Dissertations / Theses on the topic 'Ethanol Vaginosis, Bacterial Vaginosis, Bacterial'

Bacterial Vaginosis (BV) is a disorder of unknown etiology, characterized by a foul smelling vaginal discharge, loss or reduction of the normal vaginal Lactobacilli, and overgrowth of other anaerobic bacteria. Thus, it presents a formidable problem for clinicians as well as microbiologists researching its etiology, clinical course, treatment, and epidemiology. The present work focuses on the unresolved issues of the epidemiology and treatment of BV in order to provide valid methods for treatment studies of this condition and to describe the prevalence of BV in defined populations. The first study validates the use of PAP-stained smears in the diagnosis of BV. The study assesses the methods of Amsel’s clinical criteria and Nugent criteria on Gram-stain smears, against Pap-stained smears and also validates different observers. The result shows that the PAP-staining of vaginal smears is a good method in BV diagnosis; the kappa value is 0.86 (interobserver weighted kappa index) compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. This enables population based studies on archived PAP-stained smears from the screening of cervical cancer. In the second study, we use the knowledge gained from study one to investigate the prevalence of BV in a cohort from the population of Åland. The prevalences of BV on the Åland Islands were: 15.6 %, 11.9 %, 8.7 %, and 8.6% in 1993, 1998, 2003, and 2008, respectively. This means that the prevalence of BV decreased between1993-2008 from 15.6% to 8.6%. The confidence intervals are not overlapping, thus indicating a significant decrease in prevalence from 1993 to 2008. The third study is a prospective, double-blind placebo controlled treatment study of BV. After conventional treatment with clindamycin, the patients were treated with adjuvant treatment of Lactobacilli-loaded tampons or placebo. The study showed no differences between the treatment and the placebo group, indicating that the tampon does not work at all. There are a variety of possible explanations for the result, which are analyzed in this thesis. The fourth study aimed to evaluate whether clindamycin is retained for a long time in the vaginal mucosa, thus disturbing the Lactobacilli in an attempt to reimplant Lactobacilli in the probiotic treatment studies. In conventional treatment, it is also useful to know whether clindamycin is retained, especially when considering the pressure from antibiotics on the antimicrobial sensitivity pattern. In the study, we found that the clindamycin disappears rapidly. Conclusion: BV research requires effort from many different scientific disciplines and the riddle of this condition and its treatment can only be resolved by concerted actions in research and treatment. The vision for the future includes, among other factors, better molecular biology based diagnostic tools, and knowledge of population based bacterial floras.

Bacterial Vaginosis is an inbalance of vaginal flora which has been associated with increased risk of numerous gynecological and obstetric morbidities including increased risk of acquisition of HIV from an infected partner and increased risk of preterm delivery. Black race has been consistently identified as a risk factor for BV. Black women also suffer from significant disparities in most of the morbidities also associated with BV when compared to women of other ethnicities and races. Traditional predictors of BV such as douching practices and sexual behaviors do not fully account for the racial disparities in BV prevalence. Researchers have begun to explore the potential relationship between stress and BV. Also, perceived racism has been identified as a potential stressor contributing to the health outcomes of Black women. The purpose of this study was to test a predictive model of bacterial vaginosis among Black women. The Allostatic Load Model was the theoretical framework. Participants (N=94) completed a self administered questionnaire and interview including measures of perceived stress, preceived racism, behavioral responses to stress and specific behavioral responses to racism along with traditional predictors of BV. Measurement scales included the Cohen Perceived Stress Scale, the John Henryism Scale of Active Coping, the Everyday Perceived Racial Discrimination Index, the Experiences of Discrimination Scale and the Vines Telephone Administered Perceived Racism Scale (TPRS) which included a behavioral responses to racism subscale. Bacterial vaginosis was diagnosed utilizing a self-collected vaginal swab which was analyzed utilizing the BVBlue point of care testing kit. Twenty percent (N=19) of participants screened positive for bacterial vaginosis. Douching and sexual activity in the last three months and education were significantly associated with bacterial vaginosis. Age, income, hormonal contraceptive use and condom use were not associated with BV. Neither perceived stress nor perceived racism were associated with bacterial vaginosis. After logistic regression analysis, only education continued to be a significant predictor of BV. The lack of an association between BV and the main study variables may have been related to young age of the sample or the low rates of high perceived stress and high perceived racism. Perceived stress was positively associated with perceived racism and behavioral responses to stress. This association is likely a reflection of the stressful nature of perceived racism. Further research is needed to better understand how the stressful nature of racism and behavioral responses to stressors may influence health outcomes and if interventions can be utilized to promote adaptive behavioral responses.

Metagenomics is a rapidly evolving field that has facilitated the expansion of microbiology into new areas of human and environmental health. Metagenomic studies have expanded the phylogenetic tree of life by increasing taxonomic resolution in individual phyla as well as adding entirely new branches of life. This revolution in microbiology has been made possible by the introduction of second-generation high-throughput sequencing, the associated methods for preparing DNA sequencing libraries, as well as new bioinformatic algorithms for analyzing these new types of data. Because of the novelty of these methods, very few have been systematically tested for their sensitivities and specificities outside of the initial development process. As the interpretation of metagenomic studies utilizing these tools depends greatly upon their efficiencies in both detection and classification, it is essential to best determine the performance of each tool. In this study, a variety of novel techniques were utilized and tested in their abilities to characterize the microbial populations in two regions of the female genital tract: ovarian cancer tissue and the vaginal microbiome. Although a diverse microbial population was initially observed in the transcriptome sequence data for ovarian cancer using next generation sequencing, we were unable to recover these microbial sequences through PCR and Sanger sequencing approaches. Optimized methods were applied to healthy vaginal microbiome samples and tested for their ability to differentiate them from a polymicrobial disease of the vagina, bacterial vaginosis. In addition to a high correlation between a microbial scoring system for bacterial vaginosis, this novel metagenomic pipeline also revealed microorganisms not yet associated with the vaginal microbiome such as specific Bifidobacteria spp., various bacteriophage, and Debaryomyces. Collectively, both of these studies provide unique insights into each disease as well as illustrate both the limitations and potential of the rapidly growing field of metagenomics.

Vaginitis is a vaginal infection which affects many women all over the world. The disorder is characterized by an infection of the vaginal area which can cause problems like abnormal vaginal discharge, itching and redness. The two most common causes of vaginitis are bacterial vaginosis and Candida vaginitis. The prevalence of bacterial vaginosis in Sweden is around 10-20 % and approximately 75 % of all women will once in their lifetime suffer from vaginal yeast infection. The clinical symptoms of vaginal infections are not specific and the diagnosis methods of bacterial vaginosis and Candida vaginitis are subjective and depended on the acuity of the clinician. Due to the lack of standardized and objective diagnostic tools, misdiagnosis and consequently incorrect treatment may occur. As vaginal infections and symptoms impact greatly of women´s quality of life and vaginitis have been associated with serious public health consequences, it is essential to diagnose and treat the conditions correctly. Hence, there is a great need of better methods of diagnosing these conditions. The aim of this master thesis was to develop quantitative species-specific real-time PCR assays to use in diagnosing the two most common causes of vaginitis i.e. bacterial vaginosis and Candida vaginitis. Potential markers for bacterial vaginosis (Atopobium vaginae, BVAB2, Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Megasphaera type 1, Megasphaera type 2, Mobiluncus curtisii, Mobiluncus mulieris and Leptotrichia/Sneathia species) and Candida vaginitis (Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis) were chosen. Primers and probes were designed and tested on reference strains and vaginal samples. Single- and multiplex PCR reactions were successfully optimized with the designed oligonucleotides. Furthermore, standard curves with excellent linearity were created and covered more than five orders of magnitude. These developed quantitative species-specific real-time PCR assays will, in a prospective medical validation, quantify 300 vaginal samples from women visiting the RFSU Clinic in Stockholm.

Bacterial vaginosis (BV) and genital mycoplasmas are infections of the reproductive tract that play important roles in maternal and foetal health. Genital mycoplasmas include Mycoplasma genitalium, M. hominis, Ureaplasma parvum and U. urealyticum. Infection may increase a woman’s susceptibility to infection with the human immunodeficiency virus (HIV). Bacterial vaginosis associated bacteria may form biofilms that are responsible for antimicrobial resistance and about 30% of affected women will relapse within three months of treatment. Genital mycoplasmas are prone to develop point mutations, which are responsible for increased antimicrobial resistance. Infections with these bacteria become prominent during pregnancy as infection may lead to infertility and foetal death. The purpose of the study was to determine the association between genital mycoplasmas and BV in pregnant women. Pregnant women attending the antenatal and Maternal and Foetal Unit (MAFU) clinics of a tertiary academic hospital in Pretoria, South Africa were included in the study. Self-collected vaginal swab specimens were obtained from consenting women older than 18 years of age. With the aid of microscopy, the Nugent scoring system was used to diagnose BV. Genital mycoplasmas were cultured on A2 agar and were diagnosed and speciated with a multiplex polymerase chain reaction (mPCR) assay. In addition, genital mycoplasmas were diagnosed and the antimicrobial susceptibility profiles determined with the Mycofast Revolution assay. A quantitative real-time polymerase chain reaction (qPCR) was employed to quantify the BV associated bacteria Atopobium vaginae and Gardnerella vaginalis. The prevalence of BV in this study was found to be 17.7% in 220 recruited pregnant women. Threshold concentrations between 106 to 107 copies/reaction of A. vaginae and G. vaginalis were found to be the best predictors of BV. Genital mycoplasmas were poorly recovered from A2 agar media, which had a contamination rate of 54.9%. An mPCR assay revealed that genital mycoplasmas were prevalent in 2.3% to 71.4% of specimens with U. parvum being the most prevalent species. The resistance of Ureaplasma species to tetracycline and erythromycin was 73% and 80%, respectively. Minor resistance to the fluoroquinolones, levofloxacin and moxifloxacin was recorded. This study found that only the genital mycoplasmas, namely M. hominis and U. parvum, were significantly associated with BV, while M. hominis was also significantly isolated from HIV positive women. This study found that there is an association between BV and genital mycoplasmas. The high prevalence of BV and genital mycoplasmas suggests that current management and/or intervention strategies are insufficient. Bacterial vaginosis associated bacteria can form a polymicrobial biofilm, which confer protection against antimicrobial agents and host immune responses. These biofilms are present on genital sites like the endometrium, which is located close to the amniotic membranes, posing health risks for the pregnancy. Future research must focus on the study of in vitro BV biofilm models and effective treatment strategies to minimise antimicrobial resistance. In the meantime, low-cost point-of-care (POC) tests that can accurately diagnose RTIs are needed to prevent excessive and unnecessary administration of antimicrobial agents and improve maternal and foetal health in the South African health care system.
Dissertation (MSc)--University of Pretoria, 2018.
Medical Microbiology
MSc
Unrestricted

Genital inflammation associated with sexually transmitted infections (STIs) and Bacterial Vaginosis (BV) is considered a key driver in the HIV/AIDS epidemic. A new rapid point-of-care (POC) test that detects genital inflammation in women was recently developed by researchers at the University of Cape Town. The objective of this study was to establish the cost-effectiveness of this novel intervention in comparison to other relevant screening and diagnostic strategies for the management of STIs and BV in women. It follows prior research on the cost and affordability of national implementation of screening with this technology. This research indicated that it might not affordable policy option given current health budget constraints. A decision analysis model was developed to estimate the cost and health outcomes associated with five different screening and diagnostic strategies for women seeking care in the South African public health sector. A decision tree was constructed, and all cost and effectiveness parameters were obtained from published and unpublished literature. The model incorporated all clinic-level and treatment costs associated with diagnosing and treating a single episode of disease. The main outcome measure was the effectiveness of each approach in correctly diagnosing an STI or BV in women, proxied by its sensitivity measure. One-way sensitivity analyses and threshold analysis were conducted to test key uncertainties and assumptions in the model. In the base-case scenario, screening with GIFT and treating GIFT-positive cases based on syndromic management guidelines, was the most cost-effective strategy with an ICER of $2.60 per women diagnosed with an STI(s) and/or BV. This strategy remained the most cost-effective even when a variety of parameters were varied in one-way sensitivity analyses. A threshold analyses on GIFT's sensitivity revealed that the strategy would remain the most cost-effective unless the sensitivity of the test assay decreased below 14.83%. From the perspective of the South African government, screening with GIFT and treating positive cases according to syndromic management guidelines is a highly cost-effective strategy for the management of STIs and BV in women in the reproductive age, but affordability considerations cannot be ignored. The newly developed rapid POC can significantly improve the management of STIs and BV in women through identifying asymptomatic women and at the same time, reducing their risk of HIV infection, but further research is required to inform decision-making.

Sexually transmitted infections (STIs) remain a global public health concern. Together with bacterial vaginosis (BV), the association with HIV acquisition through genital inflammation in women poses a challenge towards the control of HIV/AIDS, more so in asymptomatic cases. Diagnosis of asymptomatic women using a genital inflammation screening tool, the cytokine biomarker rapid test, reduces the cases of untreated women. However, as a newly developed screening tool, there are no prior cost estimates to advocate for its funding and implementation. This study estimated the costs of genital inflammation screening of women (15-49 years) and, assessed the budget impact of providing this screening service in primary health facilities in South Africa in 2016. This thesis is a sub-study of the GIFT project (Genital Inflammation Test for HIV Prevention) whose main objective is HIV prevention through improved control of sexually transmitted infections (STIs). The micro-costing approach was used to calculate the unit cost per patient screened from a provider’s perspective at the Desmond Tutu HIV Foundation youth clinic (DTHF), and, the University of Cape Town Student Wellness Service (UCT SWS), over a 1 year period. The unit cost estimates were used to analyse the budget impact of scaling-up and providing the screening service in primary health facilities countrywide. . Sensitivity analyses were carried out to determine the robustness of the study findings. The results demonstrated that the cost per woman screened for genital inflammation was $24.26 at DTHF and $14.32 at UCT SWS. The scaled up costs ranged from $107,183,655 to $183,062,066 in South Africa. The screening intervention accounted for a significant amount of the available funds. The cost estimates were sensitive to the personnel costs, clinic utilization rates and population coverage rates. According to this study, it can be concluded that, the cost estimates of screening are high, and its implementation may not be affordable within the current budget. However, this screening tool will increase the cases detected, contributing towards better STIs management and control. Additionally, it will reduce the risk of HIV acquisition among women.

Two vaginal phylotypes of the genus Megasphaera (phylotype 1 and phylotype 2) were recently associated with bacterial vaginosis (BV), an infection characterized by vaginal dysbiosis. Through an analysis of 16S rRNA profiles of 3,986 women enrolled in the Vaginal Human Microbiome Project, we confirmed that while both phylotypes were associated with BV, Megaspheara phylotype 1 had higher specificity for the condition. Megasphaera phylotype 2 was strongly associated with trichomoniasis. Previous studies have reported that BV-associated organisms are excluded in pregnancy. We observed that Megasphaera phylotype 1, which has been associated with adverse pregnancy outcomes, exhibited a trend of increased prevalence in the pregnant cohort. We sequenced the genomes of isolates of the two phylotypes and performed comparative analyses. We demonstrate that these two phylotypes have distinct genomic features and unique potential for metabolic processes that reveal niche specialization. These findings may provide insight into their differential associations with vaginal infections.

Includes abstract.
Includes bibliographical references (leaves 55-60).
Bacterial vaginosis (BV) is a common cause of vaginal symptoms among women of childbearing age, although some women may be asymptomatic. The etiology of BV has been uncertain for many years, but several studies have related an increased frequency of BV to multiple diverse factors such as smoking, sexual behavior, vaginal douching, black race, sexually transmitted infections, and certain socio-economic factors. BV infection may lead to adverse reproductive health outcomes and may also increase the susceptibility of women to human immunodeficiency virus (HIV) and sexually transmitted infections (STIs). It is therefore of importance to know the risk factors for BV among a population so that mechanisms of prevention can be developed.

The vagina is one organ of the body which has not been studied exhaustively. Moreover, most of the studies found in the contemporary literature have been performed on women affected by a variety of genital diseases. In the present study the vaginal epithelium was examined with a histological method, morphometry, whereby cyclical changes related to hormonal variation during the menstrual cycle were demonstrated. Determination of the quantity of estrogen receptors in the vaginal epithelium on two occasions during the menstrual cycle revealed a significantly greater number in the follicular than in the luteal phase. The results of these studies indicate the presence of a menstrual variation in the vaginal epithelium comparable to that in the endometrium. Phenoxymethylpenicillin (pcV) was used as a marker substance to study the dynamics of the transport mechanisms into the vagina. PcV was found to accumulate in the vaginal fluid and high concentrations persisted for a long period of time. In hysterectomized women, the appearance of pcV in the vaginal fluid followed the same pattern. Consequently, the substance is transported through the vaginal wall and need not enter with the secretions from the internal genitalia. The greatest concentration of pcV was in the distal portion of the vagina, possibly due to the specific internal circulation of fluid within the vagina. Bacterial vaginosis as an example of an ‘ecological disease’ has been studied with regard to the formation of endotoxin, a constituent of the cell wall of Gram- negative bacteria. Large amounts of endotoxin were found and the clinical implication of this finding has been pointed out. Furthermore, the influence of pcV on the vaginal microbial flora of healthy women has been investigated. A change from a situation with predominance of lactobacilli to the appearance of Gram-negative rods was observed. In one of the women the lactobacilli disappeared completely and were replaced by E. coliand high levels of endotoxin in the vaginal fluid were found. This study demonstrates the complexity of the ecological balance of the vaginal microbial flora and illustrates the difficulty of defining a ‘normal’ vaginal condition. Is there any unquestionable state of ‘normality’ even in a healthy woman free from symptoms of genital disease?

S. 1-22: sammanfattning, s. 25-64: 6 uppsatser

digitalisering@umu

Background and Objective: Bacterial 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 Methods: Vaginal 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. Results: Relative 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. Conclusions: Quantitative 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.

The composition of vaginal microbiota in pregnancy is important as it may influence susceptibility to adverse pregnancy outcomes. Dysbiosis of the vaginal microbiota caused by the replacement of protective resident microorganisms such as Lactobacillus spp. by anaerobic bacteria is known as bacterial vaginosis (BV). BV is prevalent in women of African descent and may be a cause of higher rates of adverse birth outcomes in these women. Adverse birth outcomes have been reported to be higher in HIV-infected women both on treatment or treatment naïve compared to HIV-uninfected women. The relationship between the vaginal microbiome, HIV and pregnancy outcomes has not yet been established in Zimbabwean women, nor reported in sub Saharan Africa, where the burden of disease is high. Identification of specific organisms and immune factors associated with adverse pregnancy outcomes could lead to interventions or diagnostic algorithms to prevent and predict these outcomes in this population. We recruited 420 pregnant Zimbabwean women [48 (11.4%) HIV infected, 372 (88.6%) HIV uninfected], between 13-35 weeks of gestation with a median gestational age of 30 weeks. Vaginal swabs were collected at enrolment and women were followed until pregnancy outcome was determined. Bacterial DNA was extracted from the vaginal swabs using an optimized Phenol chloroform extraction method with an addition of an enzymatic cocktail step. Library preparation was done using the Universal primers (515F/806R) for the hypervariable V4 region of the 16S rRNA gene. For the first PCR KAPA Hotstart + primers (Roche Lifescience, UK), were used for amplification. After amplification AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used for cleaning of the PCR products at all stages. For the second PCR Nextera XT, Index Kit (Illumina) was used adding unique sequencing adapters to the amplicons. Quantitative PCR was used for library quantification and pooled libraries were the sequenced using Illumina MiSeq. Upstream analysis of sequencing data was done using QIIME and UPARSE and downstream analysis using custom R scripts on samples with good quality reads (>5000). Concentrations of 27 cytokines were measured in vaginal swab samples using a Bio-Plex Pro Human Cytokine 27- plex Assay (Bio-Rad Laboratories Inc., USA). Assay plates were read using a Bio-Plex Suspension Array Reader (Bio-Rad Laboratories Inc., USA). Data were analyzed using Bio-Plex manager software (version 4). Cytokine levels that were below the lower limit of detection of the assay were reported as the mid-point between zero and the lowest detectable level measured for that given cytokine. Pregnant women in this cohort had vaginal microbiota that clustered into 3 main community state types (CST): 109/356 (31%) CST1 - Lactobacillus iners dominant, 102/356 (29%) CST2 - Lactobacillus crispatus dominant and 145/356 (41%) CST3 - Gardnerella vaginalis dominant. There was a high prevalence of dysbiotic vaginal communities and L. iners was the predominant taxa found in > 90% of the women. When exploring whether the vaginal microbiota is associated with pregnancy outcomes, L. iners was highly associated low birth weight (LBW) and small for gestational age deliveries (SGA) while in contrast G. vaginalis was associated with normal birth outcome. There were no strong relationships identified between preterm birth (PTB) and vaginal microbiota. There was a strong correlation between vaginal microbiota and proinflammatory and adaptive cytokines. BV associated organisms (Gardnerella, Aerococcus, Prevotella Coriobacteriaceae and Dialister) were highly inflammatory while Lactobacillus spp were associated with low inflammation. Low levels of IL-13 and PDGF-BB cytokines were associated with LBW and PTB. We found that HIV status was highly associated with high vaginal microbial diversity. HIV-infected women had significantly higher alpha diversity in their vaginal microbiota, and they were more likely to have CST3. Surprisingly, having a diverse community type was not associated with high levels of vaginal inflammation. However low levels of IL13 and IL-I7 while high levels of TNF-α were associated with HIV status. In conclusion this study found that a highly dysbiotic vaginal environment is characteristics of Zimbabwean pregnant women and that vaginal microbiota is weakly associated with some poor pregnancy outcomes. The high prevalence of L. iners and its association with poor pregnancy outcomes suggests that vaginal microbiota may partly explain the high incidence of adverse pregnancy outcomes in African women. Low level of Th2 cytokines and growth factors may lead to adverse birth outcomes. Longitudinal studies of vaginal microbiota and soluble factors are needed in African women.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Pró-Reitoria de Pós-Gradução da UNESP (ProPG)
A vaginose bacteriana é o tipo mais comum de flora vaginal anormal e pode ser definida pela diminuição, ou mesmo depleção, dos lactobacilos vaginais. Tal condição está associada ao aumento do risco de parto prematuro e aquisição de diversas infecções sexualmente transmissíveis. A eficácia a curto prazo do tratamento da vaginose bacteriana com metronidazol é baixa. Portanto, o objetivo desse trabalho foi caracterizar o proteoma do fluido cérvico-vaginal de mulheres com vaginose bacteriana e comparar o perfil proteômico entre as mulheres que foram tratadas com sucesso em relação àquelas que falharam em restabelecer a microbiota lactobacilar após 7 dias de metronidazol. A presença da vaginose bacteriana foi definida de acordo com os critérios de Nugent após coloração de Gram dos esfregaços vaginais. Os perfis proteômicos do fluido cérvico-vaginal foram determinados utilizando a metodologia de shotgun LC MS/MS. As análises comparativas do proteoma das 38 mulheres com vaginose bacteriana e 39 com microbiota vaginal normal identificaram e determinaram a abundância relativa de 116 proteínas. Entre elas, catepsina G e a região BRO da cadeia pesada V-III de imunoglobulina foram exclusivas de vaginose bacteriana e o inibidor de elastase de leucócitos, involucrina e a proteína associada a diferenciação de neuroblastos AHNAK exclusivas de microbiota vaginal normal. Além disso, 20 (17.2%) proteínas foram diferencialmente expressas na vaginose bacteriana, das quais 9 são envolvidas na resposta imune. Entretanto, a comparação do proteoma do fluido cérvico-vaginal das 24 mulheres com vaginose bacteriana que foram tratadas com sucesso e 11 que persistiram com esta condição após o tratamento com metronidazol não apresentou diferença. Portanto, pudemos demonstrar que a vaginose bacteriana altera significantemente o proteoma local, mas o perfil proteômico cérvico-vaginal do hospedeiro não influencia a resposta ao...
Bacterial vaginosis is the most common type of abnormal vaginal flora and defined by the depletion of vaginal lactobacilli. This condition increases the risk of premature labor and acquisition of several sexually transmitted infections. Short-term efficacy of bacterial vaginosis metronidazole treatment is low. Thus, we aimed to characterize the cervicovaginal fluid proteome of women with bacterial vaginosis and to compare the proteomic profile between women who were successfully treated with those who failed to reestablish lactobacillar flora after the 7-days course of metronidazole. Presence of bacterial vaginosis was defined according to Nugent criteria on Gram-stained vaginal smears. Proteomic profile of cervicovaginal fluids were determined using shotgun LC MS/MS. Comparative analysis of the proteome of 38 women with bacterial vaginosis and 39 with normal vaginal flora identified and determined the relative abundance of 116 proteins. Among them, cathepsin G and Ig heavy chain V-III region BRO were exclusive of bacterial vaginosis while leukocyte elastase inhibitor, involucrin and neuroblast differentiation-associated protein AHNAK of normal flora. Moreover, 20 (17.2%) proteins were differentially expressed in bacterial vaginosis, of which 9 are involved in immune response. However, the cervicovaginal proteome of 24 women with bacterial vaginosis who were successfully treated and 11 who persisted with this condition did not differ between each other. Therefore, we showed that bacterial vaginosis significantly changes the local proteome, but cervicovaginal host's proteins do not influence the response to metronidazole treatment
FAPESP: 2012/16800-3

Orientador: Camila Marconi
Coorientador: Márcia Guimarães da Silva
Banca: Rodrigo Pauperio Soares de Camargo
Banca: Lucilene Dalazari dos Santos
Resumo: A vaginose bacteriana é o tipo mais comum de flora vaginal anormal e pode ser definida pela diminuição, ou mesmo depleção, dos lactobacilos vaginais. Tal condição está associada ao aumento do risco de parto prematuro e aquisição de diversas infecções sexualmente transmissíveis. A eficácia a curto prazo do tratamento da vaginose bacteriana com metronidazol é baixa. Portanto, o objetivo desse trabalho foi caracterizar o proteoma do fluido cérvico-vaginal de mulheres com vaginose bacteriana e comparar o perfil proteômico entre as mulheres que foram tratadas com sucesso em relação àquelas que falharam em restabelecer a microbiota lactobacilar após 7 dias de metronidazol. A presença da vaginose bacteriana foi definida de acordo com os critérios de Nugent após coloração de Gram dos esfregaços vaginais. Os perfis proteômicos do fluido cérvico-vaginal foram determinados utilizando a metodologia de shotgun LC MS/MS. As análises comparativas do proteoma das 38 mulheres com vaginose bacteriana e 39 com microbiota vaginal normal identificaram e determinaram a abundância relativa de 116 proteínas. Entre elas, catepsina G e a região BRO da cadeia pesada V-III de imunoglobulina foram exclusivas de vaginose bacteriana e o inibidor de elastase de leucócitos, involucrina e a proteína associada a diferenciação de neuroblastos AHNAK exclusivas de microbiota vaginal normal. Além disso, 20 (17.2%) proteínas foram diferencialmente expressas na vaginose bacteriana, das quais 9 são envolvidas na resposta imune. Entretanto, a comparação do proteoma do fluido cérvico-vaginal das 24 mulheres com vaginose bacteriana que foram tratadas com sucesso e 11 que persistiram com esta condição após o tratamento com metronidazol não apresentou diferença. Portanto, pudemos demonstrar que a vaginose bacteriana altera significantemente o proteoma local, mas o perfil proteômico cérvico-vaginal do hospedeiro não influencia a resposta ao...
Abstract: Bacterial vaginosis is the most common type of abnormal vaginal flora and defined by the depletion of vaginal lactobacilli. This condition increases the risk of premature labor and acquisition of several sexually transmitted infections. Short-term efficacy of bacterial vaginosis metronidazole treatment is low. Thus, we aimed to characterize the cervicovaginal fluid proteome of women with bacterial vaginosis and to compare the proteomic profile between women who were successfully treated with those who failed to reestablish lactobacillar flora after the 7-days course of metronidazole. Presence of bacterial vaginosis was defined according to Nugent criteria on Gram-stained vaginal smears. Proteomic profile of cervicovaginal fluids were determined using shotgun LC MS/MS. Comparative analysis of the proteome of 38 women with bacterial vaginosis and 39 with normal vaginal flora identified and determined the relative abundance of 116 proteins. Among them, cathepsin G and Ig heavy chain V-III region BRO were exclusive of bacterial vaginosis while leukocyte elastase inhibitor, involucrin and neuroblast differentiation-associated protein AHNAK of normal flora. Moreover, 20 (17.2%) proteins were differentially expressed in bacterial vaginosis, of which 9 are involved in immune response. However, the cervicovaginal proteome of 24 women with bacterial vaginosis who were successfully treated and 11 who persisted with this condition did not differ between each other. Therefore, we showed that bacterial vaginosis significantly changes the local proteome, but cervicovaginal host's proteins do not influence the response to metronidazole treatment
Mestre

Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is of major clinical importance due to its ability to significantly affect pregnancy outcome and enhance the transmission and acquisition of HIV. BV is characterized by a dramatic shift in the vaginal microflora; in most BV cases, the predominant bacterial species is Gardnerella vaginalis. It has been demonstrated that G. vaginalis forms an adherent biofilm on the vaginal epithelium of women with BV. Furthemore, evidence suggests that the high rate of recurrence associated with BV is related to incomplete eradication of the biofilm. The overall goal of this study was to characterize G. vaginalis virulence properties, including biofilm formation, in order to better understand the pathogenesis of BV and to improve available treatment methods. In an effort to tease apart the uncertain etiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. Only G. vaginalis demonstrated all three virulence properties, including robust biofilm formation. It has been shown that the biofilm phenotype allows its constituent bacteria to be resistant to many negative environmental stimuli. Therefore, we studied the susceptibilities of biofilm vs. planktonic cultures to H2O2 and lactic acid. Biofilms tolerated higher concentrations of both chemicals; however, when the biofilm was proteolytically disrupted, sensitivity to the chemicals returned to planktonic levels. Since our data suggested a critical role for a protein in biofilm formation, a partial genome sequence of G. vaginalis was searched for sequence homology to known biofilm adhesins using the tBLASTn program. This revealed an open-reading frame encoding a hypothetical protein with significant homology to the staphylococcal Bap protein. Antibody towards a portion of the identified gene product was produced in rabbits by inoculation of a recombinant peptide to an antigenic region of the protein. Antibody inhibition assays against biofilm formation, adherence, initial adherence and aggregation were conducted. Relative expression levels of the biofilm-associated protein were analyzed under different conditions by western blot analysis. Finally, the protein was expressed in heterologous hosts and analyzed for an increase in biofilm formation.

Preterm birth, birth prior to 37 weeks gestation, is the leading cause of neonatal mortality and morbidity worldwide. While the uterine cavity and amniotic fluid largely remain sterile throughout gestation, bacterial infections can occur and are associated with preterm birth and/or preterm premature rupture of the fetal membranes (PPROM). Sneathia amnii can be detected as a component of the vaginal flora in healthy women; however, it’s also associated with bacterial vaginosis and preterm birth. Sn35, an isolate of S.amnii, was identified and sequenced through the Vaginal Human Microbiome Project at VCU. Our objective was to classify potential virulence determinants in Sn35 and we successfully identified a putative zinc endopeptidase. The zinc endopeptidase appeared to cleave itself in a site-specific manner under calcium-depleted conditions, resulting in a truncated protein. The truncated protein did have collagenase activity and bacteriolytic activity as well.

Abstract The aim of the present study was to evaluate whether bacterial vaginosis (BV) diagnosed in early pregnancy and treated with vaginal clindamycin affects pregnancy outcome, and to investigate the predictive value of interleukins-6 (IL-6) and -8 (IL-8), and insulin-like growth factor-binding protein-1 (IGFBP-1) in cervical secretions, separately and combined by cervical measurement with transvaginal ultrasonography, on preterm delivery. A further aim was to analyze retrospectively the significance of absent or reversed end-diastolic velocity (AREDV) in the umbilical artery on perinatal outcome, and to investigate the short- and long-term outcome of infants born prematurely as a result of various causes (indicated preterm birth, preterm premature rupture of the membranes=PPROM). Bacterial vaginosis (BV) was screened in 1956 women in a low-risk population at the first antenatal visit, using Gram stain. One hundred and one of 143 BV-positive women were randomized to receive vaginal clindamycin or placebo. Seventy-seven women at 22-32 gestational weeks with premature uterine contractions, and 78 controls were recruited for assay of cervical IL-6, IL-8-, and IGFBP-1, and ultrasonographic measurements, which were repeated twice at two-week intervals. Eighty-three women with AREDV in the umbilical artery in high-risk pregnancies at less than 34 gestational weeks (e.g. pre-eclampsia, small-for-gestational age [SGA]) between the years 1988-95 were analyzed retrospectively as regards perinatal outcome. Further, for 103 women between the 24th and the 33rd week of pregnancy, delivered by cesarean section because of maternal or fetal indications, and for 103 matched women, between the years 1990-97, their infants were analyzed as regards neonatal mortality and morbidity, and the outcome at one year of corrected age. Similarly, 78 women with PPROM at gestational weeks 17-30, and 78 controls were also analyzed. The prevalence of BV was 7.3% (143/1956) and the preterm birth rate in women with BV was 9.9%. Preterm birth occurred in 21% vs. 0% according to whether or not BV persisted. The preterm birth rate was 14% in the clindamycin group vs. 6% in the placebo group. Cervical IL-6 at a concentration of 128 ng/L had a 73% sensitivity and 77% specificity in predicting preterm birth (35% vs. 6%). The combination of IL-6 and a cervical index of > 0.2 increased the specificity to 97%, the sensitivity falling to 45%. Concentrations of IGFBP-1 were most elevated (> 21 μg/mL) in cases with neonatal infections (36% vs. 2%). In cases of absent end-diastolic velocity (AEDV) the perinatal mortality (PNM) rate was 9%, compared with 36% in the reversed end-diastolic velocity (REDV) group. Respiratory distress (RDS) and hypoglycemia, and chronic lung disease (CLD; 15% vs. 3%) occurred significantly more often in the indicated than in the spontaneously preterm infants. The PPROM infants had more limb contractures (8% vs. 0%) and pulmonary hypoplasia (12% vs. 5%) and more chronic lung problems up to one year of age than the spontaneously preterm born infants without PPROM. The persistence of pregnancy BV is a risk factor for preterm birth, but vaginal clindamycin used in a low-risk population in early pregnancy is of no use in reducing the preterm birth rate in cases of BV. The level of IL-6 has a relatively low sensitivity and a limited role as a single method in clinical decision making but in combination with cervical examination by ultrasonography it seems to have a predictive role in cases of threatened preterm birth. A finding of AREDV in the umbilical artery is a warning signal of threatened fetal asphyxia. Infants born after indicated preterm delivery (for fetal or maternal reasons) or PPROM are at risk of later chronic lung disease.

Orientador: Marlus Chorilli
Coorientador: Taís Maria Bauab
Banca: Hérida Regina Nunes Salgado
Banca: Marilisa Guimarães Lara
Resumo: A candidíase vulvovaginal (CVV) é uma infecção fúngica causada principalmente pela espécie Candida albicans, sendo considerada a mais prevalente em mulheres com idade fértil, acometendo cerca de 80-85% dos indivíduos ao menos uma vez na vida. Um crescente interesse tem sido observado na utilização de compostos de origem natural, como a curcumina, de forma a atuar contra este microrganismo. Todavia, algumas de suas propriedades físico-químicas, como baixa solubilidade aquosa, dificultam a sua utilização na terapêutica. Dessa forma, a sua incorporação em sistemas líquido-cristalinos mucoadesivos (SLCM) objetivando administração vaginal pode ser uma estratégia eficiente de tratamento, de forma a potencializar a ação do fármaco, bem como oferecer maior permanência da formulação no local de ação. O objetivo deste trabalho foi desenvolver SLCM constituídos por ácido oleico e ergosterol (5:1) como fase oleosa, ácido cetílico etoxilado e propoxilado (Procetyl® AWS) como tensoativo e dispersão polimérica de quitosana (1%) como fase aquosa, acrescidos de curcumina, e caracterizá-los empregando microscopia de luz polarizada, espalhamento de raios-X de baixo ângulo (SAXS), reologia, análise de textura e mucoadesão in vitro. Posteriormente, avaliou-se a ação antimicrobiana in vitro e in vivo do sistema contra cepas de C. albicans. Pelos ensaios de microscopia de luz polarizada as formulações apresentaram estruturas semelhantes à cruz de malta e campo escuro, características de fase lamela... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Vulvovaginal candidiasis (VVC) is a fungal infection mainly caused by the Candida albicans species. The disease is being considered the most prevalent in women of childbearing age, affecting approximately 80-85% of individuals at least once in life. An increasing interest has been observed in the use of compounds of natural origin, such as curcumin, in order to act against this microorganism. However, some of its physicochemical properties, such as low aqueous solubility, make it difficult to use in therapeutics. Thus, its incorporation into mucoadhesive liquid-crystalline systems (MLCS), with vaginal administration, can be an efficient treatment strategy in order to potentiate the action of the drug, as well as to offer a longer permanence of the formulation at the site of action. The aim of this work was to develop mucoadhesive liquid-crystalline systems consisting of oleic acid and ergosterol (5: 1) as oily phase, ethoxylated and propoxylated cetylic acid (Procetyl® AWS) as a surfactant and polymer dispersion of chitosan (1%) as aqueous phase, plus curcumin and characterize them using polarized light microscopy, low-angle X-ray scattering (SAXS), rheology, texture analysis and in vitro mucoadhesion. Subsequently, the antimicrobial action in vitro and in vivo of the system against C. albicans strains was evaluated. By polarized light microscopy, the formulations presented similar structures to the cross of malt and dark field, characteristics of lamellar phase and microemulsions, respectively, the results were confirmed by low-angle X-ray scattering. In vitro mucoadhesion assays demonstrated an increase in the mucoadhesive strength of the formulations in the presence of increasing concentrations of artificial vaginal mucus (AVM), corroborating rheology results, which showed high viscosity and elasticity of the formulations in the presence of AVM... (Complete abstract click electronic access below)
Mestre

Preterm birth, defined at birth before 37 weeks gestation, affects millions of newborns worldwide every year. Preterm birth is a leading cause of infant morbidity and mortality. One major cause of preterm birth is preterm premature rupture of membranes (PPROM), which can be triggered by bacterial infection and inflammation. A bacterial species that has been implicated in preterm birth and other obstetric complications is Sneathia amnii. The goals of this study were to observe cytopathogenic effects caused by S. amnii strain Sn35 and identify putative virulence factors causing those effects. Sn35 was able to adhere to, invade, and damage/kill various host cell lines. We characterized these virulence attributes. A putative virulence determinant was identified, and a fragment of the protein was expressed for polyclonal antiserum production. Antiserum was used to characterize the expression and subcellular localization of the protein in Sn35. However, antiserum was unable to prevent cytopathogenic effects.

Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women, 
Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa
therefore, we embarked on a study to determine the 
prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics 
units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history, 
including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG) 
and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV 
status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p <
0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history 
of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV. 

About 10 % of the adult women population in Sweden are treated annually for urinary tract infections. The increasing bacterial resistance towards antibiotics is classified by WHO (World Health Organization) and ECDC (European Centre for Disease Prevention and Control) as one of the greatest treats for human health in a global perspective. To find alternatives scientists are studying the possibility to use probiotics to reduce the frequency of recurring urinary tract infections. This literature study examines five randomized double blinded placebo controlled studies where different strains of Lactobacillus have been used as a prophylactic to women suffering from recurrent urinary tract infections and bacterial vaginosis. Overall the studies do not display enough promising results to recommend the use of probiotics as a prophylax or cure.

Orientador: Paulo César Giraldo, Fernando Guimarães
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: As vulvovaginites (VV) são consideradas como agravos importantes à saúde da mulher, pois afetam suas vidas no âmbito sexual, afetivo, social, e psíquico. Muitos aspectos da fisiopatogênese destas afecções ainda precisam ser esclarecidos, entre eles os mecanismos imunes relacionados à instalação e propagação da infecção. Objetivos: Identificar e quantificar por citometria de fluxo (CF) os leucócitos presentes no conteúdo vaginal de mulheres com flora normal e VV. Avaliar a expressão do CD16 nos neutrófilos do conteúdo vaginal destas mulheres. Materiais e Métodos: Estudo de corte transversal, no período de junho de 2009 a outubro de 2010. O estudo incluiu 152 mulheres no menacme diagnosticadas com: flora vaginal normal (n=51), vaginose bacteriana (VB) (n=34), candidíase vulvovaginal (CV) (n=43) e VB associada a CV (VB+CV) (n=14). As mulheres foram submetidas a exame especular para medida de pH vaginal, teste de Whiff, coleta de material para bacterioscopia e cultura para fungos. A VB foi detectada pelos critérios de Amsel e/ou escore de Nugent ?7. A CV, pela presença de hifas ou esporos no conteúdo vaginal. VB+CV foi diagnosticada quando estas duas VV estavam presentes na mesma mulher segundo critérios já descritos. Foram consideradas com flora vaginal normal, as mulheres que apresentaram flora do tipo 1 e ausência de patógenos nos exames laboratoriais. Ao final do exame ginecológico, foi realizado um lavado da cavidade vaginal, o qual foi enviado ao laboratório para processamento. Os leucócitos foram marcados com anticorpos monoclonais conjugados a fluoróforos (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD24 e anti-CD56) e analisados por CF. Resultados: Os granulócitos neutrófilos foram as células predominantes em todos os grupos estudados. A média da porcentagem de granulócitos neutrófilos foi significativamente maior (p<0,05) na CV comparado à flora normal e VB. A média da porcentagem de granulócitos neutrófilos foi significativamente menor (p<0,05) na VB comparado à flora normal e a CV. Macrófagos e linfócitos foram detectados em percentuais bem menores que os granulócitos neutrófilos. Houve significância estatística (p<0,05) para os linfócitos TCD4 que apresentaram média da porcentagem maior na CV e VB em comparação à flora normal. Considerando-se a intensidade da expressão do CD16 nos neutrófilos, houve maior expressão nas VV (VB, CV, VB+CV) quando comparado à flora normal (p<0,05). Entre as VV, esta ocorrência foi maior na VB comparado à CV (p<0,05). Conclusões: Os granulócitos neutrófilos foram as células predominantes nos lavados vaginais e suas quantidades foram decrescentes considerando-se CV, flora normal e VB. Estes dados sugerem que, como em outros tecidos, na vagina, os neutrófilos são as principais células efetoras da resposta imune apresentando-se em maiores ou menores concentrações conforme o estímulo imunológico causado pelo micro-organismo. A maior intensidade da expressão do CD16 nos neutrófilos das mulheres com VV indica um atraso na apoptose dos neutrófilos envolvidos nestas infecções, predominando na VB
Abstract: Introduction: Vulvovaginitis (VV) is a serious health problem in women. This disease affects their lives in all aspects, be it, sexual, affective, social, and psychological. Many aspects of the pathophysiology of these infections have yet to be elucidated, including the immune mechanisms related to proliferation of microorganisms and maintenance of the infectious process. Objectives: One aim of this study was to identify and to quantify the immune cells present in the vaginal lumen of women with normal flora and VV by flow cytometry (FC). Another aim was to evaluate the expression of CD16 on neutrophils from the vaginal lumen of these women. Materials and Methods: A Cross-sectional study was performed from June 2009 to October 2010. The study included 152 women of childbearing age diagnosed with: normal vaginal flora (n=51), bacterial vaginosis (BV) (n=34), vulvovaginal candidiasis (VC) (n=43) and BV associated with VC (BV+VC) (n=14). The women underwent speculum examination for performing vaginal pH, Whiff test, Gram stain and culture for fungi. BV was diagnosed by the Amsel criteria and/or Nugent score ?7. VC was diagnosed by the presence of yeast or hyphae in the vaginal discharge. A diagnosis of BV+VC was made when both criteria were present in the same woman. Normal vaginal microflora was defined by the presence of type 1 flora and absence of pathogens in laboratorial exams. During gynecological examination, a lavage of the vaginal cavity was performed and sent to the laboratory for processing. Immune cells were labeled with fluorochrome-conjugated monoclonal-antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD24 e anti-CD56) and analyzed by FC. Results: The neutrophil granulocytes were the predominant cells in all groups. The mean of the percentage of neutrophil granulocytes was significantly higher (p <0.05) in VC compared to the normal flora and BV. On the other hand, the mean of the percentage of neutrophil granulocytes was significantly lower (p <0.05) in BV compared to the normal flora and VC. Macrophages and lymphocytes were detected in percentages far lower than the neutrophil granulocytes. The mean percentage of CD4 lymphocytes was significantly higher (p <0.05) in BV and VC compared to the normal flora. The expression of CD16 on neutrophils was higher in VV (BV, VC, BV+VC) compared to the normal flora (p <0.05). Among the VV, it was higher in BV compared to VC (p <0.05). Conclusions: Neutrophil granulocytes were the predominant cells in the vaginal lavages. Higher amount of neutrophil granulocytes was observed in VC, normal flora and BV respectively. These data suggest that neutrophils are presented in higher or lower concentrations as the immune stimulation caused by the pathogen. The highest intensity of CD16 expression on neutrophils in women with VV indicates a delay in neutrophil apoptosis in these infections, predominantly in BV
Mestrado
Fisiopatologia Ginecológica
Mestre em Ciências da Saúde

Human immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can increase susceptibility to HIV. Accordingly, one common risk factor for HIV acquisition is the microbial shift condition known as bacterial vaginosis (BV), which is characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. Our model showed good agreement with clinical BV trends; we observed a distinct cytokine and human ?-defensin-2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. We next evaluated the effect of this interaction on HIV infection by investigating the soluble effectors secreted when FRT epithelial cells were cocultured with A. vaginae. We observed increased proviral activity mediated by secreted low molecular weight effectors, and determined that this activity was not likely mediated by cytokine responses. Instead, we identified a complex mixture containing several upregulated host proteins. Selected individual proteins from the mixture exhibited HIV-enhancing activity only when applied with the complex mixture of proviral factors, suggesting that HIV enhancement might be mediated by synergistic effects. In addition to characterizing the immune interactions that mediate the enhanced HIV acquisition associated with BV, we also evaluated the safety and efficacy of RC-101, a candidate vaginal microbicide being developed for the prevention of HIV transmission. RC-101 has been effective and well tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation. Here, we treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate compatibility of this microbicide with FRT tissue and microflora. RC-101 was well tolerated by host tissues and commensal vaginal bacteria, while BV-associated bacteria were inhibited by RC-101. By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Vaginose Bacteriana (VB) é uma síndrome polimicrobiana, caracterizada pelo desequilíbrio da microbiota vaginal, associada à substituição da população bacteriana vaginal predominantemente aeróbia (Lactobacillus spp.) por uma população anaeróbia (principalmente Gardnerella vaginalis), gerando descarga vaginal anormal em mulheres em idade reprodutiva. O objetivo desse trabalho foi a avaliação de aspectos fisiológicos e moleculares de G. vaginalis em pacientes com e sem VB, atendidas na rede pública (SUS) e privada de Juiz de Fora/MG, além da determinação do perfil de susceptibilidade a antimicrobianos. Amostras de secreção vaginal foram coletadas e processadas para isolamento seletivo, conforme descrito na literatura. G. vaginalis foi identificada presuntivamente pela β-hemólise ou hemólise difusa e os testes da oxidase e catalase. A identidade bacteriana foi confirmada por reação de POR. O gene codificador para a vaginolisina (vly) foi detectado por PCR. O perfil de susceptibilidade a drogas antimicrobianas foi determinado pelo método da diluição em ágar, de acordo as recomendações do CLSI para microrganismos anaeróbios. A genotipagem foi realizada por AP-PCR. De 89 pacientes, G. vaginalis foi isolada de 42 por cultivo em meio de cultura, sendo 35 com VB, 02 com quadro intermediário e 05 saudáveis. Em 47 pacientes não foi possível realizar o isolamento de G. vaginalis por método de cultivo, sendo a reação de PCR positiva para 26 destas pacientes. Para classificar as pacientes em sintomáticas e saudáveis, foi realizado o escore de Nugent, Todos os 204 isolados de G. vaginalis submetidos à reação de PCR tiveram sua identidade confirmada, e destes, em 96,5% foi detectado o gene vly. Quanto ao perfil de susceptibilidade aos antimicrobianos, alta sensibilidade a ampicilina/sulbactam, clindamicina e cloranfenicol foi observada, e alta resistência foi observada a ampicilina, metronidazol, secnidazol e tinidazol. A cultura positiva de G. vaginalis, per se, não garante o diagnóstico de VB, já que os microrganismos fazem parte da microbiota residente. Não foi possível correlacionar a detecção do gene vly como marcador para determinação de linhagens patogênicas. A resistência bacteriana frente aos antimicrobianos mais utilizados na clínica corroboram outros estudos, e alertam para os riscos da terapia empírica rotineira. A técnica de genotipagem utilizada não permitiu o agrupamento de G. vaginalis, de acordo com sua origem de isolamento, sugerindo, assim, uma heterogeneidade populacional. Essa observação sustenta-se pelo não agrupamento bacteriano também em função da tipagem pelo perfil de susceptibilidade a antimicrobianos, embora outras técnicas de genotipagem não tenham sido utilizadas. Além disso, ainda não são descritos oligonucleotídeos iniciadores específicos para genotipagem de G. vaginalis por AP-PCR. Espera-se que esses resultados possam servir de base para suscitar discussões relacionadas ao tratamento empírico da vaginose bacteriana, considerando-se limitações na disponibilidade de diagnóstico ou mesmo terapia antimicrobiana.
Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by an imbalance in the vaginal microbiota associated with the replacement of the vaginal bacterial population predominantly aerobic (Lactobacillus spp.) by an anaerobic population (especially Gardnerella vaginalis) leading to abnormal vaginal discharge in women of reproductive age. The aim of this study was to evaluate physiological and molecular aspects of G. vaginalis in patients with and without BV, treated at public (SUS) and private services in Juiz de Fora/MG, in addition to determining the antimicrobial susceptibility patterns. Vaginal secretion samples were collected and processed for selective isolation, as described in the literature. G. vaginalis was presumptively identified by 13-hemolysis or diffuse hemolysis and oxidase and catalase negative tests. The bacterial identity was confirmed by PCR. The encoding gene for vaginolisin (vly) was detected by PCR. Antimicrobial susceptibility patterns were determined by agar dilution method, according to CLSI guidelines for anaerobic microorganisms. Genotyping was performed by AP-PCR. Out of 89 patients, G. vaginalis was isolated from 42 by cultivation in culture medium, 35 of which with VB, 02 with intermediate clinical and 05 healthy. In 47 patients it was not possible to perform the isolation of G. vaginalis by cultivation method, with a positive PCR result for 26 of these patients. To classify patients in symptomatic and healthy, Nugent score was performed. All 204 isolates of G. vaginalis subjected to PCR reaction had its identity confirmed and, of these, vly gene was detected in 96.5%. Regarding antimicrobial susceptibility patterns, high sensitivity to ampicillin/sulbactam, clindamycin and chloramphenicol was observed, and high resistance was observed to ampicillin, metronidazole, secnidazole and tinidazole. The positive culture of G. vaginalis, per se, does not ensure the diagnosis of BV, as the microorganism may be considered resident microbiota. It was not possible to correlate vly gene detection as a marker for determination of pathogenic strains. Bacterial resistance against antimicrobial drugs commonly used in clinical corroborate with others studies, and warn to the risks of empiric therapy routine. The genotyping technique used did not allow G. vaginalis grouping, according to their source of isolation, suggesting, therefore, a heterogeneous population. This observation is sustained by the impossibility of grouping using the bacterial typing by antimicrobial susceptibility profile too, although other genotyping techniques have not been used. In addition, specific primers for genotyping G. vaginalis by AP-PCR have not been described yet. It is expected that these results can serve as basis for raising discussions related to empiric treatment of bacterial vaginosis, considering limitations in the availability of diagnostic or antimicrobial therapy.

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INTRODUCTION. The balance of the vaginal microbiota is related to a woman's hormonal status, microbial metabolism, sexual stimulation and immune response. The imbalance of vaginal flora, induces elevation of pH, favoring infections and abnormal vaginal secretion. OBJECTIVES. Estimating the prevalence of the bacterial vaginosis (BV), vulvovaginal candidiasis (VVC) and trichomoniasis in cervical cytology smears, stained by the method of Papanicolaou; studying the association between sociodemographic and behavioral variables and microbiological findings; assessing the degree of agreement between the clinical characteristics of vaginal secretion and microbiological findings. METHODS. Crosssectional study performed in 302 women aged 20 to 87, referred to the Gynecology Outpatient Clinic of the Integrated Healthcare Center Chácara do Governador, in Goiânia, Goiás, from June 2012 to May 2013. All were patients interviewed and underwent a gynecology test. Cervical smear was also collected for cytology. The samples were analyzed at the Rômulo Rocha Clinical Analysis Center of the School of Pharmacy of the Federal University of Goiás - UFG. A multiple logistic regression analysis was performed to identify sociodemographic and behavioral factors associated to the microbiological findings from the cytology smear. The odds ratio (OR) and adjusted OR were calculated and the confidence interval was set at 95% (CI 95%). The kappa index was used to evaluate the degree of agreement between the characteristics of the vaginal secretion and microbiological findings. RESULTS. The prevalence of BV, VVC and trichomoniasis was 25.5%, 9.3% and 2.0%, respectively. The report of a previous STI (OR=2.27; CI 95%=1.15-4.49; p=0.018) and the use of vaginal douching were associated to the diagnosis of BV (OR=4.71; CI 95%: 2.65-8.38; p=<0.001). None of the variables studied presented a significant association with the microbiological findings of VVC and trichomoniasis. The degree of agreement between the clinical evaluation of vaginal secretion and the microbiological diagnosis of BV, candidiasis and trichomoniasis, as analyzed by the kappa index was 0.47, 0.23 and 0.28, respectively. CONCLUSION. The most prevalent microbiological diagnosis was BV. The association found between the use of vaginal douching and BV indicated that it may be a triggering factor. The clinical evaluation of vaginal secretion presented a moderate to weak degree of agreement with the microbiological diagnosis, indicating the need for complementary investigation.
INTRODUÇÃO. O equilíbrio da microbiota vaginal está relacionado ao estado hormonal, metabolismo microbiano, estimulação sexual e resposta imune da mulher. O desequilíbrio da flora vaginal, induz a elevação do pH, favorecendo infecções e secreção vaginal anormal. OBJETIVOS. Estimar a prevalência da vaginose bacteriana (VB), candidíase vulvovaginal (CVV) e tricomoníase em esfregaços citológicos do colo uterino, corados pelo método de Papanicolaou; avaliar a associação entre variáveis sociodemográficas e comportamentais e os achados microbiológicos; Avaliar o grau de concordância entre as características clínicas da secreção vaginal e os achados microbiológicos. MÉTODOS. Estudo de corte transversal em 302 mulheres de 20 a 87 anos referenciadas à consulta no Ambulatório de Ginecologia do Centro de Atendimento Integral à Saúde da Chácara do Governador, em Goiânia, Goiás. Todas foram submetidas à entrevista, exame ginecológico e coleta de esfregaço para estudo citológico, no período de junho de 2012 a maio de 2013. As amostras foram analisadas no Centro de Análises Clínicas Rômulo Rocha da Faculdade de Farmácia/UFG. Análise de regressão logística foi realizada para identificar fatores sociodemográficos e comportamentais associados aos achados microbiológicos no esfregaço citológico. Foi calculada a razão de odds (OR) e a OR ajustada com intervalo de confiança de 95% (IC95%). O grau de concordância entre as características da secreção vaginal e os achados microbiológicos foi avaliado pelo índice kappa. RESULTADOS. A prevalência da VB, CVV e tricomoníase foi de 25,5%, 9,3% e 2,0%, respectivamente. O relato IST prévia (OR=2,27; IC95%=1,15-4,49; p=0,018) e o uso de ducha vaginal associou-se ao diagnóstico de VB (OR=4,71; IC95%: 2,65- 8,38; p=<0,001). Nenhuma variável estudada apresentou associação significativa com os achados microbiológicos de CVV e tricomoníase. O grau de concordância entre a avaliação clínica da secreção vaginal e o diagnóstico microbiológico de VB, candidíase e tricomoníase, avaliados pelo índice kappa foi de 0,47, 0,23 e 0,28, respectivamente. CONCLUSÃO. O diagnóstico microbiológico de maior prevalência foi a VB. A associação entre o uso de ducha vaginal e VB aponta para seu possível fator desencadeante. A avaliação clínica da secreção vaginal apresentou grau de concordância moderado a fraco com o diagnóstico microbiológico, o que indica a necessidade de avaliação complementar.

Worldwide, more than half of all HIV-infected individuals are women. Since mucosal surfaces are the primary gateway for HIV entry, maintaining the integrity of the female reproductive tract (FRT) is essential for preventing infection. The FRT employs many immune mechanisms that serve as the first line of defense against HIV transmission. Among these are vaginal fluid secretions rich in antimicrobial peptides, and commensal bacteria that colonize the vagina and prevent infections. We sought to study vaginal fluid as an innate immune component of the FRT in the prevention of HIV infection. Additionally, we investigated the anti-HIV microbicide candidate RC-101 as a possible treatment against pathogenic bacteria that disrupt the healthy microbiota of the FRT and create a suboptimal immune state that increases host susceptibility to viruses, such as HIV. Here we report that vaginal fluid collected from healthy females inhibits HIV infection. Moreover, our studies reveal that vaginal fluid collected from Black and White women exhibit disparate anti-HIV activity, possibly rendering Black women more susceptible to HIV infection. In addition, we show that RC-101, which is active against HIV, can also inhibit pathogenic bacteria that compromise FRT innate immunity, providing a dual mechanism of protection against HIV acquisition. Overall, these findings show that vaginal fluid is an important part of female innate immunity that protects the host from heterosexual HIV acquisition. Furthermore, the microbicide RC-101 may prevent HIV infection by both directly preventing viral entry, and by restricting the growth of pathogenic bacteria that disrupt the protective commensal vaginal flora. Together, innate mechanisms and bolstered protection present a multifaceted approach to maintaining effective host immunity.
B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular and Microbiology

Grâce aux avancées de la technologie et nouvelles stratégies OMICS, de nombreuses études se sont intéressées au microbiote vaginal ces dernières années. Elles ont révélé l'impact de ce dernier sur la santé de la femme. En effet, un déséquilibre de la flore vaginale la rend vulnérable, la prédisposant à la vaginose bactérienne ainsi qu’à des complications gynéco-obstétricales sévères. La pathogénèse de la vaginose reste encore méconnue et le traitement classique par antibiothérapie échoue dans plus de 50% des cas. En analysant 50 prélèvements vaginaux provenant de patientes atteintes de vaginose et de femmes saines vivant en France et au Sénégal, nous avons constaté une plus grande diversité bactérienne chez les patientes par rapport aux témoins avec l'augmentation d'espèces telles que Gardnerella vaginalis, Atopobium vaginae ainsi que les procaryotes sensibles à l'oxygène, y compris les Cocci anaérobies à Gram-positif et les Prevotella. Les femmes saines renfermaient plus d’espèces de Lactobacillaceae et de Proteobacteria dans leurs flores. La combinaison de la métagénomique et la culturomique a permis d’identifier un complexe de 11 espèces/genres bactériens associés à la vaginose. L’utilisation de la culturomique a permis d’accroître le répertoire des bactéries humaines avec l’isolement de 27 nouvelles espèces. Le faible taux de recouvrement entre les données de métagénomique et celles de culturomique montre la nécessité de persévérer dans l’isolement des bactéries par culturomique. L’obtention d'isolats permettra d'explorer in vitro les compétitions entre les bactéries et pourrait servir également de matière première pour développer un traitement par bactériothérapie
Over the last decades, thanks to the technologic progresses including advanced molecular techniques and new OMICS strategies, many studies have focused on the vaginal microbiota. Thus, revealing the impact of the vaginal flora on women health. Indeed, the disruption of the vaginal bacterial community makes it prone to bacterial vaginosis and severe obstetrical and gynecological disorders. The pathogenesis of bacterial vaginosis is still unknown, and relapses are very frequent. Conventional treatment with antibiotic therapy fails in more than 50% of cases. The analysis of 50 vaginal samples from bacterial vaginosis patients and healthy women living in France and Senegal, showed a higher bacterial diversity in patients compared to controls with the increase of species such as Gardnerella vaginalis, Atopobium vaginae as well as oxygen-sensitive prokaryotes including Gram-positive anaerobic cocci, and Prevotella spp. Healthy women harbored more Lactobacillaceae species and Proteobacteria in their microbiota. The combination of metagenomics and culturomics has allowed the identification of a complex of 11 bacterial species/genera associated with bacterial vaginosis. The use of the culturomics approach has extended the repertoire of human-associated bacteria, with the isolation of 27 new bacterial species. The low range overlap between metagenomic and culturomics data indicates the need to continue the isolation of bacteria by culturomics. Obtaining isolates will make it possible to explore in vitro the competitions between the bacteria but can also be used as primary material for the development new treatments by bacteriotherapy

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The vagina and the cervix are the first immune and physical line of defense against sexually transmitted pathogens. However infectious process in the vagina, caused by potentially pathogenic microorganism such as bacterial vaginosis (BV), are very often. This may be associated with a morbid entity as Human Papillomavirus (HPV). It was evaluated the association between the presence of BV and HPV in the lower female genital tract, identifying the clinical and lab aspects in women who received medical attention at emergency rooms in the city of Goiânia-GO and observed the production of cytokines in endocervical secretions in cases with VB associated with HPV. This study included 173 sexually active women, between 16 and 48 years old, divided into groups: control, with BV and with high and low risk of HPV. A survey was conducted to collect the intrinsic and extrinsic factors associated with the patients. Microbial cultures, vaginal pH, identification of high and low risk of HPV by PCR, and the concentration of cytokines were performed. The result of the research data was submitted to a statistical analysis, calculating the oods ratio, confidence intervals and p of 0,05. Forty seven cases of BV were diagnosed. Besides, the presence of a vaginal pH > 4.5, showed a result statistically significant for the presence of HPV, p = 0.001 was also observed. When analyzing the multivariate logistic regression, the independent risk factors for the presence of VB were: having more than one sexual partner in the last 5 years p<0,001 and have more than 3 sexual intercourses per week p=0,002. But in HPV infection the independent risk factors were: being married p=0,029 and pH ≤ 4.5 p<0,001 and in the case of high-risk HPV the only independent factor considered was the pH ≤ 4.5 p= 0,006. Relating HPV infections with high and low risk and the presence of VB with some immune factors by the strength of local cytokines, it was found that, IL-2 and IL-12 were significantly elevated in cases of BV and HPV. The IL-6 was high only for HPV, followed by IFN-gamma and IL-10 although the last two showed a trend towards statistical significance in HPV cases. It was concluded through the univariate analysis that the factors, having more than one sexual partner in the last 5 years, a failure in the use of condoms and the presence of leucorrhoea and dyspaurenia were related to the presence of BV and level of alkaline in the vagina which could possibly predispose to infections HPV, and the immune response Th1-type cytokines in vaginal secretions in the presence of BV and/or HPV may also be similar.
A vagina e a cérvice são as primeiras linhas de defesa física e imunológica contra patógenos sexualmente transmissíveis. No entanto, os processos infecciosos na vagina causados por micro-organismos potencialmente patogênicos como nas vaginoses bacterianas (VB) são muito comuns. Podendo estar associados a uma entidade mórbida como o papilomavírus humano (HPV). Avaliou-se a relação existente entre a presença de VB e o HPV no trato genital inferior, dentificando os aspectos clínicos e laboratoriais das mulheres atendidas em um hospital e em 5 unidades básicas de saúde da cidade de Goiânia-GO e verificou-se a concentração de citocinas na secreção endocervical nos casos com VB e HPV. O estudo incluiu 173 mulheres sexualmente ativas entre 16 e 48 anos de idade, divididas em grupos: controle, com VB e com HPV de alto e baixo riscos. Aplicou-se um questionário onde foram analisados os fatores de riscos extrínsecos e intrínsecos destas pacientes. Realizou-se exames de cultura e identificação microbiana, verificando o pH vaginal, seguido da identificação do HPV de alto e baixo riscos pelo PCR e dosagem de citocinas. Os dados obtidos foram submetidos à análise estatística, com cálculos do odds ratio, intervalo de confiança e p de 0,05. Foram identificados 47 casos de VB. A presença de pH vaginal > 4,5 mostrou resultado estatisticamente significativo ao aparecimento de HPV, p=0,001. Ao se analisar a regressão logística multivariada, os fatores de risco independentes para a presença de VB foram: ter mais de um parceiro sexual nos últimos 5 anos p<0,001 e ter mais de 3 relações sexuais por semana p=0,002. Já na presença de HPV os fatores independentes foram: estado civil casado p=0,029 e pH ≤ 4,5 p< 0,001 e no caso de ser HPV de alto risco o único fator considerado independente foi o pH ≤ 4,5 p= 0,006. Ao se relacionar as infecções pelo HPV de alto e baixo risco e pela presença de VB com alguns fatores imunológicos, através da dosagem das citocinas locais, verificou-se que as IL-2 e IL-12 mostraram maior concentração nos casos de VB e HPV se comparadas ao controle. A IL-6 mostrou-se elevada somente para HPV, seguidos pelo IFN-gama e a IL-10 embora estas duas últimas próximas ao estatisticamente significativo. Concluiu-se que na análise univariada que ter mais de um parceiro sexual nos últimos 5 anos e realizar mais de 3 relações sexuais por semana são fatores independentes associados à VB e a alcalinização da vagina provavelmente seja um fator predisponente às infecções pelo HPV e a resposta imune, através das citocinas tipo Th1 da secreção vaginal na presença de VB e/ou HPV, possivelmente seja semelhante.

Orientadores: Paulo César Giraldo, Cristina Laguna Benetti Pinto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: INTRODUÇÃO: A vaginose bacteriana (VB) e a candidíase vaginal (CV) são as vulvovaginites (VV) mais frequentemente encontradas em mulheres durante o ciclo reprodutivo. Embora os tratamentos dessas VV estejam esclarecidos, a prevenção ainda é pouco estudada. Os hábitos de higiene e cuidados diários com a genitália feminina são fatores que podem influenciar o ecossistema vulvovaginal, facilitando a instalação dessas VV. OBJETIVO: Verificar os hábitos de lavagem, uso de absorventes higiênicos, práticas depilatórias, uso de piercings e tatuagens, tipo indumentária e atividades sexuais em mulheres com VV. MÉTODOS: Estudo de corte transversal utilizou questionário contendo 60 perguntas, divididas nos seguintes domínios: I ¿ Limpeza genital; II ¿ Uso de absorventes higiênicos; III ¿ Práticas depilatórias; IV ¿ Uso de piercings e tatuagens genitais; V ¿ Tipo indumentária e VI ¿ Atividades sexuais. Foram analisadas 307 mulheres de 18 a 45 anos, atendidas nos ambulatórios de um hospital universitário (Universidade Estadual de campinas, Brasil). Realizou-se exame ginecológico para diagnóstico de VB e CV por bacterioscopia e cultura de fungo, além de medir pH e realizar teste de Whiff. Os critérios de exclusão foram: uso de antibiótico até 15 dias antes da inclusão, antecedente de câncer, sorologia positiva para HIV e/ou sífilis e presença doença imunossupressora. Este estudo teve a aprovação do Comitê de Ética em Pesquisa da Unicamp sob número de protocolo 1836/2013 e todas as voluntárias assinaram o termo de consentimento livre e esclarecido previamente à participação neste estudo. A coleta de dados foi realizada no período de março a novembro de 2013. Para a análise estatística, consideraram-se dois grupos principais: mulheres sem e com vulvovaginites (presença de VB, CV e ambas) que foram comparados entre si. Também se realizou uma segunda análise de cada tipo de VV isoladamente (VB, CV e ambas associadas) versus mulheres sem vulvovaginites. Foram empregados os testes de Fischer e Qui-quadrado através do EPI INFO 0.5. Considerou-se nível de significância quando p<,05. RESULTADOS: Entre as 307 participantes, 46% foram diagnosticadas com VV presentes e 54% sem VV. Quando comparados estes grupos, não se encontraram diferenças significativas quanto à idade, IMC, escolaridade, número de gestações, número de partos, estado marital, raça, religião, uso de métodos contraceptivos, tabagismo, tempo fora de casa e queixas de dispareunia. A média de idade foi de 33,6 (±6,8) anos e de escolaridade 10,4 (±3,3) anos de estudo. A presença de VV esteve significativamente associada ao menor uso de produtos para higiene genital tais como sabonete líquido íntimo para higiene diária (p=,04) e lenço úmido para higiene pós-miccional (p=,04) e maior uso de sabonete bactericida para realização da lavagem genital durante o banho (<,0001). As mulheres com VV utilizaram mais calcinhas de tecido sintético (p<,05), apresentaram mais ciclos menstruais (p<,0001) que aquelas sem VV e apresentaram hábitos de uso de absorventes semelhantes. As mulheres com VB praticaram mais o sexo anal nos últimos 30 dias (p<,0001) e usavam mais substâncias erógenas (p<,02) que aquelas sem VV. Não houve diferenças significativas de frequência de relações sexuais, dispareunia, sexo oral e uso de lubrificantes entre os grupos estudados. A análise da depilação genital também não evidenciou diferenças significativas quanto ao método utilizado, motivação, frequência, área de depilação, irritabilidade vulvar, produtos pós-depilatórios e opinião sobre a influência da depilação genital sobre a saúde feminina. Apesar de ser um número baixo, mulheres com CV apresentaram mais tatuagens genitais que os demais grupos (p=,04) e apenas uma mulher apresentou piercing genital. CONCLUSÕES: Alguns hábitos de lavagem da genitália, a presença de ciclos menstruais, o uso de calcinhas de tecido sintético, relações sexuais anais e uso de substâncias erógenas na genitália se associaram à frequência de vulvovaginites. Especialmente estas últimas relacionadas aos hábitos sexuais estiveram associadas à presença de vaginose bacteriana. Os hábitos de uso de absorventes higiênicos e depilatórios não se associaram à presença de VV. Os adornos genitais foram raros, porém encontraram-se mais tatuagens genitais em mulheres com CV
Abstract: INTRODUCTION: Bacterial vaginosis (BV) and vaginal candidiasis (VC) are the most frequently vulvovaginitis (VV) encountered during in women at reproductive cycle. Although the treatments of VV are clear, prevention is still little studied. Hygiene habits and daily care with the female genitalia are factors that can influence the vulvovaginal ecosystem, and might facilitate the installation of these VV. MAIN: To investigate the genital washing habits, use of sanitary pads, genital hair removal, use of piercings and tattoos, clothing type and sexual activity in women with VV. METHODS: Cross-sectional study used a questionnaire containing 60 questions, divided into the following areas: I ¿ Genital Cleaning; II ¿ Use of sanitary pads; III ¿ depilatory practices; IV ¿ Use of genital piercings and tattoos; V ¿ clothing type and VI ¿ Sexual Activities. Were analyzed 307 women from 18 to 45 years, attended at two outpatient clinics of a university hospital (University of Campinas, Brazil). Gynecological exam was performed for collecting vaginal material for BV and VC diagnosis by Gram stain and culture of fungus, as well as measured pH and performed Whiff test. Exclusion criteria were: use of antibiotics within 15 days before enrollment, history of cancer, HIV positive and/or other immunosuppressive disease. This study was approved by the Ethics Committee of UNICAMP Research under protocol number 1836/2013 and all volunteers signed an informed consent form prior to their participation in this study. Data collection was conducted from March to November 2013. For statistical analysis, we considered two main groups: women with and without vulvovaginitis (presence of BV, VC and both) were compared to each other. It was also conducted a second analysis of each group of VV alone (BV, VC and both combined) versus women without vulvovaginitis. Statistical analysis used exact Fischer and chi-square tests by the EPI INFO 0.5. It was considered a significance level of p<.05. RESULTS: Among the 307 participants, 46% were diagnosed with VV and 54% without VV. When comparing these groups, there were no significant differences in age, BMI, duration of study, number of pregnancies, number of births, marital status, race, religion, use of contraceptives, smoking, time away from home and complaints of dyspareunia. The mean age was 33.6 (± 6.8) years of education and 10.4 (± 3.3) years of education. The presence of VV was significantly associated with lower use of genital hygiene products such as liquid soap for daily intimate hygiene (p=.04) and moist napkin as hygiene post urination (p=.04) and, on the other hand, increased use of antibacterial soap for daily genital wash (<.0001). Women with VV used more panties of synthetic fabric (p<.05), had more menstrual cycles (p<.0001) than those without VV and similar use of sanitary pads. Women with BV practiced more anal sex in the last 30 days (p<.0001) and used more erogenous substances (p<.02) than those without VV. There were no significant differences in frequency of sexual intercourse, dyspareunia, oral sex and using lubricants between groups. The analysis of genital hair removal also showed no significant differences in the method used, motivation, frequency, area of hair removal, vulvar irritability, post-depilatory used products, and opinion about the influence of genital waxing on women's health. Despite being a low number, women with genital VC had more tattoos than the other groups (p=.04) and only one woman had genital piercing. CONCLUSIONS: Some habits related to genitalia washing, the presence of menstrual cycles, the use of synthetic fabric underwear, anal intercourse and use of erogenous substances during sexual intercourse were associated with frequency of vulvovaginitis. Especially anal sex on the last 30 days previous to diagnosis and use of erogenous substances during sexual intercourse were specifically associated with the presence of bacterial vaginosis. Use of sanitary pads and hair removal habits were not associated with the presence of VV. The genital adornments were rare but genital tattoos were most common among women with VC
Mestrado
Fisiopatologia Ginecológica
Mestra em Ciências da Saúde

Orientadores: Paulo César Giraldo, José Eleutério Junior
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: Os mecanismos de defesa da mucosa vaginal e a fisiopatologia das vulvovaginites têm se mostrado complexos e requerem a interação entre diversas substâncias. O papel da acidez vaginal, bem como a expressão de mediadores bioquímicos na luz vaginal necessitam ser melhor investigados para esclarecer as altas prevalências e recorrências das vulvovaginites. Objetivo: Avaliar as concentrações vaginais dos isômeros L e D do ácido lático, EMMPRIN (indutor de metaloproteinase da matriz extracelular), MMP-8 (metaloproteinase da matriz extracelular-8), NGAL (lipocalina associada à gelatinase dos neutrófilos), HA (hialurona), Hyal-1 (hialuronidase-1) e hBD2 (?-defensina) em mulheres hígidas e com vulvovaginites, correlacioná-los e verificar as associações com os leucócitos presentes na cavidade vaginal. Sujeitos e Métodos: Este estudo de corte transversal envolveu 233 mulheres atendidas no Ambulatório de Infecções Genitais da Unicamp no período de maio à novembro de 2013. Foram incluídas mulheres no menacme com e sem infecções genitais. Durante o exame ginecológico, coletou-se swab vaginal para análise microbiológica (bacterioscopia vaginal e cultura para fungos), além de material endocervical para pesquisa de Chlamydia trachomatis, Neisseria Gonorrhoeae e HPV por técnica de PCR (polymerase chain reaction). Outro swab da parede vaginal lateral foi diluído em 1 ml de PBS (phosphate buffer saline), processado e congelado para posterior análise dos mediadores imunes (ácidos láticos L e D, EMMPRIN, MMP-8, NGAL, HA, Hyal-1 e hBD2) por técnica de ELISA (enzyme-linked immunosorbent assay). Para analisar as diferenças entre os mediadores nos grupos de pesquisa foram utilizados testes não paramétricos: Kruskall-Wallis seguido de Mann-Whitney, devido à distribuição assimétrica dos dados. O estudo foi aprovado pela Comissão de Ética e Pesquisa da Unicamp e obteve financiamento FAPESP. Resultados: Dos 233 casos coletados foram diagnosticados: 52 casos de candidíase vulvovaginal (22%), 43 de vaginose bacteriana (18%), 21 de vaginose citolítica (9%), outros diagnósticos (n=36) e 77 controles sem infecção (33%). Quatro casos foram excluídos por diagnóstico incompleto. Além disso, foi avaliada a magnitude do processo inflamatório representada pela contagem de leucócitos presentes nos esfregaços vaginais. Consideraram-se os grupos: inflamação ausente (0 leucócitos/campo), inflamação moderada (1-4 leucócitos/campo) e inflamação intensa (>4 leucócitos por campo). Não se observaram diferenças significativas entre os grupos quanto à idade, raça, anos de estudo, método contraceptivo, número de parceiros sexuais, média de relações sexuais/mês e tabagismo. As concentrações de ácido lático D e L foram menores no grupo com vaginose bacteriana em comparação à candidíase e grupo controle. Na candidíase, os níveis de EMMPRIN e MMP-8 foram mais altos que nos grupos vaginose bacteriana e controle. O EMMPRIN esteve fortemente correlacionado ao ácido lático L, à razão ácido lático L/D e ao MMP-8 no grupo controle e à HA em toda a amostra estudada. Os níveis de NGAL foram maiores na candidíase e menores na vaginose bacteriana em relação aos controles. Os níveis de ácido lático-L e NGAL foram correlacionados. As concentrações vaginais de EMMPRIN, MMP-8, HA, NGAL e hBD2, exceto Hyal-1, foram maiores nos grupos com inflamação comparado ao grupo sem inflamação vaginal. Todos estes resultados foram estatisticamente significativos. Conclusões: Os isômeros do ácido lático apresentam maiores concentrações em pacientes cuja flora é dominada por lactobacilos (controles, candidíase e vaginose citolítica) e mais baixos em mulheres com flora anormal (vaginose bacteriana). As concentrações vaginais adequadas de ácido lático e suas correlações com NGAL, EMMPRIN e MMP-8, são relevantes nas defesas do trato genital inferior. Os níveis de EMMPRIN, MMP-8, HA, NGAL e hBD2 sofrem alteração com a inflamação vaginal, sendo que EMMPRIN, MMP-8 e HA são fundamentais na sua modulação. Mulheres com desequilíbrios nas concentrações destes mediadores podem estar mais sujeitas a desenvolver disbioses vaginais
Abstract: Introduction: The defense mechanisms of the vaginal mucosa and the pathophysiology of vulvovaginitis are complex and require interaction between several substances. The role of vaginal acidity, as well as the expression of biochemical mediators in vaginal lumen need to be better investigated to clarify the recurrences and high prevalence of VV. Objective: To evaluate the concentrations of L and D lactic acid, EMMPRIN (extracellular matrix metalloproteinase inducer), MMP-8 (metalloproteinase-8), NGAL (neutrophil gelatinase-associated lipocalin), HA (hyaluronan), Hyal-1 (hyaluronidase-1) and hBD2 (human ?-defensin-2) from vaginal fluid of healthy women and women with vulvovaginitis, correlate them and verify their associations with leukocytes present in the vaginal cavity. Subjects and Methods: This cross-sectional study involved 233 women who were seen at Genital Infections Clinic at Unicamp from May to November 2013. Inclusion criteria: women of reproductive age with or without genital infections. During gynecological examination, vaginal swab was collected for microbiological analysis (Gram stain and culture for fungi) and, endocervical material was analysed for Chlamydia trachomatis, Neisseria gonorrhoeae and HPV by PCR (polymerase chain reaction) technique. Another swab from lateral vaginal wall was diluted in 1 ml PBS (phosphate buffered saline), processed and frozen for later analysis of immune mediators (L and D lactic acid, EMMPRIN, MMP-8, NGAL, HA, Hyal-1 and hBD2) by ELISA (enzyme-linked immunosorbent assay). To analyze the mediators¿ differences between research groups, Kruskal-Wallis followed by Mann-Whitney were used due to the asymmetric distribution of data. The study was approved by the Ethics and Research Committee at the Campinas University and received funding from FAPESP. Results: After microbiological analysis of 233 cases, 52 vulvovaginal candidiasis cases were diagnosed (22%), 43 bacterial vaginosis (18%), 21 cytolytic vaginosis (9%), other diagnoses (n=36) and 77 uninfected controls (33%). Four cases were excluded due to incomplete diagnosis. In addition, the magnitude of the inflammatory process represented by the leukocyte count present in the vaginal smears was evaluated. It was considered to be in three groups: absent inflammation (0 leukocytes per field), moderate inflammation (1-4 leukocytes per field) and intense inflammation (> 4 white cells per field). There were no significant statistical differences among groups considering age, race, years of education, contraception, number of sexual partners, average of intercourse per month and smoking. The D and L lactic acid concentrations were lower in the bacterial vaginosis group compared to candidiasis and control groups. Levels of EMMPRIN and MMP-8 were higher in candidiasis than bacterial vaginosis and control groups. The EMMPRIN was strongly correlated to L lactic acid, L / D lactic acid ratio and MMP-8 in control group, and to HA across the sample. NGAL levels were higher in candidiasis and smaller in bacterial vaginosis compared to controls. The L-lactic acid and NGAL levels were correlated. Vaginal concentrations of EMMPRIN, MMP-8, HA, NGAL and hBD2, except Hyal-1, were higher in the groups with vaginal inflammation compared to the group without inflammation. All these results were statistically significant. Conclusions: The isomers of lactic acid have higher concentrations in patients whose microflora is dominated by lactobacilli (controls, candidiasis and cytolytic vaginosis) and, lower in women with abnormal flora (bacterial vaginosis). Appropriate vaginal concentrations of lactic acid and its correlations with NGAL, EMMPRIN and MMP-8 are relevant in the lower genital tract defenses. The levels of EMMPRIN, MMP-8, HA, NGAL and hBD2 are changed by vaginal inflammation, and EMMPRIN, MMP-8 and HA are critical in modulating vaginal inflammation. Women with imbalances in the concentrations of these mediators may be more likely to develop vaginal disbioses
Doutorado
Fisiopatologia Ginecológica
Doutora em Ciências da Saúde

A microbiota vaginal saudável é constituída, majoritariamente, por espécies de lactobacilos que representam uma barreira natural contra microrganismos causadores de doenças como a candidíase vulvovaginal (CVV), vaginose bacteriana (VB) e infecções do trato urinário (ITU), que juntas acometem cerca de um bilhão de mulheres no mundo anualmente. Um melhor entendimento da ecologia microbiana vaginal pode ser útil na otimização de tratamentos existentes para as infecções urogenitais, os quais podem destruir parcialmente a microbiota autóctone, predispor a novas infecções, contribuir para a seleção de microrganismos resistentes e causar efeitos colaterais indesejáveis. A utilização de microrganismos certificadamente probióticos, Lactobacillus rhamnosus GR-1 e Lactobacillus reuteri RC-14, representa uma alternativa terapêutica promissora na abordagem de VB e CVV, uma vez que são capazes de colonizar o trato vaginal, apresentam atividade inibitória frente a diversos patógenos do trato urogenital, exibem risco mínimo para a seleção de microrganismos resistentes e podem auxiliar na restauração da microbiota vaginal. Os objetivos deste trabalho foram: (i) avaliar a prevalência de espécies de lactobacilos na microbiota vaginal de mulheres saudáveis e diagnosticadas com infecções vaginais (CVV e VB) da cidade de Ribeirão Preto (São Paulo, Brasil), (ii) avaliar a capacidade de isolados de lactobacilos produzirem peróxido de hidrogênio (H2O2) e (iii) determinar a eficácia da utilização de L. rhamnosus GR-1 e L. reuteri RC-14 no tratamento de CVV e VB, quando co-administrados com medicamentos antimicrobianos tradicionais. Participaram deste estudo, 196 pacientes voluntárias, atendidas por médicos ginecologistas de centros de saúde ligados à Universidade de São Paulo, campus de Ribeirão Preto (64 saudáveis, 68 diagnosticadas com CVV e 64 diagnosticadas com VB) e duas amostras vaginais de cada paciente foram coletadas com o auxílio de zaragatoas esterilizadas. Uma zaragatoa foi utilizada para semeadura em ágar MRS (de Man, Rogosa & Sharpe), os isolados de bactérias láticas obtidos foram analisados através da técnica de PCR-ARDRA (Reação em cadeia da polimerase Análise de restrição do DNA ribossomal amplificado) e a habilidade de Lactobacillus spp. produzir H2O2 foi determinada semi-quantitativamente. A outra zaragatoa foi utilizada para a análise das espécies de lactobacilos da microbiota vaginal pela técnica independente de cultivo PCR-DGGE (Reação em cadeia da polimerase Eletroforese em gel de gradiente de desnaturação). As leveduras do gênero Candida foram obtidas através da semeadura das amostras vaginais provenientes de pacientes saudáveis e com CVV no meio Chromagar® Candida e identificadas através de provas bioquímicas de referência. As pacientes diagnosticadas com CVV participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de fluconazol (150mg) e suplementação diária durante 28 dias com (i) duas cápsulas contendo os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 (Urex-Cap-5®) ou (ii) duas cápsulas de placebo. As pacientes com VB também participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de tinidazol (2g) e suplementação diária com cápsulas do probiótico (Urex-Cap-5®) ou placebo, conforme descrito acima. Todas as pacientes foram reavaliadas ao final do tratamento, no 28º dia. Foram realizados experimentos para averiguar o possível efeito de L. rhamnosus GR-1 e L. reuteri RC-14 na modulação in vitro da infecção por Candida albicans em culturas de células epiteliais vaginais humanas (VK2/E6E7) Os resultados mostraram que, pela técnica de PCR-ADRA, L. crispatus foi a espécie mais prevalente nos grupos de pacientes saudáveis (37,0%) e com CVV (35,9%), enquanto que L. gasseri foi predominante no grupo de pacientes com VB (34,6%). De acordo com o método de PCR-DGGE, L. iners foi o microrganismo mais prevalente nos três grupos de pacientes avaliados: saudáveis, com CVV e VB (48,7%, 44,7% e 65,0%, respectivamente). A maioria dos isolados de Lactobacillus spp. obtidos nos grupos de pacientes saudáveis (98,6%) e diagnosticadas com CVV (97,4%) foram capazes de produzir H2O2 (1 a 100mg/L), em comparação a apenas 68,2%, determinado no grupo de pacientes com VB (p<0,05). L. crispatus e L. johnsonii produziram as maiores quantidades médias de H2O2 (30mg/L). A taxa de colonização por leveduras do gênero Candida foi de 26,6% no grupo de pacientes saudáveis (C. albicans correspondeu a 52,4% de todos os isolados), enquanto que no grupo de pacientes com CVV, 89,2% dos isolados leveduriformes foram identificados como C. albicans. Para as análises estatísticas dos dados obtidos com os testes clínicos, foram consideradas 55 pacientes diagnosticadas com CVV (pela presença de sinais e sintomas da infecção e cultura positiva para Candida sp.) e foi observado que a utilização de dose única de fluconazol e suplementação diária com o probiótico, resultou em taxa de cura mais elevada para a infecção (89,7%) em comparação com aquela verificada no grupo placebo (65,4%) (p<0,05). A utilização de tinidazol em associação com a ingestão diária de Urex-Cap-5® no tratamento de pacientes com VB também resultou em taxa de cura mais elevada para a condição (87,5%), em comparação àquela observada no grupo placebo (50,0%) (p<0,05). A atividade anti-Candida de L. rhamnosus GR-1 e L. reuteri RC-14 foi observada através do modelo in vitro para infecção vaginal. Em conclusão, quando as técnicas de PCR-ARDRA e PCR-DGGE foram comparadas entre si, foi observado que ambas apresentaram limitações, evidenciando a importância do emprego de diferentes metodologias para avaliação adequada das espécies de lactobacilos presentes na microbiota vaginal. As espécies de lactobacilos vaginais determinadas nas mulheres saudáveis do presente trabalho foram semelhantes àquelas verificadas em estudos prévios descritos na literatura com pacientes com dieta e localização geográfica notadamente distintas. Os dados do presente trabalho sugerem que a presença de lactobacilos produtores de H2O2, isoladamente, não confere proteção contra CVV, enquanto que a ausência desses microrganismos pode ser um fator contribuinte para VB. Além disso, foi demonstrado que a utilização de medicamentos antimicrobianos tradicionais e suplementação com os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 foi mais eficiente no tratamento de CVV e VB em comparação com medicamentos clássicos e placebo. Estes resultados podem contribuir para prolongar a vida útil de medicamentos cuja eficácia pode ser comprometida devido à seleção de microrganismos resistentes e também reduzir o tempo de tratamento para pacientes que necessitam de terapias clássicas por períodos prolongados.
The vaginal microbiota is mainly constituted by lactobacilli species, which represent a natural barrier against microorganisms that cause vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and urinary tract infections (UTI). Together, these conditions afflict each year an estimated one billion women worldwide. A better understanding of the vaginal microbial ecology may be useful to improve the current available treatments for urogenital infections, which can partially destroy the autochthonous microbiota, predispose to other infections, contribute for the selection of resistant microorganisms and cause undesirable collateral effects. The use of microorganisms with demonstrated probiotic properties, Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, represents a promising therapeutic alternative for BV and VVC, since they are able to colonize the vaginal tract, present inhibitory against several urogenital pathogens, pose minimal risk for the selection of resistant microorganisms and can help to restore the vaginal microbiota. The objectives of this work were: (i) to evaluate the prevalence of lactobacilli species in the vaginal microbiota of healthy women and those diagnosed with vaginal infections (VVC and BV) in the city of Ribeirão Preto (São Paulo, Brazil), (ii) to evaluate the ability of the lactobacilli isolates to produce hydrogen peroxide (H2O2) and (iii) to determine the efficacy of the use of L. rhamnosus GR-1 and L. reuteri RC-14 in the treatment of VVC and BV, in co-administration with traditional antimicrobials. 196 voluntary subjects were examined by the gynecologists team from health centers affiliated with Universidade de São Paulo, campus at Ribeirão Preto (64 healthy, 68 diagnosed with VVC and 64 diagnosed with BV) and two vaginal samples from each patient were collected by the use of two sterile swabs. One swab was cultured in MRS (de Man, Rogosa & Sharpe) agar, the isolates of lactic acid bacteria obtained were analyzed by PCR-ARDRA (Polymerase chain reaction - Amplified ribosomal DNA restriction analysis) and the ability of Lactobacillus spp. to produce hydrogen peroxide was determined semi-quantitatively. The other swab was used for the analysis of lactobacilli species from the vaginal microbiota using the culture-independent PCR-DGGE (Polymerase chain reaction Denaturating gradient gel electrophoresis). The yeasts belonging to Candida genus were obtained also by culturing the vaginal material from healthy and VVC patients in Chromagar® Candida and were identified by standard biochemical tests. VVC patients were enrolled in a randomized, double-blind, placebo-controlled trial and treated with a single dose fluconazole (150mg) and daily supplementation for 28 days with (i) two capsules containing the probiotic microorganisms L. rhamnosus GR-1 and L. reuteri RC-14 (Urex-Cap-5®) or (ii) two placebo capsules. BV patients were also enrolled in a randomized, double-blind, placebo controlled trial and treated with a single dose of tinidazole (2g) and supplementation with probiotic capsules (Urex-Cap-5®) or placebo, as described above. All patients were re-evaluated at the end of treatment, on the 28th day. Experiments were also conducted to assess the possible effect of L. rhamnosus GR-1 and L. reuteri RC-14 in the in vitro modulation of vaginal infection by Candida albicans on cultures of human vaginal epithelial cells (VK2/E6E7). The results revealed that according to PCR-ADRA, L.crispatus was the most prevalent species in the groups of healthy women (37.0%) and those with VVC (35.9%), while L. gasseri was dominant in BV patients (34.6%). By PCR-DGGE method, L. iners was the most prevalent Lactobacillus species in all the three groups evaluated: healthy, VVC and BV (48.7%, 44.7% and 65.0%, respectively). The majority of the isolates of Lactobacillus spp. from healthy women (98.6%) and those with VVC (97.4%) were able to produce H2O2 (1 to 100mg/L) in comparison with only 68.2% assessed for the BV group (p<0.05). L. crispatus and L. johnsonii produced the highest average levels of H2O2 (30mg/L). Colonization rate by yeasts belonging to Candida genus was 26.6% in the group of healthy patients (C. albicans represented 52.4% of all isolates), whereas in the VVC group, 89.2% of yeast isolates were identified as C. albicans. For the performance of statistical analysis of the results obtained with the clinical trials, 55 patients diagnosed with VVC (by the presence of symptoms and signals of the infection and positive culture for Candida sp.) were taken into consideration and it was observed that the use of a single dose of fluconazole and daily supplementation with probiotics, yielded a higher cure rate (89.7%), in comparison with the placebo group (65.4%) (p<0.05). The use of tinidazole plus probiotic also resulted in higher cure rate of the infection (87.5%), compared to placebo group (50.0%) (p<0.05). An anti-Candida activity of L. rhamnosus GR-1 and L. reuteri RC-14 was observed in the in vitro model of vaginal infection. In conclusion, when PCR-ARDRA and PCR-DGGE were compared, it was verified that both presented limitations, which evidences the need of using different techniques for a better knowledge of lactobacilli species present in the vaginal microbiota. The lactobacilli species found in healthy women in this work were similar to those reported in previous studies described in the literature for patients with distinctly different diet and geographic localization. The data of the present work indicate that solely the presence of H2O2-producing isolates does not render protection against VVC, whereas the absence of those microorganisms may be a contributing factor for BV. Moreover, it was demonstrated that the use of classical medicines supplemented with the probiotics L. rhamnosus GR-1 and L. reuteri RC-14 was more efficient to treat VVC and BV in comparison with classical medicines plus placebo. These results may contribute to extend the longevity of drugs whose efficacy is compromised due to the selection of resistant microorganisms and also to shorten the length of treatment courses for patients that require long regimens with standard therapy.

L’objectif était la caractérisation moléculaire de la vaginose bactérienne (VB) et l’identification d’une relation entre anomalies moléculaires de la flore vaginale et prématurité. Nous avons élaboré un outil de quantification par PCR spécifique en temps réel ciblant 8 microorganismes impliqués dans la VB. Seule la combinaison de la quantification d’Atopobium vaginae (108 copies/mL) à celle de Gardnerella vaginalis (109 copies/mL) présentait une sensibilité (95%) et une spécificité (99%) élevées pour le diagnostic de VB. La classification des flores selon la présence d’une VB, d’après les 2 méthodes de référence (critères d’Amsel et score de Nugent), et d’après notre outil moléculaire était concordante dans 94.5% des cas (kappa=0.81, intervalle de confiance [IC] 95%: 0.70-0.81). La discordance portait sur 9 flores intermédiaires (5.5%) dont les concentrations en G. vaginalis et en A. vaginae étaient élevées. De plus, nous avons démontré une étroite corrélation entre l’auto-prélèvement vaginal et le prélèvement réalisé par le médecin pour la quantification microbienne. Cette méthode alternative semble utile pour le suivi des anomalies de la flore vaginale. Nous avons enfin établi un lien entre la composition de la flore vaginale et le risque de prématurité parmi 90 patientes hospitalisées pour une menace d’accouchement prématuré (MAP). L’analyse par courbe de survie montrait un délai raccourci entre le diagnostic de MAP et le terme de l’accouchement en présence de concentrations élevées en A. vaginae ou en G. vaginalis (hasard ratio: 3.3; IC 95%: 1.1-9.5). Notre travail a contribué à améliorer l’analyse de la flore vaginale et à l’évaluation de son lien avec la prématurité
The aim was the molecular characterization of bacterial vaginosis (BV) and the estimation of the relationship between molecular abnormalities and preterm delivery. A quantitative molecular tool targeting 8 bacterial vaginosis-related microorganisms was developed using specific real-time PCR. Only the combination of the quantification of Atopobium vaginae (108 copies/mL) and Gardnerella vaginalis (109 copies/ml) had an excellent sensitivity (95%) and specificity (99%) for the diagnosis of BV. The categorization of the vaginal flora according to the presence of BV based on the 2 reference methods (Amsel criteria and Nugent score), and our molecular tool was agree in 94.5% of the cases (kappa=0.81, 95% confidence interval [CI] 0.70-0.81). There was disagreement for 9 intermediate floras (5.5%) for which vaginal concentrations of A. vaginae and G. vaginalis were high. We also reported a good agreement for bacterial quantification in self-collected compared with practitioner-collected vaginal swabs. This method provides an alternative to practitioner-collected swabs especially for follow-up. We finally demonstrated a relationship between the high vaginal concentrations of A. vaginae and G. vaginalis and the risk of preterm delivery among 90 women with preterm labor. Survival curves analysis for preterm labor-to-delivery interval showed a significantly shorter interval for high vaginal concentrations of both A. vaginae or G. vaginalis (hazard ratio: 3.3; IC 95%: 1.1-9.5). Our work improved vaginal flora analysis and investigated the relationship with preterm delivery

Orientadores: Luiz Carlos Zeferino, Silvia Helena Rabelo dos Santos.
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Objetivo: Analisar a associação entre a coinfecção por papilomavírus humano (HPV), Chlamydia trachomatis (CT), vaginose bacteriana (VB) e resposta inflamatória (RI) com a gravidade da neoplasia cervical. Sujeitos e métodos: Estudo experimental, de corte transversal, realizado em Campinas, São Paulo e em Goiânia, Goiás, Brasil. A casuística incluiu amostras biológicas de 290 mulheres consecutivas submetidas à excisão da zona de transformação (EZT) ou conização. Para o estudo que avaliou a coinfecção entre HPV e CT e a associação com gravidade da neoplasia cervical foram selecionadas 251 (86,6%) mulheres infectadas por HPV de alto risco (HR-HPV). A detecção de HPV foi realizada utilizando os primers PGMY09/11 e a genotipagem através de hibridização reversa em pontos. A detecção da CT foi realizada por polimerase chain reaction (PCR) empregando primers cujo alvo é uma região de plasmídio críptico, gerando um fragmento de aproximadamente 512 pares de base. Para o estudo que avaliou a VB e resposta inflamatória e a associação destas condições com a gravidade da neoplasia cervical foram selecionadas 211 mulheres infectadas por HR-HPV, com esfregaços cervicais disponíveis para as análises. A presença de 20% ou mais de células indicadoras no esfregaço cervical corado pelo método de Papanicolau foi considerada positiva para VB. A resposta inflamatória nos esfregaços cervicais foi avaliada pela contagem do número de neutrófilos. O encontro de 30 ou mais neutrófilos por campo microscópico, observado sob o aumento de 1000x, foi considerado como presença de resposta inflamatória. Resultados: A prevalência de CT em mulheres HPV positivas foi de 15,1% (38/251). Foi observada uma associação significativa em mulheres CT negativas, com 30 anos ou mais, e NIC 2 ou pior diagnóstico, mas esta associação não foi observada em mulheres CT positivas. Infecções por HPV 16 e/ou 18 foram detectadas em 50% das mulheres CT negativas com menos de 29 anos e que apresentavam NIC 2 ou pior diagnóstico, e em 19,5% das mulheres CT negativas com NIC 1 ou não neoplásico. Nestas mulheres, a associação entre HPV 16 e/ou 18 e NIC 2 ou pior diagnóstico foi significativa, mas esta associação não foi observada no grupo CT positivo. Resposta inflamatória e VB foram observadas em 43,5% e 46,2% dos esfregaços cervicais de mulheres com NIC 2. Resposta inflamatória e VB foram observados em 64,2% e 32,6% dos esfregaços cervicais de mulheres com diagnóstico histológico de NIC 3. Nestas mulheres, quando infectadas pelos tipos de HPV 16 e/ou 18, foram observadas resposta inflamatória e VB, respectivamente, em 43,1% e 20% dos casos. Resposta inflamatória apresentou associação estatisticamente significante com NIC 2 ou pior diagnóstico em mulheres infectadas pelos tipos de HPV 16 e/ou 18 (OR= 6,70; 95%IC:2,32-19,31) e por outros tipos de HPV (OR=4,90; 95%IC: 1,86-12,89). Associações significativas foram observadas em mulheres com VB e NIC 2 ou pior diagnóstico, infectadas pelos tipos de HPV 16 e/ou 18 (OR= 3,38; 95% IC :1,07-10,64) e por outros tipos de HPV (OR= 3,38; 95%IC: 1,15-10,01). Conclusões: A infecção por CT detectada por PCR não mostrou associação com o aumento do risco para NIC 2 ou pior diagnóstico em mulheres HPV positivas. Em mulheres CT negativas e com menos de 30 anos de idade, os tipos de HPV 16 e/ou 18 estão associados à NIC 2 ou pior diagnóstico, resultado não observado para as mulheres CT positivas. A VB e resposta inflamatória estão associadas à NIC 2 ou pior diagnóstico em mulheres HR-HPV positivas
Abstract: Objective: To analyze the association between co-infection Human Papillomavirus (HPV) and Chlamydia trachomatis (CT), bacterial vaginosis (BV) and inflammatory response with the severity of the cervical neoplasia. Subjects and methods: This is cross-sectional experimental study carried through in Campinas, São Paulo and in Goiânia, Goiás, Brazil. The casuistic included 290 consecutive women submitted a the Excision of the Transformation Zone or conization due CIN 2 and CIN 3. For the study that evaluated the association between HPV and CT and severity of cervical neoplasia were selected 251 women who were infected with high-risk HPV (HR-HPV). HPV detection .was performed by PCR using primers PGMY09/11 and genotyping by reverse lineblot hybridization assay. The detection of CT was performed by PCR. For the study that evaluated the association between BV and inflammatory response with the severity of cervical neoplasia were selected 211 women infected with HR-HPV with cervical smears available for analysis. The presence of 20% or more clue cells in cervical smears stained by the Papanicolaou method was considered positive for VB. Inflammatory response was assessed by counting the number of neutrophils. The finding of 30 or more neutrophils per field observed under 1000x magnification was taken as presence of inflammatory response. Results: The prevalence of CT in HPV positive women was 15.1% (38/251). Significant association was observed between women with 30 years or older and CIN 2 or worse diagnosis for those CT negative, but this association was not observed for those CT positive. HPV 16 and/or HPV 18 were detected in 50% of the women ? 29 years age with CIN 2 or worse diagnosis who were CT negative, and in 19.5% for those women with CIN 1 or no neoplastic in histological diagnostic. In these women the association between HPV 16 and/or 18 and CIN 2 or worse diagnosis was significative, but this association also was not observed considering the CT positive group. Inflammatory response and BV were observed in 5.5% and 16.7% of cervical smear of women with no neoplastic diagnosis and were observed in 22.9% and 12.5% of cervical smears of women with CIN 1 in histological diagnosis. Inflammatory response and BV were observed in 43.5% and 46.2% of cervical smears of women with CIN 2 in histological diagnosis. The BV prevalence was higher in cervical smears of women infected by the types 16 and/or 18 (25.6%) and inflammatory response was more observed in cervical smears of women infected by other HPV types (25.6%). Inflammatory response and BV were observed in 64.2% and 32.6% of cervical smears of women with CIN 3 in histological diagnosis and were more observed in cervical smears of women infected types 16 and/or 18 representing respectively 43.1% and 20.0% of cases. Inflammatory response and BV were more observed in cervical smears of women infected types 16 and/or 18 in women with invasive carcinoma, representing 27.2% and 18.2% of cases respectively. Inflammatory response was significantly associated with CIN 2 or worse diagnosis in women infeted by HPV16 and/or HPV 18 (OR= 6.70; 95%CI : 2.32-19.31) and HPV other types than HPV16 and/or18 (OR=4.90; 95%CI: 1.86-12.89). Significant associations with BV and CIN 2 or worse diagnosis were observed in women infected by HPV 16 and/or 18 (OR= 3.38; 95%C1:07-10.64) and HPV types other than HPV16 and/or 18 (OR=3.38; 95%CI: 1.15-10.01). Conclusions: CT infection detected by PCR, does not increase the risk for CIN 2 or worse diagnosis in HPV positive women. HPV 16 and/or HPV 18 types in young women are associated with CIN 2 or worse diagnosis, but without obvious association with CT. BV and inflammatory response are associated with CIN 2 or worse diagnosis in women HR-HPV positives women
Doutorado
Ciencias Biomedicas
Doutora em Tocoginecologia

Orientadores: Paulo Cesar Giraldo, Ana Katherine S. Gonçalves
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Objetivo: Verificar se o uso de duchas higiênicas íntimas (DHI) pode interferir na microbiota vaginal de mulheres profissionais do sexo. Sujeitos e Métodos: Estudo de corte transversal realizado durante seis meses verificou o risco de 155 mulheres profissionais do sexo (PS), usuárias e não usuárias de DHI, apresentarem flora vaginal alterada e/ou vaginose bacteriana. As PS foram atendidas em centro de saúde (CS) localizado em zona de prostituição no município de Campinas, São Paulo, e agrupadas em usuárias e não usuárias de duchas higiênicas íntimas para análise da microbiota vaginal. A anamnese determinou os perfis sociodemográfico e sexual destas mulheres que procuraram o CS por diferentes motivos. O conteúdo vaginal foi coletado com swab estéril de Dacron e disposto em duas lâminas de vidro que foram coradas por técnica de Gram. A caracterização da microbiota vaginal pelos critérios de AMSEL adaptado foi realizada por microscopia óptica com lente de imersão. Análises univariável, bivariável com testes de qui-quadrado e exato de Fisher, além de regressão múltipla variável (stepwise) demonstraram o poder de associação entre as variáveis. Resultados: A média de idade, a etnia branca, a escolaridade básica e hábito de fumar encontrados respectivamente nas 94 usuárias e nas 61 não usuárias de DHI foram de 25,5 (± 6,2) vs. 26,0 (± 6,8)anos (p=ns), 48,9% vs. 47,5% (p=ns), 60,6% vs. 45% (p=ns) e 41,5% vs. 49,2% (p=ns) dos casos. Não houve diferença no uso regular de condom com seus parceiros sexuais fixos. Apenas o uso de lubrificantes vaginais foi significativamente maior nas usuárias de DHI (63,8%) que nas não usuárias (36,1%), p=0,0007. As prevalências de flora vaginal alterada, vaginose bacteriana, candidíase, tricomoníase e vaginose citolítica foram de 75,48%; 50,96%; 5,1%; 0,64% e 1,9%, respectivamente. Não houve diferenças significativas destas prevalências quando foram analisados estes achados entre as usuárias e não usuárias de DHI (78,7% vs. 70,5%, ns), (47,9% vs. 55,7%, ns), (5,3% vs. 4,9%, ns), (0 vs. 1,6%, ns) e (1,1% vs. 3,3%, ns). A análise de regressão múltipla tipo stepwise não identificou qualquer risco aumentado de ter flora vaginal alterada ou de vaginose bacteriana em mulheres usuárias de DHI. Conclusão: O uso de DHI não aumentou o risco de flora vaginal alterada e/ou de vaginose bacteriana em mulheres PS. Palavras-chave: profissionais do sexo, duchas higiênicas íntimas, microbiota vaginal, vaginose bacteriana, candidíase vaginal
Abstract: Aims: Verify if vaginal douching (VD) can cause vaginal flora imbalance in Female Sex Workers (FSW). Patients and Methods: A cross sectional study, carried out for six months, analysed the risk of vaginal flora imbalance and/or vaginal bacteriosis due to vaginal douching in 155 FSW. The FSW were seen at a public outpatient in a prostitution area in the city of Campinas, the state of São Paulo, Brazil. The vaginal douching (VD) users and non-users made up the two groups for vaginal microbiological analysis. The social-demographic and sexual profile of these women, who were seen for different reasons, was checked by anamnesis. Vaginal content was collected using a sterile Dacron Swab and placed on two glass slides that were stained using Gram technique. Adapted AMSEL criteria characterized the vaginal flora using optic microscope with immersion lens. Univariate, bivariate statistical analyses with X2 and exact Fisher tests apart from multiple variate regression (Stepwise) analysis determined the association between variables. Results: Mean age, white ethnicity, level of schooling and the habit of smoking analysed in the 94 users and 61 non-users of VD respectively were 25.5 (± 6.2) vs. 26.0 (± 6.8) years (p=ns), 48.9% vs. 47.5% (p=ns), 60.6% vs. 45% (p=ns) and 41.5% vs. 49.2% (p=ns) of the cases. There was no difference in the regular use of condoms with their steady sexual partners. Only the use of vaginal lubricant was significantly higher in the VD users (63.8%) than in the non-users (36.1%); p=0.0007. General prevalence of abnormal flora, bacterial vaginosis, candidiasis, trichomoniasis and cytolitic vaginosis was 75.48%, 50.96%, 5.1%, 0.64% and 1.9% respectively. There were no significant differences when analysing the findings between users and non-users of VD (78.7% vs. 70.5%; ns); (47.9% vs. 55.7%; ns); (5.3% vs. 4.9%; ns); (0 vs. 1.6% ns) and (1.1% vs. 3.3%; ns). Stepwise multiple regression analysis did not identify any increased risk of altered vaginal flora or bacterial vaginosis in VD users. Conclusion: The use of VD did not increase the risk of vaginal flora imbalance and/or bacterial vaginosis in FSW
Mestrado
Ciencias Biomedicas
Mestre em Tocoginecologia

Introduction: The quality of neonatal care is mainly determined by long-term neurodevelopmental outcome. The neurodevelopment of preterm infants is related to postnatal head growth and depends on medical interventions such as nutritional support. Head circumference (HC) is currently used as a two-dimensional measure of head growth. Since head deformities are frequently found in preterm infants, HC may not always adequately reflect head growth. Laser aided head shape digitizers offer semiautomatic acquisition of HC and cranial volume (CrV) and could thus be useful in describing head size more precisely. Aims: 1) To evaluate reproducibility of a 3D digital capture system in newborns. 2) To compare manual and digital HC measurements in a neonatal cohort. 3) To determine correlation of HC and CrV and predictive value of HC. Methods: Within a twelve-month period data of head scans with a laser shape digitizer were analysed. Repeated measures were used for method evaluation. Manually and digitally acquired HC was compared. Regression analysis of HC and CrV was performed. Results: Interobserver reliability was excellent for HC (bias-0.005%, 95% Limits of Agreement (LoA) −0.39–0.39%) and CrV (bias1.5%, 95%LoA-0.8–3.6%). Method comparison data was acquired from 282 infants. It revealed interchangeability of the methods (bias-0.45%; 95%LoA-4.55–3.65%) and no significant systematic or proportional differences. HC and CrV correlated (r2 = 0.859, p<0.001), performance of HC predicting CrV was poor (RSD ±24 ml). Correlation was worse in infants with lower postmenstrual age (r2 = 0.745) compared to older infants (r2 = 0.843). Discussion: The current practice of measuring HC for describing head growth in preterm infants could be misleading since it does not represent a 3D approach. CrV can vary substantially in infants of equal HC. The 3D laser scanner represents a new and promising method to provide reproducible data of CrV and HC. Since it does not provide data on cerebral structures, additional imaging is required.

Orientador: Paulo Cesar Giraldo
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Objetivos: Avaliar o ecossistema vaginal e a qualidade de vida (QV) de mulheres com incotinência urinária (IU) submetidas a tratamento fisioterápico com eletroestimulção intravaginal (EEIV). Métodos: Estudo de ensaio clínico realizado entre setembro de 2006 a novembro de 2008 envolveu 67 mulheres com queixa clínica de IU. O tratamento fisioterápico consistiu de 8 sessões (2x/semana) de EEIV (frequência=35Hz, largura de pulso=0,5ms e tempo=20min), orientações comportamentais e exercícios de fortalecimento do assoalho pélvico. O ecossistema vaginal foi avaliado quanto ao tipo de flora, inflamação e pH da mucosa vaginal antes e após a EEIV. O conteúdo coletado do terço médio vaginal foi corado pela técnica de Gram e analisado sob microscopia óptica em flora bacilar ou normal, intermediária e, cocóide/ cocobacilar ou vaginose bacteriana (VB). A intensidade da inflamação foi determinada contando-se o número médio de células de defesa (polimorfonucleares neutrófilos e linfócitos) encontradas em 10 campos de grande aumento (400X). A QV foi avaliada pelo questionário "Consultation on Incontinence Questionnaire-Short Form" antes e após o tratamento. Os dados foram coletados pelo mesmo investigador e analisados por apenas um microbiólogo de forma cega. O estudo foi aprovado pelo Comitê de Ética em Pesquisa etodas as pacientes assinaram termo de consentimento livre e esclarecido. Resultados: Foram selecionadas 78 mulheres, sendo 11 excluídas para a análise microbiológica e 6 para análise da QV. Das 67 mulheres incluídas, a média de idade foi de 51,3 (±11,8) anos, a maioria branca, do lar, pós-menopausada e com sintomas de IUM (76,4%) e IUE (23,6%). Antes da EEIV, 43 mulheres apresentaram microbiota normal, 24 intermediária e nenhum caso de VB ou candidíase vaginal (CV). Após as 8 sessões de EEIV não houve mudança significativa da flora vaginal, sendo que das 43 mulheres que apresentaram flora normal, 36 permaneceram na mesma categoria, 5 apresentaram flora intermediária e 2 apresentaram VB. Das 24 identificadas com microbiota intermediária, 15 permaneceram na mesma categoria, 7 foram identificadas com microbiota normal e 2 com VB. O aparecimento de 4 casos de VB foi estatisticamente significativo (p<0,05). A análise do processo inflamatório identificou que 60 mulheres não apresentavam inflamação, 4 tinham inflamação leve/moderada e 3 inflamação intensa. Após o tratamento não houve mudança significativa, sendo que 58 mulheres não apresentavam inflamação, 6 apresentaram inflamação leve/moderada e 3 inflamação intensa. Também não foram encontradas diferenças significativas nos valores do pH antes e após cada sessão de EEIV. Para a avaliação da QV foram incluídas 72 mulheres. Encontrou-se uma redução significativa dos escores da frequência de perda urinária de 3,4(±1,4) para 1,4(±5,9) [p<0,03], da quantidade de perda urinária de 3,6(±1,6) para 2,0(±1,3) [p<0,04] e do impacto da IU na QV de 7,7(±2,4) para 3,8(±2,9) [p<0,001]. O escore total do ICIQ-SF diminuiu de 14,6(±4,2) para 7,2(±4,5) [p<0,001]. Conclusões: O ecossistema vaginal neste estudo não foi significativamente influenciado pela EEIV quanto ao tipo de flora vaginal, inflamação da mucosa vaginal, mudança do pH vaginal e aparecimento de CV, apesar de terem aparecido 4 casos de VB. A EEIV proporcionou uma melhora significativa na QV de mulheres com IU.
Abstract: Objectives: To evaluate vaginal ecosystem and quality of life (QOL) of women submitted to intravaginal electrical stimulation (IVES) as physicaltherapy treatment. Methods: A clinical trial was carried out from September 2006 to November 2008 including 67 women presenting IU as symptom. Physicaltherapy treatment consisted in 8 IVES sessions during 4 weeks (frequency=35Hz, pulse width=0,5ms, duration=20min), pelvic floor muscle exercises (PFME) and behavioral orientation. Vaginal ecosystem was assed concerning type of flora, inflammation and pH before and after treatment. Vaginal bacterioscopy swab was collected from e middle third of the vagina (vaginal smear was collected for subsequent Gram stain). The inflammatory process intensity was determined by the average number of white defense cells (neutrophils and limphocytes) found in 10 observation fields (magnified 400X). Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) was used to asses QoL. All data was collected by a single investigator and analyzed by the same microbiologist. This study was approved by Institutional Ethitics Committee and every patient signed an agreement term. Results: 78 women were selected and 11 were excluded from microbiologic analyzes and 6 from QoL analyses. Of the 67 women included, the mean age was 51.3(±12.2), most were white, housewives, post menopausal, presenting symptoms of MUI (76.4%) and SUI (23.6%). Before IVES 43 women had a normal vaginal flora, 24 presented intermediary flora and none had bacterial vaginoses (BV) or vaginal candidiasis (VC). After 8 sessions of IVES no significant alteration was noticed, 43 were unaltered, 36 remained in the same category, 5 showed intermediary microbiota and 2 were identified as having VB. Of those 24 initially identified as intermediary, 15 remained in the same category, 7 were normal and 2 with VB. Four cases of BV was statistically significant (p<0,05). The inflammatory process analyses identifiyed that 60 women did not present any inflammation, 4 had slight-moderate and 3 showed it as intense. No significant changes in pH were observed in the measurements before and after each IVES session. Also, it was observed a significant reduction in scores of frequency of leakage from 3.4(±1,4) to 1.4(±5.9) [p<0.03], amount of leakage from 3.6(±1.6) to 2.0(±1.3) [p<0.04] and impact of UI in Qol from 7.7(±2.4) to 3.8(±2.9) [p<0.001]. Total score ICIQ-SF decreased from 14.6(±4.2) to 7.2 (±4.5) [p<0.001]. Conclusion: No significant influence was observed in the vaginal ecosystem, inflammatory process, and vaginal pH after IVES, but 4 cases of BV were reported. The IVES provided a significant improvement in QoL of women with UI.
Doutorado
Tocoginecologia
Mestre em Tocoginecologia

Neste estudo avaliamos o papel do metabolismo do triptofano (Trp) na homeostasia, na vaginose bacteriana e nas lesões cervicais associadas ao HPV. A importância do metabolismo do Trp se deve a sua ação na proliferação de microrganismos e de células do sistema imune. O consumo de triptofano tem sido identificado como uma forma de controlar o crescimento bacteriano limitando a infecção. Por outro lado, a oxidação de Trp produz quinurenina (QUIN), que tem papel chave na tolerância imunológica. A formação de QUIN se dá através das enzimas indoleamina 2,3-dioxigenase (IDO) e triptofano 2,3- dioxigenase (TDO). A mais estudada delas no âmbito das infecções/ imuno escape é a enzima IDO. Mais recentemente, tem-se dado ênfase ao papel da TDO no câncer. Nesta dissertação, o interesse foi avaliar a expressão da IDO no epitélio cervicovaginal de mulheres com vaginose bacteriana e de IDO e TDO em amostras cervicais de mulheres com diferentes graus de lesão cervical associada ao HPV. Foram incluídas 165 mulheres atendidas no CAISM/UNICAMP, as quais foram divididas em dois grupos: grupo caso composto por mulheres com lesão de baixo ou alto grau e carcinoma invasor (n=42) e grupo controle composto por mulheres com citologia oncológica normal, independente de apresentar infecção genital (n=123). IDO foi avaliada por imunocitoquímica em citologia em base líquida e IDO e TDO em biópsias cervicais. Mulheres com vaginose bacteriana apresentaram expressão aumentada de IDO em células escamosas em comparação às mulheres sem vaginose bacteriana (OR=7.41; IC 95%= 2.50 a 21.4; p <0.0001). No epitélio vaginal normal com ou sem infecção por HPV houve uma expressão leve de IDO em células escamosas. Na presença de lesões ou carcinoma, houve um aumento no número de células escamosas displásicas e de leucócitos IDO-positivos; aumento de IDO também pôde ser observada em culturas de pele organotípicas transduzidas com as oncoproteínas E6/ E7 do HPV16. Nas lesões cervicais, assim como visto para a IDO, a TDO esteve expressa em leucócitos, especialmente os infiltrados na região estromal e na parede dos vasos sanguíneos. A expressão basal de IDO no epitélio cervical normal e sua regulação positiva na infecção por HPV e lesões associadas sugerem a participação do metabolismo do Trp nos mecanismos imunossupressores envolvidos na doença. Embora o papel do IDO já tenha sido abordada anteriormente, até onde sabemos esta é a primeira evidência da expressão de TDO no epitélio vaginal, na neoplasia intraepitelial cervical e carcinoma de células escamosas. Ainda, em leucócitos, especialmente aqueles com morfologia típica de polimorfonucleares, parecem ser importantes fontes de IDO na cérvix uterina.
In this study we evaluated the role of tryptophan (Trp) metabolism in cervix homeostasis, bacterial vaginosis and HPV-associated lesions. The importance of Trp metabolism is due to its action on microorganisms and immune cells. Tryptophan consumption has been identified as a way to controlling bacterial growth limiting infection. On the other hand, the oxidation of Trp produces kynurenine (Kyn) which plays a key role in immunological tolerance. The formation of Kyn occurs through the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). IDO is the most studied of them within the context of infections / immune escape. More recently, TDO has also been considered in studies of cancer progression. In this thesis, we were interested in cervicovaginal epithelium IDO expression in women with bacterial vaginosis and of IDO and TDO in cervical samples of women with different degrees of cervical lesion associated with HPV. A total of 165 women attended at CAISM/UNICAMP were divided into two groups: a case group composed of women with low or high grade lesions and invasive carcinoma (n = 42) and a control group composed of women with normal cytology, independent to present genital infection (n =123). IDO was evaluated by immunocytochemistry in liquid-based cytology and IDO and TDO in cervical biopsies. Women with bacterial vaginosis had increased IDO expression in squamous cells compared to women without bacterial vaginosis (OR = 7.41, 95% CI = 2.50- 21.74; p<0.0001). In normal vaginal epithelium with or without HPV infection there was a mild IDO expression in squamous cells. In the presence of cervical intraepithelial lesions or squamous cell carcinoma, there was an increase in the number of IDO-positive dysplastic squamous cells and leukocytes; increase in IDO can also be observed in organotypic skin cultures transduced with HPV-16 E6/E7 oncoproteins. In cervical lesions, as observed for IDO, TDO was expressed in leukocytes, especially infiltrates in the stromal region and in the wall of blood vessels. The basal expression of IDO in the normal cervical epithelium and its positive regulation in HPV infection and associated lesions suggests the participation of Trp metabolism in the immunosuppressive mechanisms involved in the disease. Although some previous data have already considered the role of IDO, as far as we know this is the first evidence of the participation of TDO in the vaginal epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma. In addition, in leukocytes, especially those with a typical polymorphonuclear morphology, appear to be important sources of IDO in the uterine cervix.

Introduction: The quality of neonatal care is mainly determined by long-term neurodevelopmental outcome. The neurodevelopment of preterm infants is related to postnatal head growth and depends on medical interventions such as nutritional support. Head circumference (HC) is currently used as a two-dimensional measure of head growth. Since head deformities are frequently found in preterm infants, HC may not always adequately reflect head growth. Laser aided head shape digitizers offer semiautomatic acquisition of HC and cranial volume (CrV) and could thus be useful in describing head size more precisely. Aims: 1) To evaluate reproducibility of a 3D digital capture system in newborns. 2) To compare manual and digital HC measurements in a neonatal cohort. 3) To determine correlation of HC and CrV and predictive value of HC. Methods: Within a twelve-month period data of head scans with a laser shape digitizer were analysed. Repeated measures were used for method evaluation. Manually and digitally acquired HC was compared. Regression analysis of HC and CrV was performed. Results: Interobserver reliability was excellent for HC (bias-0.005%, 95% Limits of Agreement (LoA) −0.39–0.39%) and CrV (bias1.5%, 95%LoA-0.8–3.6%). Method comparison data was acquired from 282 infants. It revealed interchangeability of the methods (bias-0.45%; 95%LoA-4.55–3.65%) and no significant systematic or proportional differences. HC and CrV correlated (r2 = 0.859, p<0.001), performance of HC predicting CrV was poor (RSD ±24 ml). Correlation was worse in infants with lower postmenstrual age (r2 = 0.745) compared to older infants (r2 = 0.843). Discussion: The current practice of measuring HC for describing head growth in preterm infants could be misleading since it does not represent a 3D approach. CrV can vary substantially in infants of equal HC. The 3D laser scanner represents a new and promising method to provide reproducible data of CrV and HC. Since it does not provide data on cerebral structures, additional imaging is required.

INTRODUÇÃO: Poucos estudos têm focado as modificações fisiológicas que ocorrem no trato genital de mulheres menopausadas infectadas pelo HIV e sua associação com a excreção genital do vírus. Nesse estudo de corte transversal, comparou-se a excreção genital do HIV em mulheres menopausadas e em idade fértil em acompanhamento em um centro especializado em São Paulo, Brasil. Investigou-se também a associação entre a excreção genital de RNA de HIV e a viremia em ambos os grupos. Fatores associados com a intensidade da excreção genital de HIV também foram pesquisados, incluindo achados ginecológicos e marcadores de progressão da infecção por HIV. MÉTODOS: 146 mulheres infectadas pelo HIV [73 menopausadas (M)/73 em idade fértil (F)] foram selecionadas em Serviço de Extensão ao Atendimento de Pacientes com HIV/Aids Casa da Aids do Hospital das Clínicas da FMUSP, São Paulo, Brasil. As mulheres menopausadas referiram tempo médio de 8,17 anos (DP=6 anos) de menopausa. A contagem de linfócitos T CD4+ foi obtida por citometria de fluxo e a quantificação do RNA do HIV no plasma e no lavado cervicovaginal (LCV) foi realizada por RT-PCR quantitativo, utilizando-se o kit Cobas Amplicor HIV-1 Monitor Test®, no método ultrasensível. Cloreto de lítio foi introduzido no tampão para obtenção do LCV e quantificado antes e depois da coleta do lavado, a fim de determinar o fator de diluição de cada amostra. A deteção do gene SRY por PCR também foi realizada a fim de eliminar amostras com eventual contaminação espermática. A prevalência de excreção genital foi estimada para ambos os grupos e os fatores associados à intensidade da excreção viral foram investigados, utilizando-se modelo de regressão linear múltipla. As variáveis com p<0,2 na análise bivariada foram incluídas na análise multivariada, assim como o grupo em estudo (M ou F). O modelo final incluiu fatores que se mostraram independentemente associados com a intensidade da excreção genital de HIV. RESULTADOS: A prevalência de excreção genital de HIV-RNA foi similar em ambos os grupos (M: 17,8%, IC 95% 9,8 28,5; F: 22%, IC 95% 13,1 33,1, p=0,678). Similarmente, a intensidade de excreção genital do HIV também não se mostrou diferente entre os grupos (mediana - M: 1,4log/mL; F: 1,4log/mL, p=0,587). A carga viral plasmática foi detectável em 34,2% das pacientes menopausadas (IC 95% 23,5 46,3) e em 42,5% entre as pacientes em idade fértil (IC 95% 31 54,6, p=0,395). Três pacientes (2 M/1 F) exibiram excreção genital de HIV-RNA na ausência de viremia detectável. Existe evidência de correlação entre a carga viral plasmática e a genital em ambos os grupos (rM: 0,658; rF: 0,684, p<0,01). Adicionalmente, o número de células CD4+ periféricas mostrou-se negativamente correlacionada à excreção genital do HIV em ambos os grupos (rM: -0,250; rF: -0,248, p<0,05). À análise multivariada, a carga viral plasmática mostrou-se independentemente associada à ocorrência de excreção genital do HIV em ambos os grupos (OR 4,03, IC 95% 2,52 6,45, p<0,001). Já a intensidade de excreção genital mostrou-se independentemente associada ao pH vaginal (p<0,001), concentração de TNF- no LCV (p=0,01), e à carga viral plasmática (p=0,001), todos com correlação positiva. CONCLUSÕES: Apesar das modificações significativas que ocorrem na mucosa vaginal da mulher menopausada, a excreção cervicovaginal do HIV parece não ser significativamente influenciada por esse estado. A carga viral plasmática e o número de células CD4+ periféricas estão correlacionadas com a excreção genital do vírus. A frequência de excreção genital mostrouse independentemente associada à intensidade de viremia. Além disso, o aumento do pH vaginal e evidência de inflamação genital, associada à concentração de TNF- no LCV, independentemente aumentam a intensidade de excreção genital nas mulheres estudadas.
BACKGROUND: Few studies have focused on physiological modifications that occur in the genital tract of HIV-infected postmenopausal women and their association with HIV cervicovaginal shedding. In this cross-sectional study we evaluated and compared HIV genital shedding among postmenopausal and fertile-aged women under care at a specialized center in Sao Paulo, Brazil, investigating the association between HIV-RNA shedding and HIV plasma viral loads in both groups. Factors associated with higher HIV shedding were also investigated, including gynaecological features and HIV disease progression markers. METHODS: 146 women living with HIV [73 postmenopausal (PM)/73 in fertile-aged (F)] were enrolled at the HIV Clinic, University of São Paulo Medical School, Brazil. Postmenopausal women referred a mean duration of 8.17y (SD=6y) since menopause. CD4+ cell counts were obtained by flow cytometry and HIV-RNA was quantified in plasma and in cervicovaginal lavages (CVL) by RT-PCR, using Cobas Amplicor HIV-1 Monitor Ultrasensitive Test. Lithium chloride was introduced into the CVL buffer and measured before and after CVL collection in order to determine the dilution factor for each specimen. SRY gene detection by PCR was also performed in all samples in order to rule out sperm contamination. Prevalence of HIV genital shedding was estimated for both groups and factors associated with the intensity of viral shedding were investigated, using a multiple linear regression model. Variables with p<0.2 in bivariate analysis were included in multivariate analysis, as well as the study group (PM and F). The final model included factors shown to be independently associated with intensity of HIV genital shedding. RESULTS: The prevalence of HIV-RNA genital shedding was similar in both groups. (PM: 17.8%, 95%CI 9.8 28.5; F: 22%, 95%CI 13.1 33.1, p=0.678). Likewise, the intensity of HIV shedding was shown not to differ between PM and F women (means - PM: 1.4log/mL; F: 1.4log/mL, p=0.587). Plasma viral loads were detectable in 34.2% of PM patients (95%CI 23.5 46.3), as compared to 42.5% among F women (95%CI 31 54.6) (p=0.395). Three patients (2 PM/1 F) exhibited HIV-RNA genital shedding in the absence of detectable viremia. We found evidence of correlation between HIV plasma viral load and HIV cervicovaginal shedding in both groups (rPM: 0.658; rF: 0.684, p<0.01). In addition, CD4+ cell counts were shown negatively correlated to HIV shedding in both groups (rPM: -0.250; rF: -0.248, p<0.05). In multivariate analysis, HIV plasma viral load was shown independently associated with occurrence of HIV genital shedding in both groups (OR 4.03, 95%CI 2.52 6.45, p<0.001). In addition, the intensity of HIV shedding was shown independently associated with vaginal pH (p<0.001), TNF- concentrations in CVL (p=0.01), and with HIV plasma viral loads (p=0.001), all of them with positive correlation. CONCLUSION: Despite the significant changes that occur in the vaginal mucosa of postmenopausal women, HIV cervicovaginal shedding does not seem to be significantly influenced by this state. Plasma viral loads and CD4+ cell counts are correlated to HIV genital shedding. The frequency of HIV genital shedding was shown independently associated with viremia intensity. Moreover, increased vaginal pH and evidence of genital inflammation associated with TNF- concentration independently enhanced the intensity of HIV shedding in postmenopausal and fertile-aged women.