.

3-(Substituted phenyl)-5-acyloxymethyl-2H,5H-furan-2-ones related to the natural product (-)incrustoporine were synthesized and their in vitro antifungal activity evaluated. The compounds with halogen substituents on the phenyl ring displayed much higher antifungal effect against Aspergillus fumigatus than selected representatives of azole antifungal drugs. In particular, the activity (1.34 microg/mL) of the most promising derivative, 3-(3,4-dichlorophenyl)-5-pivaloyloxymethyl-2H,5H-furan-2-one, was comparable to that of amphotericin B (0.5 microg/mL). Preliminary evaluation of the toxicity of the compound was carried out as well. Considering the size and properties of these molecules in comparison with those of amphotericin B, further development of this novel group of antifungals may lead to substances with better pharmacological profiles than that of the standard anti-Aspergillus drug.