Current trend on syphilis diagnosis: issues and challenges.

Syphilis is a century old sexually transmitted infection transmitted worldwide. WHO reports 12 million new cases that were identified in 1999, with over 90% of these infections reported from patients from low-income countries. This case number of syphilis is on the rise globally in the Men have sex with Men (MSM) population. Dark field microscopy (DFM) and direct fluorescence assay (DFA) have been used in clinical laboratories for decades to demonstrate Treponema pallidum in acutely infected human tissue and/or body fluids. Molecular technologies allow detecting T. pallidum and also determine drug resistance (by identifying DNA point mutation). It is evident from the published literature that PCR is useful as an adjunct test to DFA and DFM, and is useful in confirming syphilis in genital ulcer, tissue, and other body fluid samples, providing even more sensitive detection algorithm. Serological tests remain the mainstay tests since T. pallidum is nonculturable and also because blood collection is easy. The practice of serological testing is changing rapidly from traditional nontreponemal screening followed by confirmatory treponemal testing to screening by treponemal tests referred to as "Reverse Algorithm" followed by nontreponemal testing to determine active infections. Special and further complex algorithms are essential to deal with complex issues such as neurosyphilis or congenital syphilis. Due to the huge surge of syphilis in developing countries where access to medical care is not optimal, point of care or rapid tests may play an important role. This author took an attempt to summarize the current trend of syphilis diagnosis and challenges from a global perspective.

TpF1 from Treponema pallidum activates inflammasome and promotes the development of regulatory T cells.

Human syphilis is a multistage disease, with diverse and wide-ranging manifestations caused by Treponema pallidum. Despite the fact that a cell-mediated immune response takes part in the course of syphilis, T. pallidum often manages to evade host immunity and, in untreated individuals, may trigger chronic infection. With this study, we demonstrate for the first time, to our knowledge, that Treponema pallidum induces a regulatory T (Treg) response in patients with secondary syphilis and we found that the miniferritin TpF1, produced by the bacterium, is able to expand this response and promote the production of TGF-ß. Accordingly, TpF1 stimulates monocytes to release IL-10 and TGF-ß, the key cytokines in driving Treg cell differentiation. Interestingly, we also found that TpF1 stimulates monocytes to synthesize and release several proinflammatory cytokines, such as TNF-α, IL-6, and IL-1ß, the latter following the activation of the multiprotein complex inflammasome. Collectively, these data strongly support a central role for TpF1 both in the inflammation process, which occurs in particular during the early stage of syphilis, and in the long-term persistence of the spirochete within the host by promoting Treg response and TGF-ß production.

A review of in vitro attempts to develop the axenic culture of Treponema pallidum and genomics-based suggestions to achieve this elusive goal.

To date, the axenic culture of Treponema pallidum remains a challenge in the field of microbiology despite countless attempts. Here, we conducted a comprehensive bibliographic analysis using several databases and search engines, namely Pubmed, Google scholar, Google, Web of Science and Scopus. Numerous unsuccessful empiric studies have been conducted and evaluated using as criteria dark-field microscopic observation of motile spiral shaped cells in the culture and virulence of the culture through rabbit infectivity. All of these studies failed to induce rabbit infectivity, even when deemed positive after microscopic observation leading to the misnomer of avirulent T. pallidum. In fact, this criterion was improperly chosen because not all spiral shaped cells are T. pallidum. However, these studies led to the formulation of culture media particularly favourable to the growth of several species of Treponema, including Oral Microbiology and Immunology, Zürich medium (OMIZ), Oral Treponeme Enrichment Broth (OTEB) and T-Raoult, thus allowing the increase in the number of cultivable strains of Treponema. The predicted metabolic capacities of T. pallidum show limited metabolism, also exhibited by other non-cultured and pathogenic Treponema species, in contrast to cultured Treponema species. The advent of next generation sequencing represents a turning point in this field, as the knowledge inferred from the genome can finally lead to the axenic culture of T. pallidum.

Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains.

The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle's minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication.IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum.

Investigation of Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, has been hindered by an inability to culture the organism continuously in vitro despite more than a century of effort. In this study, long-term logarithmic multiplication of T. pallidum was attained through subculture every 6 to 7 days and periodic feeding using a modified medium (T. pallidum culture medium 2 [TpCM-2]) with a previously described microaerobic, rabbit epithelial cell coincubation system. Currently, cultures have maintained continuous growth for over 6 months with full retention of viability as measured by motility and rabbit infectivity. This system has been applied successfully to the well-studied Nichols strain of T. pallidum, as well as to two recent syphilis isolates, UW231B and UW249B. Light microscopy and cryo-electron microscopy showed that in vitro-cultured T. pallidum retains wild-type morphology. Further refinement of this long-term subculture system is expected to facilitate study of the physiological, genetic, pathological, immunologic, and antimicrobial susceptibility properties of T. pallidum subsp. pallidum and closely related pathogenic Treponema species and subspecies.IMPORTANCE Syphilis, a sexually transmitted disease with a global distribution, is caused by a spiral-shaped bacterium called Treponema pallidum subspecies pallidum Previously, T. pallidum was one of the few major bacterial pathogens that had not been cultured long-term in vitro (in a test tube), greatly hindering efforts to better understand this organism and the disease that it causes. In this article, we report the successful long-term cultivation of T. pallidum in a tissue culture system, a finding that is likely to enhance our ability to obtain new information applicable to the diagnosis, treatment, and prevention of syphilis.

Sequence Variation of Rare Outer Membrane Protein ß-Barrel Domains in Clinical Strains Provides Insights into the Evolution of Treponema pallidum subsp. pallidum, the Syphilis Spirochete.

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed ß-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of ß-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) ß-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored ß-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane ß-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, ß-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the ß-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within ß-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

Prevalence of Selected Sexually Transmitted Infection (STI) and Associated Factors among Symptomatic Patients Attending Gondar Town Hospitals and Health Centers.

BACKGROUND: Sexually transmitted infection (STI) is a major global cause of acute illness, infertility, long-term disability and death, with serious medical and psychological consequences to millions of men, women and infants. Moreover, in Ethiopia, epidemiological studies on STI among STI clinic clients are limited. Therefore, the aim of this study was to determine the prevalence and associated risk factors of sexually transmitted infection (STI). METHODS: A cross sectional study was conducted between April and August 2014 among STI clinic clients in Gondar Town hospitals and health centers. One hundred twenty study participants who fulfill the criteria were included. Different laboratory methods and techniques were applied to identify the possible microorganisms. Data were entered and analyzed using SPSS version 20. Logistic regression was used to determine risk factors for STI and P values < 0.05 was considered statistically significant. RESULTS: The overall laboratory test confirmed that STIs prevalence was 74.1% with 32.5% being Candida spp., 30% T. palladium, 20.8% N. gonorrhoeae and 14.2% T. vaginalis. Two or more organisms were isolated in 20% of the study subjects. Risk factors for STI had knowledge about STI and alcohol consumption. CONCLUSION: The prevalence of N. gonorrhoeae, T. pallidum, T. vaginalis, and Candida spp. in the study area was high. It needs health education programs, promotes condom utilization and more comprehensive community based STI studies.

Neurosyphilis as a great imitator: a case report.

BACKGROUND: Neurosyphilis is defined as any involvement of the central nervous system by the bacterium Treponema pallidum. Movement disorders as manifestations of syphilis have been reported quite rarely. CASE PRESENTATION: We report a case of a 42-year-old Russian man living in Estonia with rapidly progressive dementia and movement disorders manifesting as myoclonus, cerebellar ataxia and parkinsonism. The mini mental state examination score was 12/30. After excluding different neurodegenerative causes, further diagnostic testing was consistent with neurosyphilis. Treatment with penicillin was started and 6 months later his mini mental state examination score was 25/30 and he had no myoclonus, parkinsonism or cerebellar dysfunction. CONCLUSION: Since syphilis is easily diagnosed and treatable, it should be considered and tested in patients with cognitive impairment and movement disorders.

Syphilis in children: congenital and acquired.

Syphilis rates in women and congenital syphilis rates have declined steadily in the United States in recent years. However, syphilis remains a worldwide public health problem, with more than 12 million cases in adults and more than half a million pregnancies affected yearly. Prenatal screening and treatment programs are limited or nonexistent in many developing countries. The genome of Treponema pallidum, one of the smallest among prokaryotes, has been sequenced, but methods for continuous in vitro cultivation of the microbe remain elusive. There are no promising candidates for future vaccines at this time. Serologic testing, for both specific treponemal and nontreponemal antibodies, continues to be a primary means of diagnosis. Penicillin remains the drug of choice for congenital and acquired syphilis in childhood. The diagnosis of syphilis beyond early infancy raises concerns for possible child sexual abuse, although progression of congenital syphilis may account for some cases. Syphilis is a potentially eradicable disease, but this can be achieved only with sustained international will and cooperation to fund the necessary screening and treatment programs.

Bacterial sexually transmitted infections in gay, lesbian, and bisexual adolescents: medical and public health perspectives.

Gay, lesbian, and bisexual adolescents, like all adolescents who engage in high-risk sexual behaviors, are at elevated risk for acquiring sexually transmitted infections (STIs). Personal sexual risk factors and issues related to access to care complicate the lives of youth who engage in same-gender sexual activity and who may or may not self-identify as gay, lesbian, or bisexual. Whereas epidemiologic rates of gonorrhea, chlamydia, and syphilis generally have trended downward in adolescents as a whole during the past 15 years, rates for these common reportable bacterial STIs have increased overall during recent years among men who have sex with men. This article focuses on bacterial STIs in youth with same-gender sexual activity. An understanding of trends among gay, lesbian, and bisexual youth remains incomplete, given the absence of consistent and uniform mechanisms for collecting data on sexual behaviors in adolescents and difficulties in associating these behaviors with reportable STIs. Special attention should be given to the screening, diagnosis, and treatment of bacterial STIs in those who engage in same-sex behavior, as new recommendations from the Centers for Disease Control and Prevention have been made available. It is critical that healthcare providers who work with adolescents be aware of the assortment of specific healthcare needs of gay, lesbian, and bisexual youth and address them appropriately in the clinical setting. Medical providers may be one of few true advocates for this often-marginalized adolescent population and have the power to have a positive influence on health promotion and education efforts.

Immunopathological investigations on bovine digital epidermitis.

Paraffin-embedded fragments of bovine digital skin lesions were sectioned and stained with Warthin-Starry, haematoxylin and eosin, Grocott's methenamine silver and immunohistochemical techniques. Microorganisms observed in the silver-stained sections were classified into four major morphological groups. Spirochaetes were the most prevalent organisms, but bacillary and coccoid elements were also present in most sections. Immunohistochemical probing demonstrated that approximately 80 per cent, 46 per cent and 41 per cent of the digital and interdigital dermatitis sections stained positively with polyclonal antisera to Treponema pallidum, Campylobacter jejuni and Fusobacterium necrophorum, respectively. An unidentified branching filamentous organism (presumed to be an actinomycete) was consistently present in the sections of samples from mild interdigital lesions.

Identification of functional candidates amongst hypothetical proteins of Treponema pallidum ssp. pallidum.

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.

Magic bullets need accurate guns--syphilis eradication, elimination, and control.

When cases of early syphilis are treated promptly, the spread of the bacteria within a population is interrupted. However, if complacency is induced by successful control, then upsurges in syphilis incidence can occur. The methods and aims of syphilis control in industrialised countries are reviewed in the light of the potential for regional elimination and global eradication programmes. While the medical means to eliminate syphilis are at hand, acceptable means for finding and treating cases that transmit infection need to be developed, particularly in the marginalized communities with limited access to care.

Is syphilis an incurable disease?

Structures which morphologically resemble treponemes have been found in material from patients suffering from latent or late latent syphilis, many of whom have received treatment in the past. These structures were also found in the blood or spinal fluid (SF) of asymptomatic patients with both positive and negative serological tests for syphilis. They have been found in the eye in the presence of active disease as well as in cases without inflammation ('quiet eye'). In latent syphilis, the presence of treponemes after penicillin treatment raises the question of whether the patient is completely cured of syphilis after treatment. Experimental evidence indicates that continued specific antibody production following penicillin treatment depends on the persistence of the specific antigen in the body. This raised three questions: a) Are these stimuli living treponemes? b) Are they still virulent? c) Is syphilis completely curable following penicillin treatment? Our hypothesis is that slowly dividing treponemes are not killed by penicillin. The persistence of living treponemes somewhere in the body may be the proof of this hypothesis. Cases of relapsing neurosyphilis after penicillin treatment have recently been described. We return to the old dilemma: is it a scar-tissue injury or a reactivation of the disease? Since neurosyphilis is asymptomatic, the patient is usually examined because of seroconversion. Only repeated serological analyses and clinical observations of treated cases with immediate penicillin administration can prevent relapse and damage to the central nervous system (CNS) or to other parts of the body.

Cultivation requirements for Treponema pallidum, Mycobacterium leprae and other microbial and mammalian microaerophilic cells.

Atmospheric and biological evolution progressed simultaneously and today certain cell types flourish only at oxygen tensions which were ambient 600 million years ago, i.e., at 5 to 10 mm Hg. In man, a continuous oxygen flow at these pressures is supplied in the skin where Treponema pallidum, Mycobacterium leprae and members of the genus Rickettsia grow best. In vitro studies support the microaerophilic status of these organisms and of certain other microbial and mammalian cells. Vigorous growth in pure culture will await the development of techniques which can maintain these low oxygen tensions at the cell walls of the microbes as they replicate and consume increasing amounts of oxygen. Continuing failure to consistently isolate microbes from active lesions in patients with rheumatoid arthritis or systemic lupus erythematosus may reflect the universal absence of suitable methods for isolation of microaerophilic microbes.

[Critique of the basic elements on which various syphilis treatments are founded]. / Crítica de los elementos de base en que se fundan ciertos tratamientos de la sífilis.

Certain basic treatments of syphilis depend on the following theories: 1) All treponema divide every 30 to 33 hours. Experiments have shown that this idea is only valid during the phase of exponential growth, i.e. it corresponds to the period lyinü between penetration of the germ into the organism and the onset of the initial lesion. One should also be reserved about a whole series of factors which may interfere with the rate of multiplication of treponema. 2) Blood penicillin levels of 0.03 U./ml. would destroy all treponema during cell division. It seems that the efficacy of penicillin mainly depends on levels, at the time of multiplication of the spirochetes. This level of 0.03 U./ml., according to Eagle, should be considered as an average serum concentration, the concentration of maximum efficacy is much higher, about 0.825 U./ml. In any case, it is not possible to obtain with a single dose, equal for all subjects, fixed and certain penicillin levels. For a given quantity injected, serum levels vary from one subject to another and, in the same individual, from one time to another. 3) The epidemiological results do not seem to confirm the optimistic forecasts of certain venereologists who thought that with a standard therapeutic method, it would be possible to eradicate syphilis.

MyD88 deficiency markedly worsens tissue inflammation and bacterial clearance in mice infected with Treponema pallidum, the agent of syphilis.

Research on syphilis, a sexually transmitted infection caused by the non-cultivatable spirochete Treponema pallidum, has been hampered by the lack of an inbred animal model. We hypothesized that Toll-like receptor (TLR)-dependent responses are essential for clearance of T. pallidum and, consequently, compared infection in wild-type (WT) mice and animals lacking MyD88, the adaptor molecule required for signaling by most TLRs. MyD88-deficient mice had significantly higher pathogen burdens and more extensive inflammation than control animals. Whereas tissue infiltrates in WT mice consisted of mixed mononuclear and plasma cells, infiltrates in MyD88-deficient animals were predominantly neutrophilic. Although both WT and MyD88-deficient mice produced antibodies that promoted uptake of treponemes by WT macrophages, MyD88-deficient macrophages were deficient in opsonophagocytosis of treponemes. Our results demonstrate that TLR-mediated responses are major contributors to the resistance of mice to syphilitic disease and that MyD88 signaling and FcR-mediated opsonophagocytosis are linked to the macrophage-mediated clearance of treponemes.