Evaluating the diagnostic accuracy of TpN17 and TmpA recombinant proteins in syphilis detection: a phase II study.

Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.

IgG Isotypes Targeting a Recombinant Chimeric Protein of Trypanosoma cruzi in Different Clinical Presentations of Chronic Chagas Disease.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, which leads to a spectrum of clinical presentations that range from asymptomatic to severe cardiac involvement. The host immune response plays a pivotal role in disease progression. Ig isotypes may contribute to disease pathogenesis. Investigating these components can provide insights into the immunopathogenic mechanisms underlying CD. This cross-sectional study aims to establish a correlation between the Ig profile of individuals infected with T. cruzi with the clinical forms of chronic CD. Serum samples were collected from partner institutions in different states of Brazil. Individuals diagnosed with chronic CD were categorized based on the clinical form of the disease. The indirect ELISA method using the recombinant chimeric Molecular Biology Institute of Paraná membrane protein 8.4 as the antigen was used to determine the Ig profile, including total IgG, IgG1, IgG2, IgG3, and IgG4. Ninety-seven serum samples from patients classified as negative (NEG, n = 38), indeterminate (IND, n = 24), mild cardiac (MC, n = 20), and severe cardiac (SC, n = 15) forms were analyzed. IgG1 exhibited greater levels compared with the other isotypes, showing a significant difference between the MC and IND groups. IgG3 levels were greater in individuals from the MC group compared with the SC group. IgG1 and IgG3 isotypes can serve as biomarkers to evaluate the progression of CD because they exhibit variations across clinical groups. Additional longitudinal studies are necessary to explore the relationship between antibody kinetics and the development of tissue damage.

Structural analysis of the Trypanosoma brucei EIF4E6/EIF4G5 complex reveals details of the interaction between unusual eIF4F subunits.

Recognition of the mRNA 5' end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79-166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.

Seroepidemiological Survey of Chronic Chagas Disease in a Rural Community in Southern Bahia, Brazil, Using Recombinant Chimeric Antigens.

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected tropical disease with life-threatening implications. In this study, we conducted a seroepidemiological survey to determine the prevalence and clinical profiles of CD in 217 individuals from an impoverished rural community in Southern Bahia, Brazil. The overall prevalence of CD in the studied community was 0.92%, detected through latent class analysis (LCA). Two individuals tested positive for anti-T. cruzi IgG, both being male farmers. One case was a 22-year-old man born in Camamu, with no evidence of congenital transmission, suggesting other routes of transmission such as vector-borne transmission due to migratory activities. The other case was a 69-year-old man born in São Felipe, who had lived in an adobe/brick house and had a pacemaker due to cardiac involvement caused by CD. The prevalence in this community was lower than expected, given the socioeconomic conditions and environmental factors that contribute to T. cruzi transmission. This could be attributed to the implementation of preventive measures and vector control programs by the Brazilian Government. However, continuous monitoring and surveillance are essential to sustain control efforts and detect any potential re-emergence of the disease. While the overall prevalence was low, the detection of positive cases underscores the need for continued surveillance and control measures in vulnerable populations, such as rural communities. Active surveillance, early diagnosis, and timely treatment are crucial in preventing disease progression and complications, thereby enhancing the effectiveness of screening and treatment programs.

In cellulo and in vivo assays for compound testing against Trypanosoma cruzi.

Here, we describe a combined in cellulo and in vivo approach to identify compounds with higher potential for efficient inhibition of Trypanosoma cruzi. Phase I of in cellulo assays is designed to exclude inactive or toxic compounds, while phase II is designed for accurate IC50, CC50, and selective index (SI) determination. Compounds showing high SI are tested using in vivo infection models in parallel with benznidazole to assess their efficacy relative to a reference drug used for Chagas disease treatment. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).1.

Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system.

Trypanosoma brucei belongs to a group of protozoans presenting fragmented large subunit rRNA. Its LSU rRNA equivalent to the 25S/28S rRNA of other eukaryotes is split into six fragments, requiring additional processing for removal of the extra spacer sequences. We have used a genetic complementation strategy to further investigate the T. brucei RRP44 nuclease in pre-rRNA maturation. TbRRP44 contains both a PIN and a RNB domain whose homologues are found in association with the exosome complex. We found that the exonucleolytic activity of the RNB domain as well as the physical presence of the PIN domain are essential for TbRRP44 function, while a catalytic site mutation in the PIN domain has no detectable effect on cell growth. A new endonucleolytic cleavage site in ITS1 was identified. In addition to the 5.8S rRNA 3'-end maturation, TbRRP44 is required for degradation of the excised 5'-ETS and for removal of part of ITS1 during maturation of the 18S rRNA 3'-end. TbRRP44 deficiency leads to accumulation of many LSU intermediate precursors, most of them not detected in control cells. TbRRP44 is also required for U3 snoRNA and spliced leader processing, indicating that TbRRP44 may have a wide role in RNA processing in T. brucei.

Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates.

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3'-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.

Western blot using Trypanosoma cruzi chimeric recombinant proteins for the serodiagnosis of chronic Chagas disease: A proof-of-concept study.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi. The chronic phase of CD is characterized by the presence of IgG anti-T. cruzi antibodies; and diagnosis is performed by serological methods. Because there is no reliable test that can be used as a reference test, WHO recommends the parallel use of two different tests for CD serodiagnosis. If results are inconclusive, samples should be subjected to a confirmatory test, e.g., Western blot (WB) or PCR. PCR offers low sensitivity in the chronic phase, whereas few confirmatory tests based on the WB method are commercially available worldwide. Therefore, new diagnostic tools should be evaluated to fill the gap in CD confirmatory tests. In recent years, four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) have been evaluated in phase I, II and III studies using ELISA, liquid microarray and immunochromatography with 95-100% accuracy. Given the high diagnostic performance of these antigens, the present study investigated the ability of these molecules to diagnose chronic CD using a WB testing platform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed the diagnostic potential of four chimeric antigens using 40 T. cruzi-positive, 24-negative, and three additional positive samples for visceral leishmaniasis (i.e., potentially cross-reactive) using WB as the diagnostic platform. Checkerboard titration with different dilutions of antigens, conjugated antigens, and serum samples was performed to standardize all assays. All IBMP antigens achieved 100% sensitivity, specificity, and accuracy, with the exception of IBMP-8.3, which had 100% specificity despite lack of significance, but lower sensitivity (95%) and accuracy (96.9%). No cross-reactivity was observed in samples positive for leishmaniasis. CONCLUSIONS/SIGNIFICANCE: The present phase I (proof-of-concept) study demonstrated the high diagnostic potential of these four IBMP antigens to discriminate between T. cruzi-positive and -negative samples, making them candidates for phase II and confirmatory testing with WB.

Efficient CRISPR-Cas9-mediated genome editing for characterization of essential genes in Trypanosoma cruzi.

This protocol outlines a new genetic complementation strategy to investigate gene function in Trypanosoma cruzi, the parasite causing Chagas disease. We combine CRISPR-Cas9 technology with recombination of variants of the target gene containing the desired mutations that are resistant to Cas9-cleavage, which enables detailed investigation of protein function. This experimental strategy overcomes some of the limitations associated with gene knockouts in T. cruzi. For complete details on the use and execution of this protocol, please refer to Marek et al. (2021).

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform. METHODOLOGY/PRINCIPAL FINDINGS: DAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV. CONCLUSIONS/SIGNIFICANCE: DAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

A toolkit for recombinant production of seven human EGF family growth factors in active conformation.

Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.

Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells.

Trypanosoma cruzi-the causative agent of Chagas disease-like other kinetoplastids, relies mostly on post-transcriptional mechanisms for regulation of gene expression. However, trypanosomatids undergo drastic changes in nuclear architecture and chromatin structure along their complex life cycle which, combined with a remarkable set of reversible histone post-translational modifications, indicate that chromatin is also a target for control of gene expression and differentiation signals in these organisms. Chromatin-modifying enzymes have a direct impact on gene expression programs and DNA metabolism. In this work, we have investigated the function of T. cruzi histone deacetylase 4 (TcHDAC4). We show that, although TcHDAC4 is not essential for viability, metacyclic trypomastigote TcHDAC4 null mutants show a thin cell body and a round and less condensed nucleus located very close to the kinetoplast. Sixty-four acetylation sites were quantitatively evaluated, which revealed H2AT85ac, H4K10ac and H4K78ac as potential target sites of TcHDAC4. Gene expression analyses identified three chromosomes with overrepresented regions of differentially expressed genes in the TcHDAC4 knockout mutant compared with the wild type, showing clusters of either up or downregulated genes. The adjacent chromosomal location of some of these genes indicates that TcHDAC4 participates in gene expression regulation during T. cruzi differentiation.

Stability Assessment of Four Chimeric Proteins for Human Chagas Disease Immunodiagnosis.

The performance of an immunoassay relies on antigen-antibody interaction; hence, antigen chemical stability and structural integrity are paramount for an efficient assay. We conducted a functional, thermostability and long-term stability analysis of different chimeric antigens (IBMP), in order to assess effects of adverse conditions on four antigens employed in ELISA to diagnose Chagas disease. ELISA-based immunoassays have served as a model for biosensors development, as both assess molecular interactions. To evaluate thermostability, samples were heated and cooled to verify heat-induced denaturation reversibility. In relation to storage stability, the antigens were analyzed at 25 °C at different moments. Long-term stability tests were performed using eight sets of microplates sensitized. Antigens were structurally analyzed through circular dichroism (CD), dynamic light scattering, SDS-PAGE, and functionally evaluated by ELISA. Data suggest that IBMP antigens are stable, over adverse conditions and for over a year. Daily analysis revealed minor changes in the molecular structure. Functionally, IBMP-8.2 and IBMP-8.3 antigens showed reactivity towards anti-T. cruzi antibodies, even after 72 h at 25 °C. Long-term stability tests showed that all antigens were comparable to the control group and all antigens demonstrated stability for one year. Data suggest that the antigens maintained their function and structural characteristics even in adverse conditions, making them a sturdy and reliable candidate to be employed in future in vitro diagnostic tests applicable to different models of POC devices, such as modern biosensors in development.

The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners.

The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5' end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.

Novel Genetic Constructs for Production of Recombinant HTLV-1/2 Antigens and Evaluation of Their Reactivity to Plasma Samples from HTLV-1-Infected Patients.

Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections.

Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells

Trypanosoma cruzi—the causative agent of Chagas disease—like other kinetoplastids, relies mostly on post-transcriptional mechanisms for regulation of gene expression. However, trypanosomatids undergo drastic changes in nuclear architecture and chromatin structure along their complex life cycle which, combined with a remarkable set of reversible histone post-translational modifications, indicate that chromatin is also a target for control of gene expression and differentiation signals in these organisms. Chromatin-modifying enzymes have a direct impact on gene expression programs and DNA metabolism. In this work, we have investigated the function of T. cruzi histone deacetylase 4 (TcHDAC4). We show that, although TcHDAC4 is not essential for viability, metacyclic trypomastigote TcHDAC4 null mutants show a thin cell body and a round and less condensed nucleus located very close to the kinetoplast. Sixty-four acetylation sites were quantitatively evaluated, which revealed H2AT85ac, H4K10ac and H4K78ac as potential target sites of TcHDAC4. Gene expression analyses identified three chromosomes with overrepresented regions of differentially expressed genes in the TcHDAC4 knockout mutant compared with the wild type, showing clusters of either up or downregulated genes. The adjacent chromosomal location of some of these genes indicates that TcHDAC4 participates in gene expression regulation during T. cruzi differentiation.

Correction: Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

[This corrects the article DOI: 10.1371/journal.pone.0234043.].

Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.