A Brief History of Laboratory Diagnostics for Syphilis
The Beginning of Laboratory Diagnosis of Syphilis: Direct Detection of Organisms using Microscopy
During the 500 years after syphilis was first described in Europe, no progress was made in specific laboratory diagnostics. It wasn’t until shortly after the turn of the 20th century that the etiologic agent of the disease, Treponema pallidum, was first observed in diseased tissues by German zoologist Fritz Schaudinn and dermatologist Erich Hoffman. With the identification of T. pallidum, scientists knew what to look for when developing tests to aid in the diagnosis of syphilis. Below is a photomicrograph of T. pallidum spirochetes in an infected testicular tissue, stained with modified Steiner silver stain method. This is probably not exactly what Schaudinn and Hoffman saw, but a good example of how a diseased tissue can be infested with a large number of spirochetes when examined under a microscope.
The year 1906 saw a major breakthrough in laboratory testing for syphilis when Austrian-born American immunologist Karl Landsteiner, who had made a splash in the field of hematology just a few years prior with his discovery of the ABO blood groups, along with his dermatologist colleague Viktor Mucha, introduced the use of dark-field microscopy to detect the presence of syphilitic treponemes in infected specimens.
This new technique was an important advance but ultimately proved to have significant limitations. Direct detection via microscopy works reasonably well in primary syphilis with sensitivity of approximately 80%, as high numbers of treponemes are often visible in tissue specimens from the chancre. However, darkfield sensitivity declines as the infection progresses and can also decrease if the patient has been treated with topical antibiotics. Additionally, this microscopy method cannot be used to visualize treponemes in specimens collected from the mouth, as the mouth harbors many species of non-pathogenic treponemes that are morphologically indistinguishable from T. pallidum, resulting in false positive results.
Treponemes may also be observed in specimens collected from non-oral mucosal lesions and condylomata lata (secondary syphilitic lesions that are usually smooth, moist, and flat). Nasal discharge specimens from newborns with snuffles (syphilitic rhinitis) collected from congenital syphilis cases are often teeming with treponemes, which can also easily be visualized with a dark-field microscope. Below is a photomicrograph taken using dark field visualization technique, demonstrating the presence of T. pallidum spirochetes. Those who are curious about what these conditions and lesions look like may go to the CDC Public Health Image Library and search using “syphilis” as a keyword.
The sensitivity of direct visualization assays depend largely on proper specimen collection, which involves obtaining serous exudates from lesions while avoiding blood or pus that may be close to or even over the lesion, followed by placement of collected specimens on a slide which needs to be examined with a dark-field microscope within 20 minutes after collection. The method has largely been abandoned in routine clinical practice due to the requirement for a special microscope and a trained microscopist close to the site where specimens are collected. As a result, most healthcare facilities and clinical laboratories do not have the capability to perform this type of testing.
In Germany around the same time, bacteriologist August Paul von Wassermann was working with 2 dermatologists/venereologists, Albert Neisser (after which Neisseria gonorrhoeae was named) and Carl Bruck, to develop the first serologic test for the diagnosis of syphilis. Their assay, published in 1906, utilized the complement fixation method developed by Belgian scientists Jules Bordet and Octave Gengou. (One brownie point to anyone who can comment with the organism that was named in honor of Bordet, which grows in the culture medium developed by these 2 microbiologists.) The presence of antibodies “specific” to syphilis in patients’ sera, aptly named Wassermann antibodies at the time, would lead to formation of immune complexes and depletion of complement factors, resulting in indicator erythrocytes remaining intact (a positive Wassermann reaction.) The figure below depicts the Wassermann test and how to interpret results based on the observation of red blood cells.
Wassermann and colleagues tried a few different antigens for the assay, and a liver extract of a syphilitic fetus was much more reactive compared to other diseased monkey or human tissues. Upon initial evaluation, the assay was exclusively positive on sera from syphilis patients, but further testing of more specimens began to reveal false positivity among non-syphilitic individuals. It was not until 1907 when Landsteiner elucidated the previously unknown mechanism behind the Wassermann reaction via identification of lipoid “autotoxic” substances, with which Wassermann antibodies form immune complexes in the presence of lecithin and cholesterol. The identification of these substances in non-syphilitic tissues including the heart suggested that these antibodies, named “reagin" by Landsteiner, were not specific to T. pallidum; an assay to detect these antibodies, now known as anti-phospholipid antibodies, would be considered what we now call a non-treponemal test. It was discovered later that reagins are also produced in response to acute or chronic non-treponemal diseases where tissue damage occurs.
Biological false-positive non-treponemal test results are associated with several non-syphilitic conditions, including other infections (such as Human Immunodeficiency Virus), autoimmune disorders, pregnancy, immunizations, injection-drug use, and older age. Despite its pitfalls, the Wassermann test was one of the first serological tests for the diagnosis of infectious diseases and the basis for subsequent non-treponemal tests for the diagnosis of syphilis.
In the 1940s, the major substance used as an antigen in the Wassermann test was identified as a diphosphatidylglycerol called cardiolipin, which is commonly found in normal non-syphilitic tissues, including the heart. Why the detection of anti-phospholipid, non-treponemal antibodies could indicate syphilis has been a subject of debate for the past 6 decades. A study in 2018 using a rabbit infection model demonstrated that T. pallidum itself contained a cardiolipin antigen but with weak immunogenicity, while active T. pallidum induced higher non-treponemal antibody production in the host. The increase in anti-phospholipid antibody production was suggested to be a result of the combined effects of both the T. pallidum cardiolipin antigen and the damaged host-cell cardiolipin antigen during syphilis infection, the latter of which probably plays a major role in this induction process.
Antigens used in older generations of the non-treponemal assays were crude extracts of diseased tissues processed using various extraction methods, making them difficult to standardize. In 1941, Mary Pangborn et al. published a method for isolation of cardiolipin and lecithin from beef heart. The use of a combination of purified cardiolipin, lecithin, and cholesterol as an antigen for non-treponemal antibody tests allowed for better standardization of assays and improved reproducibility within and between laboratories. Nowadays, synthetic cardiolipin and lecithin are used to prepare reagents for non-treponemal assays instead of extracts from beef heart.
Another technique that played an important role in the development of non-treponemal antibody testing for syphilis is flocculation, which was introduced by German scientists Hans Sachs and Walter Georgi. Flocculation occurs when antigens used in the assay form a complex with non-treponemal antibodies in a patient’s specimen, resulting in clumping of antigens that can be visually observed (macro- or microscopically). Non-treponemal tests utilizing flocculation were much less tedious than those relying on complement fixation, all of which became obsolete by the 1940s. Many non-treponemal tests based on the flocculation method have been developed. One of the first generation of flocculation tests was the Khan Test. This assay, published in 1922, utilized an antigen consisting of beef heart extract and cholesterol suspended in a salinized solution. In 1927, an American bacteriologist/pathologist, William Augustus Hinton, (who later became the first African-American professor at Harvard University) published an improved flocculation test for which the choleterolized beef heart extract antigen was suspended in a glycerinated saline solution instead of regular saline. This new assay saw an increase in sensitivity compared to the Khan test, and the method was adopted for routine patient testing for many years. (A second brownie point to those who can comment with the name of a culture medium that Dr. Hinton’s daughter, veterinarian Jane Hinton, co-developed while working as a laboratory technician at Harvard.)
The sensitivity and ease-of-use of reagin-type flocculation tests led to the development of many other tests that relied on similar principles. These included the Price Precipitation Reaction (PPR), the Unheated Serum Reagin (USR), Automated Reagin Test (ART), and the Toluidine Red Unheated Serum Test (TRUST). However, only the Venereal Disease Research Laboratory (VDRL) Test) and the Rapid Plasma Reagin Test (RPR) are still commonly performed in clinical and public health laboratories. Read about these assays in detail as part of the syphilis diagnostic algorithms discussed in the next article on syphilis diagnostics.
Treponemal Antibody Testing
The identification of T. pallidum as the etiologic agent for syphilis allowed scientists to develop diagnostic tests to detect the presence of treponemal antibodies. In addition to his work on identification of lipoid substances that serve as antigen in the Wassermann test, Landsteiner and his colleagues also reported that serum of syphilis patients inhibited the movements of T. pallidum. This observation formed the basis for the T. pallidum Immobilization (TPI) test, which was introduced in 1949 by American scientists, Manfred Mayer and Robert Nelson. Despite the complicated and tedious test procedures, TPI became one of the first treponemal antibody tests and was adopted as a confirmatory test for specimens positive for non-treponemal tests.
The fluorescent antibody test for syphilis was first reported in 1957 by Deacon, Falcone, and Harris. In this test, patient sera were incubated on slides that contained antigen smears prepared from an inactivated organism suspension, followed by washing and staining with a fluorescein isothiocyanate (FITC) conjugated antibody. The presence of treponemal antibodies would result in fluorescence of the organism on the slide. The first generation of the assay suffered from a high false positivity rate, potentially due to cross-reactivity with non-pathogenic Treponema species. To lower the false positivity rate, the investigators experimented with further dilutions of the specimens as an attempt to dilute out non-specific reactivity. However, this approach came at the expense of assay sensitivity. In 1964, the FTA with absorption procedure (FTA-ABS) was published. The addition of an absorption step, where the specimen was pre-treated with ultrasonically disintegrated non-pathogenic Reiter treponemes, resulted in a significant increase in specificity while retaining the sensitivity of the assay. FTA-ABS is still being performed in some clinical and public health labs as a treponemal test, but it may produce variable results due to variation in equipment, reagents and subjective interpretation.
The Treponema pallidum Hemagglutination Assay (TPHA) was introduced in 1966 by Takayuki Tomizawa and Shigeo Kasamatsu, who developed the test with a goal to simplify the process of treponemal antibody testing. In this test, patient sera were pre-treated with regular (un-sensitized) sheep red blood cells. Pre-treated sera were incubated with red cells that had been sensitized with antigen prepared from T. pallidum Nichols strain. Hemagglutination indicated the presence of treponemal antibodies in the specimen. A newer generation of this test is called the T. pallidum particle agglutination (TP-PA) test. This test utilizes the same antigen as TPHA, but gelatin particle carriers are used instead of red blood cells.
In addition to the tests mentioned above, many enzyme immunoassays (EIA) and chemiluminescence immunoassays (CIA) have been developed and are commercially available for the detection of treponemal antibodies. One of the advantages of EIAs and CIAs is that they can be automated for high-throughput screening, while minimizing human error and inter-operator variation. Interestingly, utilization of EIAs and CIAs has increased over the past few years due to the adoption of the reverse screening algorithm for syphilis among clinical and public health laboratories. This will be discussed in my next article on syphilis diagnostics.
What about Molecular Testing for Syphilis?
Many PCR assays have been developed and published, but the methods are not standardized, and none are available commercially . However, molecular testing could be useful in the diagnosis of congenital syphilis, neurosyphilis and early primary syphilis. The Centers for Disease Control and Prevention (CDC) perform molecular detection of T. pallidum in many specimen types, but providers are required to first consult with their health department and obtain approval to submit specimens to the CDC via their local or state public health laboratories.
For my next article in this series on syphilis diagnostics, I will discuss the advantages and disadvantages of non-treponemal and treponemal tests and recommended testing algorithm.
The statements and opinions expressed in this article are those of the author and do not necessarily reflect those of Los Angeles County Department of Public Health and Public Health Laboratories nor of the American Society for Microbiology.