Dataset Information

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Treponema pallidum DNA in the Peripheral Blood of Secondary Syphilis Patients.

ABSTRACT: Secondary syphilis (SS) has always been puzzling for the clinicians because of the similarity of the appearance of skin rashes with other dermatoses. Serological assays are useful, but less sensitive at an early stage of SS or when patients are immunodeficient. Therefore, there is an urgent need to develop a rapid and effective tool for the diagnosis of SS, which may play an important role in the control of epidemic syphilis outbreaks. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay, targeting gene encoding the basic membrane protein of Treponema pallidum, to detect the presence of circulating T. pallidum DNA in the blood of SS patients. The specificity of LAMP was validated using three strains of Spirochaetales and six common clinical bacteria. The clinical applicability of LAMP assay was assessed using 642 blood samples from clinically suspected SS patients and 80 samples from healthy blood donors, showing a sensitivity of 82.1% and a specificity of 100.0% in the diagnosis of SS. Thus, our results indicate that the LAMP can be used as a supplementary method for the diagnosis of SS.

SUBMITTER: Xiao Y

PROVIDER: S-EPMC5805031 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of <i>Treponema pallidum</i> DNA in the Peripheral Blood of Secondary Syphilis Patients.

Xiao Yongjian Y Xie Yafeng Y Xu Man M Liu Shuangquan S Jiang Chuanhao C Zhao Feijun F Zeng Tiebing T Liu Zhuoran Z Yu Jian J Wu Yimou Y

The American journal of tropical medicine and hygiene 20170928 6

Secondary syphilis (SS) has always been puzzling for the clinicians because of the similarity of the appearance of skin rashes with other dermatoses. Serological assays are useful, but less sensitive at an early stage of SS or when patients are immunodeficient. Therefore, there is an urgent need to develop a rapid and effective tool for the diagnosis of SS, which may play an important role in the control of epidemic syphilis outbreaks. In this study, we evaluated a loop-mediated isothermal ampli ...[more]

PMID: 29016285

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Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Project description:IntroductionYaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi (HD), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes.Methods and analysisThe LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum (TP) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d'Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts.Ethics and disseminationWe have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization.Trial registration numberNCT04753788.

Project description:BackgroundThe 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 findingsTo 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.ConclusionsThis 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.

Dataset Information

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Treponema pallidum DNA in the Peripheral Blood of Secondary Syphilis Patients.

Publications

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of <i>Treponema pallidum</i> DNA in the Peripheral Blood of Secondary Syphilis Patients.

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