Clinical outcomes of syphilis in HIV-negative and HIV-positive MSM: occurrence of repeat syphilis episodes and non-treponemal serology responses.

OBJECTIVES: HIV-positive men who have sex with men (MSM) may be at a higher risk of repeat syphilis, have different clinical manifestations and have a different serological response to treatment compared with HIV-negative MSM. The objective of this study was to assess whether HIV-negative and HIV-positive MSM with infectious syphilis (primary, secondary or early latent) differed in history of previous syphilis episodes, disease stage and non-treponemal titre of initial and repeat episodes, and the titre response 6 and 12 months after treatment. Furthermore, determinants associated with an inadequate titre response after treatment were explored. METHODS: This retrospective analysis used data of five longitudinal studies (four cohorts; one randomised controlled trial) conducted at the STI clinic in Amsterdam, the Netherlands. Participants were tested for syphilis and completed questionnaires on sexual risk behaviour every 3-6 months. We included data of participants with ≥1 syphilis diagnosis in 2014-2019. Pearson's χ² test was used to compare HIV-negative and HIV-positive MSM in occurrence of previous syphilis episodes, disease stage of initial and repeat syphilis episode and non-treponemal titre treatment responses. RESULTS: We included 355 participants with total 459 syphilis episodes. HIV-positive MSM were more likely to have a history of previous syphilis episodes compared with HIV-negative MSM (68/90 (75.6%) vs 96/265 (36.2%); p<0.001). Moreover, HIV-positive MSM with repeat syphilis were less often diagnosed with primary syphilis (7/73 (9.6%) vs 36/126 (28.6%)) and more often diagnosed with secondary syphilis (16/73 (21.9%) vs 17/126 (13.5%)) and early latent syphilis (50/73 (68.5%) vs 73/126 (57.9%)) (p=0.005). While not significantly different at 12 months, HIV-negative MSM were more likely to have an adequate titre response after 6 months compared with HIV-positive MSM (138/143 (96.5%) vs 66/74 (89.2%); p=0.032). CONCLUSIONS: In repeat syphilis, HIV infection is associated with advanced syphilis stages and with higher non-treponemal titres. HIV infection affects the serological outcome after treatment, as an adequate titre response was observed earlier in HIV-negative MSM.

Short communication: Informative value of an ELISA applied to bulk tank milk to assess within-herd prevalence of digital dermatitis in dairy herds.

The objective of this study was to assess whether an antibody ELISA applied to bulk tank milk (BTM) could be used to accurately estimate within-herd prevalence of digital dermatitis (DD). The ELISA was designed for the detection of antibodies against Treponema phagedenis-like strain V1 (PrrA antigen). The hind feet of all lactating cows from 40 commercial French dairy herds with a history of DD were scored by an observer in the milking parlor, using the 4 M-stage system. After milking, a BTM sample was collected and tested for anti-Treponema phagedenis-like antibodies using the antibody ELISA. Within-herd DD prevalence at the cow level was determined using 2 different approaches: (1) having DD lesion on at least 1 hind foot (Prev; prevalence of affected cows), and (2) having an M1 or M2 lesion on at least 1 hind foot (PrevA; prevalence of cows affected by DD in an active stage). Receiver operating characteristic analysis was used to determine both optimal within-herd DD prevalence and BTM sample to positive (S/P) ratio cut-off values. Two optimal cut-off values were identified. Herds with an S/P ratio of BTM ≤0.2 had a Prev ≤10% (sensitivity = 0.97, specificity = 1), whereas herds with an S/P ratio of BTM >0.38 had a Prev >40% (sensitivity = 0.94, specificity = 0.86). In the same way but with a slightly lower specificity, an S/P ratio >0.38 corresponds also to a PrevA >18% (sensitivity = 0.92, specificity = 0.70). The BTM antibody ELISA shows great promise for screening purposes during DD management programs.

Estimation of Full-Length TprK Diversity in Treponema pallidum subsp. pallidum.

Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

A retrospective study on nested PCR detection of syphilis treponemes in clinical samples: PCR detection contributes to the diagnosis of syphilis in patients with seronegative and serodiscrepant results.

Syphilis, caused by Treponema pallidum ssp. pallidum (TPA), is a persisting global health problem. Although syphilis diagnostics relies mainly on serology, serological tests have some limitations, and it is recommended that the final diagnosis be supported by additional tests. The purpose of this study was to analyze the relationship between serology and PCR in syphilis diagnostics. From the year 2004 to May 2019, a total of 941 samples were taken from 833 patients suspected of having syphilis, in Czech Republic. In all these samples, both nested PCR detection of TPA and serology testing were performed. Of the 941 samples, 126 were seronegative, 651 were seropositive, and 164 were serodiscrepant. Among seronegative samples (n = 126), 11 were PCR-positive (8.7%). Among seropositive samples (n = 651; i.e., samples positive for both non-treponemal and treponemal serology tests), 368 samples were PCR-positive (56.5%). The remaining 164 serodiscrepant samples included RPR negative and treponemal serological test-positive samples (n = 154) and a set of 10 RPR-positive samples negative in treponemal serological tests. While the first group revealed 73 PCR-positive samples (47.4%), the second revealed 5 PCR positive samples (50.0%). PCR detection rates were highest in primary syphilis, with lower rates in the secondary and undetermined syphilis stages. As shown here, the nested PCR can improve diagnostics of syphilis, especially in seronegative patients and in patients with discrepant serology.

Putative ß-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative ß-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted ß-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as ß-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly ß-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal ß-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

VITREOUS TREPONEMAL ANTIBODY AS A SUPPLEMENTARY TEST TO SEROLOGY FOR THE CONFIRMATION OF SYPHILITIC CHORIORETINITIS.

PURPOSE: To report the novel application of nontreponemal and treponemal antibody to confirm diagnosis of ocular syphilis from vitreous samples. METHODS: Two distinct case reports emphasizing the importance of confirmatory vitreous treponemal antibody. Multimodal imaging of patients was also applied. RESULTS: We report two distinct cases with positive serum treponemal antibody but opposing vitreous treponemal antibody results. One case with a positive vitreous test responded well to antisyphilitic treatment. By contrast, a case with a negative vitreous result was changed to serpiginous choroiditis, eventually cured by immunomodulatory treatment. CONCLUSION: Intraocular fluid analysis of nontreponemal and treponemal antibody may play an important role in ruling out suspected ocular syphilis in settings without a polymerase chain reaction facility, especially immunocompromised patients who are at risk of multiple infections. Further studies are needed to establish the sensitivity and specificity of nontreponemal and treponemal antibody test on vitreous samples.

Clinical Performance of the BioPlex 2200 Syphilis Total & RPR Assay at a Tertiary Medical Center with a High Rate of Syphilis.

Manual treponemal and nontreponemal serologic testing has historically been used for the diagnosis of syphilis. This approach is simple and reproducible but labor intensive. Recently, the FDA cleared the fully automated BioPlex 2200 Syphilis Total & RPR assay for the detection of treponemal and nontreponemal antibodies. We evaluated the clinical performance of this assay at a tertiary medical center with a high syphilis prevalence. Prospective consecutively collected (n = 400) and known RPR-positive (n = 100) specimens were compared using predicate manual rapid plasma reagin (RPR) and fluorescent treponemal antibody absorption (FTA) methods and the BioPlex 2200 Syphilis Total & RPR assay. Positive and negative percent agreements (PPA and NPA, respectively) between the assays were calculated. The PPA and NPA between the manual and BioPlex 2200 RPR results for the prospective population were 85% (17/20; 95% confidence interval [CI], 69% to 100%) and 98% (373/380; 95% CI, 97% to 99%), respectively. The PPA for the manual RPR-positive population was 88% (88/100; 95% CI, 82% to 94%). Overall, the manual and BioPlex 2200 RPR titers demonstrated 78% (99/127) concordance within ±1 dilution and 94% (120/127) within ±2 dilutions. An interpretation of the syphilis serologic profile using the traditional algorithm showed a concordance of 99.5% in the prospective population and 85% in the manual RPR-positive cohort. The performance of the BioPlex 2200 Syphilis Total & RPR assay is comparable to those of manual methods. The high NPA of this assay combined with the ability to automate a historically labor-intensive assay is an appealing attribute for syphilis screening in a high-volume laboratory.

Complement Immune Evasion by Spirochetes.

The complement system plays an important role in the innate and acquired immune response against pathogens. A sophisticated network of activating and regulating proteins allows the distinction between intact and damaged host and non-host surfaces such as bacteria and other parasites. Non-host structures trigger the alternative pathway which may lead to their elimination by phagocytosis or cell lysis. In addition, complement proteins such as C1q, mannose binding lectin (MBL), and ficolins act as pathogen pattern-recognition molecules. Biological functions such as opsonization, activation of B lymphocytes and production of antibodies, degranulation of mast cells and basophils, and cell lysis that are important for elimination of microorganisms are dependent on complement activation. However, several pathogens including spirochetes have developed several specialized mechanisms to evade the complement system, thereby contributing to survival in the host. In this review, we give a brief overview of complement activation and regulation, and discuss in detail the strategies used by spirochetes from the genera Borrelia, Leptospira, and Treponema to overcome complement activation.

Comparison of Six Automated Treponema-Specific Antibody Assays.

Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology.

Comparison of a combined nontreponemal (VDRL) and treponemal immunoblot to traditional nontreponemal and treponemal assays.

BACKGROUND: Serology is the mainstay for the diagnosis and management of patients with syphilis. Newer technologies such as immunoblotting are now available for the diagnosis of syphilis. METHODS: A commercial IgM/IgG immunoblot assay that detects both nontreponemal (VDRL-Venereal Disease Research Laboratory) and treponemal antibodies was compared with standard nontreponemal and treponemal assays. The immunoblot and T. pallidum particle agglutination assay (TP-PA) were performed on 198 samples. Ninety-seven samples were Rapid plasma reagin (RPR)-positive and one hundred one were RPR-negative. Positive RPR samples were titered by VDRL. RESULTS: The agreement, sensitivity, and specificity of the IgM/IgG VDRL results of the immunoblot compared to RPR were 74.2% (95% CI: 67.2-80.2), 77.3% (95% CI: 70.2-83.4), and 71.3% (95% CI: 64.4-77.1), respectively. The agreement, sensitivity, and specificity of the IgM/IgG treponemal immunoblot compared to TP-PA were 100% for all parameters, if the ten equivocal results were not used in the calculation. CONCLUSION: The treponemal portion of the ViraBlot IgM/IgG immunoblot compared well with the treponemal confirmation assay and could be a useful supplemental method to fluorescent treponemal antibody or TP-PA for the confirmation of syphilis. The addition of the detection of nontreponemal antibodies to the immunoblot assay, however, may not be of added benefit to the overall assay, due to decreased sensitivity and specificity compared to standard assays.

Analysis of the complement sensitivity of oral treponemes and the potential influence of FH binding, FH cleavage and dentilisin activity on the pathogenesis of periodontal disease.

Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola's dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of Treponema socranskii, Treponema medium, Treponema pectinovorum and Treponema maltophilum are also serum resistant, whereas Treponema vincentii and Treponema amylovorum are serum sensitive. Although T. denticola's ability to evade complement-mediated killing is strictly dependent on FH binding, other serum-resistant treponemal species lack FhbB and do not bind FH, indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. All T. denticola strains bound FH and were serum resistant, but differences in binding kinetics, dentilisin activity and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease.

Prevalence of antibodies against transfusion-transmissible infections (TTI) in blood donors from the Colombian eastern region.

BACKGROUND: The main purpose of this study was to establish the prevalence of antibodies against five transfusion-transmissible infections (TTIs) in blood donors from one of the most important blood banks in Colombia. METHODS: A cross-sectional, descriptive and case control study was performed from a database of Higuera-Escalante blood bank, for a period of a year. Serum was used for donor screening. Surface antigens for hepatitis B (HbsAg), anti-hepatitis C antibodies, Chagas disease, syphilis, and HIV were identified. Chemiluminescent Microparticle Immunoassay (CMIA, Abbott Diagnostics) was performed. RESULTS: From 41,575 total donors analyzed, 1,226 were reactive for any of the infectious markers (total prevalence of 2.95%). The prevalence of specific infections was: Chagas disease 0.49%, HbsAg 0.21%, HCV 0.45%, HIV 0.12%, and syphilis 1.68%. Reactivity was more frequent in men (n = 785, 64%) with a mean age of 36.35 years. HIV was present in the youngest donors with a mean age of 26.5 years (IC 95%: 23.6 - 27.6); on the other hand, Chagas disease was found in the oldest donor population, with a mean age of 40 years (IC 95%: 39.1 - 41.3). CONCLUSIONS: Identifying the prevalence of circulating antibodies against transfusion transmissible infections allows us to establish an epidemiological profile of donors inhabiting the geographic catchment area of our blood bank. Total prevalence in this study was 2.95% for any of the five markers. Syphilis prevalence demonstrates its high distribution within the blood donor population of our country, although this result could be influenced by the high rate of false-reactive test. Chagas disease is endemic in Santander, Colombia, which correlates with the results obtained in this study.

Comparative investigation of the genomic regions involved in antigenic variation of the TprK antigen among treponemal species, subspecies, and strains.

Although the three Treponema pallidum subspecies (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum), Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence. tprK heterogeneity is generated by nonreciprocal gene conversion between the tprK expression site and donor sites. Although each of the above-mentioned species and subspecies has a functional tprK antigenic variation system, it is still unclear why the level of expression and the rate at which tprK diversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as the tprK expression sites, among eight T. pallidum subsp. pallidum isolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), three T. pallidum subsp. pertenue isolates (Gauthier, CDC2, and Samoa D), one T. pallidum subsp. endemicum isolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain of T. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that the tprK recombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression of tprK variants among these Treponema isolates.

It is time to use treponema-specific antibody screening tests for diagnosis of syphilis.

Assays that detect treponema-specific antibodies, which are either automated or can be done as point-of-care tests, have been developed, some of which are FDA approved. These assays have the advantage of being easily performed and demonstrate high sensitivity, both key features of an infectious disease screening test. As a result, many high-volume clinical laboratories have begun to offer a reverse syphilis testing algorithm where a treponema-specific test is used for screening, followed by a nontreponemal test (i.e., rapid plasma reagin [RPR]) to assess disease activity and treatment status. Concerns about physicians being able to understand and apply this new testing algorithm have been expressed (8). In this point-counterpoint, Michael Loeffelholz of the University of Texas Medical Branch at Galveston explains why his laboratory has adopted this reverse algorithmic approach. Matthew Binnicker of the Mayo Clinic, Rochester, MN, explains why the reverse algorithm may not be suitable for all clinical laboratories and every clinical situation.

Identification of immunogenic proteins in Treponema phagedenis-like strain V1 from digital dermatitis lesions by phage display.

Digital dermatitis (DD) is a contagious claw disease causing lameness in cattle, affecting both animal welfare and economics. In this study, shotgun phage display was used to identify immunogenic proteins in a strain (V1) of the Treponema phylotype closely related to Treponema phagedenis, indicated as a key agent in the pathogenesis of DD. A genomic phage library was constructed and selected against antibodies from a rabbit immunized with live strain V1 bacteria. A homolog to the immunogenic protein TmpA of Treponema pallidum subsp. pallidum was identified, as well as a putative phage tail tape measure protein (Ttm), and a putative proline-rich repeat lipoprotein (PrrA). The complete amino acid sequences of these proteins were predicted from a genomic sequence of strain V1 generated by 454 Sequencing™. The presence of these genes in ten Treponema spp. field isolates was investigated by PCR. The tmpA and ttm genes were detected in all T. phagedenis-like isolates while prrA was detected in four out of seven. None of the genes were detected in the three Treponema pedis isolates investigated. Recombinant proteins were produced and used in indirect ELISAs. For all three proteins, a majority of serum samples from cattle with DD (n=8) showed higher optical density values than samples from cattle without DD (n=7).

[Evaluation of latex agglutination test for anti-treponemal antibody in comparison with chemical luminescence tests].

The performance of a latex agglutination test (Mediace TPLA) in the detection of anti-treponemal antibody was evaluated in comparison with chemical luminescence tests (LumipulsII-N and Architect TPAb) in 346 cases. Anti-treponemal antibody was further determined by immunochromatography and immunoblotting tests and additionally evaluated by a serological test for syphilis with lipoidal antigens. The total concordance rate between the latex agglutination test and chemical luminescence tests ranged from 96% to 97%: the positive concordance rate ranged from 96% to 97%, and the negative concordance rate, from 97% to 98%. The latex agglutination test showed two false positive cases, and each chemical luminescence test showed two false positive cases, respectively. In eight cases, only the latex agglutination test showed negative results; all specimens contained anti-treponemal antibodies. However, none of these was considered to be a false positive and each was treated as syphilis based on the results of confirmatory analysis with immunochromatography and immunoblotting tests and a serological test for syphilis. The discordant results in the latex agglutination test and chemical luminescence tests may be caused by the different antigenisity of each test. With detailed analysis of those sera treated as syphilis, each specimen was found to contain various antibodies against syphilitic antigens, suggesting that there was a different specificity of native and recombinant antigens. Based on the present results for the comparison between the latex agglutination test and chemical luminescence tests, it was considered that further investigation is necessary to clarify the anti-treponemal antibody profile of syphilis at the disease stage.