Orientador: Paulo César Giraldo, Fernando Guimarães / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T17:52:48Z (GMT). No. of bitstreams: 1 Ferreira_JozianiBeghiniJunqueiradeCarvalho_M.pdf: 1296627 bytes, checksum: ecb2857b529cc3c0e322efcb11c882e7 (MD5) Previous issue date: 2011 / 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

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.

>Magister Scientiae - MSc / 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. / South Africa

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

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.

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 β- 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 iii 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.

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.

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.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-09-12T18:15:05Z No. of bitstreams: 2 Dissertação - Kélvia Cristina de Camargo - 2014.pdf: 1810460 bytes, checksum: 3f5dbf06427d9e09b4ebb202a6433de2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-09-12T18:15:17Z (GMT) No. of bitstreams: 2 Dissertação - Kélvia Cristina de Camargo - 2014.pdf: 1810460 bytes, checksum: 3f5dbf06427d9e09b4ebb202a6433de2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-12T18:15:17Z (GMT). No. of bitstreams: 2 Dissertação - Kélvia Cristina de Camargo - 2014.pdf: 1810460 bytes, checksum: 3f5dbf06427d9e09b4ebb202a6433de2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-06-18 / 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.

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No. of bitstreams: 1 danielemariaknuppdesouza.pdf: 8224549 bytes, checksum: bc3a45c095a6504198c18b63f1922033 (MD5) Previous issue date: 2013-04-25 / 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.