O diagnóstico de neurosífilis é freqüentemente dependente dos resultados dos testes serológicos e alterações no líquido cefalorraquidiano, mas a confiabilidade desses resultados em pacientes com infecção pelo HIV-1 tem sido questionada especialmente em pacientes assintomáticos com sífilis latente. O estudo se propõe avaliar a presença de DNA do T. pallidum no LCR de pacientes assintomáticos infectados pelo HIV, com o diagnóstico de sífilis. Amostras de LCR foram coletadas de 12 pacientes infectados pelo HIV atendidos em um terciário localizado no sul do Brasil , durante o período de 2012 a 2013. A presença de DNA do T. pallidum foram analisadas nas amostras de LCR pelo método de PCR “seminested”. Dados demográficos dos pacientes, parâmetros bioquímicos, celularidade e VDRL do LCR e linfócitos T-CD4 também foram analisados. Nas amostras de LCR de cinco dos 12 pacientes (40%) foram detectados o DNA do T. pallidum . Inesperadamente, nestes doentes, os níveis de contagem de células, proteína e glicose no LCR foram normais. Além disso , nenhuma destas cinco amostras de CSF apresentou uma reacção positiva VDRL. Os títulos de VDRL no soro foram semelhantes entre pacientes positivos e negativos para a presença T. pallidum DNA no LCR. A maioria dos pacientes com DNA de T. pallidum detectável apresentaram baixos títulos de VDRL no soro. O VDRL sérico elevado com título de 1:64 foi observada em apenas um paciente. Nossos resultados demostraram que os pacientes assintomáticos infectados pelo HIV com evidência de sífilis latente e LCR normais podem apresentar DNA de T. pallidum detectável no LCR. A detecção do DNA do T. pallidum pelo nosso seminested PCR pode fornecer informações adicionais além da análise convencional do LCR para o diagnóstico de neurossífilis. presença do DNA de T. pallidum no LCR em pacientes infectados pelo HIV com sífilis latente e resultados de LCR normais pode determinar uma mudança terapêutica do uso de penicilana benzatina intramuscular para o de penicilina cristalina intravenosa aquosa para o tratamento da sífilis. / Neurosyphilis diagnosis is frequently dependent upon the results of serological tests and cerebrospinal fluid abnormalities, but the reliability of findings in patients with HIV-1 infection has been questioned, especially asymptomatic patients with latent syphilis, We present the data on the presence of T. pallidum DNA in CSF from asymptomatic HIV-infected patients with the diagnosis of syphilis. CSF and serum samples were collected from 12 HIV-infected patients attending a tertiary care located in southern Brazil, during the period 2012 to 2013. In CSF samples from five of 12 patients (40%), we detected T. pallidum DNA. Unexpectedly, in these patients, CSF cell count, protein and glucose levels were normal. In addition, none of these 5 CSF samples presented a positive VDRL reaction. Serum VDRL titers were similar between patients with positive and negative CSF T. pallidum DNA. Most patients with detectable T. pallidum DNA presented low serum VDRL titers. Serum VDRL titer of 1:64 was observed in one patient. Our results have shown that asymptomatic HIV-infected patients with evidence of latent syphilis and normal CSF might present detectable T. pallidum DNA in the CSF. The detection of T. pallidum DNA by our seminested PCR provide additional information beyond conventional CSF analysis for diagnosis of neurosyphilis. The detection of T. pallidum DNA in the CSF despite normal CSF findings in HIV-infected patients could also provide a different therapeutic approach including the use of intravenous aqueous crystalline penicillin.

This study had two objectives: to achieve suspension cultivation of Sf1Ep cells and to develop procedures for achieving the replication of T. pallidum in those cell cultures. Sf1Ep cells have been the sole cell line used for the in vitro cultivation of T. pallidum. A study was undertaken to determine if other cell lines can support growth of T. pallidum. Rabbit skin fibroblasts (RAB-9), nude mouse ear (NME) cells, and normal rebbit testis fibroblasts (RT) were compared to Sf1Ep cells for their ability to support in vitro multiplication of T. pallidum. RAB-9 cells supported multiplication of treponemes equal to that of Sf1Ep cells. NME and RT cells also supported growth but to a lesser extent than Sf1Ep cells. Utilization of alternative cell lines may lead to improved in vitro growth of T. pallidum including possible serial passage.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-93).

EVALUATION AND DETERMINATION OF THE SENSITIVITY AND SPECIFICITY OF A Treponema pallidum DRIED BLOOD SPOT (DBS) METHOD FOR SEROLOGIC DIAGNOSIS OF SYPHILIS Background: Syphilis is a sexually transmitted infection (STI) caused by Treponema pallidum subspecies pallidum. Syphilis is known as the "great imitator" due to the similarity of clinical signs and symptoms to other infectious diseases. The primary diagnosis of syphilis relies on clinical findings, including the examination of treponemal lesions, and/or serologic tests. Serologic tests are divided into nontreponemal and treponemal tests. Nontreponemal tests are useful for screening, while treponemal tests are used as confirmatory tests. Methods: A total of 200 serum and DBS specimens collected from patients at the Los Angeles Municipal Sexually Transmitted Disease Clinics were tested by the DBS and enzyme immunoassay (EIA) methods. These samples were sent to the Syphilis Diagnostics Laboratory, Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia for testing. Samples were blindly evaluated by the TREP-SPOTTM DBS and the TREP- SURETM EIA methods for the detection of anti-treponemal IgG- and IgM-class antibodies. Results: The sensitivity of the DBS method was 83% (95% CI, 73.89 - 89.50) and specificity was 100% (95% CI, 95.39 - 100)). The positive predictive value and negative predictive values were 100% (95% CI, 94.48 - 100) and 85% (95% CI, 77.43 - 91.0), respectively. The efficiency of the DBS method was 91.5%. The kappa value for the agreement between the DBS method and EIA assay was 0.83 (95% CI, 0.754 - 0.906). The correlation coefficient (r2) between the anti-treponemal antibody assay results obtained from DBS and serum samples was 0.94. Conclusion: DBS is an optimal choice to be used as a screening tool for the detection of anti-treponemal antibodies for the diagnosis of syphilis. The detection of anti-treponemal antibodies (TREP-SPOTTM DBS EIA) compared favorably to the results of serum-base assay (TREP-SURETM EIA), with an overall concordance of 91.5%. Dried blood spots are technically easier to obtain and are suitable blood samples for primary health care centers.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 112-126).

Yes / Background. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism. / This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/.

Made available in DSpace on 2016-08-10T10:54:12Z (GMT). No. of bitstreams: 1 RONALDO PORTELA.pdf: 1248321 bytes, checksum: 32236d6c47873061a20e4e8cd5b4e4b3 (MD5) Previous issue date: 2014-03-28 / Background: Syphilis is an infectious disease that in Brazilian prison systems has optimal conditions to increase the risk of transmission among inmates. Objectives: Determine prevalence and assess risk situations associated with transmission of syphilis among inmates of Prison Complex Aparecida de Goiânia/GO in the period from February to September 2011. Methods: cross-sectional study exploratory of quantitative approach. Study participants were 1.173 inmates that answered a questionnaire containing variables on behaviors risk for T. pallidum infection. The seroprevalence was found using serological tests screening VDRL and ELISA as confirmatory. Data were tabulated and the variables were analyzed using the EPI INFO and SPSS (Statistical Package for Social Sciences).Were calculated rates of seroprevalence by age and educational level, and relative risks (odds ratios) associated with risk behaviors. Results: The seroprevalence of syphilis found in this population was 2.22% (IC95%). Of the study participant s total, 83.4% were aged between 18 and 39 years. This age group contributes with 85% of cases found positive for serological screening. The seroprevalence among those with low education contributed 58.2% of the population seroprevalence. For the variables of risk situations the homosexual relationship had an odds ratio of 3.44 (IC 0,77 - 15,29 and p=0,084), use of tattoo 3.05 (IC 1,14 - 8,14 and p=0,019), condom use 1.84 (IC 0,40 - 8,43 and p=0,356)and use of injecting drugs 3.71 (IC 1,35 - 10,13 and p=0,006).Conclusions: The seroprevalence rate was lower than those found in studies performed in other prisons in the country. Of the variables evaluated in the study the use of tattoos and the use of injectable drugs were statistically significant risk factors associated with seropositivity to syphilis. The combination of risk situations evaluated in this study can contribute to the transmission of syphilis on the Brazilian prisons. / Introdução: A sífilis é uma doença infectocontagiosa que encontra no ambiente prisional brasileiro condições que podem aumentar o risco da sua transmissão entre a população carcerária. Objetivos: Determinar a soroprevalência e avaliar as situações de risco associadas à transmissão da sífilis entre os presidiários do Complexo Prisional de Aparecida de Goiânia/GO no período de fevereiro a setembro de 2011. Metodologia: Estudo de base exploratória transversal com abordagem quantitativa. Um total de 1.173 participantes do estudo responderam a um questionário contendo variáveis sobre comportamentos e situações de risco para infecção pelo T. pallidum. A soroprevalência foi encontrada após a triagem sorológica utilizando o teste VDRL e ELISA como confirmatório. Os dados obtidos foram tabulados e as variáveis foram analisadas através dos programas EPI INFO e SPSS (Statistical Package for Social Sciences). Foram calculadas as taxas de soroprevalência por faixa etária e nível de escolaridade, e os riscos relativos (odds ratio) associados a comportamentos de risco. Resultados: A soroprevalência encontrada para sífilis na população estudada foi de 2,22% (IC95%). Do total de participantes do estudo, 83,4% tinham idade entre 18 e 39 anos. Esta faixa etária contribui com 85% dos casos positivos encontrados na triagem sorológica. A soroprevalência entre àqueles que possuem escolaridade abaixo do nível médio contribuiu com 58,2% da soroprevalência encontrada na população. Para as variáveis de situações de risco a relação homossexual teve uma razão de chances de 3,44 (IC 0,77 - 15,29 e p= 0,084), uso de tatuagem 3,05 (IC 1,14 - 8,14 e p= 0,019), uso de preservativos 1,84 (IC 0,40 - 8,43 e p =0,356) e uso de drogas injetáveis 3,71 (IC 1,35 - 10,13 e p= 0,006). Conclusões: A taxa de soroprevalência encontrada foi menor do que as encontradas em estudos realizados em outros presídios do País. Das variáveis avaliadas no estudo o uso de Tatuagens e o uso de Drogas Injetáveis foram os fatores de risco estatisticamente significantes associados à soropositividade para a sífilis. A combinação das situações de risco avaliadas pode contribuir para a transmissão da sífilis nos presídios brasileiros.

The major goal of this study was to achieve sustained cultivation of virulent T. pallidum in vitro. The putatuive binding of host proteins to the outer membrane (OM) of intact, virulent T. pallidum subsp. pallidum has been investigated. A major breakthrough was the development of a filtration assay, usinglow protein-binding membrane filters, for the measurement of substances bound to or incorporated into th eOM of T. pallidum. This avoided the conventional manipulations which can damage the fragile OM of T. pallidum. Using this filtration assay, studies on the binding of host serum proteins demonstrated that intact treponemes did not bind host proteins as previously reported. It also indicated that previous studies were probably performed with damaged by this research. The studies on the binding of polyclonal and monoclonal antibodies to intact and detergent treated treponemes provided evidence of the low level binding of antibody to intact treponemes which was greatly enhanced but the removal of the outer membrane with 0.1% Triton X. This research research corroborated that of others which suggests that the outer membrane of T. pallidum contains very little protein or surface exposed antigen.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-01T15:54:13Z No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-01T15:54:40Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-01T15:54:40Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Crack users are at high risk for syphilis, due to multiple risk behaviors, such as inconsistent condom use, exchanging sex for money and/or drugs and multiple sexual partners. The aim of this study was to investigate the epidemiology of syphilis in institutionalized crack users in Goiânia, Goiás. Between 2012 and 2013, a cross-sectional study was conducted with 600 users of a reference unit for chemical dependency treatment of Goiás. All participants were interviewed regarding socio-demographic characteristics and risk factors for syphilis. Blood samples were then collected for the detection of anti-Treponema pallidum antibodies using enzyme-linked immunosorbent assay (ELISA). Positive samples were retested by the Venereal Disease Research Laboratory (VDRL). Active syphilis was considered when there was a positive ELISA result and VDRL titers ≥ 1:8. Of the 600 samples tested by ELISA, 13.8% (95% CI: 11.3 to 16.8%) were positive and 4.5% (95% CI: 3.1 to 6.5%) had titles VDRL ≥ 1:8. The prevalence of exposure to Treponema pallidum and active syphilis was three times greater in women than in men (p < .001). In multiple regression analysis, female sex (adjusted prevalence ratio [APR]: 3.73; p < .001), exchange sex for money and/or drugs (APR: 3.22; p = .040) and a history of genital ulcers (APR: 4.12; p = .010) were associated with active syphilis. Furthermore, it was observed that the prevalence of infection decreased by 12% for each year of formal study (APR: 0.88; p = .030). The results of this research show high prevalence of syphilis in the investigated crack users and indicate the need to implement strategies for prevention and control of this infection in these individuals, including health education activities, diagnosis of infection in clinical treatment for chemical dependency and treatment of positive cases. / Usuários de crack apresentam elevado risco para sífilis, devido aos múltiplos comportamentos de risco, como uso inconsistente do preservativo, troca de sexo por dinheiro e/ou drogas e múltiplas parcerias sexuais. O objetivo desse estudo foi investigar a epidemiologia da sífilis em usuários de crack institucionalizados em Goiânia, Goiás. Entre 2012 e 2013, um estudo de corte transversal foi conduzido em 600 usuários de uma unidade de referência em tratamento de dependência química de Goiás. Todos os participantes foram entrevistados sobre características sociodemograficas e comportamentos de risco para sífilis. A seguir, foram coletadas amostras sanguíneas para detecção de anticorpos anti-Treponema pallidum pelo ensaio imunoenzimático (ELISA). As amostras positivas foram retestadas pelo Venereal Disease Research Laboratory/Laboratório de Pesquisa de Doenças Venéreas (VDRL). Considerou-se sífilis ativa positividade no ELISA e títulos de VDRL ≥ 1:8. Das 600 amostras testadas pelo ELISA, 13,8% (IC 95%: 11,3-16,8%) foram positivas e 4,5% (IC 95%: 3,1- 6,5%) apresentaram títulos de VDRL ≥ 1:8. A prevalência de exposição ao Treponema pallidum e de sífilis ativa foi três vezes maior em mulheres do que em homens (p < 0,001). Em análise de regressão múltipla, sexo feminino (Razão de Prevalência Ajustada [RPaj]: 3,73; p < 0,001), troca de sexo por dinheiro e/ou drogas (RPaj: 3,22; p = 0,040) e antecedentes de úlceras genitais (RPaj: 4,12; p = 0,010) foram associados à sífilis ativa. Além disso, observou-se que a prevalência da infecção diminuiu 12% a cada ano de estudo formal (RPaj: 0,88; p = 0,030). Os resultados dessa investigação evidenciam elevada prevalência de sífilis nos usuários de crack investigados e indicam a necessidade de implementação de estratégias de prevenção e controle dessa infecção nesses indivíduos, incluindo ações de educação em saúde, diagnóstico da infecção em clínicas de tratamento para dependência química e tratamento dos casos positivos.

T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.