Differential Serodiagnostics of Latent Stages of Syphilis Based on Measuring IgG and IgM Levels towards Extended Panel of Recombinant Antigens of T. pallidum.

Immunochips containing 12 recombinant antigens of T. pallidum (Тр15, Тр17, Тр47, TmpA, Тр0163, Тр0277, Тр0319, Тр0453, Тр0684, Тр0965, Тр0971, and Тр1038) were prepared to assay for IgG and IgM in serum samples (n=68) of healthy individuals and patients with the latent stages of syphilis. The linear discriminant analysis of detected IgG and IgM differentiated three groups of serum samples as 1) early latent syphilis; 2) seroresistant early latent syphilis; and 3) late latent syphilis with overall differentiation potency of 95.6% (88.9-100%). The samples of all syphilis patients were differentiated from the samples of healthy individuals with 100% specificity.

[Сomparison of immunoarrays for syphilis diagnostics produced by co-polymerization immobilization and non-contact printing techniques.]

The aim of the study was to investigate the characteristics of immunoarrays (microarrays) produced by co-polymerization immobilization and non-contact printing techniques for enhancing the capacities of syphilis diagnostics. In diagnostic context immunoarrays of both protein immobilization techniques have shown high sensitivity and specificity together with potency to differentiate syphilis stages in serologic assays. The article discloses the advantages and limitations of non-contact printing techniques as well as the results and problems revealed in the study. Solution of these problems in future may provide the development of new serodiagnostic tools with higher accuracy of the results.

[Diagnostic imminoarray assay for characterization of immunoglobulin igg and igm level in syphilis patients serum towards 12 recombinant antigens of t. pallidum before and after the therapy.]

The aim of the study was to characterize the dynamics of immunoglobulin IgG and IgM level in syphilis patients serum at different stages of the disease before and after the therapy towards 12 diagnostic antigens of T. pallidum in an microarray assay and to evaluate these data as possible prognostic markers. The dynamics of immunoglobulin IgG and IgM level was measured in the reaction of indirect immunofluorescence using microarray and compared to the results of non-treponemal RPR test and treponemal tests as EIA and reaction of passive hemagglutination. In microarray assay diagnostically high level of IgM in patients with primary, secondary and early latent and late latent syphilis decreased dramatically to zero after the successful therapy. Continuously high level of IgM after the therapy proposes the persistence of infection agents in the organism and points out the need of additional antimicrobial treatment. In most of the cases anti-treponemal IgG level also declined after the successful therapy and this confirms the appropriate treatment. The results of microarray assay coincide with the results of other mentioned laboratory tests for syphilis diagnostics. Microarray assay with the recombinant T. pallidum antigens gives the perspective for creating methods with wider spectrum of diagnostic and therapy control options using the IgM immunoglobulin level as a marker for successful syphilis treatment.

Whole-Genome Sequencing of Russian Neisseria Gonorrhoeae Isolates Related to ST 1407 Genogroup.

The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for ß-lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.

Immunochip for Syphilis Serodiagnostics with the Use of Extended Array of Treponema pallidum Recombinant Antigens.

An immunochip for multiple parallel detection of specific serum IgG in serological screening for syphilis is based on the use of an extended array of Treponema pallidum recombinant proteins and includes traditionally used immunodominant antigens (Tp15, Tp17, Tp47, and TmpA) and new synthetic proteins (Tp0277, Tp0319, Tp0453, Tp0684, Tp0965, and Tp1038). The use of individual antigens has demonstrated high analytical value of Tp0277 (periplasmatic C-terminal protease), Tp0319 (cytoplasmic membrane-associated lipoprotein TmpC), and external membrane-associated protein Tp0453 with transporting function, all of them improving significantly the efficiency of screening for syphilis in comparison with the traditional array of antigens. Multiparametric analysis of the results obtained on the immunochip with the use of linear discriminant analysis confirmed the efficiency of extended array of T. pallidum diagnostic antigens. Due to proposed modification, the "positive" and "negative" sera are clearly differentiated: the serological study showed 94.1% sensitivity and 100% specificity.

[Evaluation of the Recombinant Protein Tp0965 of Treponema Pallidum as Perspective Antigen for the Improved Serological Diagnosis of Syphilis].

UNLABELLED: BACKGRAUND. Treponemal tests based on the detection of antibodies against the Treponema pallidum antigens are the most specific methods for serological diagnosis of syphilis. Due to the inability to cultivate this bacterium in vitro, the most promising sources of antigens for diagnostics are recombinant proteins of T. pallidum. Evaluation of the analytical value of certain T. pallidum proteins is the approach to improve sensitivity, specificity, and reproducibility of syphilis serological tests, including possibilities of differential diagnosis of various forms of the disease. OBJECTIVE: The aim of the research was to evaluate the analytical values (sensitivity and specificity) of recombinant protein Tp0965 of T. pallidum as a candidate antigen for serological diagnosis of syphilis. METHODS: tp0965 gene was cloned into the expression vector pET28a and the construct was used for the transformation of E. coli BL-21 (DE3) cells and further expression and purification of the recombinant protein. The collected protein was used as T. pallidum antigen for serum analysis (ELISA) of groups of patients with various forms of syphilis (n=84) and the group of healthy donors (n = 25). RESULTS: High frequency of positive ELISA results was shown with serum of patients with syphilis, compared to the group of healthy donors. The sensitivity of serological reactions using recombinant protein Tp0965 was 98.8%, specificity--87.5%. The highest sensitivity (100%) was detected in the groups of patients with primary, secondary and early latent syphilis while in the group of patients with late latent syphilis it decreased to 95.2%. CONCLUSIONS: We concluded that due to its specificity T. pallidum recombinant protein Tp0965 can be used as a novel perspective antigen for development of syphilis serological diagnostic assays (for primary and early latent forms).