[INVESTIGATION ON ANTIFUNGAL SUSCEPTIBILITY OF CANDIDA YEASTS IN PREGNANT PATIENTS WITH CONFIRMED VULVOVAGINAL CANDIDIASIS AND THEIR NEWBORNS.]

Background Vulvovaginal candidiasis (VVU) is considered as a special risk factor during pregnancy, with important influence on the reproductive function of the patients and on the morbidity in the newborns from mothers with VVC. Maternal VVC is a major risk factor for the development of candida-colonization of the infant, which in turn is the first step towards the development of mucocutaneous or systemic candidiasis and Candida-septicemia in the newborn. In pregnant patients, the possible applicable local and systemic medications are limited, while the therapeutic resistance in chronic recurrent forms of VVC increases, facts that require precision of the diagnosic approach to optimize the therapeutic recommendations in pregnant patients, considered as a high risk group. The aim of this study was to investigate in vitro antifungal susceptibility of Candida yeasts to current antifungal agents in pregnant patients with confirmed VVC before the act of birth. Material and Methods Vaginal secretions of 23 healthy pregnant women with proven Candida vaginitis were taken within 48 hours before birth and the presence of yeasls of Candida was confirmed by culture examination. Between 47-72 hours after birth, samples were taken for Candida colonization of the oralmucosa and feces of their newborns. Samples were plated on Sabouraud agar and cultured in an incubator for 2 to 3 days at a temperature of 25° C. Species identification of the isolated yeasts were performed by commercial API Candida test - API 20C AUX (BioMerieux, Marcy-l'Etoile, France). Part of the isolates was identified by commercial whale AUXACOLOR (BioRad, Mames la Coquette, France). Antifungal sensitivity of isolated strains was examined by applying commercial solicitation ready kit and methods of disc diffusion and E-test, as the aim of the authors was to assess their potential for use in the diagnosis, and the correlation between them. Results Candida albicans was the prevalent etiological agent in pregnant patients with VVC immediately before birth (n = 22, 91.67 +/- 0.06%). Positive Candida colonization was detected in 14 (58.33%) of their newborns (n = 24), as no statistically significant difference was established, depending on the mode of delivery. The investigated antifungal susceptibility with test Fungifast (ELITech Microbiology Reagents), found 100% sensitivity of Candida albicans to Amphotericin B, Flucytosin and Voriconazole. Intermediate susceptibility to Itraconazole was found in 6 of 23 (26%) maternal isolates, and 5 of 23 (22%) isolates were moderately sensitive to Fluconazole. Candida krusei showed complete resistance to Fluconazole and Itraconazole. Within the group of antifungals for topical application (Econazole, Ketoconazole, Miconazole, Nystatin), the results established that 100% of the studied fungi were sensitive to Nystatin, while within the groups of azoles for vaginal and topical use - C.krusei was 100% resistant, as the sensitivity of C. albicans varied between 60-80%. Conclusion Our recommendation, based on the esablished results is that in pregnant with uncomplicated VVC as a first-line therapy should be considered the group of vaginal azoles and Nystatin, while the systemic therapy should be considered carefully and only after the firSt trimester. In cases of oral and intestinal candidiasis in neonatology, we recommend a therapy with minimal absorbable antifungals as Nystatin and miconazole (amphotericin B is available in our country), while systemic antifungal should be initiated only as a second choice. The exact etiological diagnosis is especially important because in our country there is a tendency for increased incidence of non-albicans fungus resistant to therapy, and that changes the therapeutic behavior.

[The effect of vaginal sildenafil citrate on uterine blood flow and endometrium in the infertile women].

UNLABELLED: Evaluation of endometrial receptivity remains a challenge in clinical practice. Ultrasound evaluation of endometrial thickness and texture and measurement of uterine artery blood flow has been used for endometrial assessment. AIM: To investigate the role of combination of sildenafil citrate and serophene on endometrial thickness, endometrial volume, endometrial FI and VFI on Angiohistogram, RI and PI to a. uterine on the day of hCG, in prediction of IUI outcome in infertile women. MATERIALS AND METHODS: Forty two patients were selected randomly who had anovulatory infertility. In Sildenafil citrate plus Serophene group (Group I), patients got 25 mg sildenafil citrate (Silden) vaginally and Serophene 100-150 mg orally, and in Serophene group (Group II), 100-150 mg of Serophene was given orally. RESULTS: Mean endometrial thickness and endometrial volume was 11.8 +/- 2.6 v/s 10.2 +/- 2.8 and 5.2 +/- 1.4 v/s 3.6 +/- 1.8 respectively in group I and in group II (p < 0.05). There was significant decrease in PI and RI to a. uterina in group I. CONCLUSIONS: Combination of Sildenafil citrate and Serophene is an effective agent as a first-line of treatment for ovulation induction.

[Stillbirth rate and perinatal mortality--criteria for quality of prenatal obstetric care].

AIM: To determine the frequency and structure of stillbirth rate, its relative share compared to PM, the trends for the period of 2007-2012 and their dependence to birth weight of the fetus and newborn. MATERIAL AND METHODS: The birth rate, stillbirth rate and PM for the period of 2007-2012 have been followed up retrospectively according to data provided by the department of Obstetrics & Gynecology in University hospital "St. George" Plovdiv. The trends of those two indicators have been established, their relationship to and dependence of fetal and newborn birth weight. Representative and correlative analyses have been used. RESULTS: During the period being analyzed 13 558 deliveries have been registered. 13.4 per thousand (183) of them are stillbirths, and 92.3% (169) died antenatal. After 2010 the birth rate decreases. Stillbirth rate increases from 13 per thousand in 2009 to 20.6 per thousand in 2012. It is mostly due to prematurity, which for the past two years are mostly with extremely low birth weight (57.2%). Antenatal stillbirth rate prevails. Intranatal fetal death is being observed significantly less, but in those cases where it is, the premature with weight 600-999 grams play a major role. Increased stillbirth rate maintains a high PM and constitute more than 2/3 of it. CONCLUSIONS: Against the backdrop of lower birth rate, the stillbirth rate and PM remain high. In the last 2 years they are presented mainly by premature weighing 600-999 grams. The prevalence of antenatal stillbirth rate means good control of delivery but refers to the prenatal period, which is related to the quality of the obstetric care.

[Pain in the neonatal period. I -- physiological aspects, causes, response, diagnosis and long-term effects of neonatal pain].

UNLABELLED: Entering of high-tech methods used in the treatment of premature infants carries the risk of increased invasiveness which causes the pain and stress in them. The aim of this literature review is to present the achievements in the study of neonatal pain, causes and responses to it, the short-term effects on their status and their later development, as well as the ways of pain's assessment. The newborn after 26-30 weeks of gestation has functionally mature pain receptor apparatus. Numerous invasive procedures cause in the prematurely born baby psycho-physiological abnormalities (changes in heart rate, breathing, blood pressure and oxygen saturation) and behavioral abnormalities (facial expression, motion activity of the body and extremities, sleep disturbances, characteristics of crying). The last mentioned are basis for creating a rating scale for procedural and prolonged pain. Future studies are aimed at creating an universal scales. CONCLUSION: At the time of intensive treatment, the premature infant is under the adverse effects of acute or repeated pain incentives. They worsen the status, prolong the hospital stay and influence the later neurological development and outcome of the premature infant. In everyday clinical practice, prevention of pain, establishing its severity and its evaluation reduce its negative effects on the newborn.

[Dynamics in levels of procalcitonin in healthy term newborns during the first 24 hours of life].

UNLABELLED: Procalcitonin /PCT/ is a reliable marker for the diagnosis of early onset neonatal sepsis but its sensitivity and specificity in literature vary widely mostly due to existing unclarity on its normal values and kinetics in the postnatal period. Aim of our research is to study the normal values of PCT and its dynamics during the first 24 hours of life for the Bulgarian population. MATERIALS & METHODS: 70 healthy neonates were prospectively enrolled and separated in the following 3 groups (Group 1- immediately afterbirth; Group 2- 0-12 h afterbirth; Group 3- 12-24 h afterbirth) regarding the age at the time of PCT testing. The selected method of PCT testing is direct chemiluminescence. Results were analyzed with Statistical Package for Social Science (SPSS). RESULTS: For group 1 and group 2 the mean PCT level was 0.06 and 0.59 ng/ml respectively. In group 3 PCT concentrations reached peak mean values of 3.35ng/ml. In the last group also the greatest variations were observed PCTmin - 0.31 ng/ml, PCTmax - 23.81 ng/ml. No correlation between PCT concentrations, sex, mode of delivery and parity was found. CONCLUSION: During the first 24 hours of life PCT values in healthy term neonates show broad variations, most marked in the interval 12-24 h. The dynamics of PCT concentrations is characterized by a gradual increase from birth reaching peak levels approximately 24 hours later.

[Pain in the neonatal period II. non-pharmacological and pharmacological treatment].

UNLABELLED: In their postnatal development the newborns are often exposed to the influence of procedural and repetitive painful stimuli that worsen their status. This requires the implementation of non-pharmacological and pharmacological treatment. Aim - to explore the literature data on the possibilities of non-pharmacological and pharmacological treatment methods which are implemented to reduce and control pain in neonatal period. Some of the non-pharmacological strategies are the priority of the personnel who gives care of newborns (swaddling, nonnutritive sucking, usage of sweet solution, etc.) Ever more is discussed the participation of mother(parents)in the care of her child's comfort and pain reduction. The breastfeeding and "kangaroo" care additionally reduce negative effects of pain. The importance of the music continues to be explored. The principles of pharmacological therapy include: control of procedure pain, its treatment during mechanical ventilation and at the time and after surgical intervention which is based on analgesia. Specificities of the pharmacotherapy of newborns and premature infants require careful application of the medications and additional studies on these children. CONCLUSION: The elimination of neonatal pain and its negative effects on the newborn is achieved by applying different strategies. Non-pharmacological methods reduce pain stimuli and ensure the child's comfort and the pharmacological methods block and eliminate the pain. Most often a combination of them is used in practice.

CHEK2 gene alterations independently increase the risk of death from breast cancer in Bulgarian patients.

Checkpoint kinase 2 (CHEK2) is a DNA damage-activated protein kinase implicated in cell cycle checkpoint control. The significance of CHEK2 alterations for breast cancer incidence and clinical behavior is not clear. In this study we determined the mutational spectrum and the level of promoter hypermethylation of CHEK2 gene in a group of 145 Bulgarian patients with breast cancer. A special emphasis was put on the clinical impact of CHEK2 alterations for breast cancerogenesis. PCR-SSCP-sequencing analysis of the entire coding sequence of CHEK2 gene was performed to estimate the mutational profile of tumor samples. Methylation-sensitive SSCP was applied to determine the methylation status in CpG clusters implicated in CHEK2 silencing. Clinical significance of CHEK2 alterations was evaluated using standard statistical methods. Mutations in CHEK2 were identified in 9.65 % of the patients. Two novel missense substitutions Thr476Met (C >T) and Ala507Gly (C>G), and a novel silent variant Glu79Glu (A>G) were registered. However, hypermethylation was not found in any of the studied cases. Comparison with clinical characteristics showed that CHEK2 positive women have predominantly lobular type of breast carcinoma (р=0.04) and PR+ status (p=0.092). CHEK2 mutations correlated significantly with ATM+ status (p=0.046). All patients with the Glu79Glu variant were progesterone receptor positive (p=0.004). A decrease in overall survival (p = 0.6301) and a threefold increased independent risk of death (HR = 3.295, 95%CI 0.850-12.778, p = 0.085) in CHEK2+patients was found. Our data indicate the significance of CHEK2 gene alterations in contrast to promoter hypermethylation in breast cancerogenesis. Specificity of CHEK2 mutational profile for the Bulgarian population was found. Though CHEK2 mutational status correlated with more favorable clinical characteristics, including positive progesterone receptor and lobular histological type, it independently increased the risk of death in these patients.

Breast cancer patients with hypermethylation in the promoter of BRCA1 gene exhibit favorable clinical status.

Promoter hypermethylation was shown to be involved in human cancerogenesis through silencing gene expression. Several studies were dedicated to explore the frequency and clinical significance of BRCA1 hypermethylation in sporadic breast cancer to identify a specific molecular and clinico-pathological phenotype. However the available data are limited and rather too heterogeneous. In this study we investigated the level of methylation in the promoter region of BRCA1 and its correlation with clinico-pathological and molecular characteristics in a group of 135 Bulgarian patients. Methylation specific PCR was applied to determine methylation status of tumor samples. Clinical impact of BRCA1 hypermethylation was estimated using standard statistical methods including Fisher's exact and the Chi-squared tests, the Kaplan-Meier method, the univariate and multivariate Cox proportional hazards regression model. We found that hypermethylation was present in 17.04% of the cases (23/135). Patients with hypermethylation in BRCA1 displayed favorable clinical status as their tumors were smaller in size (P = 0.066), lacked p53 gene mutations (P = 0.073) and were of lobular type (P = 0.046). The presence of hypermethylation was weakly associated with better overall survival (P = 0.2) with a hazard ratio of 0.47 (95% CI 0.14-1.54, P= 0.213). Our study provides the first data on the BRCA1 hypermethylation of Bulgarian patients and contributes to elucidation of its clinical significance in sporadic breast cancer.

A novel SOX9 nonsense mutation, q401x, in a case of campomelic dysplasia with XY sex reversal.

Campomelic dysplasia (CD, MIM 114290) is a rare, often lethal, dominantly inherited, congenital skeletal dysplasia, associated with male-to-female autosomal sex reversal and due to de novo mutations of the SOX9 gene, a tissue-specific transcription factor gene involved both in skeletogenesis and male sexual differentiation. Here we report on a 4 months-old 46,XY sex reversed infant with typical clinical features for CD due to a novel mutation of the SOX9 gene, Q401X, leading to synthesis of a truncated SOX9 protein that completely lacks the C-terminal transactivation domain.

Vibrational circular dichroism signature of hemiprotonated intercalated four-stranded i-DNA.

The four-stranded intercalated DNA structure exemplified by the oligonucleotide 5'-d(CCCCCCCCCCCC) (d(C)12) was studied at acidic pH by infrared absorption (IR) and vibrational circular dichroism (VCD) spectroscopy and compared with spectra of the same oligonucleotide at neutral pH to establish distinct VCD markers for the intercalation motif. The most striking feature is a new absorption at 1694 cm(-1) and its corresponding VCD couplet with reversed sign. These are unique for the intercalated structure and have not been observed for other parallel stranded duplexes. Significant characteristic features resulting from the spatial arrangement of the sugar-phosphate backbone are also clearly present for d(C)12 at acidic pH. An extensive network of CH...O bonds twists the backbone such that multiple through-space vibrational coupling occurs among neighbouring sugar-phosphate residues resulting in unusual VCD signals.

Optimized polymerase chain reaction-based single-strand conformation polymorphism analysis of p53 gene applied to Bulgarian patients with invasive breast cancer.

During the last few decades a substantial amount of evidence has accumulated proving that the abrogation of the normal p53 pathway is a critical step in the initiation and progression of tumors. Decoding the genetic mechanisms involved in carcinogenesis requires screening for consistent genetic tumor alterations, including those concerning the p53 gene. Thus, practical, efficient, and inexpensive techniques for accurate determination of p53 mutational status are needed. Polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis is considered to be a useful tool to investigate the role of the p53 gene in the development and progression of human cancers. The sensitivity of the method can be increased considerably by varying the experimental conditions. Here we demonstrate a scheme of PCR-SSCP optimization for detection of p53 gene mutations of patients with various cancers. Optimal conditions for PCRSSCP of p53 exons 4-9 are reported. Such PCR-SSCP optimization could allow an increase in the sensitivity and reproducibility of the technique and facilitates screening of large series of patients to assess the clinical significance of p53 mutations in human cancers. Using the optimized PCR-SSCP analysis we screened Bulgarian patients with invasive breast cancer for p53 gene mutations and registered a 33.33% frequency of mutations. To date, there are no data concerning the p53 status of Bulgarian breast cancer patients. Screening for p53 gene mutations enables an accurate and routine determination of the p53 status of patients with cancer and may be applied in clinical oncology to cancer diagnosis, prediction of prognosis and response to treatment.

Afferent and efferent phases of allergic contact dermatitis (ACD) can be induced after a single skin contact with haptens: evidence using a mouse model of primary ACD.

Allergic contact dermatitis is a T cell-mediated delayed type hypersensitivity reaction that occurs upon hapten challenge in sensitized individuals. The inflammatory response in classical allergic contact dermatitis requires both a sensitization phase and an elicitation phase responsible for the recruitment and activation of specific T cells at the site of hapten skin challenge. Conversely, previously unsensitized patients may develop a "primary allergic contact dermatitis" after the first skin contact with potent contact sensitizers leading to a skin inflammation with all the features of classical allergic contact dermatitis. In this study we used an experimental murine model, referred to as contact hypersensitivity, to study the pathophysiology of primary allergic contact dermatitis and its relationship to classical allergic contact dermatitis. We show that one epicutaneous application of a nonirritant dose of hapten (2,4-dini-trofluorobenzene, fluorescein isothiocyanate) was sufficient to induce an optimal allergic contact dermatitis reaction at the site of primary contact with the hapten without subsequent challenge. As in classical allergic contact dermatitis, the skin inflammation in primary allergic contact dermatitis was mediated by interferon-gamma producing, CD8+ effector T cells that were induced in the draining lymph nodes at day 5 postsensitization and downregulated by CD4+ T cells. Reverse transcription-polymerase chain reaction analysis revealed that the primary allergic contact dermatitis reaction was mediated by a recruitment of CD8+ T cells at the sensitization skin site at day 6 postsensitization. Analysis of the fate of the hapten fluorescein isothiocyanate applied once on the skin revealed its persistence in the epidermis for up to 14 d after skin painting. These results suggest that the development of primary allergic contact dermatitis (i.e., without secondary challenge) is associated with persistence of the hapten in the skin, which allows the recruitment and activation of CD8+ T cells at the site of the single hapten application.

Contact dermatitis II. Clinical aspects and diagnosis.

Contact dermatitis (CD) is an altered state of skin reactivity induced by exposure to an external agent. "Eczema" and "dermatitis" are often used synonymously to denote a polymorphic pattern of inflammation of the skin characterized, at least in its acute phase, by erythema, vesiculation and pruritus. Substances that induce CD after single or multiple exposures may be irritant or allergic in nature. The clinical presentation may vary depending on the identity of the triggering agent and the reactivity of the subject, but in all cases the lesions are primarily confined to the site of contact. According to the mechanism of elicitation, the following types of contact reactions may be distinguished: (1) allergic contact dermatitis (ACD); (2) irritant contact dermatitis (ICD); (3) phototoxic and photoallergic contact dermatitis, and (4) immediate type contact reactions. The present review will focus on allergic contact dermatitis. ACD is the clinical presentation of contact sensitivity in humans. The pathophysiology of the contact sensitivity reaction has been reviewed in a preceding issue of this journal [1].

Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity.

Contact hypersensitivity (CHS) is a T cell-mediated skin inflammation induced by epicutaneous exposure to haptens in sensitized individuals. We have previously reported that CHS to dinitrofluorobenzene in mice is mediated by major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. In this study, we show that CD8(+) T cells mediate the skin inflammation through their cytotoxic activity. The contribution of specific cytotoxic T lymphocytes (CTLs) to the CHS reaction was examined both in vivo and in vitro, using mice deficient in perforin and/or Fas/Fas ligand (FasL) pathways involved in cytotoxicity. Mice double deficient in perforin and FasL were able to develop hapten-specific CD8(+) T cells in the lymphoid organs but did not show CHS reaction. However, they did not generate hapten-specific CTLs, demonstrating that the CHS reaction is dependent on cytotoxic activity. In contrast, Fas-deficient lpr mice, FasL-deficient gld mice, and perforin-deficient mice developed a normal CHS reaction and were able to generate hapten-specific CTLs, suggesting that CHS requires either the Fas/FasL or the perforin pathway. This was confirmed by in vitro studies showing that the hapten-specific CTL activity was exclusively mediated by MHC class I-restricted CD8(+) T cells which could use either the perforin or the Fas/FasL pathway for their lytic activity. Thus, cytotoxic CD8(+) T cells, commonly implicated in the host defence against tumors and viral infections, could also mediate harmful delayed-type hypersensitivity reactions.

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation.

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.

[Physiopathology of eczema]. / Physiopathologie de l'eczéma.

Eczema is a skin inflammatory reaction mediated by antigen-specific T cells. Two main disorders belong to this group: contact dermatitis and atopic dermatitis. The pathophysiological mecanisms involved in both disorders are similar and include, at the cellular level, three elements: the antigen (hapten or environmental antigen), antigen-presenting cells belonging to the group of dendritic cells and antigen-specific T cells. The Langerhans cell responsible for antigen handling and presentation to specific T cells appears to play a central role for the generation of the inflammatory reaction.