Dissertations / Theses on the topic 'Genital tract infections'

Thesis (MScMedSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The basic semen analysis plays a pivotal role in the diagnosis of male infertility and makes a significant contribution to the diagnostic process in andrology, gynecology and clinical urology. In 1902, the man considered to be ―the founding father of modern andrology‖ Edward Martin, proposed that an analysis of a semen sample should be incorporated into all infertility assessments. Following this suggestion in 1956, the scientist John MacLeod advanced the basic semen analysis from beyond a mere observation and introduced the importance of certain semen parameters such as morphology, motility and viscosity. The present day examination includes the analysis of certain established semen parameters, which can provide key information about the quality of a patient‘s semen and the functional competence of the spermatozoa. A semen analysis is also a valuable diagnostic tool in assessing possible disorders of the male genital tract and the secretory pattern of the male accessory sex glands. This information can help to determine the reproductive capacity of the male and can be used in conjunction with the partner to indicate the impact of male genital pathophysiology in the assessment of a couple‘s prospect for fertility. Patients attending the andrology laboratory at Tygerberg Academic Hospital for a semen analysis are referred based on primary, secondary or idiopathic infertility. Amongst these patients, an increase in semen viscosity has been observed over a period of time and created the need to assess the possible causes behind this trend. Despite viscosity being included in a routine spermiogram, it raises a considerable amount of concern as it is assessed semi-quantitatively. In the first part of this study, the possible correlation between seminal hyperviscosity and leukocytospermia was assessed. To achieve the most comprehensive assessment of viscosity, a new approach was used, which is a highly quantitative method to record viscosity in the international unit, centipoise (cP). The analysis of semen samples for possible leukocytospermia was approached by three methods the first of which was cytological. During this method granulocyte grading was performed on stained semen smears during the normal determination of morphology. The same approach was taken for the second method, whereby white blood cell concentrations were quantified with a leukocyte peroxidase test in the total sample group (n=200). Viscosity was compared between the samples classified as leukocytospermic positive or negative, according to the set reference values of the World Health Organisation (WHO). Correlation analysis between the two variables was also performed. In the biochemical approach of detecting leukocytospermia, an enzyme-linked immunoabsorbant assay (ELISA) was used to quantify the concentration of the extracellular polymorphonuclear (PMN) enzyme released from leukocytes. This test was performed on 124 randomly selected samples. All samples were fractionated before storage in liquid nitrogen, to allow for multiple assessments to be performed on each sample. The PMN elastase concentration was assessed against viscosity to investigate a possible correlation and relationship with the presence of leukocytospermia. All three methods of detecting possible infection showed a significantly positive relationship with increased viscosity in semen samples. The second approach in the study was to assess increased viscosity and leukocytospermia against parameters included in the spermiogram. An evaluation of hyperviscosity and its correlations to the various other semen parameters can allow for a detailed study into the effects that this anomaly may elicit. With the assessment of each of the sperm parameters against the leukocyte count and viscosity (cP), volume, concentration and morphology showed significance. To further the study, the third angle was to investigate a possible correlation between viscosity and the functional status of the male accessory sex glands. The biochemical approach of assessing the secretory patterns of the prostate and seminal vesicles against markers of infection can possibly further the understanding behind hyperviscous semen and leukocytospermia. Citric acid and fructose, secretory products of the prostate and seminal vesicles respectively, showed no significance when assessed against the leukocyte count and viscosity. However, this project was a pilot study and this approach offers an exciting avenue for further research. These research findings may provide a more comprehensive assessment of a man‘s fertility status. Seen in the context of patients attending the andrology laboratory of Tygerberg Academic Hospital, this is greatly needed as the majority of these patients cannot afford advanced assisted reproductive therapies. The introduction of a more accurate method of quantifying viscosity may possibly help to identify, diagnose and treat patients suffering from leukocytospermia in order to ultimately enhance their fertility potential.
AFRIKAANSE OPSOMMING: Die basiese semenanalise speel 'n belangrike rol in die diagnose van manlike infertiliteit en maak dus 'n betekenisvolle bydrae tot die diagnostiese proses in andrologie, ginekologie en kliniese urologie. In 1902 het Edward Martin, wat deur sommige navorsers as die vader van moderne andrologie beskou word, voorgestel dat 'n semenanalise deel moet vorm van alle infertiliteitsondersoeke. In 1956 het die wetenskaplike John MacLeod aanvoorwerk gedoen om die grondslag van 'n basiese semenanalise daar te stel, wat beteken het dat, in plaas van net 'n observasie studie te doen, 'n semenmonster kwantitatief analiseer moes word en dat parameters soos spermmorfologie, motiliteit en viskositeit as deel van die volledige analise gedoen moet word. Die hedendaagse analise sluit, behalwe die basiese semenparameters, ook inligting in oor die funksionele aspekte van spermatozoa. Die semenanalise is dus ook ‗n belangrike diagnostiese hulpmiddel om inligting rakende moontlike abnormaliteite in die manlike genitale traktus en die sekretoriese funksies van die manlike bykomstige geslagskliere te verskaf. Hierdie inligting kan help om 'n moontlike diagnose van die man se fertiliteitspotensiaal te maak. Terselftertyd kan dit ook tesame met die metgesel se reproduktiewe inligting meer lig werp op die impak van die man se genitale patofisiologie op die paartjie se fertilitetspotensiaal. Pasiënte wat die andrologielaboratorium van die Tygerberg Akademiese Hospitaal besoek word verwys op grond van primêre, sekondêre of idopatiese infertiliteit. Gedurende die laaste aantal jare is daar ‗n toename in voorkoms van verhoogde semenviskositeit onder hierdie groep pasiënte waargeneem. Dit het die behoefte laat ontstaan om die moontlike redes hiervoor te ondersoek. Ten spyte van die feit dat viskositeit deel vorm van die roetine semenanalise is dit tog kommerwekkend aangesien dit op 'n semi-kwantitatiewe manier bepaal word. In die eerste deel van hierdie studie is 'n moontlik korrelasie tussen seminale hiperviskositeit en leukositospermie ondersoek. Om die beste moontlike verwantskap te kon bepaal is 'n nuwe en hoogs kwantitatiewe metode gebruik om viskositeit in numeriese waardes volgens internasionale standaarde in centipoise (cP) te meet. Daar is van drie metodes gebruik gemaak om die teenwoordigheid van leukositospermie in 'n semenmonster te ondersoek. Die eerste metode was die sitologiese metode waar die teenwoordigheid van granulosiet op die gekleurde semensmeer tydens die standaard morfologie beoordeling bepaal word. Die tweede was deur middel van 'n leukosietperoksidase toets waarmee daar 'n kwantitatiewe telling gedoen kan word, soos teenwoordig in 'n voorbereide semenmonster. Hierdie twee bepalings is op die totale studiepopulasie van 200 pasiënte gedoen. Die viskositeit van monsters met of sonder die teenwoordigheid van leukositospermie, soos bepaal met die voorafgaande metodes en gebaseer op die WGO riglyne, is met mekaar vergelyk. Korrelasies is ook tussen hierdie twee veranderlikes en verskeie semenparameters van hierdie twee groepe gedoen. Die derde metode was 'n biochemiese ontleding met behulp van 'n ensiemgekoppeldeimmuunsorberende essai (ELISA) vir die bepaling van die ekstrasellulêre konsentrasie van polimorfonukleêre (PMN) elastase ensiem in die seminale plasma. Hierdie toets is op 124 lukraak gekose semenmonsters uitgevoer. Alle monsters is gefraksioneer voor berging in vloeibare stikstof om meervoudige analises van elke monster moontlik te maak. Die PMN elastase konsentrasies is vergelyk met die viskositeit van die semenmonsters vir 'n moontlike korrelasie en verwantskap met die teenwoordigheid van leukositospermie. Die resultate van al drie hierdie metodes, vir die moontlike bepaling van infeksie, het 'n betekenisvolle positiewe verwantskap met die toename in graad van viskositeit in semenmonsters aangetoon. Die tweede benadering van hierdie studie was om die viskositeitsgradering en die kwantitatiewe leukositopermie waardes te vergelyk met die semenparameters wat bepaal is tydens die semenanalise. Die doel van hierdie benadering was om enige verwantskap of effek van viskositeit asook die teenwoordigheid van witbloedselle op die semenparameters te ondersoek. Daar is betekenisvolle verwantskappe gevind tussen die viskositeitstatus van 'n semenmonster, die teenwoordigheid van witbloedselle en die semenparameters, soos motiliteit, morfologie en spermatosoa konsentrasie. Die derde benadering was om 'n ondersoek te doen na die moontlike verwantskap tussen viskositeit en die sekretoriese funksies van die manlike bykomstige geslagskliere, te wete die prostaat en seminale vesikula. Die biochemiese ondersoek na die sekresies van hierdie twee organe, naamlik fruktose en sitroensuur, is gedoen om te bepaal of die teenwoordigheid van infeksies van die manlike traktus, en waargeneem as leukositospermia, ook in verband gebring kan word met die viskositeitstatus van 'n semenmonster. Daar is geen verband gevind tussen die sekresies van hierdie twee kliere en die viskositeit van die semenmonsters nie. Aangesien hierdie deel van die studie net as 'n loodsprojek beskou is, is die biochemiese bepalings slegs op 'n beperkte aantal semenmonsters uitgevoer en kan hierdie tipe ondersoek as 'n moontlike verdere studie onderneem word. Hierdie navorsingsresultate kan lei tot ‗n meer omvattende assessering van mans se fertiliteitstatus. Dit is uiters noodsaaklik in die konteks van omstandighede van die pasiënte wat die andrologielaboratorium van die Tygerberg Akademiese Hospitaal besoek aangesien die meerderheid nie gevorderde in vitro behandeling kan bekostig nie. Die akkurate bepaling van 'n semenmonster se viskositeit kan dus moontlik waarde toevoeg tot die identifisering, diagnose en behandeling van pasiënte met leukositospermie om sodoende hulle fertiliteitspotensiaal te verbeter.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

Infertility is estimated to affect approximately 9-12% of reproductive aged couples worldwide. The causes of infertility can be attributed to several pathological conditions affecting one or both partners. The introduction of “Assisted Reproduction Technologies” (ART) has allowed remarkable scientific and medical advances in the field of assisted reproduction. ART consists of different strategies to overcome some infertility factors, thereby improving reproduction efficacy. Infections of the urogenital tract may contribute to infertility with different sexually transmitted diseases (STDs) being directly or indirectly associated to infertility. Among the pathogens that have been associated to infertility, there is Enterococcus faecalis. This microorganism, previously considered just as a member of the gut microbiota of both animals and humans, is now acknowledged as an important human pathogen responsible for a variety of infections, including infections of the urogenital tract, but also life-threating infections such as sepsis and endocarditis. The presence of acquired resistance to major antibiotic classes, in addition to natural intrinsic resistance, is a relevant issue for the treatment of enterococcal infections. Aminoglycosides in combination with β-lactams is the frontline drug combination therapy for severe enterococcal infections. Of special concern is the acquisition, by horizontal transfer, of genes coding for aminoglycoside modifying enzymes (AMEs) which confer resistance to high levels of aminoglycosides (HLA) and eliminate the synergistic bactericidal activity with β-lactams. Despite genital tract infections can affect human fertility, there are no consensus guidelines available on their management (i.e., microbial screening, antibiotic treatment, therapy outcome) in infertile couples undergoing ART treatment. In the present thesis, an attempt was made to better understand how infections impact on couple fertility. We have also explored the effect of hormone therapy on vaginal microbiota and reproductive outcomes of females undergoing in vitro fertilization (IVF). In addition, we have characterized the antibiotic susceptibility and population structure of a collection of E. faecalis clinical strains isolated from the genital tract of infertile couples to provide clinicians with relevant data to implement management of urogenital infections in infertile couples. Chapter 2 is a general introduction to infertility-associated infections with a special focus on E. faecalis. In the first section, virulence determinants, disease pathogenesis and clinical manifestations of E. faecalis are described. Then, treatment of enterococcal infections with a broad overview on action and resistance mechanisms of major antibiotic classes is provided. Chapter 3 explores the role of urogenital infections on couple fertility starting from a collection of vaginal/endocervical swabs and semen samples from 285 infertile couples. The impact of different bacterial species on the outcome of IVF was examined. The results showed the negative impact of E. faecalis on sperm quality and the association of different bacterial pathogens with reduced levels of vaginal lactobacilli. Interestingly, the presence of E. faecalis together with Ureaplasma urealyticum/Mycoplasma hominis in genital samples of infertile couples was predictive for a negative outcome of IVF. Chapter 4 describes the phenotypic and genotypic features of 41 “infertility-associated E. faecalis” (IAF) strains described in chapter 3. Antibiotic susceptibility of different drugs was carried out (Vitek, MIC and disk diffusion methods) and assessed using EUCAST guidelines. The majority of IAF isolates were susceptible to clinically relevant antibiotics, except for 8 strains that were resistant to HLA and 1 which was also resistant to fluoroquinolones. In order to characterize the IAF isolates, whole genome sequences were used to get insights into the IAF population structure and analyse the genetic bases of antimicrobial resistance. Multi-locus sequence typing (MLST) showed a high diversity of the IAF population. However, a clonal structure of HLA resistant strains was found, as 6 out of 8 resistant IAF isolates belonged to the same clonal complex (CC)/sequence type (ST) CC16/ST480. The work described in chapter 5 evaluates the effect of treatment with exogenous gonadotropins on the vaginal microbiota of 108 infertile women undergoing controlled ovarian stimulation prior to being subjected to IVF. A significant increase of vaginal diamines was observed following hormonal treatment. Analysis of vaginal swabs revealed that a shift occurred after hormone treatment from a Lactobacillus-based microbiota to a microbial population mostly constituted by streptococci, enterococci, enterobacteria, staphylococci and yeasts. A highly significant association between reduced amounts of vaginal lactobacilli and the presence of above mentioned pathogens was found. Finally, IVF outcome was significantly decreased in the patients whose vaginal samples were positive for high levels of diamines and presence of bacterial pathogens, suggesting that a link may exist between vaginal microbiota dysbiosis due to hormonal treatment and IVF failure. Chapter 6 contains a short paragraph with the main conclusions of this Ph.D. thesis.

Chlamydia trachomatis is the most prevalent agent of bacterial sexually transmitted infections in the world. However, a profuse number of cases are unreported, as the infection is often asymptomatic. Sequelae such as pelvic inflammatory disease, an increased risk of cervical cancer, premature birth, and perinatal infections in pregnant women can occur. Inflammation occurs in the body in response to infection or injury. Although inflammation can lead to some unwanted secondary effects, such as pain, it serves to return the body to homeostasis by restoring injured tissues and eliminating pathogens. One recently identified connection between the central nervous system and the immune system that regulates inflammation is the cholinergic anti-inflammatory pathway (CAP). In the CAP, pathogen-associated molecular patterns stimulate the vagus nerve to activate the pathway, which ultimately results in acetylcholine (ACh) release, which down regulates inflammation. We hypothesized that genital chlamydial infection would increase the expression of choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, in the female murine genital tract, therefore down regulating inflammation and promoting chlamydial infection. Transgenic female mice carrying a ChAT-promoter driven GFP reporter gene were vaginally infected with C. muridarum. Mice were sacrificed on days 3, 9, 15, and 21 post infection; cervical, uterine horn, and ovarian tissues were removed and embedded in paraffin. Small sections of each tissue were cut and mounted onto slides. The tissue sections were then stained for the expression of ChAT using immunohistochemical techniques. Finally, tissue sections were viewed under a microscope for positive staining and the data was analyzed. The results indicated that there is a significant increase in the number of cells that express ChAT in genital tract of chlamydia-infected mice versus non-infected mice.

Background. In sub-Saharan Africa, which has the highest prevalence of HIV-1 worldwide, most newHIV-1 infections occur by sexual transmission to women. Recent studies in non-human primates have demonstrated that pro-inflammatory cytokine production in the genital tract is necessary for immune cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The aims of this study were to evaluate the relationships between inflammation in the female genital tract and (i) susceptibility to HIV-1 infection and (ii) subsequent disease progression in women who became infected. Additionally, genital inflammation was investigated as a mechanism for breakthrough HIV-1 infections in women who became infected even though they were using 1% tenofovir (TFV) microbicide. In the systemic compartment, the level of T cell activation and soluble markers of immune activation during HIV-1 infection are associated with disease outcome. Therefore, the relationships between plasma cytokine concentrations during early HIV-1 infection and disease progression were evaluated Methods. The participants of this study included 230 HIV-uninfected women from the CAPRISA 002cohort who were followed longitudinally for HIV-1 infection, 49 women who were enrolled during acuteHIV-1 infection and followed until 12 months post-infection and 166 HIV-uninfected women who were enrolled in the CAPRISA 004 1% TFV microbicide trial (62 of whom later became HIV-1-infected).Cytokine concentrations were measured in cervicovaginal lavage (CVL) and plasma samples from these women using Luminex and ELISA.

In uno studio caso-controllo, sono state selezionate 196 pazienti (di et¨¤ compresa tra 45 e 92 anni) nell¡¯arco di 18 mesi. Sono stati costituiti due gruppi: il gruppo di studio comprendeva 98 pazienti donne, in amenorrea post-menopausale da almeno due anni, affette da prolasso del segmento anteriore, con score H.W.S. ¡Ý2, sintomatiche; il gruppo di controllo era costituito da 98 pazienti, in amenorrea post-menopausale da almeno due anni, affette da patologie ginecologiche che non presentavano difetti del supporto del pavimento pelvico. Dai due gruppi sono state escluse le pazienti con patologie maligne in atto o pregresse. In entrambi i gruppi vi erano donne che presentavano sintomi di UTI (26,5% vs. 21,4%; p=0,40) e che avevano urinocolture positive (14,5% vs. 42,9%; p<0.01). Il patogeno di pi¨´ frequente riscontro nei due gruppi era Escherichia coli (8,4% vs. 31,4%; p<0.01) seguito da Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis, Proteus penneri, Streptococcus agalactiae. Nelle pazienti sottoposte ad intervento chirurgico per riduzione del colpocele anteriore, il 78,4% delle urinocolture era negativo e in quelle positive i patogeni isolati erano Escherichia coli (6,1%), Streptococcus agalactiae (1,5%), Proteus mirabilis (3%), Enterococcus faecalis (1,5%). In 16 pazienti del gruppo di studio il valore del residuo vescicale post-minzionale era ¡Ü 50 cc, in 5> 200cc, 8 avevano valori intermedi. Tra le pazienti operate, 5 avevano un residuo vescicale patologico, e di queste solo 1 aveva urinocoltura positiva ma non mostrava sintomatologia per UTI. In base ai risultati, si pu¨° concludere che il colpocele anteriore di vario grado costituisce uno dei fattori di rischio per UTI e avendo riscontrato anche nel gruppo di controllo un¡¯alta percentuale di donne affette da UTIs ricorrenti, possiamo desumere che la menopausa con tutti i suoi fattori di rischio associati (disordini degenerativi, incontinenza urinaria, ridotto svuotamento vescicale, deficit di estrogeni) predispone alle UTIs. Nei due gruppi non sempre c¡¯era corrispondenza tra segni e sintomi di UTI e i risultati delle urinocolture. Inoltre, non possiamo affermare che il residuo vescicale post-minzionale costituisce un fattore di rischio aggiuntivo per UTI nelle pazienti analizzate.

Introdução: a infecção do trato urinário (ITU) é uma das complicações mais frequentes na gravidez, repercute negativamente sobre os índices de morbidade e mortalidade perinatal. Objetivo: o objetivo desta pesquisa foi verificar a associação entre as práticas de higiene genital e sexual e a ocorrência de ITU na gravidez. Casuística e método: foi realizado um estudo transversal, exploratório e descritivo de base hospitalar. Os dados foram coletados com 220 (N) puérperas que receberam assistência ao parto em um hospital público localizado na Cidade de São Paulo. Um formulário estruturado foi utilizado para coletar os dados com as puérperas que foram introduzidos em um banco de dados do Epi Info e analisados no Programa estatístico Statistical Package for Social Sciences (SPSS) for Windows versão 12.0. O Teste Qui-Quadrado foi feito para verificar a existência de associação entre as variáveis independentes e a ocorrência da ITU na gravidez. Foram consideradas significativas todas as associações, cujos resultados apresentaram p<0,05. Conclusões e considerações finais: Quanto às características sociodemográficas das puérperas, a maior proporção tinha idade entre 20 a 29 anos (51,8%), estudou até o ensino médio (46,4%), era católica (48,7%), tinha filhos (60%) e parceiro fixo (91,8%). Seus parceiros apresentaram características semelhantes. Não foi identificada existência de associação significativa (p<0,05) entre as características sociodemográficas da gestante e seu parceiro, da assistência pré-natal, paridade e tipo de parto, disponibilidade de banheiro, higienização das roupas íntimas, hábito de uso de absorventes higiênicos, práticas de higiene genital das puérperas e parceiros antes e após as eliminações vesicointestinais e no coito, hábitos sexuais e a ocorrência da ITU na gravidez. A ocorrência desta patologia na gravidez foi autorreferida por 33,2% das puérperas. Chamou atenção o fato de algumas puérperas (0,9%) não realizarem higiene genital, após as eliminações intestinais. Informações sistematizadas sobre os hábitos de higiene genital devem ser obtidas para que as demandas individuais sejam identificadas e atendidas. A inexistência de associações significativas entre as variáveis estudadas nesta pesquisa e a ocorrência da ITU na gravidez indicou que outras dimensões da vida de gestante devem ser enfocadas nas futuras pesquisas
Introduction: Urinary tract infection (UTI), which is one of the most frequent complications during pregnancy, negatively affects perinatal morbidity and mortality ratios. Objective: This research aimed to verify the association between genital and sexual hygiene practice and the occurrence of UTI during pregnancy. Cases and method: A cross-sectional, exploratory and descriptive hospital-based study was carried out. Data were collected from 220 (N) puerperal women who received delivery care at a public hospital in São Paulo City, Brazil. A structured form was used for data collection. Data were fed into an Epi Info database and analyzed in Statistical Package for Social Sciences (SPSS) for Windows version 12.0. The Chi-Square Test was performed to check for associations between the independent variables and the occurrence of UTI in pregnancy. All associations with p<0.05 were considered significant. Conclusions and final considerations: As to these womens sociodemographic characteristics, a majority was between 20 and 29 years old (51.8%), finished secondary education (46.4%), was catholic (48.7%), had children (60%) and a fixed partner (91.8%). Their partners presented similar characteristics. No significant association (p<0.05) was identified between the sociodemographic characteristics of the pregnant woman and her partner, prenatal care, parity and delivery type, availability of bathroom, washing of intimate clothing, habit to use sanitary towels, genital hygiene practices of the puerperal women and their partners before and after urinary-intestinal eliminations and after coitus, sexual habits and the occurrence of UTI during pregnancy. The occurrence of this disease during pregnancy was self-referred by 33.2% of the women. It was remarkable that some women (0.9%) did not perform genital hygiene after intestinal eliminations. Systemized information on genital hygiene habits should be obtained with a view to identifying and responding to individual demands. The lack of significant associations between the research variables and the occurrence of UTI during pregnancy indicated that other dimensions of the pregnant womans life should be focused on in future research

Além da deficiência física, a diminuição ou perda da sensibilidade geniturinária é um dos maiores impactos para mulheres com Lesão Medular (LM). Devido à perda da mobilidade funcional e as barreiras arquitetônicas, estas muitas vezes não tem acesso aos cuidados adequados para a saúde ginecológica. Como aproximadamente 80% das lesões da medula espinal acometem indivíduos do sexo masculino, os estudos raramente focam as necessidades e questões referentes às mulheres. Objetivo: Avaliar a prevalência de infecções genitais não virais em mulheres com deficiência física por lesão medular, comparativamente às mulheres saudáveis Método: Estudo de corte transversal, caso controle. Foram estudadas 52 mulheres com LM (grupo estudo) e 57 mulheres saudáveis (grupo controle). Todas responderam a um questionário estruturado e foram submetidas à coleta de conteúdo vaginal para pesquisa de Trichomonas vaginalis e leveduras, bacterioscopia com coloração pelo método Gram, cultura geral (meio ágar sangue), cultura para fungos (meio Sabouraud) e coleta de conteúdo endocervical para pesquisa de Chlamydia trachomatis e Neisseria gonorrhorae (reação em cadeia da polimerase) e cultura para Mycoplasmas sp (meios U9, A7). Resultados: As mulheres com lesão medular, comparativamente ao grupo controle, apresentaram maior frequência de Candida sp no exame micológico direto (p= 0,017); entretanto não foi observada diferença estatisticamente significativa na frequência de isolamento de espécies fúngicas entre os grupos. O grupo estudo apresentou maior frequência de isolamento de Escherichia coli (p= 0,002) e de Corynebacterium sp (p= 0,023) e menor frequência de Lactobacillus sp (p < 0,001) em conteúdo vaginal. Em ambos os grupos não foram encontrados casos positivos para Trichomonas vaginalis. A avaliação do escore de Nugent para diagnóstico de vaginose bacteriana demonstrou maior freqüência de flora intermediária (Nugent 4-7) no grupo estudo (p= 0,039). As pesquisas de Chlamydia trachomatis e Neisseria gonorroheae foram negativas em todas as mulheres. Com relação ao isolamento de Mycoplasmas sp, os resultados foram semelhantes em ambos os grupos. Conclusão: A menor freqüência de isolamento de Lactobacillus sp e a maior freqüência de isolamento de Corynebacterium sp e de Escherichia coli na vagina em mulheres com LM, assim como a elevada frequência de flora intermediária pelo escore de Nugent verificada nas mesmas, fortemente sugerem um desequilíbrio da microbiota vaginal, diferente de uma flora dominada por Lactobacillus sp em tais mulheres. Desde que os Lactobacillus sp são essenciais para a manutenção da flora vaginal e a inibição do crescimento de outras bactérias, sua ausência relativa em mulheres com LM pode influenciar a ocorrência de infecções do trato urogenital. Adicionalmente, a mais elevada frequência de detecção de fungos pela microscopia em mulheres com LM sugere que estas podem albergar uma maior concentração vaginal desses microorganismos do que outras mulheres
Besides their physical disability, decreased or absent genitourinary sensitivity has a huge impact in women with spinal cord injury (SCI). Due to the absence of functional mobility and the architectonic barriers these women frequently do not have access to adequate gynecological care. Since about 80% of spinal cord injuries affect men, studies have rarely focused on the needs of women with SCI. Objective: To evaluate the prevalence of non-viral genital infections in women with SCI compared to mobile women. Methods: Fifty two women with SCI (study group) and 57 mobile women (control group) were evaluated in a case-control study. All answered a structured questionnaire and were submitted to the following microbiological tests: fresh examination of vaginal secretions for Trichomonas vaginalis and yeasts, Gram stain, general culture (agar-blood medium), yeast culture (Sabouraud medium) and endocervical sampling for Chlamydia trachomatis and Neisseria gonorrhorae (polymerase chain reaction) and Mycoplasmas sp. (U9, A7 medium). Results: A higher percentage of women with SCI had Candida sp detected by direct mycological examination than did women in the control group (p= 0.017). However there were no significant differences between the two groups in the frequency of yeast-positive cultures. The study group had a higher isolation frequency from the vagina of Escherichia coli (p= 0.002) and Corynebacterium sp (p= 0.023) and a lower frequency of Lactobacillus sp (p < 0.001). In both groups, there were no cases positive for T. vaginalis, C. trachomatis or N. gonorrhoeae. The evaluation of Nugent score for bacterial vaginosis showed a higher frequency of intermediate flora (Nugent score 4-7) in the study group (p= 0.039). Related to Mycoplasma sp isolation, the results were similar in both groups. Conclusion: The lower frequency of Lactobacillus sp isolation and the higher frequency of Corynebacterium sp and Escherichia coli isolation from the vagina in women with SCI, and the higher frequence of intermediate Nugent score, strongly suggests a disequilibrium of the vaginal microbiota away from a Lactobacillus sp dominated flora in these women. Since lactobacilli are essential for maintaining vaginal health and inhibiting growth of other bacteria, their relative absence in women with SCI may influence the occurrence of urogenital tract infections in these women. The higher frequency of yeast detection by microscopy in women with SCI suggests that these women may harbor a higher vaginal yeast concentration than do other women

Objetivo: este estudo teve como objetivo avaliar as infecções por papilomavírus humano (HPV) e Chlamydia trachomatis (CT) em pacientes com artrite reumatoide (AR) pré e pós-terapia anti-TNF. Método: foram avaliadas 50 pacientes do sexo feminino com AR (preenchiam os critérios do Colégio Americano de Reumatologia) que eram elegíveis para terapia anti-TNF. Cinquenta pacientes foram incluídas no estudo de forma prospectiva. Destas, 45 pacientes foram reavaliadas após 6 meses de terapia anti-TNF. Inicialmente as 50 pacientes com AR foram comparadas com 50 controles saudáveis pareadas por idade. Foram avaliados dados demográficos, avaliação ginecológica (citologia cervical e avaliações histológicas), função sexual, parâmetros de doença e tratamento atual da AR. Os testes para detecções dos DNAs do HPV e CT nas espécimes cervicais foram realizados utilizando a captura híbrida II. Resultados: na avaliação inicial, a mediana da idade das pacientes com AR e controles foi de 49 (18-74) vs. 49 (18-74) anos, p = 1,0. Observou-se uma tendência de menor frequência de infecção por HPV nas pacientes com AR pré anti-TNF em relação aos controles (14% vs. 30%, p = 0,054). Adicionalmente, realizou-se avaliação das pacientes com AR com infecção positiva e negativa por HPV antes da terapia anti-TNF que demonstrou que o primeiro grupo apresentou maior frequência de relações sexuais (100% vs. 48%, p = 0,014), maior número de parceiros sexuais [1 (1-1) vs. 0 (0-1), p = 0,032] e maior frequência de citologia cervical anormal (43% vs. 7%, p = 0,029). A idade atual, a duração da doença, os parâmetros da doença e os tratamentos foram semelhantes em ambos os grupos (p > 0,05). Após 6 meses de terapia anti-TNF, a infecção por HPV permaneceu inalterada em cinco pacientes, enquanto que dois tornaram-se negativos e uma paciente adicional tornou-se positiva (p = 1,0). A infecção por CT foi uniformemente negativa nas pacientes com AR pré e pós-TNF, assim como nas controles. Conclusões: a infecção por HPV observada nas pacientes sexualmente ativas com AR antes da terapia anti-TNF foi leve, sem evidência de infecção por CT. A terapia anti-TNF não aumentou o risco de exacerbação e/ou progressão das infecções por HPV e CT em pacientes com AR
Objective: to evaluate human papillomavirus (HPV) and Chlamydia trachomatis (CT) infections in rheumatoid arthritis (RA) patients pre- and post-TNF blocker. Methods: fifty female RA patients (American College of Rheumatology criteria), who were eligible to anti-TNF therapy, [n = 50 at baseline (BL) and n = 45 after 6 months of treatment (6M)] and 50 agematched healthy controls were prospectively enrolled. They were assessed for demographic data, gynecologic, sexual, cervical cytology and histological evaluations, disease parameters and current treatment. HPV DNA and CT DNA testing in cervical specimens were done using Hybrid Capture II assays. Results: at BL, the median current age of RA patients and controls was 49(18-74) vs. 49(18-74) years, p = 1.0. A trend of lower frequency of HPV infection was observed in AR patients pre anti-TNF compared to controls (14% vs. 30%, p = 0.054). Further evaluation of AR patients with and without HPV infection before anti-TNF therapy showed that the former group had higher frequency of sexual intercourses (100% vs. 48%, p = 0.014), higher median number of sexual partners [1(1-1) vs. 0(0-1), p=0.032] and higher frequency of abnormal cervical cytology (43% vs. 7%, p = 0.029). Current age, disease duration, disease parameters and treatments were alike in both groups (p > 0.05). At 6M after TNF blockage, HPV infection remained unchanged in five patients, whereas two became negative and one additional patient turn out to be positive (p = 1.0). CT infection was uniformly negative in RA patients pre- and post-TNF blockage and in controls. Conclusions: anti-TNF does not seem to increase short-term risk of exacerbation and/or progression of HPV and CT infections in RA patients

L’emtricitabine est une molécule centrale dans la stratégie de lutte contre l’infection à VIH. En effet, elle est recommandée en première intention pour le traitement de l’infection chez l’adulte, ainsi que pour la prévention de la transmission de l’infection. Bien que l’emtricitabine soit une molécule clé, elle reste jusqu’à présent peu étudiée. Il est donc primordial d’analyser et de caractériser la pharmacocinétique de l’emtricitabine dans les différentes populations susceptibles d’être exposées à cette molécule. Une approche de population a été utilisée pour réaliser les analyses pharmacocinétiques, nous permettant de décrire la pharmacocinétique avec peu de prélèvements par individu (contraintes éthiques, difficulté de recueil des prélèvements…) et nous permettant d’expliquer la variabilité observée. L’étude rapportant la pharmacocinétique de l’emtricitabine chez l’adulte a mis en évidence une modification du profil cinétique selon l’état de la fonction rénale. Le modèle développé nous a permis d’évaluer les recommandations posologiques actuelles. Afin d’optimiser l’efficacité, la simulation de schémas thérapeutiques alternatifs chez les patients avec une insuffisance rénale modérée a été réalisée. Dans le cadre de la prévention de la transmission mère-enfant, l’étude décrivant la pharmacocinétique de l’emtricitabine chez les femmes enceintes a mis en évidence une modification de la pharmacocinétique au cours de la période gestationnelle. Toutefois, il ne semblait pas nécessaire d’adapter la posologie au cours de la grossesse. La dernière étude a permis d’étudier la pharmacocinétique de l’emtricitabine dans le tractus génital masculin. L’étude de la pénétration dans le compartiment génital est d’un intérêt capital pour le traitement mais aussi pour la prévention de la transmission de l’infection à VIH dans le cadre de la prophylaxie pré-exposition. Notre étude a rapporté une distribution importante de l’emtricitabine dans le tractus génital, avec des concentrations séminales supérieures aux concentrations plasmatiques
Emtricitabine is a key antiretroviral drug in the strategy to fight against HIV infection. Indeed, emtricitabine is recommended in first-line treatment for HIV infection in adults, as well as for the prevention of HIV transmission. Although emtricitabine is a key molecule, it remains poorly studied until now. It is therefore essential to analyze and characterize the pharmacokinetics of emtricitabine in the different populations that may be exposed to this drug. A population approach was used to perform pharmacokinetic analyses, allowing us to describe the pharmacokinetics with few samples per individual (ethical constraints, difficulty of samples collection...) and allowing us to explain the observed variability. The study reporting emtricitabine pharmacokinetics in adults highlighted a change in emtricitabine kinetic profile depending on the state of renal function. The developed model allowed us to evaluate the current dosing recommendations. To optimize efficiency, simulations of alternative regimens in patients with moderate renal impairment were performed. As part of the prevention of mother to child transmission, the study describing emtricitabine pharmacokinetics in pregnant women showed a change in pharmacokinetics during the gestational period. However, it did not seem necessary to adjust the dosage during pregnancy. The latest work focused on emtricitabine pharmacokinetics in the male genital tract. Studying the penetration in the genital tract is of major interest for treatment but also for prevention of HIV transmission in the context of pre-exposure prophylaxis. Our study reported a significant distribution of emtricitabine in the genital compartment, with concentrations in seminal plasma higher than concentrations in blood plasma

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

Includes bibliographical references (leaves 85-103).
T helper (Th) 17 cells have recently been implicated in regulating gut mucosal immunity during HIV infection by sustaining gut mucosal barrier integrity, although they do not respond to HIV directly. Depletion of Th17 cells from the gut mucosa during HIV infection has been suggested to contribute to elevated microbial translocation and immune activation. The role of Th17 cells in regulating genital mucosal immunity during HIV infection is less well described. The aims of this study were (1) to compare the frequency and phenotype of Th17 cells in the female genital tract and blood in uninfected compared to HIV-infected women; and (2) to investigate the role of inflammatory/regulatory cytokines and bacterial burden in modulating Th17 cell frequencies in genital secretions and plasma.

Avaliar a associação entre as infecções genito-urinárias, o comprimento do colo uterino e a prematuridade é importante para determinar quais podem ser os fatores preditivos para o parto pré-termo. Foi realizado estudo tipo coorte de conveniência, prospectivo, avaliando 1370 gestantes na cidade de Ribeirão Preto, com idade gestacional entre 20 e 25 semanas. Aplicou-se questionário sócio demográfico com história reprodutiva para a identificação do histórico obstétrico, idade materna, paridade, tabagismo e antecedente de parto pré-termo. Foi realizada ultrassonografia endovaginal para a avaliação do comprimento do colo uterino segundo as diretrizes da Fetal Medicine Foundation (FMF). Foram coletadas amostras de urina e conteúdo vaginal para avaliar a presença de infecção urinária e vaginose bacteriana respectivamente. A associação entre infecções, comprimento do colo uterino e parto pré-termo (PPT) foi realizada mediante teste não paramétrico e o cálculo do Risco Relativo das diferentes variáveis, por meio do ajuste de modelos log-binomiais. Das 1370 mulheres grávidas avaliadas, 132(9,63%) cursaram com parto pré-termo (<37 semanas), sendo que 19 (14,4%) dos partos pré-termo ocorreram em mulheres com colo <= 2,5 cm. O estudo microbiológico determinou que no grupo das mulheres que cursaram com parto pré-termo, 15 apresentaram ITU, 19 apresentaram vaginose bacteriana (VB) e uma apresentou ITU e VB. Avaliando as 75 pacientes com PPT espontâneos, 10 apresentaram ITU e 14, VB. Após a análise destes dados, foi possível concluir que o colo uterino curto entre 20 e 25 semanas de gestação está associado ao PPT e que ITU e VB rastreadas nesta idade não se associaram ao encurtamento do colo nem ao PPT. No entanto a ITU, mesmo assintomática apresentou relação com o PPT espontâneo.
To evaluate the association between the genito-urinary infections, cervical length and preterm birth is important to determine which can be predictive factors for preterm birth. It was conducted cohort study of convenience, prospective, evaluating 1370 pregnant women in the city of Ribeirão Preto, with gestational age between 20 and 25 weeks. Applied socio-demographic questionnaire with reproductive history to identify the obstetric history, maternal age, parity, smoking and preterm birth (PTB) history. Transvaginal ultrasonography was performed for evaluation of cervical length in the guidelines of the Fetal Medicine Foundation (FMF). Urine and vaginal discharge samples were collected to evaluate the presence of urinary tract infection (UTI) and bacterial vaginosis (BV) respectively. The association between infections, cervical length and preterm delivery was performed using non-parametric test and calculate the relative risk of different variables, by adjusting log-binomial model. Of the 1370 evaluated pregnant women, 132 (9.63%) presenting with preterm delivery (<37 weeks), and 19 (14.4%) of preterm deliveries occurred in women with cervix <=2.5 cm. The microbiological study found that the group of women presenting with preterm birth(PTB), 15 had UTI, 19 had BV and one presented UTI and VB. Evaluating 75 patients with spontaneous PTB, 10 had UTI and 14, BV. After the analysis of these data, it was concluded that the short cervix between 20 and 25 weeks of gestation is associated with the PTB and UTI and VB screened at this age not associated to the shortening of the cervix or the PTB. However, the UTI even asymptomatic were related to the spontaneous PTB.

There is a relative paucity of information about the immune system within the female lower genital tract. This study had three aims. Firstly, to investigate the distribution and function of immunocompetent cells within the ectocervix and to establish the components of humoral immunity within the normal female lower genital tract. Secondly, to determine the effects of HIV infection on these parameters. Thirdly, to identify alterations in cellular and humoral immunity in the context of cervical intraepithelial neoplasia, with and without HIV co-infection. Cervical tissue samples were studied using standard immunohistological techniques and cervicovaginal secretions were analysed by radial immunodiffusion, and by the ELISA method. Normal cervical tissue showed high proportions of primed memory CD4+ T-cells (CD45RO+) and cytotoxic CD8+ cells (CD8+TIA-1+). The majority of epithelial macrophages were of the inducer-type (D1+D7-) and the majority of stromal macrophages were mature phagocytes (D1-D7+) with very few suppressive macrophages (D1+D7+). Although the cytokines IL-ip, TNF-a and TGF-Pi were detected in vaginal secretions, only TNF-a appeared cell-associated in cervical tissues. Relatively high concentrations of IgG and IgA occurred in cervicovaginal secretions. This suggests that the normal female genital tract possesses a reactive immune system with a high proportion of primed activated cells. Cervical biopsies from HIV-infected women showed reversal of the CD4+:CD8+ T-cell ratio. Despite greater proportions of activated T-cells and epithelial macrophages, there was no increase in cytolytic potential. There was an increase in suppressive macrophages and a fall in Langerhans' cell numbers. These changes may facilitate the sexual transmission of HIV infection. The emergence of CIN was associated with greater proportions of activated and cytolytic T-cells. The CIN+HIV+ group showed lower epithelial inducer macrophage proportions and higher suppressive cells. The combination of these factors may contribute to the susceptibility of HIV-infected women to develop CIN, as well as to the rapid progression of CIN in this group.

The distinctive feature of the Human Immunodeficiency Virus (HIV) epidemic in the 21st century is the burden it places on women. Scientists believe that the best opportunities for successful interventions to prevent sexual HIV transmission lie in the initial stages of infection at the portal of entry, the genital tract (GT), which offers the greatest host advantages and viral vulnerabilities. However, understanding of the correlates of protection/vulnerability and innate immunity at the portal of entry is poor. First and foremost, there is no agreement about which GT sub-compartment is the primary site of HIV/SIV infection. Second, the epithelium, previously studied solely for its function as a barrier, has hardly been investigated for its role in innate immunity in the context of SIV/HIV infection. MIP-3α, a chemokine secreted by epithelial cells, was previously proposed to have a role in amplifying the early Simian Immunodeficiency Virus (SIV) infection events in the GT of female macaques. Specifically, MIP-3α was shown to be secreted by epithelial cells of the endocervix, accumulating subepithelially within the first 24 hours post exposure, following deposition of an intravaginal inoculum of SIV. Similar studies in humans have not been reported. We hence undertook to study MIP-3α for its role in early HIV infection events in the endocervix of humans. In order to achieve this, we first characterised MIP-3α constitutive secretion patterns in different sub-compartments of the GT before proceeding to determine its induced secretion patterns, stimulating with HIV-1 and various Toll-like receptor ligands. For completeness we determined constitutive and induced secretion patterns of multiple soluble proteins (SPs) and antimicrobial peptides (AMPs) in the endocervices of humans and macaques. The GT being an immunohormonal system, we further studied the influence of endogenous hormonal changes on the stability of MIP-3α and that of other innate immune markers. We quantified MIP-3α with a sandwich Elisa, and SPs and AMPs with the Luminex multiplex bead assay. Our results showed that the GT is a rich source of MIP-3α with its levels being among those of the highest SPs in the GT. Constitutive levels were highest in the endocervical sub-compartment of all the sub-compartments studied. Further, the GT is an inflammatory environment, which would explain the high levels of MIP-3α. The primary driver of MIP-3α levels appears to be inflammation rather than hormonal levels. MIP-3α levels are significantly higher in the GT of humans than in macaques. There was no evidence that MIP-3α levels are elevated on exposure to HIV and SIV in humans and macaques, respectively. We therefore concluded that since the endocervix is unlikely to respond to HIV/SIV by secreting MIP-3α in vivo, contrary to the previous reports, MIP-3α is hence not a key player in amplifying early events in infection. And as such, it should not be a prime target for preventive therapy. Further, the human GT having a pre-existing inflammatory profile may explain the high rates of HIV sexual transmission. Lastly, we concluded that the infection mechanisms described in the macaque model (i.e. the 'outside-in' signaling) are likely not required for human infection.

Philosophiae Doctor - PhD
Heterosexual transmission of HIV-1 via the female genital tract is the leading route of HIV infection in sub-Saharan Africa. Viruses then traffic between the cervical compartment and blood ensuring pervasive infection. Previous studies have however reported the existence of genetically diverse viral populations in various tissue types, each evolving under separate selective pressures within a single individual, though it is still unclear how compartmentalization dynamics change over acute and chronic infection in the absence of ARVs. To better characterize intrahost evolution and the movement of viruses between different anatomical tissue types, statistical and phylogenetic methods were used to reconstruct temporal dynamics between blood plasma and cervico-vaginal lavage (CVL) derived HIV-1 subtype C gp120 envelope sequences. A total of 206 cervical and 253 blood plasma sequences obtained from four treatment naïve women enrolled in the CAPRISA Acute Infection study cohort in South Africa were evaluated for evidence of genotypic and phenotypic differences between viral populations from each tissue type up to 3.6 years post-infection. Evidence for tissue-specific differences in genetic diversity, V-loop length variation, codon-based selection, co-receptor usage, hypermutation, recombination and potential N-linked glycosylation (PNLG) site accumulation were investigated. Of the four participants studied, two anonymously identified as CAP270 and CAP217 showed evidence of infection with a single HIV-1 variant, whereas CAP177 and CAP261 showed evidence of infection by more than one variant. As a result, genetic diversity, PNLGs accumulation and the number of detectable recombination events along the gp120 env region were lowest in the former patients and highest in the latter. Overall, genetic diversity increased over the course of infection in all participants and correlated significantly with viral load measurements from the blood plasma in one of the four participants tested (i.e. CAP177). Employing a structured coalescent model approach, rates of viral migration between anatomical tissue types on time-measured genealogies were also estimated. No persistent evidence for the existence of separate viral populations in the cervix and blood plasma was found in any of the participants and instead, sequences generally clustered together by time point on Bayesian Maximum Clade Credibility (MCC) trees. Clades that were monophyletic by tissue type comprised mostly of low diversity or monotypic sequences from the same time point, consistent with bursts of viral replication. Tissue-specific monophyletic clades also generally contained few sequences and were interspersed among sequences from both tissue-types. Tree and distance-based statistical tests were employed to further evaluate the degree to which cervical and blood plasma viruses clustered together on Bayesian MCC trees using the Slatkin-Maddison (S-M), Simmonds Association index (AI), Monophyletic Clade (MC), Wright’s measure of population subdivision (FST) and Hudson’s Nearest Neighbour (Snn) statistics, in the presence and absence of monotypic and low diversity sequences. Statistical evidence for the presence of tissue-specific population structure disappeared or was greatly reduced after the removal of monotypic and low diversity sequences, except in CAP177 and CAP217, in 3/5 of longitudinal tree and distance-based tests. Analysis of phenotypic differences between viral populations from the blood plasma and cervix revealed inconsistent tissue-specific patterns in genetic diversity, codon-based selection, co-receptor usage, hypermutation, recombination, V-loop length variation and PNLG site accumulation during acute and chronic infection among all participants. There is therefore no evidence to support the existence of distinct viral populations within the blood plasma and cervical compartments longitudinally, however slightly constrained populations may exist within the female genital tract at isolated time points, based on the statistical findings presented in this study.

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

Includes bibliographical references (leaves 124-150).
The female genital tract serves as the major portal of entry for human immunodeficiency virus (HIV). Local immune factors unique to the mucosal micro-environment such as the genital tract cytokine milieu or the activation/differentiation status of T cells may play a significant role in heterosexual transmission of HIV and subsequent pathogenesis. Elucidation of the mechanisms underlying the persistent recruitment, activation and differentiation of mucosal T cells will give crucial insight into potential therapeutic targets to restore effective local immunity.

博士
國立臺灣大學
臨床醫學研究所
84
The prevalence rates of HPV 16 and 18 DNA in 82 cervical carci- noma tissues by Southern blot hybridization (SBH) were 56.1% and 36.6%, respectively. Concurrent infection of HPV 16 and 18 were noted in 19.5% of the patients. The overall prevalence rate of HPV 16 or 18 was 73.2%, which was ten times larger than that of the control group. The overall prevalence rate of HPV 6, 11, 16, 18 and 33 infection in patients of general out-patient-clinic by dot blot hybridization was 15.3%, while that of Pap. smears by in situ hybridization was 14.9%.The prevalence rates of multiple HPV infections by PCR in patient with normal cervix, low-grade SIL, high-grade SIL and cervical carcinomas were 22.2%, 61.8%, 41.5% and 21.3% respectively, while those of a single-type infection were 36.1%, 21.8%, 30.2%, and 61.7% respectively. HPV 16 was the predominant single-type infection (46.8%) in patients with cervi- cal carcinomas. The prevalence of HPV 16 DNA in cervical cancer by nested PCR was significantly larger than that by SBH (90% vs 57.1%, p<0.001). The concordance between SBH and nPCR was 61.4% only and the discrepancy resulted mainly from 25 cases (35.7%) that were positive by nested PCR but negative by SBH. Patients having parametrial invasion, lymph node metastasis or having both integrated/episomal HPV 16 DNA or integrated HPV 16 DNA of rela- tively larger copy number had worse prognosis than those without. Moreover, patients with integrated HPV 16 infection had bigger tumors and more frequent parametrial invasion, while those with relatively larger copy number of HPV 16 infection had lymph node metastasis more frequently. Serum antibodies against recombinant HPV 16,18 and 33 E7-encoded proteins were more frequently seen in patients with cervical cancer than in those having CIN (50% vs 13%,p<0.05). Large scale seroepidemiological study on serum anti- bodies against the recombinant HPV specific proteins will be con- ducted in the future.

Magister Scientiae (Medical Bioscience) - MSc(MBS)
Aim: Leukocytes are the major source of reactive oxygen species (ROS) in the ejaculate and contribute to up to 30% of male infertility. ROS have been associated with markers of apoptosis such as sperm DNA damage, externalization of phosphatidylserine and caspase-3 activation. Therefore, this study aimed at investigating the impact male genital tract infections/inflammations on the induction of apoptosis in spermatozoa.Materials and Methods: Semen samples were obtained from 60 men consulting for fertility problems at the Reproductive Biology Unit, University of Stellenbosch at Tygerberg Academic Hospital, and Vincent Pallotti Hospital (Cape Town, South Africa). To investigate the relationship between male genital tract infection and sperm apoptosis, the following were measured: semen parameters including sperm count, motility and forward progression; oxidative status in the ejaculate by evaluating the concentration of seminal leukocytes, ROS production in the ejaculate,generation of O2-• and H2O2 by spermatozoa, and the activity of reduced glutathione(GSH) in sperm; sperm apoptotic markers by measuring mitochondrial membrane potential (Δψm), caspase-3/7 activation, and DNA fragmentation (TUNEL).Results: The concentration of seminal leukocytes had a significant positive correlation with ROS production in the ejaculate (ρ=0.378; P=0.0064), sperm O2-• production (ρ=0.336; P=0.0098), and caspase-3/7 activation in sperm (ρ=0.527;P<0.0001). Furthermore, at the cutoff value of ≥0.25×106 leukocytes/mL of semen,the concentration of peroxidase-positive cells correlated significantly with sperm GSH activity (ρ=0.718; P=0.008), the percentage of sperm with disrupted Δψm(ρ=0.465; P=0.043), caspase-3/7 activation in sperm (ρ=0.794; P=0.001), and the percentage of sperm with fragmented DNA (ρ=0.563; P=0.017). ROS production in the ejaculate, besides the association with seminal leukocytes, was also correlated with the sperm count (ρ= -0.296; P=0.033), sperm GSH activity (ρ=0.577; P<0.0001),caspase-3/7 activation in sperm (ρ=0.487; P=0.0005), and sperm DNA fragmentation(ρ=0.331 P=0.0171). Caspase-3/7 activation was strongly correlated with oxidative stress in both, the ejaculate and in spermatozoa; although this parameter was not correlated with sperm Δψm and DNA fragmentation. Sperm O2-•, which had a link with seminal leukocyte concentration, was significantly correlated to sperm Δψm(P=0.0098), as was sperm GSH activity (P=0.0055). Sperm DNA fragmentation was positively correlated with ROS in the ejaculate and sperm H2O2-production(P=0.039). Conclusions: Excessive ROS in the ejaculate, mainly a consequence of seminal leukocytes, is not only linked to internal generation of O2-•, but also to sperm DNA fragmentation and the activation of effector caspases. Moreover, even in nonleukocytospermic patients with ≥0.25×106 leukocytes/mL of semen, oxidative stresscan occur which can trigger apoptosis, caspase-3/7 activation, and induce sperm DNA fragmentation. Therefore, it is possible that male genital tract infection, the major cause of leukocyte infiltration in the male reproductive tract, can induce apoptosis, of which the observed sperm DNA fragmentation is a late feature.