Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP Faculdade de Medicina de Botucatu Juliano Novak Estudo da associação entre o microbioma vaginal com variáveis sociodemográficas e de hábitos comportamentais de mulheres brasileiras em idade reprodutiva Dissertação apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Botucatu, para obtenção do título de Mestre em Patologia. Orientadora: Profa. Dra. Camila Marconi Coorientadora: Profa. Dra. Márcia Guimarães da Silva Botucatu 2019 Juliano Novak Estudo da associação entre o microbioma vaginal com variáveis sociodemográficas e de hábitos comportamentais de mulheres brasileiras em idade reprodutiva Dissertação apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Botucatu, para obtenção do título de Mestre em Patologia. Orientadora: Profa.Dra. Camila Marconi Coorientadora: Profa.Dra. Márcia Guimarães da Silva Botucatu 2019 Dedico este trabalho á minha mãe Elvira, por seu exemplo de vida. Agradeço a todos que fizeram parte da minha formação durante a elaboração deste trabalho, pois todos foram essenciais. Agradeço também as agências de fomento à pesquisa, FAPESP pelo auxílio pesquisa e CAPES pela bolsa de mestrado. “Aqueles que passam por nós, não vão sós, não nos deixam sós. Deixam um pouco de si, levam um pouco de nós”. Antoine de Saint-Exupéry SUMÁRIO 1. RESUMO ............................................................................................................ 6 2. ABSTRACT ........................................................................................................ 8 2. REVISÃO DE LITERATURA ........................................................................... 11 3. REFERÊNCIAS BIBLIOGRÁFICAS ................................................................ 19 4. ARTIGO CIENTÍFICO ...................................................................................... 28 5. ANEXOS .......................................................................................................... 46 6 1. RESUMO A microbiota vaginal normal é composta predominantemente por Lactobacillus spp. que conferem proteção contra infecções por patógenos, por meio da produção de ácido lático, peróxido de hidrogênio e bacteriocinas. Diferentemente, a vaginose bacteriana (VB) é caracterizada pela substituição da microbiota de Lactobacillus spp. por bactérias anaeróbias em sua maioria. A VB é a alteração de microbiota vaginal mais comum em mulheres de idade reprodutiva, acometendo aproximadamente 30% dessa população. Além disso, a VB é fator de risco para aquisição de infecções sexualmente transmissíveis (IST). Diversas características da população já foram associadas à VB, como idade, etnia, comportamentos sexual e de higiene. Entretanto, a real composição da microbiota vaginal só foi possível em 2011 com estudo utilizando o sequenciamento de nova geração do gene bacteriano RNA ribossômico 16S. Foi demonstrado que o microbioma vaginal pode ser classificado em cinco tipos de comunidades bacterianas (community-state types, CST). Quatro dessas CSTs tem predomínio de Lactobacillus: L. crispatus (CSTI), L. gasseri (CST II), L. iners (CST III) e L. jensenii (CST V), enquanto que a CST IV apresenta grande diversidade bacteriana e engloba a maioria dos casos de VB. Apesar de quatro CSTs apresentarem predomínio de Lactobacillus, o papel protetor da CST III, dominada por L. iners, contra aquisição de IST tem se demonstrado menor que os demais. Embora os estudos de microbioma tenham possibilitado conhecer melhor a relação entre as espécies bacterianas com as IST, não existe na literatura informações acerca dos fatores sociodemográficos, comportamentais e clínicos das mulheres associados às CSTs. Desta forma, o objetivo desse estudo foi identificar as características sociodemográficas, comportamentais e clínicas da população associadas à CST III. Incluímos neste estudo transversal 609 mulheres brasileiras, que buscaram unidades de saúde para realização do exame preventivo do câncer de colo do útero. As participantes responderam a um questionário estruturado para obtenção de dados sociodemográficos, comportamentais e clínicos. Para caracterização do microbioma vaginal, amostras vaginais foram submetidas ao sequenciamento das regiões V3-V4 do RNA ribossômico16S em plataforma Miseq (Illumina, San Diego, CA). Um modelo de 7 regressão logística utilizando o método stepwise foi utilizado para testar a associação entre o microbioma dominado por L. iners com todas as variáveis avaliadas, considerando suas razões de chances (OR) e intervalos de confiança de 95% (IC95%) em software Stata (Stata Corp, College station, TX). A maior prevalência foi de CST III foi de 36,5% (n = 222), das demais CST foram: CST I, 30,5% (n=186), CST II 4,4% (n = 27) e CST V 1,2% (n = 7). As demais 167 (27,4%) participantes apresentaram CST IV e não foram considerados na análise. A análise multivariada mostrou que as participantes que relataram ter dois ou mais parceiros sexuais no ano anterior ao estudo apresentaram um risco aumentado para CST III (OR: 3,27; IC 95% 1,50-7,11), bem como o achado microscópico de Candida spp. em esfregaços vaginais (OR: 2,24; IC 95% 1,02- 4,89). Além disso, três fatores foram protetores para esse tipo de microbioma: uso de preservativo (OR: 0,59; IC 95% 0,38-0,91), ensino médio completo (OR: 0,61; IC 95% 0,41-0,91) e consumo diário de leite ou derivados (OR: 0,43; IC 95% 0,20- 0,90). Dessa forma, concluímos que o microbioma vaginal dominado por L. iners é independentemente associado a características da população anteriormente associadas à VB como baixa escolaridade, múltiplos parceiros sexuais e sexo desprotegido. Palavras-chave: Microbioma vaginal; Lactobacillus iners; Infecções sexualmente transmissíveis; Vaginose bacteriana; Sequenciamento de nova geração. 8 2. ABSTRACT The normal vaginal microbiota is predominantly composed of Lactobacillus spp. which confer protection against pathogen infections through the production of lactic acid, hydrogen peroxide and bacteriocins. Differently, bacterial vaginosis (BV) is characterized by the replacement of the microbiota of Lactobacillus spp. by anaerobic bacteria for the most part. BV is the most common vaginal microbiota alteration in women of reproductive age, affecting approximately 30% of this population. In addition, BV is a risk factor for the acquisition of sexually transmitted infections (STIs). Several characteristics of the population have already been associated with BV, such as age, ethnicity, sexual and hygiene behaviors. However, the actual composition of the vaginal microbiota was only possible in 2011 with study using the new generation sequencing of the bacterial 16S ribosomal RNA gene. It has been shown that the vaginal microbiome can be classified into five types of community-state types (CST). Four of these CSTs have a predominance of Lactobacillus: L. crispatus (CSTI), L. gasseri (CST II), L. iners (CST III) and L. jensenii (CST V), while CST IV shows great bacterial diversity and involve most cases of BV. Although four CSTs have a predominance of Lactobacillus, the protective role of CST III, dominated by L. iners, against IST acquisition has been shown to be lower than the others are. Although microbiome studies have made it possible to know better the relationship between bacterial species and STIs, there is no information in the literature about the socio- demographic, behavioral and clinical factors of women associated with STIs. Thus, the objective of this study was to identify the sociodemographic, behavioral and clinical characteristics of the population associated with CST III. We included in this cross-sectional study 609 Brazilian women, who sought health units for the cervical cancer screening. Participants answered a structured questionnaire to obtain sociodemographic, behavioral and clinical data. To characterize the vaginal microbiome, vaginal samples were submitted to the sequencing of the V3-V4 regions of 16S ribosomal RNA in Miseq platform (Illumina, San Diego, CA). A logistic regression model using the stepwise method was used to test the association between the L. iners dominated microbiome with all the variables evaluated, considering its odds ratios (OR) and 95% confidence intervals (95% CI) 9 in software Stata (Stata Corp., College Station, TX). The highest prevalence of CST III was 36.5% (n = 222); the other CSTs were CST I, 30.5% (n = 186), CST II 4.4% (n = 27) and CST V 1.2% (n = 7). The remaining 167 (27.4%) participants presented CST IV and were not considered in the analysis. Multivariate analysis showed that participants who reported having two or more sex partners in the year prior to the study had an increased risk for CST III (OR: 3.27; 95% CI 1.50-7.11), as well as the finding microscopic study of Candida spp. in vaginal smears (OR: 2.24; 95% CI 1.02-4.89). In addition, three factors were protective for this type of microbiome: condom use (OR: 0.59, 95% CI 0.38-0.91), complete high school (OR: 0.61, 95% CI, 41-0.91) and daily consumption of milk or derivatives (OR: 0.43; 95% CI 0.20-0.90). Thus, we conclude that the vaginal microbiome dominated by L. iners is independently associated with characteristics of the population previously associated with BV such as low education level, multiple sexual partners and unprotected sex. Keywords: Vaginal microbiome; Lactobacillus iners; Sexually transmitted infections; Bacterial vaginosis; Next generation sequencing. 10 Revisão de Literatura 11 2. REVISÃO DE LITERATURA A microbiota vaginal foi descrita pela primeira vez em 1892 por Albert Döderleine, já neste momento, foi considerada como saudável quando encontrada a predominância de espécies do gênero Lactobacillus1. Ao longo dos anos, a literatura constantemente tem confirmado a importância da colonização por tais microrganismos2. Espécies de Lactobacillus são responsáveis pela manutenção do ambiente vaginal equilibrado, visto que conferem defesa contra patógenos3. Dentre as linhagens de Lactobacillus spp., grande parte é capaz de liberar peróxido de hidrogênio e bacteriocinas, conferindo importante capacidade antimicrobiana4,5. Outro importante fator se dá pela capacidade que os Lactobacillus spp. possuem de produzir ácido lático utilizando o metabolismo do glicogênio armazenado nas células epiteliais vaginais sob a influência do estrogênio do hospedeiro, dessa forma esses microrganismos contribuem para a manutenção do pH vaginal normal ácido, que também é considerado importante fator na manutenção da microbiota vaginal normal6,7. Além disso, ao romper membranas celulares bacterianas o ácido lático estimula a imunidade do hospedeiro na presença de lipopolissacarídeo presente na parede bacteriana de algumas espécies8. Curiosamente, há uma pequena porcentagem de mulheres que podem apresentar microbiota vaginal diversa com pH local ácido, composta por espécies também produtoras de ácido lático9,10, embora os métodos utilizados para identificação bacteriana nesses casos sejam atualmente questionáveis. Devido à capacidade protetora das diferentes espécies de Lactobacillus, vários trabalhos tiveram por objetivo realizar avaliação individual de cada espécie. Um dos primeiros estudos, ao avaliar a capacidade de produção de peróxido de hidrogênio demonstrou que apenas 9% dos isolados de L. iners apresentaram 12 essa capacidade, enquanto que 95% dos isolados de L. crispatus foram capazes de produzi-lo11. Com relação à produção de ácido lático, já foi demonstrado que L. iners produz majoritariamente a isoforma L-lactato, enquanto que L. crispatus produz maiores quantidades de D-lactato12. A contribuição da isoforma L-lactato no ambiente vaginal ainda não é completamente compreendida, no entanto estudos têm mostrado que L. iners tem a capacidade de manter o pH vaginal relativamente baixo, mas não tão eficientemente quanto L. crispatus13-15. Ainda com relação ao pH, recentemente foi demonstrado que somente o pH baixo não é suficiente para proteção contra a infecção pelo vírus da imunodeficiência humana (HIV), mas a isoforma mais produzida pelo L. crispatus é a que oferece melhor proteção contra esse vírus por dificultar a permeabilidade ao muco cérvico- vaginal16. A literatura é consistente ao demonstrar que mulheres com predomínio de Lactobacillus spp. estão menos propensas à aquisição de infecções sexualmente transmissíveis (IST), sejam as bacterianas por Chlamydia trachomatis, Neisseria gonorrhoeae, bem como as infecções virais, como pelo papilomavírus humano (HPV) e HIV1-3; 16. Quando há depleção de espécies de Lactobacillus, tem-se a principal alteração de microbiota vaginal, denominada vaginose bacteriana (VB), neste caso Lactobacillus spp. são parcialmente ou totalmente substituídos por outras espécies bacterianas, principalmente anaeróbias17. Esta alteração foi descrita pela primeira vez em 1954 por Gardner e Dukes, como sendo causada por um único agente bacteriano, o bacilo Gram-variável e anaeróbio facultativo, denominado Gardnerella vaginalis18. No entanto, os estudos posteriores demonstraram que na verdade a VB está associada ao aumento de várias outras 13 espécies bacterianas como Mobiluncus sp., Mycoplasma sp., Peptostreptococcus sp., Prevotella sp., Bacteroides fragilis, Ureaplasma sp.,Staphylococcus aureus e Streptococcus sp., e, portanto, tal alteração passou a ser considerada como de etiologia polimicrobiana17, 19-20. A prevalência de VB é de aproximadamente 30% em mulheres em idade reprodutiva em todo o mundo, incluindo estudos realizados no Brasil, mas pode variar conforme características populacionais como idade, etnia e características comportamentais21-22. Deste modo, os estudos já demonstraram a associação entre VB e fatores como hábitos sexuais como histórico de mais de um parceiro sexual na vida, relato de novo parceiro sexual ou início de atividade sexual em idade mais jovem, além do uso de duchas vaginais e estresse crônico22-25. É bem estabelecido que há associação entre a VB e diversas complicações obstétricas e ginecológicas levando a efeitos deletérios para a saúde, visto que, mulheres que apresentam essa alteração de microbiota têm risco aumentado para desenvolver doença inflamatória pélvica (DIP) e aquisição e transmissão de IST. No contexto da associação negativa entre Lactobacillus spp. e IST, a VB já foi associada à infecção por C. trachomatis, N. gonorrhoeae, HPV e HIV. Além destas, a instalação dessa condição também aumenta o risco de infecção pelo parasita Trichomonas vaginalis26-30. Com relação à infecção pelo HPV, sabe-se que a persistência desta infecção é condição necessária para o desenvolvimento das lesões neoplásicas no colo uterino e nesse sentido já foi demonstrado que a VB aumenta o tempo de clearance viral31. Dentre as mulheres grávidas, tal condição é particularmente deletéria, visto que a VB durante a gestação pode levar a complicações como aborto 14 espontâneo, corioamnionite, trabalho de parto pré-termo, além de ser um fator de risco para apresentar baixo peso ao nascimento32-35. O padrão-ouro para o diagnóstico da VB se dá pela observação microscópica dos morfotipos bacterianos presentes no esfregaço do conteúdo vaginal após coloração pela técnica de Gram desenvolvida por Nugent et al.13 Desta forma, baseando-se na semiquantificação dos morfotipos presentes, são atribuídos escores de 0 a 10, onde a microbiota normal recebe escores de 0 a 3, a intermediária de 4 a 6 e escores superiores são compatíveis com VB36. Devido às diversas complicações ocasionadas pela VB e as limitações das técnicas microscópicas, vários estudos direcionaram seus esforços a fim de se conhecer a composição bacteriana exata do ambiente vaginal, bem como as interações existentes entre as espécies. Desta forma, os primeiros estudos realizados com métodos de cultura bacteriana contribuíram para o conhecimento da composição bacteriana do ambiente vaginal, no entanto tais métodos não permitiam cultivar bactérias fastidiosas ou não-cultiváveis, com o avanço das técnicas de biologia molecular, inicialmente pela reação em cadeia da polimerase (PCR) e depois por clonagem pôde-se ter conhecimento das espécies presentes nesta alteração, embora houvesse limitação com relação ao número de clones recuperados37-40. Embora as técnicas acima citadas já tivessem permitido avanços no conhecimento da composição bacteriana da microbiota vaginal, o marco para a determinação não só do microbioma vaginal, bem como do microbioma humano como um todo foi o desenvolvimento das técnicas de sequenciamento de nova geração. Neste sentido, tais técnicas permitiram conhecer a real composição bacteriana no ambiente vaginal bem como os estudos tornaram possível avaliar as transições de microbiota vaginal saudável e VB13, 41. Tal técnica consiste no 15 sequenciamento de porções do gene bacteriano RNA ribossômico 16S, permitindo a identificação das espécies bacterianas presentes, além de determinar qual a participação de cada espécie na comunidade bacteriana total42. A partir da utilização dessa técnica demonstrou-se que grande parte da microbiota vaginal é composta por organismos fastidiosos e até então nunca cultivados em laboratório13, 43. Empregando o pirosequenciamento do gene bacteriano RNAr 16S, Ravel et al. (2011)41, classificaram pela primeira vez o microbioma vaginal. Este estudo demonstrou que 75% das mulheres norte-americanas apresentaram microbiota vaginal constituída predominantemente por uma ou mais espécies de Lactobacillus, além do fato de que apesar da grande diversidade microbiana vaginal, o microbioma vaginal de mulheres em idade reprodutiva pode ser agrupado em cinco principais comunidades bacterianas (CST, Community-state type) conforme predominância de determinadas espécies. Destas cinco comunidades, quatro são caracterizadas pela prevalência de uma das espécies de Lactobacillus: L. iners, L. crispatus, L gasseri e L. jensenii. Enquanto que a comunidade remanescente não apresenta predomínio de Lactobacillus spp., mas sim grande diversidade de espécies, embora em alguns casos possa existir a presença de Lactobacillus spp. Neste ponto, vale destacar que é justamente nesta comunidade onde há diversidade e heterogeneidade bacteriana que estão incluídos a maioria dos casos de VB, quando a microbiota é classificada pelo método de Nugent, além do fato de que a microbiota vaginal intermediária pode estar distribuída entre as cinco comunidades descritas, mas a maior parte delas concentra-se naquela dominada por L. iners. 16 Lactobacillus iners foi descrito pela primeira vez em 1999 sendo isolado do trato genital inferior e urinário44. Este microrganismo foi descrito como bacilo Gram-positivo, com arranjos individuais, em pares ou ainda em cadeias curtas. O crescimento é lento, aproximadamente 24 horas, e as colônias são pequenas, lisas e não pigmentadas44. Posteriormente foi demonstrado que a espécie nem sempre é Gram-positiva, visto que alguns isolados se apresentam como cocobacilo e coloração Gram-negativa. Tal fator possivelmente dificultou seu reconhecimento em esfregaços vaginais 45. Essa diferença na morfologia celular e nas propriedades de coloração de Gram do L. iners deve ser ainda explorada, visto que muitas determinações diagnósticas dependem dessas características, por exemplo, o escore de Nugent, que é amplamente usado na rotina diagnóstica de alterações da microbiota vaginal. Tal método se baseia na coloração pelo método de Gram do conteúdo vaginal permitindo diferir Lactobacillus spp. como bastonetes Gram-positivos de outros morfotipos bacterianos proporcionando o diagnóstico, principalmente da VB, sendo um método de diagnóstico dependente da morfologia vista ao microscópio, o comportamento diferente do L. iners comparado as outras espécies lactobacilar, pode ser um fator confundidor36. O microbioma com predomínio de L. iners corresponde a um dos mais prevalentes em mulheres em todo o mundo, chegando a quase 40% dependendo da população estudada46-49 sendo que em gestantes essa prevalência pode ser ainda maior, visto que estudo encontrou predomínio desta espécie em 51% da população avaliada49. Visto que L. iners figura entre as espécies mais prevalentes colonizando o ambiente vaginal, estudos direcionaram seus objetivos a entender melhor a sua participação na microbiota vaginal. Desta forma, alguns autores apontam que a 17 comunidade com predomínio de L. iners é mais instável e variável com relação à variedade de outras espécies co-habitando o mesmo ambiente, dessa forma pode-se assumir que o microbioma com predomínio de L. iners favorece o aumento da beta-diversidade, enquanto que as comunidades com predomínio de L. crispatus e L. gasseri tendem a ser mais estáveis ao longo do tempo13, 50. De fato, estudos prévios já demonstraram associação entre o L. iners e a VB39-40,51-52. Além da associação com VB, o microbioma vaginal dominado por L. iners já demonstrou ter associação com ISTs, como no caso da infecção pelo HPV, além disso, este estudo demonstrou que o tempo do clearance viral foi maior, diferente do que ocorreu no microbioma dominado por L. gasseri50. No caso da infecção bacteriana por C. trachomatis, o microbioma dominado por L. iners é fator de risco para aquisição, enquanto que o microbioma dominado por L. crispatus apresenta associação contrária, diminuindo as chances de se adquirir essa infecção53. De fato, estudo in vitro demonstrou que L. crispatus é capaz de diminuir a adesão e a infectividade da C. trachomatis devido a sua capacidade de produção de D-lactato54-55. Pouco se sabe sobre a interação entre microbioma vaginal e presença de Candida spp., causadora de vaginites como a candidíase vulvovaginal, que acomete entre 30 a 50% de mulheres ao menos uma vez durante a vida56-57, e compartilha de fatores de risco já associados ao microbioma, como frequência de relação sexual, ducha vaginal e uso de contraceptivos58-59. Neste sentido, estudo apontou que embora não houvesse associação estatisticamente significativa, houve maior prevalência de mulheres com candidíase vulvovaginal na comunidade com predomínio de L. iners definida pelo microbioma60. 18 Até o presente momento existem diversos estudos associando fatores sociodemográficos, comportamentais e clínicos à VB, possibilitando saber quais características populacionais são fatores de risco para o desenvolvimento desta alteração que pode levar a efeitos deletérios a saúde da mulher22-25. Sabe-se que a composição do microbioma humano está relacionada a fatores ambientais, de comportamento e dieta do hospedeiro. No entanto, a literatura ainda carece de dados acerca da relação entre o microbioma vaginal e variáveis sociodemográficas, comportamentais e clínicas. Principalmente, no que se refere ao microbioma vaginal com predomínio de L. iners, que embora seja o mais frequente em diversas populações e apresente similaridades com àquelas observadas na alteração da microbiota vaginal, não existem informações sobre sua relação com características da população. 19 3. REFERÊNCIAS BIBLIOGRÁFICAS 1.Doderlain A. Das scheiden sekretund seine Bedeuting fur das puerperal fieber. Leipzig: Verloag von Edgard Besold. Jan 1892. 2.Hillier SL, Krohn MA, Rabe LK, Klebanoff SJ, Eschenbach DA. The normal vaginal flora, H2O2-producing lactobacilli, and bacterial vaginosis in pregnant women. Clin Infect Dis 1993; 4: 273-81. 3.Graver MA, Wade JJ. The role of acidification in the inhibition of Neisseria gonorrhoeae by vaginal lactobacilli during anaerobic growth. Ann Clin Microbiol Antimicrob 2011; 10:8. 4.McLean NW, Rosenstein IJ. Characterization and selection of a Lactobacillus species to re-colonise the vagina of women with recurrent bacterial vaginosis. J Med Microbio 2000; 49: 543-52. 5.Eschenbach DA, Davick PR, Williams BL, Klebanoff SJ, Young-Smith K, Critchlow CM, et al. Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 1989; 27: 251-6. 6.Boskey ER, Telsch KM, Whaley KJ, Moench TR, Cone RA. Acid production by vaginal flora in vitro is consistent with the rate and extent of vaginal acidification. Infect Immun 1999; 67: 5170-5. 7.Amsel R, Totten PA, Spiegel CA, Chen KCS, Eschenbach DA, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74: 14-22. 8.Bradshaw CS, Brotman RM. Making inroads into improving treatment of bacterial vaginosis-striving for long-term cure. BMC Infect Dis 2015; 15: 292. 20 9.Petrova MI, van den Broek M, Balzarini J, Vanderleyden J, Lebeer S. Vaginal microbiota and its role in HIV transmission and infection. FEMS Microbiol Rev 2013; 37: 762–92. 10.Aldunate M, Srbinovski D, Hearps AC, Latham CF, Ramsland PA, Gugasyan R, et al. Antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis. Front Physiol 2015; 6: 164. 11.Antonio MAD, Hawes SE, Hillier SL. The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 1999; 180: 1950-6. 12.Witkin SS, Mendes-Soares H, Linhares IM, Jayaram A, Ledger WJ, Forney LJ. Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections. mBio 2013; 4: 00460-13. 13.Gajer P, Brotman RM, Bay G, Sakamoto J, Schutte UME, Zhong X, et al. Temporal dynamics of the human vaginal microbiota. Sci Transl Med 2012; 4: 132- 52. 14.Srinivasan S, Hoffman NG, Morgan MT, Matsen FA, Fiedler TL, Hall RW, et al. Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. PLoS One 2012; 7: 37818. 15.Hummelen R, Fernandes AD, Macklaim JM, Dickson RJ, Changalucha J, Gloor GB, et al. Deep sequencing of the vaginal microbiota of women with HIV. PLoS One 2010; 5: 12078. 21 16.Nunn KL, Wang YY, Harit D, Humphrys MS, Ma B, Cone R, et al. Enhanced trapping of HIV-1 by human cervicovaginal mucus is associated with Lactobacillus crispatus-dominant microbiota. M Bio 2015; 6(5): 01084-15. 17.Spiegel CA. Bacterial vaginosis. Clin Microbiol Rev 1991;4:485-502. 18.Gardner HL, Dukes CD. New etiologic agent in non-specific bacterial vaginitis. Science 1954; 120: 853. 19.Newton ER, Piper JM, Shain RN, Perdue ST, Peairs W. Predictors of the vaginal microflora. Am J Obstet Gynecol 2001; 184: 845-55. 20.Larsen B, Monif GR. Understanding the bacterial flora of the female genital tract. Clin Infect Dis 2001; 32: 69-77. 21.Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J, Sutton M, et al. The prevalence of bacterial vaginosis in the United States, 2001-2004; associations with symptoms, sexual behaviors, and reproductive health. Sex Transm Dis 2007; 34: 864-9. 22.Marconi C, Duarte MT, Silva DC, Silva MG. Prevalence of and risk factors for bacterial vaginosis among women of reproductive age attending cervical screening in southeastern Brazil. Int J Gynaecol Obstet 2015; 131: 137-41. 23.Schwebke JR, Richey CM, Weiss HL. Correlation of behaviors with microbiological changes in vaginal flora. J Infect Dis 1999; 180: 1632-6. 24.Culhane JF, Rauh V, McCollum KF, Elo IT, Hogan V. Exposure to chronic stress and ethnic differences in rates of bacterial vaginosis among pregnant women. Am J Obstet Gynecol 2002; 187: 1272-6. 25.Gallo MF, Macaluso M, Warner L, Fleenor ME, Hook EW, Brill I, et al. Bacterial vaginosis, gonorrhea, and chlamydial infection among women attending a sexually 22 transmitted disease clinic: a longitudinal analysis of possible causal links. Ann Epidemiol 2012; 22: 213-20. 26.Wiesenfeld HC, Hillier SL, Krohn MA, Landers DV, Sweet RL. Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection. Clin Infect Dis 2003; 36: 663-8. 27.Myer L, Denny L, Telerant R, Souza M, Wright TC Jr, Kuhn L. Bacterial vaginosis and susceptibility to HIV infection in South African women: a nested case-control study. J Infect Dis 2005; 192: 1372-80. 28.Sewankambo N, Gray RH, Wawer MJ, Paxton L, McNaim D, Wabwire-Mangen F, et al. HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet 1997; 350: 546-50. 29.Martin HL, Richardson BA, Nyange PM, Lavreys L, Hillier SL, Chohan B, et al. Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 1999; 180: 1863-8. 30.Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis 2011; 11: 10. 31.King CC, Jamieson DJ, Wiener J, Cu-Uvin S, Klein RS, Rompalo AM. Bacterial vaginosis and the natural history of human papillomavirus. Infect Dis Obstet Gynecol 2011; 2011: 319460. 32.Donders GG, Van Bulk B, Caudron J, Londers L, Vereecken A, Spitz B. Relationship of bacterial vaginosis and mycoplasmas to the risk of spontaneous abortion. Am J Obstet Gynecol 2000; 183:431-7. 33.Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000; 342: 1500-7. 23 34.Platz-Christensen JJ, Pernevi P, Hagmar B, Andersson E, Brandberg A, Wiqvist N. A longitudinal follow-up of bacterial vaginosis during pregnancy. Acta Obstet Gynecol Scand 1993; 72: 99-102. 35.Leitich H, Bodner-Adler B, Brunbauer M, Kaider A, Egarter C, Husslein P. Bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis. Am J Obstet Gynecol 2003; 189: 139-47. 36.Nugent RP, Krohn MA, Hillier SL. Reability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29: 297-301. 37.Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis: a newly defined specificinfection previously classified non-specific vaginitis. Am J Obstet Gynecol 1955; 69: 962-76. 38.Spiegel CA, Eschenbach DA, Amsel R, Holmes KK. Curved anaerobic bacteria in bacterial (nonspecific) vaginosis and their response to antimicrobial therapy. J Infect Dis1983; 148: 817-22. 39.Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Simaey LV, et al. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 2004; 21:16-26. 40.Fredricks DN Fiedler TL, Marrazzo JM. Molecular identification of bacteria associatedwith bacterial vaginosis. N Engl J Med 2005; 353: 1899-911. 41.Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011; 108: Suppl1:4680-7. 24 42.Fadrosh DW, Ma B, Gajer P, Ott S, Sengamalay N, Brotman RM, et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq Platform. Microbiome 2014; 24: 2-6. 43.Drell T, Lillsaar T, Tummeleht L, Simm J, Aaspõllu A, Väin E, et al. Characterization of the vaginal micro- and mycobiome in asymptomatic reproductive-age Estonian women. PLoS One 2013; 8(1): 54379. 44.Falsen E, Pascual C, Sjodén B, Ohlen M, Collins MD. Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp. nov. Int J Syst Bacteriol 1999; 49: 217-21. 45.De Backer E, Verhelst R, Verstraelen H, Alqumber MA, Burton JP, Tagg JR, et al. Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners. BMC Microbiol 2007; 19: 115. 46.Mitra A, MacIntyre D A, Lee YS, Smith A, Marchesi JR, Lehne B, et al. Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity. Sci Rep 2015; 5: 16865. 47.Tamarelle J, de Barbeyrac B, Le Hen I, Thiébaut A, Bébéar C, Ravel J, et al. Vaginal microbiota composition and association with prevalent Chlamydia trachomatis infection: a cross-sectional study of young women attending a STI clinic in France. Sex Transm Infect 2018; 0: 1–3. 48.Balle C, Lennard K, Dabee S, Barnabas SL, Jaumdally SZ, Gasper MA, et al. Endocervical and vaginal microbiota in South African adolescents with asymptomatic Chlamydia trachomatis infection. Sci Rep 2018; 8: 11109. 49.Nasioudis D, Forney LJ, Schneider GM, Gliniewicz K, France MT, Boester A, et al. The composition of the vaginal microbiome in first trimester pregnant women 25 influences the level of autophagy and stress in vaginal epithelial cells. J of Rep Immun 2017; 123: 35–39. 50.Brotman RM, Shardell MD, Gajer P, Tracy JK, Zenilman JM, Ravel J, et al. Interplay between the temporal dynamics of the vaginal microbiota and human papillomavirus detection. J Infect Dis 2014; 210: 1723–33. 51.Mitchell C, Fredricks D, Agnew K, Hitti J. Hydrogen peroxide-producing lactobacilli are associated with lower levels of vaginal interleukin-1beta, independent of bacterial vaginosis. Sex Transm Dis 2015; 42: 358-63. 52.Santiago GL, Cools P, Verstraelen H, Trog M, Missine G, Aila NE, et al. Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles. PLoS One 2011; 6: 28180. 53.van Houdt R, Ma B, Bruisten SM, Speksnijder AGCL, Ravel J, de Vries HJC, et al. Lactobacillus iners-dominated vaginal microbiota is associated with increased susceptibility to Chlamydia trachomatis infection in Dutch women: a case-control study. Sex Transm Infect. 2018; 94: 117–123. 54.Nardini P, Ñahui Palomino RA, Parolin C, Laghi L, Foschi C, Cevenini R, et al. Lactobacillus crispatus inhibits the infectivity of Chlamydia trachomatis elementary bodies, in vitro study. Sci Rep 2016: 6: 29024. 55.Gong Z, Luna Y, Yu P. & Fan, H. Lactobacilli inactivate Chlamydia trachomatis through lactic acid but not H2O2. PLoS One 2014: 9: 107758. 56.Corsello S, Spinillo A, Osnengo G, Penna C, Guaschino S, Beltrame A, et al. An epidemiological survey of vulvovaginal candidiasis in Italy. Eur J Obstet Gynecol Reprod Biol 2003; 110(1): 66-72. 26 57.Foxman B, Muraglia R, Dietz JP, Sobel JD, Wagner J. Prevalence of recurrent vulvovaginal candidiasis in 5 European countries and the United States: results from an internet panel survey. J Low Genit Tract Dis 2013; 17(3):340-5. 58.Geiger AM, Foxman B. Risk factors for vulvovaginal candidiasis: a case-control study among university students. Epidemiology 1996; 7(2): 182-7. 59.Foxman B. The epidemiology of vulvovaginal candidiasis: risk factors. Am J Public Health 1990; 80(3): 329-31. 60.Gautam R, Borgdorff H, Jespers V, Francis SC, Verhelst R, Mwaura M, et al. Correlates of the molecular vaginal microbiota composition of African women. BMC Infectious Diseases 2015; 15:86. 27 Artigo científico Manuscrito apresentado de acordo com as normas para submissão no periódico SexuallyTransmitted Infections (Fator de impacto: 3.346). 28 4. ARTIGO CIENTÍFICO FACTORS ASSOCIATED TO Lactobacillus iners-DOMINATED VAGINAL MICROBIOME J Novak,1 CST Ferreira,1 MG Silva1 C Marconi1,2 ¹São Paulo State University (UNESP), Botucatu Medical School, Department of Pathology, Botucatu-SP, Brazil 2Universidade Federal do Paraná (UFPR), Sector of Biological Sciences, Department of Basic Pathology, Curitiba-PR, Brazil. ABSTRACT Objective: Given the high prevalence of Lactobacillus iners-dominated microbiome and the instability of this type of vaginal microbiota, we aimed to test the association between the L. iners-microbiome with sociodemographic, behavioral and clinical variables in Brazilian women of reproductive age. Participants and Methods: This cross-sectional study included 609 Brazilian women attending clinics in five cities throughout the country for routine Pap- testing. Participants answered a structured questionnaire for assessment of sociodemographic, behavioral and clinical data. For characterization of vaginal microbiota, vaginal samples were evaluated by microscopy using Nugent criteria and sequenced forV3-V4 16S rRNA genes in Miseq platform (Illumina, San Diego, CA) for microbiome analysis. A logistic regression model using stepwise method was used to test association between L. iners-dominated microbiome with all assessed variables, considering their odds ratios (OR) and 95% confidence intervals (95% CI). Results: The prevalence of microbiome dominated by L. iners was 36.5% (n=222), while the remaining participants had their microbiome dominated by L. crispatus (30.5%, n=186), L. gasseri (4.4%, n=27) and L. jensenii (1.2%, n=7). The 167 (27.4%) participants with Lactobacillus-depleted microbiome were excluded of the analysis. The multivariable analysis showed that participants who reported having two or more sexual partners in the year prior to enrollment had an increased risk of presenting L. iners microbiome (OR: 3.27; CI 95% 1.50-7.11), as well as the microscopic finding of Candida spp. on vaginal smears (OR: 2.24; CI 95% 1.02-4.89). Three variables showed protective against having L. iners- dominated: condom use (OR: 0.59; CI 95%0.38-0.91), high school degree (OR: 29 0.61; CI 95% 0.41-0.91) and daily intake of milk or dairy (OR: 0.43; CI 95%0.20- 0.90). Conclusion: The vaginal microbiome dominated by L. iners is independently associated with several population characteristics, such as low education level, multiple sex partners and unprotected sex. Such characteristics were already associated with Lactobacillus-depleted microbiota. INTRODUCTION The studies concerning the structure of vaginal microbiome into community- state types (CST) were initiated by Ravel et al. (2011) and allowed to determine the exact composition of vaginal microbiota. It has been widely accepted that vaginal microbiome may be classified into five CSTs, according to the dominance and diversity of bacterial species found in this niche. Four out of the five CSTs are dominated by Lactobacillus spp.: L. crispatus (CST I), L. gasseri (CST II), L. iners (CST III) and L. jensenii (CST V). Women with the remaining CST (IV) have partial or total depletion of Lactobacillus, accompanied by an increased microbial beta- diversity. In this latter community, most of bacterial vaginosis (BV) cases stand [1]. This condition is the main type of abnormal vaginal microbiota that affects around 30% of reproductive aged-women, which main complains are genital malodor and increased vaginal discharge [2]. It is widely recognized that BV increases the risk for prevalence and incidence of sexually transmitted infections (STI) including, but not limited to Chlamydia trachomatis, human papillomavirus (HPV) and human immunodeficiency virus (HIV) [3-5]. Several characteristics of the population were already associated with microscopically-detected BV, such as ethnicity, sexual behavior, use of hormonal contraceptives, among others [2; 6-7]. The recent studies on vaginal microbiome have confirmed such associations with Lactobacillus-depleted microbiome [7]. Despite most of microscopic BV cases are placed into CST IV, many of them are categorized as CST III [8-11]. In addition, longitudinal studies on vaginal microbiome have shown that L. iners-dominated CST III frequently shifts to CST IV and vice-versa, suggesting the instability of this type of microbiome [12-13]. 30 The first description of L. iners dates to 1999 [14], thus despite the studies conducted since still much remains to be established regarding its role for vaginal microbiota. Recent studies have suggested that L. iners may offer diminished protection against important pathogens as C. trachomatis, HPV and HIV [13; 15- 16]. This is particularly troublesome given the high prevalence of L. iners- dominated microbiome, which is found in more than one-third of women in reproductive age [1; 17-19] and is even higher in pregnant women (reaching up to 51.8% prevalence) [20-21]. Considering the high prevalence of women with a L. iners-dominated microbiome and the fact that it was already shown to be rather unstable and associated with STI, our aim was to identify sociodemographic, behavioral and clinical characteristics of the population that is associated with this type of microbiome. METHODS Study design and population Recently, our group characterized the vaginal microbiome of Brazilian woman in reproductive age [22]. Briefly, we cross-sectionally included 609 Brazilian women belonging to the five geographic regions of country (South = 109; Southeast = 140; Midwest = 119; North = 133; Northeast = 108). Women enrolled were aged between 18 and 50 years-old, were not pregnant, and did not report use of intrauterine device, infection or urinary loss, less than five days after the end of the menstrual period, less than 72 hours of last sexual intercourse use of local or systemic antibiotics or antifungal drugs within 45 days prior to enrollment. These women were approached when attending primary healthcare clinics for routine screening of cervical cancer (Pap-testing), except for one center (South) where enrollment was taken place at a referral University hospital. The study was reviewed and approved by the Ethics Committee of Botucatu Medical School (Approval number: 3.094.514). After being informed of the aims and procedures of the study, all participating women signed an informed consent term. 31 Data collection and sampling procedures Upon enrollment, women answered a questionnaire that included information regarding sociodemographics, behavioral characteristics, and clinical history. Women answered the questionnaire individually during an interview with one training member of the research team. During physical examination, a non-lubricated speculum was used and two sterile swabs were used for sampling the middle third of the vaginal wall. The first swab was stored in Amies liquid transport medium (Copan, Brescia, Italy) at -800C until microbiome analysis. The other swab was smeared onto glass slides for microscopic analysis and after wards used for KOH testing that was reported as positive, inconclusive or negative by the nurse/physician. Vaginal pH was assessed by allowing the contact of a pH strip with vaginal wall (Merck, Darmstadt, Germany) for one minute. Cervical brush samples were taken for Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis testing by PCR according to methods previously described [23-25]. Microbiota analysis and study groups For microbiota assessment and characterization, both microscopic analysis and bacterial DNA sequencing were performed. Using Nugent criteria, vaginal smears were Gram-stained and scored from 0 to 10, according to the semiquantification of bacterial morphotypes observed by microscopy. They were further classified in normal (scores 0-3), intermediate (scores 4-6) and BV (scores >6) [26]. The presence of Candida pseudohyphae/hyphae was also evaluated using the Gram-stained smears, as well as the number of polymorphonuclear inflammatory cells by field at 1000x magnification. Analysis of the bacterial DNA was performed for microbiome characterization. Aimes medium-containing samples were subjected to DNA extraction using MoBio Powersoil Kit (MoBio Lab Inc, Carlsbad, CA) according to manufacturer's instructions. The microbiome analyzes were carried out at the Institute for Genome Sciences of the University of Maryland, Baltimore, MD, using methodology developed by Fadrosh et al. (2014) [27]. In brief, such samples were submitted to sequencing of V3 to V4 region of the bacterial 16S rRNA gene on 32 Illumina MiSeq platform (Illumina Inc.) using barcoded primers. Sequences other than bacterial were trimmed and demultiplexed using QIIME software (version 1.8.0) [28] (for details, please refer to Fadrosh et al. [27]). Taxonomic assignments were performed using UCHIME (v5.1) [29] and the greengenes database of 16S rRNA sequences (Aug, 2013 vers.) [30]. Each participant’s sequences were further clustered Markov Chain model according to similarity in terms of composition and relative abundance of species, in order to obtain the five CSTs. Statistical analysis Descriptive statistical analyses were calculated for sociodemographic, behavioral and clinical variables. These characteristics were compared by CST using Chi-squared and Mann-Whitney tests for categorical and continuous variables, respectively, with P-value<0.05 considered as statistically significant. We performed univariate logistic regression models to assess the association between CST III with population characteristics. For that, crude and age- and region-adjusted odds ratios (OR) were estimated, as well as their corresponding 95% confidence intervals (CI). A multivariable logistic regression analysis was carried out using a forward stepwise model selection process (variables retained at p-value ≤0.15) to identify variables independently associated with CST III. All analyses were performed using Stata/SE, version15.1 (Stata Corp, College Station, TX). RESULTS Of the 609 women recruited, 167 (27.4%) did not have Lactobacillus- dominance (CST IV), and therefore were excluded of the analysis. Thus, this study population consisted of the 442 with Lactobacillus-dominated microbiome. Among them 186 (30.5%) belonged to CST I (L. crispatus), 27 (4.4%) had CST II (L. gasseri), 222 (36.5%) had CST III (L. iners) and 7 (1.2%) had CST V (L. jensenii). The sociodemographic and behavioral data of the 442 women included in the study are displayed in Table 1. We compared these variables between the two study groups: (1) participants that had CST III (n=222) and (2) those with the other Lactobacillus-dominated CST (n=220).The mean age was 34 years at both study 33 groups most of the women self-reported as black or pardo (n=229), not differing between the groups. Having completed the high school, daily intake milk and dairy, and condom use were more frequent in women with dominance of other Lactobacillus spp. than L. iners, while having two or more sexual partners were more frequent in CST III group. The microbiota classified by microscopy and the presence of 5 or more inflammatory cells and Candida morphotypes also differed between the groups (Table 1). Table 2 shown the distribution of normal microbiota, intermediate and bacterial vaginosis according to the CST defined by the microbiome analysis and highlights that the majority of women with abnormal vaginal microbiota (i. e. intermediate or BV) belonged to CST III group. The prevalence of C. trachomatis, N. gonorrhoeae and T. vaginalis among the groups Lactobacillus-dominated was 4.1% (n=18), 0.4% (n=2) and 0.2 (n=1), respectively. Odds ratios (OR) for the association between sociodemographic, behavioral and clinical factors and the vaginal microbiome dominated by L. iners (CST III) are shown in Table 2. Crude analysis showed that education level, milk and dairy intake, number of sexual partners and condom use were significantly associated with CST III. When adjusting for age and region, all previous variables remained associated with CST III. Finally, the multivariable analysis showed having a CST III-microbiome was independently associated with reporting two or more sexual partners in the previous year and the detection of Candida morphotypes on vaginal smears. On the other hand, some sociodemographics and behavioral characteristics such as having at least the high school degree, milk and derivates consumption and regular condom use were protective against L. iners-dominated microbiome. DISCUSSION Literature findings on the vaginal colonization by L. iners have demonstrated that this species can be found in the microbiota of healthy women and also in those with BV, even in the presence of BV-related symptoms [1, 8-11]. In fact, studies have demonstrated that L. iners colonization persists during the temporal shifts of the normal vaginal microbiota to BV [12, 31]. As well as in BV, L. iners-dominance in the vaginal microbiome may have serious consequences for 34 women’s health, such as increased susceptibility to STI [13, 15-16]. Thus, identifying the population characteristic associated with this condition is of particular importance and, up to our knowledge, this was the first study carried out with this goal. For that, we limited our study population in women regularly menstruating in order to avoid possible bias due to hormonal influences. Research on microbiome has pointed out that the influences environment has influence on the microbial composition [32]. Factors seen in poverty, diet and social conditions may contribute to a less healthy vaginal environment, such as STI [33]. For the first time, we demonstrate that education level, one of mainly social indicators, was a factor that may influence the composition of the microbiome. In this way, our group of women who had completed high school presented lower odds of having vaginal microbiome dominated by L. iners. When looking at our data regarding the sexual behavioral characteristics, we found a significant association of microbiome dominated by L. iners with sexual habits previously associated with BV [34]. Condom use had a protective factor against CST III, while having two or more sexual partner per year increased the risk of having this type of microbiota. Literature is consistent in demonstrating that women with two or more sexual partners per year have higher chances to have BV and there is a strong inverse association between bacterial vaginosis and condom use [34]. However, up to now little is known about how a woman’s sexual and habits may influence the microbiome, or vice versa. Similarly to our results, Vodstrcil et al. (2017) also showed that young women who had unprotected sex were more likely to have vaginal microbiome dominated by L. iners or the BV- associated Gardnerella vaginalis [35]. Corroborating with this findings, other study demonstrated that the consistent condom use increases the number of L. crispatus colonizing the vaginal environment [36]. When adjusted by age and region, BV history was a risk factor for having CST III, but the multivariate analysis did not show this association. As it has already been shown that in the vaginal microbiome dynamics, CST IV frequently shift to CST dominated by L. iners, the same situation occurs inversely [13], and beyond this transition, we now demonstrate that these communities share the same associations related to sexual behavior. 35 In relation to dietary behavioral characteristics studied, we found inverse association between the milk and dairy intake with L. iners-dominated microbiome. To our knowledge, this is the first time that association is described in studies on vaginal microbiome. Some studies have already shown the interaction between some vitamins and minerals, which are found in milk, and BV. Bodnar et al. [37] shown that maternal vitamin D deficiencies associated with BV in the first trimester of pregnancy, although a longitudinal study have no found this association [38]. Neggers et al. [39] demonstrated inverse association between bacterial vaginosis and an increased intake of vitamin E and calcium, both milk components. Prior study with the microbiome gut, which has been more studied as the relation between bacterial composition and host demonstrated that diet may be common determinants of microbial community composition [40]. This away, studies about vaginal microbiome also demonstrated this association, although the direct relationship between diet and cervicovaginal microbial composition is not as clear as in the gut, thus one study showed the influence of diet as well as probiotics in the treatment of BV, in this sense, Laue et al. [41] indicate that the administration of a yoghurt containing probiotics had better effects on the treatment of BV. Another study showed that Saccharomyces cerevisiae-based probiotic had antagonistic effect against G. vaginalis colonization in vaginal environment, such effect was associated with the fact that it inhibited sialidase, an important virulence factor of G. vaginalis [42]. Among the clinical characteristics we found significant difference between the CSTs when the number of polymorphonuclear cells per field was evaluated, being the microbiome dominated by L. iners statistically associated with increased number of cells. The increase of polymorphonuclear cells is seen in aerobic vaginitis, which was first characterized in 2002 by Donders et al., this condition is accompanied by more inflammatory changes than BV and the presence of mainly aerobic enteric commensals or pathogens [43]. Until now, there are no studies reporting the relationship between L. iners and increased polymorphonuclear cells and even aerobic vaginitis, as all studies have focused in the relation between L. iners and BV. A previous study demonstrated that the rupture or depletion of Lactobacillus was not associated with vulvovaginal candidiasis [44], for the first time, we shown 36 association between vaginal microbiome L. iners-dominated and presence of Candida morphotypes. In fact, a study conducted in three African countries with heterogeneous group of women, including pregnant and not pregnant, has already demonstrated only high prevalence of vaginal candidiasis, detected on wet mount, in the community with a predominance of L. iners, although did not reach statistical significance [45]. Little is known about the interaction between the bacterial species found in the vagina and colonization by Candida species, this way, the association showed may be elucidate in future studies in order to get better results in the treatment of candidiasis, such as alternatives treatment with probiotics, as emerging experimental studies have shown promise [46]. CONCLUSION Lactobacillus iners-dominated microbiome and bacterial vaginosis share the same association with some sociodemographic, behavioral characteristics. Considering that the protective role of L. iners has been questioned, the identification of women with this type of microbiota might be of clinical interest. Further studies are also necessary to address which factors are associated with the transition between L. iners and Lactobacillus depletion. This further knowledge may contribute for identifying strategies of vaginal microbiota stabilization. REFERENCES 1.Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011; 108: 4680–7. 2.Marconi C, Duarte MT, Silva DC, Silva MG. Prevalence of and risk factors for bacterial vaginosis among women of reproductive age attending cervical screening in southeastern Brazil. Int J Gynaecol Obstet 2015; 131(2): 137-41. 3.Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis 2011; 11:10. 4.Gallo MF, Macaluso M, Warner L, Fleenor ME, Hook EW, Brill I, et al. Bacterial vaginosis, gonorrhea, and chlamydial infection among women attending a sexually 37 transmitted disease clinic: a longitudinal analysis of possible causal links. Ann Epidemiol 2012; 22: 213-20. 5.Cu-Uvin S, Hogan JW, Caliendo AM, Harwell J, Mayer KH, Carpenter CC, et al. Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract. Clin Infect Dis 2001; 33: 894–6. 6.Schwebke JR, Richey CM, Weiss HL. Correlation of behaviors with microbiological changes in vaginal flora. J Infect Dis 1999; 180: 1632-6. 7.Lewis FMT, Bernstein KT, Aral SO. Vaginal Microbiome and Its Relationship to Behavior, Sexual Health, and Sexually Transmitted Diseases. Obstet Gynecol 2017; 129: 643–54. 8.Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, et al. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 2004; 4: 16. 9.Fredricks DN, Fiedler TL, Marrazzo JM. Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005; 353: 1899-911. 10.Mitchell C, Fredricks D, Agnew K, Hitti J. Hydrogen peroxide-producing lactobacilli are associated with lower levels of vaginal interleukin-1beta, independent of bacterial vaginosis. Sex Transm Dis 2015; 42: 358-63. 11.Santiago GL, Cools P, Verstraelen H, Trog M, Missine G, Aila NE, et al. Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles. PLoS One 2011; 6: 28180. 12.Gajer P, Brotman RM, Bay G, Sakamoto J, Schutte UME, Zhong X, et al. Temporal dynamics of the human vaginal microbiota. Sci Transl Med 2012; 4: 132- 52. 13.Brotman RM, Shardell MD, Gajer P, Tracy JK, Zenilman JM, Ravel J, et al. Interplay between the temporal dynamics of the vaginal microbiota and human papillomavirus detection. J Infect Dis 2014; 210: 1723–33. 14.Falsen E, Pascual C, Sjodén B, Ohlen M, Collins MD. Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp.Int J Syst Bacteriol 1999; 49: 217-21. 15.Hummelen R, Fernandes AD, Macklaim JM, Dickson RJ, Changalucha J, Gloor GB, et al. Deep sequencing of the vaginal microbiota of women with HIV. PLoS One 2010; 5: 12078. 16.van Houdt R, Ma B, Bruisten SM, Speksnijder AGCL, Ravel J, de Vries HJC, et al. Lactobacillus iners-dominated vaginal microbiota is associated with increased 38 susceptibility to Chlamydia trachomatis infection in Dutch women: a case-control study. Sex Transm Infect 2018; 94: 117–123. 17.Mitra A, MacIntyre D A, Lee YS, Smith A, Marchesi JR, Lehne B, et al. Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity. Sci Rep. 2015; 17: 5:16865. 18.Tamarelle J, de Barbeyrac B, Le Hen I, Thiébaut A, Bébéar C, Ravel J, et al. Vaginal microbiota composition and association with prevalent Chlamydia trachomatis infection: a cross-sectional study of young women attending a STI clinic in France. Sex Transm Infect 2018; 0:1–3. 19.Balle C, Lennard K, Dabee S, Barnabas SL, Jaumdally SZ, Gasper MA, et al.Endocervical and vaginal microbiota in South African adolescents with asymptomatic Chlamydia trachomatis infection. Sci Rep 2018; 8:11109. 20.Nasioudis D, Forney LJ, Schneider GM, Gliniewicz K, France MT, Boester A, et al. The composition of the vaginal microbiome in first trimester pregnant women influences the level of autophagy and stress in vaginal epithelial cells. Journal of Reproductive Immunology 2017; 123: 35–39. 21.Romero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Nikita L, et al. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome 2014: 2:4. 22.Marconi C, Ravel J, Gajer P, et al. Characterization of vaginal microbiome in women of reproductive-age from five regions in Brazil. Artigo em fase de submissão. 23.Marconi C, Santos-Greatti MM, Parada CM, Pontes A, Pontes AG, Giraldo PC et al. Cervicovaginal levels of proinflammatory cytokines are increased during chlamydial infection in bacterial vaginosis but not in lactobacilli-dominated flora. J Low Genit Tract Dis 2014; 18(3): 261-5. 24.Ho BS, Feng WG, Wong BK, Egglestone SI. Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples. J. Clin. Pathol. 1992; 45: 439–442. 25.Riley DE, Roberts MC, Takayama T, Krieger JN. Development of a Polymerase Chain Reaction-Based Diagnosis of Trichomonas vaginalis. J of Clin Micr 1992; 30: 465-472. 26.Nugent RP, Krohn MA, Hillier SL. Reability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29: 297-301. 27.Fadrosh DW, Ma B, Gajer P, Ott S, Sengamalay N, Brotman RM, et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq Platform. Microbiome 2014; 24: 2-6. 39 28.Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK,et al. QIIME allows analysis of high-throughput community sequencing data.NIH Public Access 2011; 7: 335–336. 29.Edgar, RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. 2011; 27: 2194–2200. 30.McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J 2011; 6: 610–618. 31.Srinivasan S. et al. Temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. PLoS One 2010; 5: 10197. 32.Pflughoeft KJ, Versalovic J. Human microbiome in health and disease. Annu Rev Pathol 2012; 7: 99-122. 33.Holtgrave DR, Crosby RA. Social capital, poverty, and income inequality as predictors of Gonorrhoea, syphilis, Chlamydia and AIDS case rates in the United States. Sex Transm Infect 2003; 79: 62–4. 34.Fethers KA, Fairley CK, Hocking JS, Gurrin LC, Bradshaw CS. Sexual risk factors and bacterial vaginosis: a systematic review and meta-analysis. Clin Infect Dis 2008; 47: 1426–35. 35.Vodstrcil LA, Twin J, Garland SM, Fairley CK, Hocking JS, Law MG, et al. The influence of sexual activity on the vaginal microbiota and Gardnerella vaginalis clade diversity in young women. PLoS One 2017; 12(2): 0171856. 36.Ma L, Lv Z, Su J, Wang J, Yan D, Wei J, et al. Consistent condom use increases the colonization of Lactobacillus crispatus in the vagina. PLoS One 2013; 8(7): 70716. 37.Bodnar LM, Krohn M, Simhan H. Maternal vitamin D deficiency is associated with bacterial vaginosis in the first trimester of pregnancy. J Nutr 2009; 139: 1157– 61. 38.Klebanoff MA, Turner AN. Bacterial vaginosis and season, a proxy for vitamin D status. Sex Transm Dis 2014; 41: 295–9. 39.Neggers YH, Nansel TR, Andrews WW, Schwebke JR, Yu KF, Goldenberg RL, et al. Dietary intake of selected nutrients affects bacterial vaginosis in women. J Nutr 2007; 137: 2128–33. 40.Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. Human gut microbiome viewed across age and geography. Nature 2012; 486: 222–227. 40 41.Laue C, Papazova E, Liesegang A, Pannenbeckers A, Arendarski P, Linnerth B, et al. Effect of a yoghurt drink containing Lactobacillus strains on bacterial vaginosis in women – a double-blind, randomised, controlled clinical pilot trial. Beneficial Microbes 2018; 9(1): 35–50. 42.Sabbatini S, Monari C, Ballet N, Mosci P, Decherf AC, Pélerin F, et al. Saccharomyces cerevisiae–based probiotic as novel anti-microbial agent for therapy of bacterial vaginosis. Virulence 2018; 9: 954–966. 43.Donders GG, Vereecken A, Bosmans E, Dekeersmaecker A, Salembier G, Spitz B. Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG 2002; 109(1): 34–43. 44.Zhou X, Westman R, Hickey R, Hansmann MA, Kennedy C, Osborn TW, et al. Vaginal microbiota of women with frequent vulvovaginal candidiasis. Infect Immun 2009; 77: 41305. 45.Gautam R, Borgdorff H, Jespers V, Francis SC, Verhelst R, Mwaura M, et al. Correlates of the molecular vaginal microbiota composition of African women. BMC Infectious Diseases 2015; 15:86. 46.Pericolini E, Gabrielli E, Ballet N, Sabbatini S, Roselletti E, Cayzeele Decherf A, et al. Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis. Virulence 2017; 8(1): 74–90. 41 Table 1. Sociodemographic, behavioral and clinical variables grouped into CST I, II and V versus CST III. CST I, II and V (n=220) CST III (n=222) P value* SOCIODEMOGRAPHIC Region South 59 (26.82) 41 (18.47) 0.13 Southeast 45 (20.45) 51 (22.97) Central 36 (16.36) 44 (19.82) Northeast 34 (15.45) 47 (21.17) North 46 (20.91) 39 (17.57) Residence Urban 217 (98.64) 219 (98.65) 0.991 Rural 3 (1.36) 3 (1.35) Age+ (mean) 34 (18-50) 34 (18-51) 0.91 Age++ 18-35 years 115 (52.27) 113 (50.90) 0.773 ≥35 years 105 (47.73) 109 (50.90) Ethnicity Black/pardo 112 (50.91) 117 (52.70) 0.793 White 96 (43.64) 97 (43.70) Other 12 (5.45) 8 (3.60) Living with partner No 75 (34.09) 82 (36.94) 0.532 Yes 145 (65.91) 140 (63.06) Completed high schoola No 74 (33.64) 105 (47.30) 0.003* Yes 146 (66.36) 117 (52.70) Has personal income? No 72 (32.73) 81 (36.49) 0.406 Yes 148 (67.27) 141 (63.51) BEHAVIORAL Milk and dairy intake No 12 (5.45) 26 (11.71) 0.019* Yes 208 (94.55) 196 (88.29) Smoking No 200 (90.91) 198 (89.19) 0.546 Yes 20 (9.09) 24 (10.81) Alcohol consumption No 162 (73.64) 158 (71.17) 0.562 Yes 58 (26.36) 64 (28.83) Intimate soap, daily use No 111 (50.45) 115 (51.80) 0.777 Yes 109 (49.55) 107 (48.20) Douching No 188 (85.45) 194 (87.39) 0.553 Yes 32 (14.55) 28 (12.61) Sitz bathing No 203 (92.27) 202 (90.99) 0.627 Yes 17 (7.73) 20 (9.01) Daily use of pantyliners No 210 (95.45) 215 (96.85) 0.447 Yes 10 (4.55) 7 (3.15) Number of sexual partnersb 0 7 (3.18) 15 (6.76) 0.009* 1 202 (91.82) 182 (81.98) 42 CST I, II and V (n=220) CST III (n=222) P value* 2+ 11 (5.00) 25 (11.26) New sexual partnerc No 204 (92.73) 202 (90.99) 0.505 Yes 16 (7.27) 20 (9.01) Number of sexual intercourses/week 0 31 (14.10) 43 (19.37) 0.301 1-2 112 (50.90) 102 (45.94) 3+ 77 (35.00) 77 (34.69) Hormonal contraceptive No use 120 (54.55) 131 (59.01) 0.181 Oral 84 (38.18) 68 (30.63) Injectable 16 (7.27) 23 (10.36) Condom use No 132 (60.00) 157 (70.72) 0.018* Yes 88 (40.00) 65 (29.28) CLINICAL Body mass index+ Median 25.1 (17.97- 50.64) 24.84 (16.18 - 47.94) Body mass index++d Underweight/normal 110 (50.00) 115 (51.80) 0.923 Overweight 69 (31.36) 68 (30.63) Obese 41 (18.64) 39 (17.57) At least once pregnant No 56 (25.45) 50 (22.52) 0.470 Yes 164 (74.55) 172 (77.48) Phase Cycle Luteal 102 (46.37) 95 (42.79) 0.337 Follicular 93 (42.27) 108 (48.65) Supressed 25 (11.36) 19 (8.56) STD historye No 190 (86.36) 192 (86.49) 0.970 Yes 30 (13.64) 30 (13.51) BV historye No 126 (57.27) 109 (49.10) 0.085 Yes 94 (42.73) 113 (50.90) Dyspaneuria No 144 (65.45) 142 (63.96) 0.743 Yes 76 (34.55) 80 (36.04) Cervicitis No 194 (88.18) 205 (92.34) 0.140 Yes 26 (11.82) 17 (7.66) Pruritus No 189 (85.91) 181 (81.53) 0.213 Yes 31 (14.09) 41 (18.47) Abnormal discharges No 135 (61.36) 125 (56.31) 0.280 Yes 85 (38.64) 97 (43.69) pH <=4.5 134 (60.91) 124 (55.86) 0.281 >4.6 86 (39.09) 98 (44.14) Whiff test 0.179 Negative 166 (75.45) 151 (68.02) Inconclusive 16 (7.27) 25 (11.26) Positive 38 (17.27) 46 (20.72) Nugent cat 43 CST I, II and V (n=220) CST III (n=222) P value* Normal 192 (87.27) 160 (72.07) 0.000* Intermediate 14 (6.36) 35 (15.77) BV 14 (6.36) 27 (12.16) PMNs (5 or more per field) No 211 (95.91) 198 (89.19) 0.007* Yes 9 (4.09) 24 (10.81) Candidiasis No 209 (95.00) 200 (90.09) 0.049* Yes 11 (5.00) 22 (9.91) +Continuous variable ++Categorical variable aGrades completed; b12 months prior to enrollment; c2 months prior to enrollment; dCategorized according to the World Health Organization into underweight/normal (<25.0 unit), overweight (25.0-29.9) and obese (≥30.0); eSelf reported. Table 2: Distribution of vaginal microbiota classified by the Nugent scoring criteria, according to the community state-type (CST) determined by the vaginal microbiome in the 442 participants of the study. Normal (n=352) Intermediate (n=49) Bacterial vaginosis (n=41) CST I 168 (47.7) 8 (16.3) 10 (24.4) CST II 19 (5.4) 5 (10.2) 3 (7.3) CST III 160 (45.5) 35 (71.4) 27 (65.9) CST V 5 (1.4) 1 (2.1) 1(2.4) 44 Table 3. Odds ratios and 95% confidence intervals for the association between sociodemographic and behavioral factors and the vaginal microbiome dominated by Lactobacillus iners (CST-III). Crude Age- and region- adjusted Multivariable SOCIODEMOGRAPHIC Region South 1.00 1.00 -- Southeast 1.63 (0.92 - 2.87) 1.62 (0.92 - 2.86) Central 1.75 (0.97 - 3.18) 1.76 (0.97 - 3.19) Northeast 1.98 (1.09 - 3.60) 1.98 (1.09 - 3.60) North 1.22 (0.68 - 2.18) 1.22 (0.68 - 2.18) Age <35 years 1.00 1.00 -- ≥35 years 1.05 (0.72 - 1.53) 1.31 (0.66 - 2.63) Ethnicity Black 1.00 1.00 -- White 0.96 (0.65 - 1.41) 1.09 (0.71 - 1.69) Other 0.63 (0.25 - 1.61) 0.58 (0.22 - 1.50) Living partner No 1.00 1.00 -- Yes 0.88 (0.59 - 1.30) 0.85 (0.56 - 1.29) Education levela ≤11 years 1.00 1.00 1.00 >11 years 0.56 (0.38 - 0.82) 0.58 (0.39 - 0.87) 0.61 (0.41 - 0.91) Has personal income? No 1.00 1.00 -- Yes 0.84 (0.57 - 1.25) 0.87 (0.58 - 1.31) BEHAVIORAL Milk and dairy intake No 1.00 1.00 1.00 Yes 0.43 (0.21 - 0.88) 0.46 (0.22 - 0.97) 0.43 (0.20 -0.90) Smoking No 1.00 1.00 -- Yes 1.21 (0.64 - 2.26) 1.18 (0.62 - 2.24) Alcohol consumption No 1.00 1.00 -- Yes 1.13 (0.74 - 1.71) 1.23 (0.80 - 1.90) Intimate soap daily use No 1.00 1.00 -- Yes 1.08 (0.70 - 1.65) 1.17 (0.75 - 1.82) Douching No 1.00 1.00 -- Yes 0.84 (0.49 - 1.46) 0.89 (0.51 - 1.55) Sitz bathing No 1.00 1.00 -- Yes 1.18 (0.60 - 2.32) 1.32 (0.62 - 2.80) Daily pantyliner use No 1.00 1.00 -- Yes 0.68 (0.25 - 1.82) 0.92 (0.32 - 2.62) Number of sexual partnersb 0 2.37 (0.94 - 5.96) 2.51 (0.98 - 6.39) 2.34 (0.90 - 6.07) 1 1.00 1.00 1.00 2+ 2.52 (1.20 - 5.27) 2.58 (1.21 - 5.49) 3.27 (1.50 - 7.11) New sexual partnerc No 1.00 1.00 -- Yes 1.26 (0.63 - 2.50) 1.42 (0.69 - 2.91) 45 Crude Age- and region- adjusted Multivariable Number of sexual intercourses/week 0 1.52 (0.89 - 2.59) 1.65 (0.95 - 2.85) -- 1-2 1.00 1.00 3 + 1.09 (0.72 - 1.66) 1.04 (0.68 - 1.59) Condom use No 1.00 1.00 1.00 Yes 0.62 (0.41 - 0.92) 0.59 (0.39 - 0.90) 0.59 (0.38 - 0.91) Hormonal contraceptive No use 1.00 1.00 -- Oral 0.74 (0.49 - 1.11) 0.74 (0.47 - 1.15) Injectable 1.31 (0.66 - 2.61) 1.20 (0.59 - 2.44) CLINICAL Phase cycle Luteal 1.24 (0.84 - 1.84) 1.31 (0.88 - 1.96) -- Follicular 0.81 (0.42 - 1.57) 0.84 (0.42 - 1.64) STD history 0.98 (0.57 - 1.70) 1.02 (0.59 - 1.78) BV history 1.38 (0.95 - 2.02) 1.62 (1.08 - 2.43) 1.45 (0.98 - 2.15) Candidiasis 2.09 (0.98 - 4.42) 1.98 (0.92 - 4.25) 2.24 (1.02 - 4.89) Body mass indexd Underweight/normal 1.00 1.00 -- Overweight 0.94 (0.61 - 1.44) 0.91 (0.59 - 1.42) Obese 0.90 (0.54 - 1.51) 0.82 (0.48 - 1.41) aGrades completed; b12 months prior to enrollment; c2 months prior to enrollment; dCategorized according to the World Health Organization into underweight/normal (<25.0 unit), overweight (25.0-29.9) and obese (≥30.0). 46 5. ANEXOS 1. Parecer do Comitê de Ética em Pesquisa (CEP) 47 48 1. Resumo 2. abstract 2. REVISÃO DE LITERATURA 3. Referências bibliográficas 4. Artigo Científico Factors associated to Lactobacillus iners-dominated vaginal microbiome Abstract Methods results Discussion Conclusion References 5. ANEXOS