Treponema pallidum: Classification, Structure, Pathogenicity and Treatment


The History of Discovery


  • Syphilis, the most notorious venereal or sexually transmitted disease (STD) has been known for many ages. Until quite recently two main theories of syphilis origin existed: pre-Columbian, which supposed syphilis to emerge in ancient times in Central Africa with farther spread towards Europe and Asia, and Columbian one, relied upon syphilis epidemic rise in Europe after Columbus voyage.
  • According to multiple investigations of fossils the first theory was preferentially supported for a long time.
  • Nonetheless, in 2008-2011 the thorough phylogenetic analysis of evolution of various groups of treponemas elaborated by K. Harper and colleagues has proven the agent of syphilis to be phylogenetically youngest from all other treponemas.
  • The most probably it has been developed from some non-venereal treponema subspecies and later caused epidemics of syphilis in Europe after the voyage of Columbus.
  • It is generally believed that syphilis was named by G. Fracastoro in 1530 after a mythical shepherd, Syphilus, described in his poem “Syphilis or the French Disease”.
  • Only in 1905 F. Schaudinn and E. Hoffmann discovered Treponema pallidum to be the causative agent of syphilis. They revealed spirochetes in Giemsa-stained fluid smears from secondary syphilitic lesions. Year later A. Wassermann proposed complement fixation test for serological diagnosis of syphilis.
  • The first chemical drugs for syphilis treatment were introduced into clinical practice still by P. Erlich (organic arsenical compound salvarsan). In 1943 J. Mahoney demonstrated the effectiveness of penicillin for syphilis therapy, and it remains to be the most preferable drug for disease treatment.

Classification of Pathogenic Treponemas


  • T. pallidum belongs to the order Spirochaetales, family Spirochaetaceae, and genus Treponema.
  • Pathogenic species T. pallidum has 3 subspecies and 1 closely related species:
  • T. pallidum subsp. pallidum, which causes venereal syphilis;
  • T. pallidum subsp. endemicum that causes endemic syphilis or bejel;
  • T. pallidum subsp. pertenue that produces yaws;
  • T. carateum, the agent of pinta.
  • These microbial pathogens are very similar; their DNA homology exceeds 95%.

Structure and Properties of T. pallidum


Morphology

  • T. pallidum are gram-negative thin corkscrew-shaped bacteria about 0.2 μm in diameter and 5 to 20 μm in length with 6-20 regular small coils with tapered ends. The cytoplasmic membrane lends treponemas a spiral shape.
  • Microbial body consists of an axial filament and cytoplasm wound spirally around the filament. Cytoplasmic membrane is covered by three-layer outer membrane.
  • It covers basal bodies with attached 3-4 bacterial endoflagella or fibrils localized in periplasmic space. Endoflagella provide active variable motility of bacteria.
  • Treponemas don’t produce spores or capsules. Old treponema cultures form cyst-like structures.
  • The bacteria stain pale-pink with Romanowsky-Giemsa method as they poorly stained with aniline dyes due to the large lipid contents. Treponemas can be detected by silver impregnation method, dark field and phase contrast microscopy.

Cultivation

  • T. pallidum are extremely fastidious microaerophilic bacteria that maintain viability in presence of 1-4% oxygen. They can’t propagate in ordinary media.
  • When cultivated at 37°C on rich artificial media with ascitic fluid and brain tissue under anaerobic conditions they gradually lose their virulence (cultural treponemas).
  • Nevertheless, T. pallidum grows well and maintain virulence by animal inoculation, e.g., in rabbit testicular tissue (tissue treponemas). However, rabbit infectivity test is long lasting and requires from 3 to 6 months for cultivation.
  • Cultural and tissue treponemas demonstrate various antigenic properties.

Biochemical properties

  • The bacteria have slow metabolism, which is not ascertained in details. Genome sequence revealed treponemas to be unable to synthesize necessary growth factors (enzyme cofactors, fatty acids, nucleotides, and others).
  • On the contrary, treponemas carry multiple transport proteins, specific to various substrates, to compensate the lack of nutrients. Carbohydrates serve as energy source in microbial metabolism owing to the presence of all glycolytic pathway enzymes in bacterial cell.
  • T. pallidum doesn’t produce superoxide dismutase, catalase, or peroxidase.

Antigenic structure

  • Antigenic characteristics of T. pallidum are also not completely elucidated. Bacteria are considered to have many lipid and protein antigenic substances mostly with haptenic activity.
  • More than 100 protein antigens have been found in treponemas. Among them three core proteins of endoflagella are similar with other bacterial flagellin proteins.
  • Lipid antigens include phospholipid cardiolipin that shows mimicry with bovine heart lipid antigens. It is important for syphilis serological diagnosis.
  • Multiple lipid and protein antigens of T. pallidum cause hypersensitivity reactions of host immune system.

Virulence factors

  • T. pallidum bears membrane proteins that may function as porins and adhesins. These microorganisms are not shown to produce LPS endotoxins or clear exotoxins, but can develop cytotoxic activity against various cell cultures.
  • Bacteria render hemolytic activity encoded by genes of five hemolytic proteins, and may produce hyaluronidase, which promotes microbial invasiveness.
  • Pathogenesis of syphilis is closely associated with host autoimmune reactions triggered by microbial antigens.

Resistance

Spirochetes are very sensitive to drying, heating, and action of chemical disinfectants. For instance, heating at 55°C kills them in 15 minutes. Nevertheless, they stay viable for a meaningful time in tissues especially at low temperatures. For example, bacteria survive for one day and even more in blood or plasma, stored at 4°C.


Pathogenesis and Clinical Findings in Syphilis


  • Syphilis is an anthroponotic disease with cyclic chronic course. It is an actual example of social disease, where economic and social conditions, the state of healthcare service as well as personal lifestyle and mode of behaviour play decisive role in disease spread.
  • Syphilis is transmitted predominantly by sexual intercourse; transmission by direct contact or via medical manipulations seems negligible.
  • T. pallidum penetrates through small lesions in the skin or mucosals. Infectious dose for disease is minimal: as little as 1-5 microbial cells can cause the disease.
  • Incubation period depends on inoculated dose. A large inoculum, e.g., about 107 bacterial cells, results in disease appearance in 5-7 days.
  • There are several consequent stages in syphilis course.
  • After 7-90 days of incubation with an average of about 3 weeks a hard chancre, essential tissue lesion of primary syphilis, appears. It is followed by regional lymphadenopathy.
  • Chancre evolves at the primary site of microbial entry. In men it usually affects penis. Anorectal chancres emerge in homosexual men.
  • In women it predominantly occurs on vulva. Hard chancre is a painless ulcer about 0.5-3 cm with sharp margins, clean base, induration, and sometimes with purulent discharge.
  • In most cases chancre heals spontaneously within about 6 weeks. Nevertheless, in several weeks the disease comes into the stage of “secondary syphilis”, which results from lymphogenous and hematogenous microbial dissemination.
  • Secondary syphilis is characterized by skin rashes, headache, fever, malaise, lymphadenopathy, mucosal lesions, and CNS disorders. It lasts from 2-3 months to more than 1 year.
  • Primary and secondary syphilitic lesions contain great amount of spirochetes, being highly infectious.
  • In the secondary stage specific immune reactions against spirochetes arise (seropositive syphilis). Primary syphilis is regarded as seronegative, but the end of primary syphilis might be seropositive as well.
  • Meanwhile, hypersensitivity response doesn’t provide complete microbial elimination without antimicrobial treatment; and after latent period of various duration (about 1 year and more) tertiary syphilis develops.
  • Tertiary syphilis affects various body’s organs and tissues, especially cardiovascular system and CNS. Syphilitic aortitis damages the ascending aorta. It may happen between 10 and 30 years after primary infection.
  • Specific slow indurative injuries (gummas) emerge in central nervous system and parenchimatous organs. They are followed by necrosis and connective tissue proliferation.
  • The latest period of disease is characterized by profound CNS disorders (neurosyphilis). This period is usually regarded as seronegative because spirochetes are absent in bloodstream and antibody titers are low.
  • The disease results in meningovascular syphilis, pareses, and tabes dorsalis. Tabes dorsalis ensues from the severe injury of dorsal roots and columns of spinal cord.
  • Congenital syphilis results from vertical disease transmission from mother to fetus with a rate of 70 to 100% for primary syphilis. Congenital syphilis influences pregnancy outcome, thus it is often followed by spontaneous abortion, or perinatal death.
  • The infected infants may be asymptomatic or show various early and late manifestations, such as lymphadenopathy, hepatomegaly; skeleton and teeth lesions, CNS disorders like deafness (Hutchinson’s triad), asymptomatic neurosyphilis, etc.
  • The immunity in syphilis is not sterile. It always causes patient’s hypersensitivity. Immune response usually doesn’t prevent disease progression, but autoimmune reactions accelerate tissue damage aggravating the disease course.


Laboratory Diagnosis of Syphilis


  • Laboratory diagnosis rests on microscopical examination of lesion specimens for treponemas, and/or serological tests for specific antibodies.
  • Microscopy is the main diagnostic method for primary syphilis diagnosis. It also may be used in secondary syphilis.
  • Specimens are collected from chancre discharge, rash elements, lymph node aspirates, etc.
  • Romanowsky-Giemsa stain, silver impregnation, dark field microscopy and direct fluorescent-antibody testing for T. pallidum (DFA-TP) are used.
  • Serological testing is the cornerstone for laboratory diagnosis for latent, secondary, and tertiary syphilis.
  • The methods of analysis include nontreponemal and treponemal reactions.
  • Nontreponemal reactions are employed for mass screening, whereas treponemal tests are confirmatory.
  • Widespread nontreponemal tests comprise two similar reactions – Venereal Disease Research Laboratory test (VDRL test) and Rapid Plasma Reagin test (RPR test).
  • Both tests are based on flocculation reaction. In these methods a complex antigen containing lecithin, cholesterol, and purified cardiolipin is used to reveal host antibodies against cardiolipin that arise in syphilis.
  • The method is cheap and rapid but of limited sensitivity; and it can give false-positive data in autoimmune diseases, patients with malignancies, tuberculosis, leprosy, viral and parasitic infections, pregnancy, etc.
  • Wasserman reaction devised from complement fixation test can use both nontreponemal cardiolipin antigen and specific treponemal antigens for detection of anti-treponemal antibodies.
  • Treponemal tests comprise serum fluorescent treponemal antibody absorption test, as well as T. pallidum immobilization test, microhemagglutination test and ELISA for detection of antibodies against T. pallidum.
  • These tests use pathogenic killed or live T. pallidum cultures or the filtrate of virulent tissue treponemas as an antigenic source.
  • ELISA test is regarded as the most convenient and universal for routine laboratory diagnosis of syphilis.
  • PCR for T. pallidum DNA is a swift, sensitive and reproducible method; it becomes available now in wide clinical practice.
  • Clinical diagnosis of primary syphilis is confirmed by positive results of microscopy and/or positive results of one nontreponemal and one treponemal test.
  • Secondary syphilis is diagnosed by positive data from one nontreponemal and one treponemal test.
  • Tertiary syphilis should be confirmed by two or more treponemal tests.
  • Congenital syphilis is diagnosed by clinical, serological, and direct microscopic methods. Detection of immunoglobulin M antibodies by fluorescent treponemal antibody absorption test or ELISA confirms the diagnosis.

Treatment and prophylaxis of syphilis


  • As T. pallidum has no genetic resistance to beta-lactam drugs, benzylpenicillin and its long-acting derivatives (e.g., benzathine penicillin) remain the drugs of choice for syphilis treatment. Tetracyclines and macrolides can be used in case of patient’s allergy to beta-lactam antibiotics.
  • Prophylaxis of syphilis is non-specific. It requires public education, screening for syphilis, timely recognition of syphilitic cases, their adequate treatment, and improvement of socioeconomic conditions.