Chlamydia is the most commonly reported sexually transmitted infection in the US, with an estimated 4 million new cases in 2018 alone. In addition to humans, Chlamydia infects other animals including mice, and mice have become a popular model for the study of Chlamydia infection. Female sex hormones (FSH) estrogen (E2) and progesterone (P4) rise and fall in a cyclic fashion in both humans and mice, and it is well established that these hormones affect the establishment and progression of genital chlamydial infection. Prior studies that used a co-culture model of human endometrial epithelial cells (IK cells) grown on extracellular matrix-coated inserts over human stromal cells (SHT cells) showed that E2 treatment enhanced initial chlamydial infection and production of progeny Chlamydia compared to hormone free (HF), P4 or combination E2’E2/P4 treatment. This led to the hypothesis that the treatment of ovariectomized (OVX) mice with E2 would enhance chlamydial infection compared to mice treated with no hormone, P4, or a combination of E2 and P4. We ordered OVX mice from Jackson Laboratories and surgically implanted silastic capsules that contained E2, P4, E2/P4, or no hormone diluted in sesame oil. A gas chromatography method was developed to test E2 and P4 concentration in mouse serum, ensuring that hormone levels were physiologically relevant. 8 days after the implantation of the capsules, mice were vaginally-inoculated with C. muridarum¸ a chlamydial species that mimics human chlamydial infection in mice. Every 3 days post infection (pi), for 21 days, we vaginally swabbed mice to determine how much C. muridarum each mouse shed and created a graphical representation of chlamydial shedding. A subset of mice were sacrificed on day 10pi so that presence and identity of immune cells could be analyzed by flow cytometry. Surprisingly, E2 alone and E2/P4 treatment completely protected mice from chlamydial infection. HF-treated mice peaked in chlamydial shedding on day 3pi, and P4-treated mice peaked on day 9pi. Flow cytometry data showed that E2-treated mice had a significantly reduced T cell presence in the genital tract. Thus far, our data suggest that FSH affect chlamydial infection in mice differently than in humans. This observation could have important implications for a field that is heavily reliant on murine studies.

archives@tulane.edu / Background: Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection in the United States. The rate among Black Americans is disproportionately higher than that of White Americans. Ct acquisition is influenced by factors at different levels of the social-ecological model. Methods: Data was collected through Check It, a community venue-based screening study for Black men aged 15-24 who have sex with women, in New Orleans, Louisiana between 05/17/2017-03/16/2020. Latent class analysis identified classes (patterns) of behaviors and relationship traits. Regression mixture modeling assessed associations between covariates and distal outcomes within classes. Path analysis examined mediation of neighborhood factors on Ct by sexual behaviors. Results: At the individual level, five classes of risk behaviors—including substance use, condomless sex, and multiple recent partners—were identified among 1872 men; Ct prevalence ranged from 7.3%-13.6%. Age and education beyond high school were significant risk factors for two classes and health insurance was significantly protective against Ct for two classes. Among 2906 partners reported, five relationship classes emerged with Ct prevalence ranging from 10.1%-18.3%. Community members provided diverse descriptions of the classes. Age, education, substance use, multiple partners, health insurance, and time in a detention facility were predictive of class membership. The effects of three neighborhood and institutional level factors (everyday discrimination, neighborhood safety, and time in a detention facility) on Ct prevalence were mediated by behaviors (substance use, multiple partners, and condomless sex). The three neighborhood and institutional factors were highly interrelated. Conclusion: This work describes the unique characteristics of this population and can be utilized in risk assessment and delivering sexual health services to young Black men who have sex with women. Understanding risk factors across levels of the social-ecological model supports the need for policy changes that address unequal environments and opportunities that increase the risk of Ct acquisition. / 1 / Megan Clare Craig-Kuhn

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

The obligate intracellular bacterium Chlamydia trachomatis requires iron in order to complete its developmental cycle. Addition of an iron-chelating drug, Desferal (deferoxamine mesylate), to infected cell culture causes Chlamydia to enter persistence. Here, we explore the ability of a stably-transfected cell line with inducible over-expression of the eukaryotic iron efflux protein ferroportin to starve C. trachomatis serovar E for iron. Ferroportin-induced iron removal is perhaps a more direct method of removing iron from the intracellular compartment versus exposure to an exogenous chemical chelator. Following induction, ferroportin-green fluorescent protein (Fpn-GFP) was detected in the plasma membrane, and cells expressing Fpn-GFP remained viable throughout the timescale required for Chlamydia to complete its developmental cycle. Following Fpn-GFP induction in infected cells, chlamydial infectivity remained unchanged, indicating chlamydiae were not in persistence. Ferritin levels indicate only a small decrease in cellular iron following Fpn-GFP expression relative to cultures exposed to Desferal. These data indicate that expression of Fpn-GFP in chlamydiae-infected cells is not capable of reducing iron below the threshold concentration needed to cause chlamydiae to enter persistence.

Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia is the most frequently reported bacterial sexually transmitted infection in the world. Most urogenital chlamydia infections in men and women are asymptomatic, but these infections can lead to irreparable damage in the reproductive system and other tissues. Apart from the urogenital chlamydial infections, we know that chlamydia infects the gastrointestinal tract (GIT) in humans and can colonize the GIT for extended intervals without eliciting pathology. We are interested in investigating tissue tropism determinants in Chlamydia spp. because these could be targeted to development live-attenuated vaccines. Recently, we generated mutagenized isolates of the mouse pathogen Chlamydia muridarum, a close relative of the human pathogen Chlamydia trachomatis which causes chlamydia. One mutant that we isolated is significantly attenuated in murine gastrointestinal tissues compared to wild type, but retains its pathogenicity in the murine urogenital tract. Using novel genetic techniques, whole-genome sequencing, and complementation using newly developed vector systems we identified a chromosomal factor, tc0600, that we believe mediates the altered tissue tropism phenotype of this mutant in mice. Notably, the Chlamydia trachomatis ortholog of tc0600 has been linked to chlamydial GIT tropism in humans.

Asthma is the most common chronic respiratory disease affecting young children and adults all over the world. An estimated 34.1 million Americans have reported asthma in their lifetime and the disease costs ~US $56 billion dollars to treat each year. Current treatment is based on a paradigm of asthma as a non-infectious atopic condition whose root cause is inflammation. Chronically administered anti-inflammatory medications, primarily inhaled corticosteroids (ICS), ameliorate asthma symptoms in many patients. However, up to 50% of asthmatics, characterized by neutrophil infiltration, IL-17 secretion and increased risk of fatality are refractory to ICS treatment. Chlamydia pneumoniae, a ubiquitous, obligate intracellular pathogen with an innate propensity to persist and cause chronic infections, along with Mycoplasma pneumoniae have been implicated in the development of chronic, refractory asthma. C. pneumoniae infections are common in infants and young children, often coinciding with the development of early onset asthma in the population. These facts lead the Webley lab to evaluate the carriage of Chlamydia in pediatric respiratory disease patients and the work confirmed that respiratory infections caused by Chlamydia is a significant risk factor in asthma development and live Chlamydia was isolated from the lungs of children with chronic asthma. However, the exact mechanism underlying chlamydial involvement in the disease remained unknown and we believed that a better understanding could shed important light on expanded treatment options and mechanisms of this infectious asthma phenotype. The work presented here provides new insight into how (1) early life chlamydial infection can lead to asthma initiation and exacerbation (2) respiratory chlamydial infection induces cellular and chemical immune responses that support asthmatic inflammation (3) other respiratory pathogens (eg. Mycoplasma) can drive similar immunological responses resulting in significant lung pathology.

Chlamydia is a sexually transmitted disease caused by Chlamydia trachomatis, commonly affecting sexually active college-aged adults. Presently, opportunistic testing, self-testing, and information campaigns are methods to screen vulnerable populations and raise awareness about chlamydia. Chlamydia remains underdiagnosed and undertested due to a lack of participation by individuals who may have been exposed to it. Wastewater-based epidemiology is a rising biomonitoring tool that detects the presence of disease- and drug-specific biomarkers in a community's wastewater. In this study, wastewater-based epidemiology was used to detect the presence of C. trachomatis on the University of Central Florida campus. Wastewater samples were collected from two locations on campus from January 2022 to December 2022. The samples were pasteurized and filtered. DNA was extracted from the filters and was subsequently quantified using qPCR. C. trachomatis was detected at both sites of the UCF campus, with peaks corresponding to periods of the academic semester at which students arrived on campus or had fewer academic responsibilities. It was concluded that wastewater-based epidemiology provided a low-cost and non-invasive tool to notify the public of potential chlamydia outbreaks and encourage testing. Exploration in wastewater-based epidemiology should continue in research of C. trachomatis detection.

The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors. / Der Krankheitserreger Chlamydia trachomatis (CT) - ein gramnegatives Bakterium - ist verantwortlich für die häufigste sexuell übertragene Infektionskrankheit weltweit, die CT basierte Chlamydiose. Sie wird von der Weltgesundheitsorganisation zu den vernachlässigten Krankheiten gezählt. CT Infektionen können unter anderem zu Unfruchtbarkeit, Erblindung und reaktiver Arthritis führen. CT agiert als Krankheitserreger mittels seiner Fähigkeit, die Immunantwort des Wirts zu umgehen. Dies umfasst unter anderem die Schwächung und Störung der NF-κB vermittelten Entzündungsantwort und des Mcl1 pro-Apoptoseweges über ihr deubiquitinierendes, deneddylierendes und trans-acetylierendes Enzym ChlaDub1 (Cdu1). Die Expression von Cdu1 ist aber auch mit der Fragmentierung des Golgi-Apparates des Wirtes verknüpft, ein Schlüsselprozess bei Infektionen mit CT. Cdu1 ist daher vermutlich ein attraktives Zielprotein für die Entwicklung von Wirkstoffen, um CT Infektionen zu behandeln. Eine Leitstrukturverbindung zur Entwicklung neuer wirksamer Inhibitoren war bislang jedoch noch nicht bekannt. Sequenzvergleiche und phylogenetische Untersuchungen verorten Cdu1 im CE Clan der Cysteinproteasen. Die Adenovirus-Protease (Adenain) gehört ebenfalls diesem Clan an und besitzt strukturelle Ähnlichkeit mit Cdu1. Unter Ausnutzung der topologischen Ähnlichkeiten der aktiven Zentren von Cdu1 und Adenain wurde ein Target-Hopping Ansatz mit einem klar definierten und fokussierten Satz von bei Novartis entwickelten Adenain-Inhibitoren verfolgt. Die hierbei identifizierten Cyano-Pyrimidine stellen die ersten kovalenten Inhibitoren von Cdu1 dar, die an das aktive Zentrum von Cdu1 binden und es direkt adressieren. Hochauflösend wurden Kristallstrukturen sowohl von Komplexen von Cdu1 mit kovalent gebundenen Cyano-Pyrimidinen als auch mit Cdu1’s natürlichem Substrat Ubiquitin bestimmt. Die Kristallstrukturdaten dieser Doktorarbeit in Kombination mit Enzymassays und kovalenten Docking-Studien liefern wertvolle Hinweise bezüglich der Aktivität des Enzyms, der molekularen Substraterkennung, der Flexibiliät der Proteintasche rund um das aktive Zentrum und potentielle Hotspots für die Wechselwirkung mit Liganden. Ein strukturbasiertes Wirkstoffdesign erlaubte die Optimierung des Cyano-Pyrimidin-basierten Molekülgerüstes, die zu der Entwicklung der HJR108 Verbindung führte. Es ist das erste Molekül seiner Art, das speziell dazu entworfen wurde Cdu1 zu inhibieren. Strukturen potentiell noch wirksamerer und selektiver Cdu1 Inhibitoren werden in dieser Arbeit vorgeschlagen. Diese Dissertationsschrift liefert somit wertvolle Beiträge zum Verständnis der strukturellen Grundlagen der molekularen Erkennung von Ubiquitin durch Cdu1 und Hinweise, die die Entwicklung hoch-spezifischer kovalenter Cdu1 Inhibitoren erlauben sollten.

Bacteria adapt to stress conditions by altering their physiology, behavior, and gene expression in re- sponse to external challenges. They use evolved mechanisms to thrive in environments with various stress factors including temperature changes, nutrient shortages, and toxins. Specific stress-response pathways are activated upon encountering stress, regulating genes to cope and maintain cellular func- tions. This thesis focuses on a bioinformatical analysis of two bacterial pathogens: a) Staphylococcus aureus and b) Chlamydia trachomatis during stress. To understand the role of S. aureus proteins during infection, gene expression data from two strains (NewHG and NCTC 8325) were analyzed, and flux changes were examined during the middle and late exponential growth phases. The aim was to identify metabolic variations between the wild-type (WT) and various knockout mutants, including the Ser/Thr Kinase PknB, its phosphatase Stp, and the double knockout PknB/Stp. Both S. aureus strains were cultured in nutrient-poor medium to simulate infection conditions similar to those in an abscess of infected individuals. Subsequently, a comprehensive meta- bolic model was constructed using time-resolved transcriptome data, which was validated by qRT-PCR. This study highlights the critical role of PknB-mediated phosphorylation on Ser/Thr residues in regulat- ing amino acid catabolism and promoting gluconeogenesis, ensuring the cell's supply of essential com- ponents. Both PknB and Stp play crucial roles in multiple cellular processes, such as peptidoglycan, nucleotide, and aromatic amino acid synthesis, as well as aspartate transaminase catabolism. Deletion of stp signif- icantly impaired pyrimidine synthesis, while functional loss of PknB had a minor impact. In double knock-out experiments, genes responsible for synthesizing peptidoglycan, purines, and aromatic amino acids from glucose showed increased activity, while pyrimidine synthesis from glucose was less active compared to the WT. Furthermore, this thesis extensively explores the regulatory modules associated with the glmR/yvcK regulon and the cdaA-cdaR-glmM-glmS module. The study emphasizes the conservation of the glmR/yvcK regulon and its core genes, namely yvcJ, glmR/yvcK, and whiA, across various bacteria and cellular processes. Notably, the presence of structural and phosphorylation site similarities in glmR sequences among different bacteria suggests a complex interactome involving PknB/Stp and these regulatory modules, potentially leading to broader implica- tions for bacterial adaptation and virulence. The aim of the second part of this thesis was to investigate how Chlamydia trachomatis adapts to stress induced by the host and evades immune defenses by manipulating human cells. The study introduces an innovative in silico model that captures the dynamic regulatory processes of C. trachomatis and its in- teractions with host signaling proteins during infection. The network model gives particular emphasis to chlamydial chaperones like ClpB, ClpC, and Dnak, along with related proteins such as UhpC, PyrG, inclusion membrane proteins, and the host proteins associated with various signaling pathways. The analysis of multiple time points and subsequent statistical data analysis pinpointed significantly up- and down-regulated genes and proteins, which were used as the basis for dynamic modeling and interac- tome analysis. Network models of predicted and validated protein interactions were used to study the time course of pathogen-host interactions, particularly focusing on chlamydial membrane proteins and chaperones. Through transcriptomic data (GSE104166, GSE147538, and GSE165628) analysis and pathway enrichment, the study identifies key host proteins, including PI3K, MAP kinases, MDM2, c- Myc, and hexokinases, that play significant roles in C. trachomatis pathogenesis. The infection substan- tially modulates multiple signaling pathways, including Interferon-α and -γ response, TNF-α signaling via NFκB, and inflammatory responses. Gene set enrichment analysis provides valuable insights into the host's response to C. trachomatis in- fection, showing distinct gene enrichment patterns at different time points. The integration of pathways from multiple transcriptome datasets enhances the specificity of the network model. Additionally, the research underscores the importance of metabolic reprogramming, particularly involving glutamine uti- lization, for chlamydial survival and the transition between the two different morphological forms. Developed bioinformatic pipeline and systems biology model used in this study offer valuable tools for identification of essential genes/proteins for bacterial survival in the stress environment, virulence profiling, and understanding host-pathogen interactions, with potential applications in various research areas beyond the scope of this study. / Bakterien passen sich an Stressbedingungen an, indem sie ihre Physiologie, ihr Verhalten und ihre Genexpression als Reaktion auf externe Herausforderungen verändern. Sie nutzen weiterentwickelte Mechanismen, um in Umgebungen mit verschiedenen Stressfaktoren wie Temperaturschwankungen, Nährstoffmangel und Toxinen zu gedeihen. Beim Auftreten von Stress werden spezifische Stressreaktionswege aktiviert, die Gene regulieren, um mit dem Stress fertig zu werden und die zellulären Funktionen aufrechtzuerhalten. Diese Arbeit konzentriert sich auf eine bioinformatische Analyse zweier bakterieller Krankheitserreger: a) Staphylococcus aureus und b) Chlamydia trachomatis. Um die Rolle von S. aureus-Proteinen während der Infektion zu verstehen, wurden Genexpressionsdaten von zwei Stämmen (NewHG und NCTC 8325) analysiert und Flussänderungen während der mittleren und späten exponentiellen Wachstumsphase untersucht. Ziel war es, metabolische Variationen zwischen dem Wildtyp und verschiedenen Knockout-Mutanten zu identifizieren, nämlich Ser/Thr-Kinase PknB, ihre Phosphatase Stp und der Doppel-Knockout PknB/Stp. Beide S. aureus-Stämme wurden in nährstoffarmem Medium kultiviert, um Infektionsbedingungen zu simulieren, die denen in einem Abszess infizierter Personen ähneln. Anschließend wurde anhand von zeitaufgelösten Transkriptom-Daten ein umfassendes Stoffwechselmodell erstellt, das durch qRT-PCR Messungen validiert wurde. Diese Studie unterstreicht die kritische Rolle der PknB-vermittelten Phosphorylierung an Ser/Thr-Resten bei der Regulierung des Aminosäurekatabolismus und der Förderung der Gluconeogenese, wodurch die Versorgung der Zelle mit essentiellen Komponenten gewährleistet wird. Sowohl PknB als auch Stp spielen eine entscheidende Rolle bei zahlreichen zellulären Prozessen wie der Peptidoglykan-, Nukleotid- und aromatischen Aminosäuresynthese sowie dem Aspartat-Transaminase-Katabolismus. Die Deletion von stp beeinträchtigte die Pyrimidin-Synthese erheblich, während der Funktionsverlust von PknB nur geringe Auswirkungen hatte. In doppelten Knock-out-Experimenten zeigten die Gene, die für die Synthese von Peptidoglykan, Purinen und aromatischen Aminosäuren aus Glukose verantwortlich sind, eine erhöhte Aktivität, während die Pyrimidinsynthese aus Glukose im Vergleich zum Wildtyp weniger aktiv war. Darüber hinaus werden in dieser Arbeit die regulatorischen Module im Zusammenhang mit dem glmR/yvcK-Regulon und dem cdaA-cdaR-glmM-glmS-Modul eingehend untersucht. Die Studie unterstreicht die Konservierung des glmR/yvcK-Regulons und seiner Kerngene, nämlich yvcJ, glmR/yvcK und whiA, über verschiedene Bakterien und zelluläre Prozesse hinweg. Insbesondere das Vorhandensein von Struktur- und Phosphorylierungsstellen in glmR-Sequenzen zwischen verschiedenen Bakterien deutet auf ein komplexes Interaktionssystem hin, an dem PknB/Stp und diese regulatorischen Module beteiligt sind, was möglicherweise zu umfassenderen Auswirkungen auf die bakterielle Anpassung und Virulenz führt. Ziel des zweiten Teils dieser Arbeit war es, zu untersuchen, wie sich C. trachomatis an den vom Wirt ausgelösten Stress anpasst und der Immunabwehr entgeht, indem es menschliche Zellen manipuliert. In der Studie wird ein innovatives in silico Modell vorgestellt, das die dynamischen Regulationsprozesse von C. trachomatis und seine Interaktionen mit Wirtssignalproteinen während der Infektion abbildet. Das Netzwerkmodell legt besonderes Augenmerk auf Chlamydien-Chaperone wie ClpB, ClpC und Dnak sowie auf verwandte Proteine wie UhpC, PyrG, Einschlussmembranproteine und Wirtsproteine, die mit die mit verschiedenen Signalwegen assoziiert sind. Mehrere Zeitpunkte und statistische Datenanalysen ermittelten signifikant hoch- und herunterregulierte Gene und Proteine, die als Grundlage für die dynamische Modellierung und Interaktomanalyse verwendet wurden. Mit Hilfe von Netzwerkmodellen für vorhergesagte und validierte Proteininteraktionen wurde der zeitliche Verlauf von Pathogen-Wirt-Interaktionen untersucht, wobei der Schwerpunkt auf chlamydialen Membranproteinen und Chaperonen lag. Die Analyse von Transkriptom-Daten (GSE104166, GSE147538 und GSE165628) und die Anreicherung von Signalwegen identifizierte wichtige Wirtsproteine, darunter PI3K, MAP-Kinasen, MDM2, c-Myc und Hexokinasen, die eine wichtige Rolle in der Pathogenese von C. trachomatis spielen. Die Infektion moduliert im Wesentlichen mehrere Signalwege, darunter die Interferon-α- und -γ-Antwort, die TNF-α -Signalisierung über NFκB und Entzündungsreaktionen. Die Analyse der Anreicherung von Gensätzen liefert wertvolle Einblicke in die Reaktion des Wirts auf die Infektion mit C. trachomatis und zeigt unterschiedliche Muster der Genanreicherung zu verschiedenen Zeitpunkten. Die Integration von Signalwegen aus mehreren Transkriptom-Datensätzen erhöht die Spezifität des Netzwerkmodells. Darüber hinaus unterstreicht die Forschung die Bedeutung der metabolischen Reprogrammierung, insbesondere der Glutaminverwertung, für das Überleben von Chlamydien und den Übergang zwischen den beiden morphologisch unterschiedlichen Lebensformen. Sowohl die entwickelten bioinformatischen Pipelines als auch die systembiologischen Modelle bieten wertvolle Werkzeuge zur Identifizierung wesentlicher Gene/Proteine für das Überleben von Bakterien in der Stressumgebung, zur Erstellung von Virulenzprofilen und das Verständnis von Wirt-Pathogen-Interaktionen, mit potenziellen Anwendungen in verschiedenen Forschungsbereichen, die über den Rahmen dieser Studie hinausgehen.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:56:09Z No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:57:32Z (GMT) No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T13:57:32Z (GMT). No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-12-18 / Infection with Chlamydia trachomatis (CT) may be associated with persistent carcinogenic HPV types and the development of cervical neoplasia. There are indications that detection of CT serum antibodies rather than DNA is a better measure of cumulative exposure to CT or of exposure occurring several years prior to the development of cervical disease. The objective of this study was to compare the positivity for CT by ELISA and PCR and to correlate with the severity of cervical neoplasia in women with abnormal cervical smear. Between February 2007 and March 2009, 136 women were referred to the colposcopy clinic at the Santa Casa de Misericordia in Goiânia-GO. HPV DNA was detected by the polymerase chain reaction (PCR) and genotyping was performed by reverse line-blot hybridization assay. CT seropositivity was tested by ELISA for the detection of IgG antibodies and the detection of CT was done by PCR to amplify a sequence in the cryptic plasmid generating a fragment of 512 base pairs. The total prevalence of HPV infection was 85.2%. Seropositivity for CT was 26%. Thirty-one women 26.7 were tested positive for CT antibodies and HPV-DNA. Of these 10.3% had diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) or cervicitis, while 16.3% had histological diagnosis of CIN2 worse diagnosis. When employed PCR test positivity was found to be 8.8%. Eleven women 9.48% were tested positive for CT and HPV DNA. Of these 5.1% had diagnosis of cervicitis or CIN1 and 4.3% had a diagnosis of CIN2 or worse diagnosis. The agreement between serology and PCR tests for CT was considered poor (kappa=0.10 IC 95% 0.69-7.9). Taking as reference the cases negatives for HPV and CT, a positivity for HPV and CT seropositivity was significantly associated with a diagnosis of CIN2 or worse diagnosis, for all HPV types (OR=11.9 IC=2.00-91.5 p=0.0009) and types 16 and 18 (OR=7.50 IC=0.91-76.28 p=0.02). Significant association was observed after adjustment for HPV. A Borderline significance was observed considering other HPV types (OR=7.50 IC=0.91-76.28 p=0.02). CT seropositivity was associated with CIN2 worse diagnosis in women infected by HPV, mainly when the types 16 and 18 were involved. This study did not show any association between CT infection detected by PCR and CIN2 or worse diagnosis. These data support the hypothesis that seropositivity for CT compared to PCR positivity in HPV positive women, especially for types 16 and 18, is a better measure of previous exposure, which reflects a higher probability of association with the severity of cervical neoplasia. / A infecção por Chlamydia trachomatis (CT) pode estar associada com a persistência dos tipos de Papilomavírus humano (HPV) oncogênicos e desenvolvimento da neoplasia cervical. Há indicações de que a detecção de CT por sorologia seja uma melhor medida de exposição cumulativa ou da exposição passada quando comparada a detecção pela reação da polimerase em cadeia (PCR). O objetivo deste estudo foi comparar a positividade para CT pelos métodos de ELISA e PCR e relacionar com a gravidade da neoplasia cervical em mulheres com anormalidades citológicas. Entre fevereiro de 2007 e março de 2009, 136 mulheres, foram encaminhadas à Clínica de Colposcopia na Santa Casa de Misericórdia em Goiânia-GO por exame citológico alterado. A detecção de DNA do HPV foi realizada por PCR utilizando os iniciadores PGMY09/PGMY11, e a genotipagem foi realizada por hibridização reversa em pontos. A positividade para CT foi avaliada por ELISA para detecção de anticorpos IgG e por PCR empregando iniciadores cujo alvo é uma região de plasmídeo críptico, gerando um fragmento de aproximadamente 512 pares de bases. A prevalência total da infecção por HPV foi 85,2%. A positividade para CT por sorologia foi de 25%. Trinta e uma amostras 26,7% foram positivas para HPV e CT. Destas 10,3% tinham diagnóstico de neoplasia intraepitelial cervical grau 1 (NIC1) ou cervicite, enquanto 16,3% tinham diagnóstico histológico de NIC 2 ou pior diagnóstico. Quando empregado o teste de PCR a positividade encontrada foi de 8,8%. Onze amostras 9,48% foram positivas para HPV e CT por PCR, sendo que 5,1% das pacientes apresentavam diagnóstico de NIC1 ou cervicite e 4,3% tinham diagnóstico de NIC 2 ou pior diagnóstico. A concordância entre os testes de sorologia e PCR para CT foi considerado ruim (kappa=0,10 IC 95% 069-7.9). Tomando como referência casos negativos para HPV/CT, a positividade para HPV/CT por sorologia foi significantemente associada com diagnóstico de NIC2 ou pior diagnóstico, para todos os tipos de HPV (OR=11.9 IC=2.00-91.5 p=0.0009) e para os tipos 16 e 18 (OR=16.25 IC=2.28-148.57 p=0.0005). Uma associação limítrofe foi observada considerando outros tipos de HPV (OR=7.50 IC=0.91-76.28 p=0.02). Houve associação estatisticamente significante após o ajustamento para infeção por HPV entre as infecções pelos tipos 16 e 18 e soropositividade para CT com a gravidade da neoplasia cervical. Quando empregado o teste de PCR, não houve associação entre a coinfecção HPV/CT e a gravidade da neoplasia cervical. Estes dados reforçam a hipótese de que a soropositividade para CT quando comparada a positividade por PCR em mulheres HPV positivas, especialmente para os tipos 16 e 18, é uma melhor medida de exposição anterior, o que reflete maior probabilidade de associação com a gravidade da neoplasia cervical.