RESUMO
Treponema pallidum subsp. pallidum is a fastidious spirochete and the etiologic agent of syphilis, a sexually transmitted infection (STI). Syphilis diagnoses and disease staging are based on clinical findings and serologic testing. Moreover, according to most international guidelines, PCR analysis of swab samples from genital ulcers is included in the screening algorithm where possible. It has been suggested that PCR might be omitted from the screening algorithm due to low added value. As an alternative to PCR, IgM serology might be used. In this study, we wanted to establish the added value of PCR and IgM serology for diagnosing primary syphilis. Added value was defined as finding more cases of syphilis, preventing overtreatment, or limiting the extent of partner notification to more recent partners. We found that both PCR and IgM immunoblotting could aid the timely diagnosis of early syphilis in ~24% to 27% of patients. PCR has the greatest sensitivity and can be applied to cases with an ulcer with suspected reinfection or primary infection. In the absence of lesions, the IgM immunoblot could be used. However, the IgM immunoblot has better performance in cases with suspected primary infection than in reinfections. The target population, testing algorithm, time pressures, and costs should determine whether either test provides sufficient value to be implemented in clinical practice.
Assuntos
Testes Diagnósticos de Rotina , Imunoglobulina M , Sífilis , Humanos , Immunoblotting/normas , Imunoglobulina M/análise , Reação em Cadeia da Polimerase/normas , Sífilis/diagnóstico , Sífilis/imunologia , Sífilis/microbiologia , Treponema pallidum/genética , Testes Sorológicos/normas , Testes Diagnósticos de Rotina/economia , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Treponema pallidum (Tp) is a widespread and destructive pathogen that leads to syphilis. As the acknowledged executor of host immunity, macrophage plays vital roles in combating the invasion and migration of Tp. However, the mechanisms of these processes are largely unknown, especially the critical driver genes and associated modifications. OBJECTIVE: We aimed to systematically dissect the global N6-methyladenosine (m6A) RNA modification patterns in Tp-infected macrophages. METHODS: The RNA of Tp-infected/non-infected macrophage was extracted, followed by mRNA sequencing and methylated RNA immunoprecipitation (MeRIP) sequencing. Bioinformatics analysis was executed by m6A peaks and motifs identification, Gene ontology and signaling pathways analysis of differentially expressed genes, and comprehensive comparison. The m6A levels were measured by RNA Methylation Assay, and m6A modified genes were determined by qPCR. RESULTS: Totally, 2623 unique and 3509 common m6A peaks were proved along with related transcripts in Tp-infected macrophages. The common m6A-related genes were enriched in the signals of oxidative stress, cell differentiation, and angiogenesis, while unique genes in those of metabolism, inflammation, and infection. And differentially expressed transcripts revealed various biological processes and pathways associated with catabolic and infection. They also experienced comprehensive analysis due to hyper-/hypo-methylation. And the m6A level of macrophage was elevated, along with qPCR validation of specific genes. CONCLUSION: With a particular m6A transcriptome-wide map, our study provides unprecedented insights into the RNA modification of macrophage stimulated by Tp in vitro, which partially differs from other infections and may provide clues to explore the immune process for syphilis.
Assuntos
Sífilis , Treponema pallidum , Humanos , Treponema pallidum/genética , Sífilis/genética , Transcriptoma , Adenosina , MacrófagosRESUMO
OBJECTIVE: To discover novel serodiagnostic candidates for the serological diagnosis of syphilis. METHODS: Two recombinant Treponema pallidum proteins Tp0100 and Tp1016 were expressed, purified, and identified by Western Blotting. A total of 600 clinical serum samples were tested with the Tp0100-based ELISA, the Tp1016-based ELISA, and the commercial LICA Syphilis TP kit (ChIVD, Beijing, China). The sensitivities were determined by testing 340 samples from individuals with clinically diagnosed primary, secondary, latent, and tertiary syphilis. The specificities were determined by screening 260 samples from healthy controls and individuals with potentially cross-reactive infections, including leptospirosis, Lyme disease, hepatitis B, tuberculosis, rheumatoid arthritis, systemic lupus erythematosus. Kappa (κ) values were applied to compare the agreement between clinical syphilis diagnosis and the Tp0100-based ELISA, the Tp1016-based ELISA, or the LICA Syphilis TP test. RESULTS: Using clinical syphilis diagnosis as the gold standard, Tp0100 exhibited an overall sensitivity of 95.6% and specificity of 98.1% for testing IgG antibody while Tp1016 demonstrated only an overall sensitivity of 75.0% and specificity of 79.6%. In contrast, the LICA Syphilis TP test revealed an overall sensitivity of 97.6% and specificity of 96.2%. In addition, the overall percent agreement and corresponding κ values were 96.7% (95% CI 95.6%-97.8%) and 0.93 for the Tp0100-based ELISA, 77.0% (95% CI 74.3%-79.7%) and 0.54 for the Tp1016-based ELISA, and 97.0% (95% CI 96.0%-98.0%) and 0.94 for the LICA Syphilis TP test, respectively. CONCLUSION: The recombinant T. pallidum protein Tp0100 shows promise as a novel diagnostic antigen in the serological tests for syphilis.
Assuntos
Sífilis , Treponema pallidum , Anticorpos Antibacterianos , Antígenos de Bactérias , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Recombinantes , Sensibilidade e Especificidade , Testes Sorológicos , Sífilis/diagnóstico , Sorodiagnóstico da Sífilis , Treponema pallidum/genéticaRESUMO
BACKGROUND: Sexually transmitted diseases (STDs) and in particular genital ulcer disease (GUD) have a major impact on morbidity and mortality in developing countries. The World Health Organization recommends the use of syndromic guidelines for the treatment of sexually transmitted infections (STIs) in resource-constrained countries. Surveillance of autochthonous etiologies provides epidemiological information contributing to the prevention and treatment of STIs. We investigated the etiology and factors associated with GUD among male patients attending a STD clinic in Havana, Cuba. METHODS: Swabs from genital ulcers of 113 male patients, collected from May 2012 to June 2015, were analyzed using PCR for herpes simplex virus types 1 and 2, Treponema pallidum, Haemophilus ducreyi, and Chlamydia trachomatis. We also investigated the clinical and epidemiological characteristics associated with the presence of these pathogens in GUD. RESULTS: At least one of the pathogens was detected in 70% of patients. The occurrence of the pathogens was herpes simplex virus type 2 (HSV-2) (51.3%), T. pallidum (29.2%), and C. trachomatis (1.8%). Co-infections occurred as follows: T. pallidum-HSV-2 (10.6%), C. trachomatis-HSV-2 (0.9%) and C. trachomatis-T. pallidum (0.9%). Herpes simplex virus type 1 and H. ducreyi were not detected. Ages 15 to 40 years, HIV-positive serostatus, and no condom use were significant risk factors for the presence of HSV-2 in genital ulcers. CONCLUSIONS: Our preliminary results highlight the predominance of HSV-2 and T. pallidum as the leading GUD etiologies in the study population and identified risk factors associated with HSV-2. This information should help to inform guidelines for better management of GUD in Havana, Cuba.
Assuntos
Doenças dos Genitais Masculinos/etiologia , Herpesvirus Humano 2/isolamento & purificação , Infecções Sexualmente Transmissíveis/etiologia , Treponema pallidum/isolamento & purificação , Úlcera/etiologia , Adolescente , Adulto , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Coinfecção , Cuba/epidemiologia , Doenças dos Genitais Masculinos/epidemiologia , Doenças dos Genitais Masculinos/virologia , Soropositividade para HIV , Haemophilus ducreyi/genética , Haemophilus ducreyi/isolamento & purificação , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Infecções Sexualmente Transmissíveis/epidemiologia , Infecções Sexualmente Transmissíveis/virologia , Treponema pallidum/genética , Úlcera/epidemiologia , Úlcera/virologia , Adulto JovemRESUMO
Syphilis is a multistage, sexually transmitted disease caused by the spirochete, Treponema pallidum (Tp). A significantly high incidence of syphilis has been reported in several countries, including China, and there is an urgent need for the development of efficacious vaccines against syphilis. DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines. Animal model studies of Tp DNA vaccines have not been reported elsewhere but our previous reports describe the development of a single-gene Tp DNA vaccine and preclinical immunization study. In this study, chitosan (CS) nanoparticles were used as a vector and an interleukin-2 expression plasmid (pIL-2) as an adjuvant to enhance a TpGpd DNA vaccine candidate (pTpGpd) in a rabbit Tp skin challenge model. At week 8 after the first immunization, three rabbits from each group were used to determine cytokine measurements and spleen lymphocyte proliferation assay. pTpGpd in combination with pIL-2 wrapped with CS led to the greatest enhancement of anti-TpGpd antibodies and T-cell proliferation. During infection, levels of anti-TpGpd antibodies and T-cell proliferation were measured. Both the serum special IgG and IL-2, interferon-γ were significantly increased by the co-injection of the IL-2 plasmid compared with the injection of TpGpd DNA alone (P<0.05). Furthermore, IL-2 plasmid coinjection efficiently enhanced the antigen-specific lymphocyte proliferation response. Additionally, the ratios of positive skin lesions and ulcer lesions in groups immunized with pTpGpd were significantly lower than those of the pIL-2, CS or pIL-2 mixed with CS control groups (P<0.001). CS vectored and pIL-2 adjuvanted pTpGpd immunized animals exhibited the lowest rates of positive skin tests (8.33%) and ulcer lesions (4.17%) and the fastest recovery (42 d). These experiments indicate that co-injection of a pIL-2 plasmid with pTpGpd DNA vaccine wrapped with CS can significantly strengthen the long-term stability of immune response during infection, efficiently improve the protective effect against T. pallidum spirochetes infection and attenuate syphilitic lesion development.
Assuntos
Vacinas Bacterianas/administração & dosagem , Treponema pallidum/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Quitosana/administração & dosagem , Modelos Animais de Doenças , Genes Bacterianos , Células HeLa , Humanos , Interferon gama/metabolismo , Interleucina-2/administração & dosagem , Interleucina-2/genética , Interleucina-2/metabolismo , Nanopartículas/administração & dosagem , Coelhos , Sífilis/imunologia , Sífilis/prevenção & controle , Linfócitos T/imunologia , Treponema pallidum/genética , Treponema pallidum/patogenicidade , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Mathematical models are an important tool to explain and comprehend complex phenomena, and unparalleled computational advances enable us to easily explore them without any or little understanding of their global properties. In fact, the likelihood of the data under complex stochastic models is often analytically or numerically intractable in many areas of sciences. This makes it even more important to simultaneously investigate the adequacy of these models-in absolute terms, against the data, rather than relative to the performance of other models-but no such procedure has been formally discussed when the likelihood is intractable. We provide a statistical interpretation to current developments in likelihood-free Bayesian inference that explicitly accounts for discrepancies between the model and the data, termed Approximate Bayesian Computation under model uncertainty (ABCmicro). We augment the likelihood of the data with unknown error terms that correspond to freely chosen checking functions, and provide Monte Carlo strategies for sampling from the associated joint posterior distribution without the need of evaluating the likelihood. We discuss the benefit of incorporating model diagnostics within an ABC framework, and demonstrate how this method diagnoses model mismatch and guides model refinement by contrasting three qualitative models of protein network evolution to the protein interaction datasets of Helicobacter pylori and Treponema pallidum. Our results make a number of model deficiencies explicit, and suggest that the T. pallidum network topology is inconsistent with evolution dominated by link turnover or lateral gene transfer alone.
Assuntos
Algoritmos , Proteínas de Bactérias/genética , Evolução Molecular , Modelos Biológicos , Proteínas de Bactérias/metabolismo , Teorema de Bayes , Biologia Computacional/métodos , Bases de Dados de Proteínas , Transferência Genética Horizontal , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Funções Verossimilhança , Método de Monte Carlo , Mapeamento de Interação de Proteínas , Treponema pallidum/genética , Treponema pallidum/metabolismoRESUMO
OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.
Assuntos
DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sífilis/diagnóstico , Treponema pallidum/genética , Adulto , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Farmacorresistência Bacteriana/genética , Métodos Epidemiológicos , Feminino , Soropositividade para HIV , Humanos , Masculino , Gravidez , Análise de Sequência de RNA , Sífilis/sangue , Sífilis/urina , Sorodiagnóstico da Sífilis , Treponema pallidum/isolamento & purificação , Sexo sem ProteçãoRESUMO
The most probable secondary structure of an RNA molecule, given the nucleotide sequence, can be computed efficiently if a stochastic context-free grammar (SCFG) is used as the prior distribution of the secondary structure. The structures of some RNA molecules contain so-called pseudoknots. Allowing all possible configurations of pseudoknots is not compatible with context-free grammar models and makes the search for an optimal secondary structure NP-complete. We suggest a probabilistic model for RNA secondary structures with pseudoknots and present a Markov-chain Monte-Carlo Method for sampling RNA structures according to their posterior distribution for a given sequence. We favor Bayesian sampling over optimization methods in this context, because it makes the uncertainty of RNA structure predictions assessable. We demonstrate the benefit of our method in examples with tmRNA and also with simulated data. McQFold, an implementation of our method, is freely available from http://www.cs.uni-frankfurt.de/~metzler/McQFold.
Assuntos
Biologia Computacional/métodos , Cadeias de Markov , Modelos Químicos , Conformação de Ácido Nucleico , RNA Mensageiro/química , Método de Monte Carlo , RNA Mensageiro/genética , Software , Treponema pallidum/genéticaRESUMO
The incidence of infectious syphilis in men who have sex with men and human immunodeficiency virus-infected patients has increased steadily in Victoria, Australia, since 2002. A TaqMan real-time PCR assay targeting the polA gene of Treponema pallidum (TpPCR) was developed. The analytical sensitivity of the assay was estimated to be 1.75 target copies per reaction. Initially, the assay was used to test a variety of specimens (excluding blood) from 598 patients. Of the 660 tests performed, positive PCR results were obtained for 55 patients. TpPCR results were compared with serology results for 301 patients being investigated for early syphilis. Of these patients, 41 were positive by both TpPCR and serology, 246 were negative by both TpPCR and serology, 4 were TpPCR positive but negative by serology, and 10 were TpPCR negative but showed evidence of recent or active infection by serology. Directly compared with serology, TpPCR showed 95% agreement, with a sensitivity of 80.39% and a specificity of 98.40%. Potential factors leading to the discrepant results are discussed. Concurrent serology on 21 patients with TpPCR-positive primary syphilitic lesions demonstrated that a panel of current syphilis serological tests has high sensitivity for the detection of early syphilis. We found that TpPCR is a useful addition to serology for the diagnosis of infectious syphilis. Direct comparison with other T. pallidum PCR assays will be required to fully assess the limitations of the assay.
Assuntos
Reação em Cadeia da Polimerase/métodos , Sífilis/diagnóstico , Treponema pallidum/isolamento & purificação , Proteínas de Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Masculino , Sensibilidade e Especificidade , Sífilis/microbiologia , Sorodiagnóstico da Sífilis , Taq Polimerase/metabolismo , Treponema pallidum/genéticaRESUMO
Protection in experimental rabbit syphilis has been previously assessed by lesion development following intradermal challenge with Treponema pallidum. We have recently reported that passive immunization using monoclonal antibody M131 conveys partial protection as evidenced by significant lesion delays following intradermal challenge. To determine whether such delays in time to lesion appearance corresponded to decreases in the numbers of spirochetes, we used real-time PCR to quantitate T. pallidum genomic DNA copy numbers in lesion biopsies taken throughout the course of lesion development. Three groups of animals were given one prechallenge passive immunization with immune rabbit serum (IRS), M131, or control monoclonal antibody (CMAb) and then challenged with treponemal admixtures of IRS or monoclonal antibody in normal rabbit serum (NRS). As compared to the CMAb NRS controls, delays in the mean time to lesion appearance of 5.8 days for IRS and 8.8 days for M131 were observed. At the earliest time point (10 days postchallenge), real-time PCR showed a mean T. pallidum DNA copy number per mug of rabbit DNA in the CMAb NRS group of 7.65 x 10(3) copies, while no T. pallidum DNA could be detected in the M131 group. At approximately the mean time to lesion appearance in the IRS and M131 groups (17 and 20 days, respectively), the numbers of T. pallidum DNA copies were still 5- and 30-fold less, respectively, than those in the control group at these times. By 30 days postchallenge, the T. pallidum DNA copy numbers were similar in all three groups. These findings indicate that the delays in appearance of syphilitic lesions conferred by IRS and M131 corresponded to a marked decrease in treponemal numbers during the course of lesion development.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunização Passiva , Sífilis/imunologia , Sífilis/prevenção & controle , Animais , Quimiocinas CC , Modelos Animais de Doenças , Dosagem de Genes , Reação em Cadeia da Polimerase , Coelhos , Treponema pallidum/genética , Treponema pallidum/imunologia , Proteínas ViraisRESUMO
Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.