A review of the biology and the laboratory diagnosis of Treponema pallidum infections.
Treponema pallidum has been infecting the human population for centuries, causing the disease known as syphilis. Untreated syphilis infections can result in serious disease sequelae including increased morbidity and mortality associated with cardiovascular, neuronal, and general systemic complications. Syphilis is an eradicable disease because of its lack of a known animal reservoir and its ability to be diagnosed and easily cured (1). Despite these factors, syphilis remains a significant public health burden globally, and infection rates have begun to rise in developed nations within the past decade. The potential complications of syphilis and recent rises in infection rates among vulnerable populations provide evidence that medical professionals need to better understand the available diagnostic methods and disease management options in order to provide more effective patient care. Here, the possible clinical manifestations of syphilis are outlined and the most common diagnostic methods are reviewed. In particular, the recommendations made by a meeting of experts from the Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories (APHL) to improve laboratory diagnosis of syphilis are emphasized (2) .
A newly recognized disease plagued Europe in the 15th century (3). Due to the coincidence of this epidemic and Columbus's return from the New World in 1493, many believe that the infectious agent responsible for this new illness was brought back from the Caribbean by Columbus and his sailors (1). Conversely, supporters of the "Old World Hypothesis" believe that the disease originated in central Africa and was present in Europe before Columbus's voyage in 14924. Regardless of its origin, the disease spread through Europe at a rate which many believe was augmented by the era's wars and the subsequent travel of military personnel (1). By 1498, the disease had reached India and in 1505, China joined the many countries which had fallen victim to the illness (5). Because of the wide distribution of this infectious illness of unknown origin, the disease was given a variety of names. In order to differentiate this infection from smallpox, many referred to the disease as "Great Pox" (5), while more colloquial names such as the Spanish, German, Italian, Polish, or the French disease were also used due to the cultural embarrassment associated with the disease and the desire to blame rival nations (5). The term "syphilis" was ultimately the name that remained throughout centuries to identify the illness. Historically, it is believed that the name originated from a poem written by Hieronymus Fracastorius in 1503, entitled Syphilis Sive Morbus Gallicus, which translates as "Syphilis or the French Disease" (5).
It was not until four centuries after the disease's debut in Europe that scientists and public health professionals were successful at identifying the etiologic agent responsible for the infection. Researchers believed for many years that gonorrhea and syphilis were the same infection. This confusion was, in part, caused by an experiment conducted by John Hunter, in which he allegedly inoculated himself with exudates from a lesion of a patient suspected to have syphilis. After developing combined symptoms of both syphilis and gonorrhea, Hunter concluded that the diseases were in fact caused by the same infectious agent. Unfortunately, the patient had in fact been infected with both gonorrhea and syphilis, thus confusing his findings. It was not until 1838, when Philippe Ricord completed a study based on 2,500 human inoculations, that syphilis and gonorrhea were accepted to be distinct infections (3,5.) In 1905, Schaudin and Hoffman identified spirochetes in Giemsa-stained smears from the fluid of syphilitic lesions, demonstrating the association between syphilis and the infectious organism now called Treponema pallidum (5).
Treponema pallidum subspecies pallidum, the bacterial agent responsible for the disease syphilis, is a member of the order Spirochaetales, the family Spirochaetaceae, and the genus Treponema (6). Also in this genus are three other human pathogens: T. pallidum subspecies pertenue, the causative agent of the disease known as yaws; T. pallidum subspecies endemicum, which is responsible for nonvenereal endemic syphilis, also known as bejel; and T. carateum, which causes the disease called pinta (4, 6.) All four pathogens have similar morphology, a high degree of DNA homology, and comparable antigenicity. These characteristics make it possible for other species and subspecies of treponemes to cross-react in T. pallidum diagnostic tests. However, differences such as unique genomic sequences, pathogenic potential, host tissue specificity, and geographic distribution make it possible to distinguish between these organisms and the diseases they cause (1). T. pallidum subspecies pallidum, and therefore venereal syphilis, has a worldwide distribution, whereas T. pallidum subspecies endemicum is frequently found in the more arid areas of Africa and the Middle East. T. pallidum subspecies pertenue is more commonly found in tropical or desert regions of Africa, South America, and Indonesia, while T. carateum is found in the semi-arid, warm areas of Central and South America (4, 7).
Morphology and Structure
As with all spirochetes, the members of the Treponema genus have a unique bacterial morphology, for which these pathogens are named. T. pallidum is unicellular and has a slender, tightly coiled, helical morphology (4, 6). Due to its resemblance to a twisted thread, this pathogen was given the name "Treponema," a term with Greek origins roughly translating as "turning thread" (6). The name "pallidum" refers to the pale color of the bacterial body. On average, the cellular helix winds around six to fourteen times and is observed to have pointed ends, which differ from the normal hook shape that is characteristically seen in other spirochetes (5). The organism measures approximately six to fourteen micrometers in length and is 0.25 to 0.3 micrometers wide (6). It is enclosed within a cytoplasmic membrane, which is then loosely surrounded by an associated outer membrane (3). Between these two membranes lies a thin layer of peptidoglycan, which provides structural stability to the organism (3). Due to the nature of the peptidoglycan layer, this bacterium stains Gram-negative but because of the width of the organism, T. pallidum cannot be visualized using normal light microscopy.
Pathogenesis and Virulence Factors
Mode of Infection: T. pallidum is unique in that it has no known animal reservoir (3). Instead, it maintains its life-cycle through its pathogenic relationship with humans. The primary transmission route between humans is through sexual contact, where the pathogen enters the human host through compromised skin or by invading intact mucosal layers (4). The second most common mode of transmission is from mother to infant, most often when treponemes pass through the placental lining resulting in transmission to the fetus. Other, less common routes of transmission include contact with contagious lesions, often by health care workers or infants during the delivery process, contaminated blood transfusion from donors positive for syphilis, and participation in needle-sharing practices (4).
Host-Dependence: The lack of any other natural animal reservoir reinforces T. pallidum's dependence on its human host. In large part, this dependence stems from the limited metabolic capabilities of the organism's small genome (3). The 1.14 Mb circular chromosome of T. pallidum encodes a little over 1,000 putative proteins and includes the enzymes required to carry out glycolysis (1). However, the enzymes necessary for both the tricarboxylic acid (TCA) cycle and those involved in the electron transport chain are lacking. In addition, the T. pallidum genome is also lacking the necessary components for amino acid and fatty acid synthesis pathways (4). Due to these limitations, the organism is incapable of synthesizing the nutrients it requires and is therefore completely dependent on its host for its nutritional requirements.
Virulence Factors: The T. pallidum genome does not encode for some of the more common virulence factors found in pathogenic bacteria (3). For example, T. pallidum does not express lipopolysaccharide (LPS), the endotoxin commonly found in Gram-negative bacteria that is responsible for many of the damaging effects of infectious by these organisms (3,8). Additionally, there are no true cytolytic enzymes or cytotoxins encoded in the genome (3). However, the organism does produce other lipoproteins that may induce inflammation. Many researchers believe that such a host inflammatory response is responsible for the tissue damage seen during T. pallidum infections (3).
Despite the apparent lack of the more common virulence factors seen in other pathogenic bacteria, T. pallidum is not without mechanisms for enhancing its pathogenic potential. For example, T. pallidum displays multiple immune evasion techniques, which assist the pathogen in escaping recognition and clearance by the host immune system. Importantly, the organism has few antigenic targets on its surface, particularly lacking the outer membrane proteins that are often targeted by the innate immune cells (1,8,9). For some of the few antigens that are present, studies have demonstrated the potential for gene conversion mechanisms that allow for antigenic variation of these particles, which would help prevent immune recognition (1,8). However, T. pallidum's lack of surface antigens and antigen variation are not the only mechanisms by which the organism remains hidden from the immune system. In host tissues, the number of organisms is low, most likely due to the slow metabolism of the organism, thereby allowing T. pallidum to maintain a chronic state of infection without reaching the critical antigenic mass necessary for recognition and elimination by the immune system (3).
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While immune evasion is an important factor for the pathogenesis of T. pallidum, there are multiple barriers that must be overcome before the organism even encounters immune effector cells. Primarily, the organism must be able to invade and establish an infection in its host. As highlighted above, T. pallidum is capable of passing from host to host, invading both intact and compromised tissue (10). After invasion, the pathogen rapidly disseminates throughout the host and can spread to a variety of tissues (3). Research has shown that the spirochete is capable of attaching to epithelial, fibroblast-like, and endothelial cells, as well as capillary, kidney, and abdominal wall tissue in ex vivo studies (3,11). This attachment, which is thought to involve adhesin molecules on the treponeme and complementary ligands and extracellular matrix components of the host cell, facilitates the pathogen movement into the tissue (3,12). Moreover, studies have shown that nonpathogenic treponemes are unable to adhere to cells, further supporting the idea that successful adhesion plays a role in pathogenesis (3,11).
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Adhesion is not the only factor facilitating invasion; motility also helps the organism disseminate to and infect host tissue. Like all spirochetes, T. pallidum has endoflagella, made up of filamentous proteins, within the periplasmic space (3). Movement of these endoflagella rotates the organism along its longitudinal axis in a corkscrew fashion, which facilitates movement through gel-like matrices and allows the organism to invade tissues that other bacterial pathogens may not be able to infect.
After invading and infecting the host tissue, T. pallidum must be capable of surviving in the environment of the human tissue. Despite the fact that human blood is rich in iron, the amount of free iron is actually quite low. Within the human host, the majority of iron is tightly bound to hemoglobin and other proteins such as transferrin and lactoferrin. For many organisms, this resulting iron-deficient environment is enough to inhibit growth and pathogenesis. However, T. pallidum is able to overcome host iron sequestration due, in part, to the fact that very few of the pathogen's enzymes require iron and may instead use other metal co-factors, such as zinc or manganese, in order to function (3).
In the mid-1800s, Philippe Ricord first described the infection with T. pallidum, and the subsequent clinical manifestations, as having distinct stages which include primary, secondary, latent, and tertiary syphilis (3). Recognition of these stages is important not only because of their unique clinical manifestations, but also because of the implications such differences have for diagnosis, treatment, and prevention of both disease sequelae and spread.
The first stage of the disease is characterized by the appearance of a syphilitic lesion, or chancre, which usually appears approximately three weeks after exposure (3). While this is the typical time frame, studies have shown that the incubation period of syphilis can range from ten to ninety days. The chancre forms at the site of the treponeme inoculation and is usually a single lesion, although multiple lesions have been reported to occur at the same time (4). The lesion is most often painless, indurated, and a nonpurlent ulcer with a clean base that ranges in size from 0.3 to 3 centimeters. While these general characteristics are found in many cases, approximately 60% of cases present with atypical lesions. Chancres may appear on genital surfaces of both men and women, but it is not uncommon for the lesion to appear at an inconspicuous site, such as the pharynx, cervix, urethra, or rectum (4). These lesion sites, combined with the usual painless nature of the chancre, often result in the lesion going undetected and therefore undiagnosed (4). However, during this stage, infected individuals are highly infectious and are at high risk for transmitting disease. In addition to primary chancres, approximately 80% of primary syphilis cases present with regional lymphadenoapathy within seven to ten days after lesion formation. Within four to six weeks after chancre appearance, the lesion may spontaneously heal without treatment, but it is also possible for the lesion to remain through progression into secondary syphilis (3).
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During the initial stages of infection, T. pallidum rapidly disseminates to a wide variety of tissues (13). However, despite the rapid early spread of the organism, the clinical manifestations that are indicative of secondary syphilis are not usually apparent until three months after initial infection (3). These clinical symptoms can be quite subtle and can involve a variety of organ systems, making diagnosis difficult. A rash is the most common symptom of secondary syphilis, which can vary in severity and can include the palms and soles of feet (4). Typically, this nonpuritic rash is characterized by three to ten millimeter pink or red macules that are initially symmetrical (4). Without treatment, the rash will usually resolve over several weeks without scarring.
Other common symptoms of secondary syphilis include alopecia, inconspicuous secondary genital lesions which may go undetected (3), and the appearance of white gray mucous lesions, called condylomata lata, which range in size from five to ten millimeters (4). Condylomata lata usually appear in warm, moist areas, such as the perineum and anus, and are highly infectious (3). While not as common, secondary syphilis can involve multiple systems to varying degrees, resulting in a wide assortment of symptoms. General systemic symptoms include sore throat, headache, fever, muscle aches, weight loss, and either slight malaise to prostration and cachexia (3,5). Gastrointestinal symptoms can include anorexia, nausea, and occasional vomiting (5). Syphilis of the stomach can also occur, resulting in mu cosal erosions, rugal hypertrophy, or shallow ulcers. Syphilis of kidneys is possible but rare, as is syphilitic hepatitis, even though jaundice has been reported during secondary syphilis. Vague bone and joint pain can also be present, but osteitis and arthritis are rarely seen (5). T. pallidum infections can also cause ocular complications, involving almost any possible inflammatory process. Finally, bilateral tinnitus and deafness have also been reported in cases of secondary syphilis (5).
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Potential CNS Involvement: During these early stages of disease, there is the potential for the infection to penetrate the central nervous system (CNS) (3,14). After such invasion, there are a few possible outcomes. The majority of individuals with cases of CNS involvement are able to successfully clear or control the treponeme invasion (4). If this is not the case, it is possible for the invasion to develop into a condition known as neurosyphilis, which is defined as increased protein and leukocytes in the cerebrospinal fluid (CSF) as well as a positive, reactive serology test for syphilis (3). This neurosyphilis can be asymptomatic in some cases, but can also progress to more advanced disease states, such as syphilitic meningitis or late neurosyphilis (4). Syphilitic meningitis usually occurs within the first six months of infection or at the time of the secondary rash (4). Symptoms of actue early meningitis include fever, headache, nausea, vomiting, and a stiff neck. Other symptoms stem from cranial involvement, resulting in visual disturbances, hearing loss, facial weakness, ocular inflammation, and numbness or pain in the extremities (3,4). Memory loss and mental confusion can also be symptoms of early syphilitic meningitis, but are not usually seen (4). Late neurosyphilis is considered to be a tertiary complication of syphilis.
After the symptoms of secondary syphilis resolve, the patient may be asymptomatic for a vari are associated with increased mortality and decreased longevity (4). Late complications of syphilis, known as tertiary syphilis, can manifest as three general categories of symptoms. These categories are divided into gummatous syphilis, also known as late benign syphilis, cardiovascular syphilis, or late neurosyphilis.
Gummatous/Late Benign Syphilis: As early as two years after initial infection, patients infected with syphilis can present with gummatous syphilis, which is characterized by a primary lesion called a gumma (4). This lesion, which is granulomatous in nature, is a nodular lesion with variable central necrosis (3). Such lesions usually occur in locations such as bone or skin, and therefore, as the name implies, these late stage manifestations are usually benign (3). However, if lesions occur in other sites, such as the liver, heart, brain, stomach, or upper respiratory tract, their size and presence can disrupt normal tissue function, resulting in serious complications (3,4).
Cardiovascular: Clinical manifestations of tertiary syphilis can include the cardiovascular system, but this occurs much less commonly than other late complications. The most common manifestation of this rare occurrence is aortitis, which most often affects the ascending aorta (4). However, such involvement of the ascending aorta is often uncomplicated and is usually asymptomatic (4).
Neurosyphilis: Early treponemal invasion of the CNS can progress to late neurological complications resulting in multiple clinical manifestations. Within five to ten years of an untreated initial infection, patients can develop meningovascular syphilis. Such progression results in symptoms such as vertigo, insomnia, personality changes, loss of consciousness, and possible seizures. Additionally, there can be either widespread or focal arterial involvement, culminating in possible complications due to cerebrovascular accidents (3). Within two to three decades after infection, patients with syphilitic CNS involvement can develop late parencymatous syphilis. Symptoms can include general paresis, which is characterized by personality changes, emotional instability, memory impairment, hallucinations, and hyperactive reflexes. Such a disease state also presents with tabes dorsalis, which can result in sensory ataxia in the lower extremities, paresthesia, and sudden-onset of vomiting or abdominal pain (3).
Pregnant mothers may transmit syphilis to the fetus during any stage of disease, resulting in a condition known as congenital syphilis (3). However, according to a commonly observed principle, known as Kassowitz's Law, the longer the interval between infection and pregnancy, the more benign is the out able amount of time (3). This period, known as latent syphilis, can be divided into two different stages based on an approximation of the time of infection and the duration of time that a patient is asymptomatic before relapsing with symptoms of secondary syphilis. Early latent syphilis refers to the first year after infection, while late latent syphilis includes asymptomatic infections that maintain for either longer than one year or for an unknown duration (5). Approximately 90% of first relapses, or the recurrence of secondary manifestations, occur within one year, falling into the early latent syphilis stage, while 94% occur within two years, categorizing them as late latent syphilis (5). The remainder of relapses usually occurs over a four year post-infection period (5). During this time, infected individuals will test positive for syphilis, but in most cases, are unlikely to transmit the infection through sexual contact (3). One important exception is that pregnant mothers, when infected with T. pallidum, may infect the fetus during any stage of the disease, regardless of the presence of symptoms or not (3,15). Latent syphilis ends in all patients when either curative antibiotic therapy is administered or when manifestations of tertiary syphilitic disease present (3).
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Due to increased diagnostic and treatment methods, the natural progression of syphilis does not occur as frequently and is therefore more difficult to study and define. However, the combined results from three major clinical studies that were completed before current disease management were well developed have helped public health officials better understand the possible disease progression of syphilis in untreated patient populations (4). Generally, these studies uncovered that approximately one-third of patients with untreated syphilis present with late complications of the disease and that such infections come in the infant (5). Studies have shown that if a woman is infected with syphilis within one year of pregnancy, the result is almost always fetal infection (3, 5). Researchers believe the inflammatory response to the infecting T. pallidum results in adverse effects on fetal development. The most drastic results of such infection can lead to spontaneous abortion, stillbirth, or death of the neonate (3).
Even if the child survives the pregnancy, post-delivery complications of congenital syphilis include low-birth weight, pulmonary hemorrhage, secondary bacterial infection, and severe hepatitis (3). After the birth of an infected infant, there are two stages of congential syphilis, known as early or late manifestations. Early manifestations fall within the first two years of life, with symptoms appearing two to ten weeks after delivery (3). During this stage, the infant is infectious and presents with symptoms similar to adult secondary syphilis. "Snuffles," a symptomatic response to persistent rhinitis, is the most common clinical presentation during this stage, with skin lesions, condylomata lata, mucous patches, anemia, hepatosplenomegaly, renal involvement, and jaundice also being reported as possible symptoms (3). The late manifestation stage, which occurs after two years of life, is primarily characterized by interstitial keratitis. This condition presents with symptoms such as damage to the cornea and iris, tooth deformities, saddle nose, and saber shins. Another possible symptom of this late stage congenital syphilis is neurosyphilis, which can be either symptomatic or asymptomatic (3). While congenital syphilis is still a prevalent disease, antibiotic treatment of infected mothers during the first two trimesters can greatly decrease the risk of negative outcomes related to congenital syphilis (3).
Syphilis has been prevalent in global populations for many centuries and so to has the quest to develop effective treatment options. In 1497, mercury treatment was one of the first therapies to be used against syphilis infections (5,16). Over four centuries later, in 1909, Paul Ehrlich introduced the compound arsphenamine, more commonly referred to as Salvarsan, as an alternative therapy to replace mercury treatment. However, in order to reach a clinical cure using this treatment, infected individuals required repeated injections over an 18-month period. Such a regimen was associated with rather toxic effects; therefore, researchers examined whether other heavy metal compounds, such as bismuth, could provide an anti-syphilis therapy with less harmful side effects (5).
In 1917, Julius Wagner von Jauregg, a Viennese psychiatrist, introduced an alternative therapy for treating late neurosyphilis in T. pallidum-infected individuals (3). During this treatment regimen, patients were inoculated with malaria-infected blood, which resulted in a cycling of host-mediated fevers. These fevers, responsible for the basis of this heat-therapy, killed the infectious treponeme. The patients were then given quinine in order to treat the malaria infection. Even though this therapy ultimately killed approximately 10% of patients, it was used to treat neurosyphilis for more than a quarter of a century (3).
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With the discovery of penicillin in the mid-20th century, anti-syphilis treatment made a significant advance. In the early 1940's, syphilis was among the first infections to be treated with penicillin when John Mahoney et al. were able successfully to treat the disease in four patients (5,15-17). Treating with penicillin increases the rate of resolution of clinical manifestations of early infections and helps prevent both progression to later stages of disease and transmission to other individuals (1). To date, penicillin regimens remain the preferred treatment option for syphilis, but the dosage form and duration of therapy varies depending on the patient population and the stage of disease being treated (4). For patients with penicillin allergies, oral doxycycline is often recommended as an alternative treatment option (3).
Despite the effectiveness of current treatment methods, the global population has been unable to successfully eliminate the threat of this disease. Throughout the history of the relationship between T. pallidum and human populations worldwide, there have been multiple cycles of syphilis infections (5). In the United States, the incidence of primary and secondary syphilis fell from 66.4 cases per 100,000 persons in 1947 to 3.9 cases per 100,000 persons in 1956, a decline that many researchers attribute to the availability of penicillin, changes in sexual behavior, and improved public health measures (5). In 1999, the Centers for Disease Control and Prevention mounted the National Plan to Eliminate Syphilis from the United States (18). These efforts reduced rates to the lowest level in recorded history, when in 2000, syphilis rates reached an all-time low of 2.1 cases per 100,000 persons (5). Unfortunately, these numbers have begun to rise in the past decade. This rising trend in syphilis infection rates has been reported throughout global populations, and is in large part attributed to continued high prevalence in developing nations. In 1999, the World Health Organization (WHO) estimated that approximately 12 million new cases of syphilis arise each year, with more than 90% of these cases occurring in developing nations (15). However, the United States, despite public health outreaches and the availability of effective antibiotic treatment, is not immune to this rise in syphilis infections. Instead, syphilis rates in the U.S. are 50 to 100 times higher than in most other industrialized nations (5). In 2008, the rate of primary and secondary syphilis infections was 4.5 cases per 100,000 people, a rise of 18.4 percent compared to the previous year. The number of total cases reported in the United States, which included all stages of disease, rose by 13.1 percent from 2007 to 2008, with an increase from 40,921 to 46,277 cases (18).
Public health officials attribute the increases in syphilis in the United States to changes in the at-risk patient populations. Studies have shown an increased prevalence of syphilis in men who have sex with men (MSM), with more than 60% of new cases occurring in this population (4). Researchers believe this rise can be connected to increases in risk behaviors, such as unprotected sexual contact, possibly due to the development and use of more effective anti-retroviral therapies which reduce the perceived risk of infection with the human immunodeficiency virus (HIV), another common sexually transmitted infection (4).
The public health phenomenon of a shared at-risk population for both syphilis and HIV infections has significant implications for the rise in incidence in syphilis infections in the United States. Studies have shown that co-infections with both syphilis and HIV are common, due to these similar risk behaviors (4). Beyond similar behavioral risks, many researchers have concluded that infection with one pathogen directly increases the risk of infection with the second pathogen. According to the CDC, there is an estimated two- to five-fold increased risk of acquiring an HIV infection if exposed to the virus while infected with syphilis (18). Syphilis-induced genital tract inflammation or lesions can disrupt the innate mucosal barriers that help to prevent HIV infections, introducing additional portals of entry for the virus and increasing the risk of infection (3,4). Additionally, the immune cells that the virus targets to facilitate viral replication and dissemination are present in syphilis lesions in order to combat the T. pallidum infection, thus increasing the likelihood of viral infection (3). Furthermore, there is some evidence that lipoproteins expressed in T. pallidum actually up-regulate HIV replication rate (3). HIV infections also alter normal immune responses by decreasing the number of functional immune effector cells. This immunodeficient state creates an environment that is more favorable for T. pallidum infections, thus increasing the likelihood of infection (4).
Clinically, syphilis has been referred to as "the great imitator" due to variability of the symptomatic manifestations of the different stages of the disease. Many of these signs and symptoms are shared between multiple treponemal and nontreponemal diseases (19). For example, a clinical presentation of symptoms seen during primary syphilis can be explained by a differential diagnosis including balanitis, trauma, erysipelas, genital herpes, and chancroid. The systemic manifestations of secondary syphilis can often be considered as influenza-like symptoms, while the secondary lesions and rash can often result in a differential diagnosis including pityriasis rosea, rosacea, erythema multiforme, and psoriasis, especially when symptoms are atypical. Adding to the frustration, syphilis itself may be asymptomatic during many stages of the disease or the present chancres are painless, atypical, or located in an inconspicuous anatomical site, thus confounding the clinical diagnostic capabilities (19). Therefore, the key to clinical diagnosis is a high index of suspicion, due to patient history, and relies heavily on supplemental laboratory diagnostic methods (5).
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Laboratory techniques play an important role in the effective and efficient diagnosis of T. pallidum infections. However, there are multiple aspects of this infectious spirochete that make traditional diagnostic methods more difficult or impossible. The small size of the pathogen makes it difficult to visualize using the standard Gram-stained smears (4). Another obstacle for traditional laboratory diagnosis of syphilis is that T. pallidum cannot be successfully cultured (4). The organism does not survive outside of a mammalian host and loses its infectious capability within a few hours to days after harvest. This organism's limited growth has been partially attributed to its slow generation time, likely due to the pathogen's dependence on glycolysis for energy, its sensitivity to oxygen due to a lack of catalase and oxidase, which are necessary for protection against reactive oxygen species, and its limited stress response (3). While it is possible to propagate the pathogen in rabbits, this is a lengthy process and can only be achieved to limited quantities (3).
Because of these limitations in traditional diagnostic methods, the process of developing techniques to detect T. pallidum in patient specimens has relied heavily on two basic diagnostic approaches which have been modified and improved over the years. These diagnostic approaches are divided into direct detection methods and serologic techniques, which take advantage of antibody detection as an indication of T. pallidum infections. Serology diagnosis can be further divided into nontreponemal and trepomenal assays, depending on the type of antibody being targeted.
Direct Detection Methods for T. pallidum
Rabbit Infectivity Testing (RIT): While it is not a natural host for T. pallidum, rabbits can serve as an incubator for propagation of the spirochete. The disease can then be studied in infected rabbits, as it presents with clinically and histologically similar symptoms in rabbits as it does in humans with primary and secondary syphilis (3, 20). Patient specimens such as blood, CSF, amniotic fluid, primary and secondary lesion exudates, and lymph node aspirates can be used to inoculate rabbits, with a subsequent infection indicating a sample that is positive for syphilis. This assay is incredibly sensitive and specific, with the ability to detect as few as one to two organisms (2). However, disadvantages of this technique include a need for animals, availability in a limited number of research settings, a requirement for a very long incubation time after inoculation, taking several weeks to months for results to be reportable, and a variation in rabbit susceptibility to infection (2, 19). Therefore, the RIT is not considered to be a practical tool for syphilis diagnosis (2).
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Dark Field Microscopy: While T. pallidum is too small to be visible under normal light microscopy, dark field microscopy techniques allow sufficient visualization of the organism (21). This method becomes particularly important when the sensitivities of the serology tests fail to detect disease, such as during early syphilis or in immunodeficient patient populations (19). Using this method, the distinctive undulating movements of spirochetes can be identified, thus indicating a positive infection. However, care must be taken when using this microscopic technique, as other pathogenic and commensal spirochetes may present with similar motility. Therefore, the specificity of the method is dependent on the skill of the microscopist analyzing the sample. Because of this reliance on skilled personnel, many believe that the necessity for quality assurance becomes more critical and must be addressed by laboratory professionals on a national level (2,22).
Dark field microscopy can be used to detect organisms in specimens such as lymph node aspirate, CSF, amniotic fluid, and exudates of primary and secondary lesions, except oral lesions due to presence of spirochetes in the mouth other than T. pallidum (1,11). The sensitivity of this examination method approaches 80%, but depends largely on the state of lesion development. Despite this high sensitivity, a negative dark field test does not necessarily mean that the patient is free of syphilis (19). Negative tests can result from a variety of explanations, such as too rapid drying of the slide prep, too much fluid on the slide which makes the motility of the organism difficult to observe, refractile elements in the sample, and improper thickness of the cover glass making focusing difficult (19). However, the potential for the use of this technique as a point-of-care test is significant, and therefore should still be considered a valuable diagnostic tool (2).
Direct Fluorescent Antibody Test for T. pallidum (DFA-TP): This direct detection technique takes advantage of antibody-labeling with the use of fluorescent isothiocyanate (FITC)-labeled anti-T. pallidum amtibodies. In order to obtain the most treponemepathogen specific antibodies, mixtures of antibodies are filtered using non-pathogenic spirochetes, thus identifying and removing cross-reactive antibodies and reducing the risk of false-positive tests (19). The DFA-TP detection method can be used to test a variety of specimens, including lesion smears, concentrated fluids, tissue brushings, and fixed or unfixed tissue samples. It has a similar sensitivity as dark field microscopy, which depends largely on the concentration of organisms in the sample (2). The specificity of this test relies on the specificity of the antibody and can vary depending if polyclonal, mono-specific, or monoclonal antibodies are used to label the specimen. Despite the potential for this assay, there is currently no FDA approved DFA-TP test for the diagnosis of syphilis (2).
Polymerase Chain Reaction (PCR): Polymerase chain reactions use molecular technology to amplify specific nucleic acid sequences to the extent that these sequences can ultimately be isolated and their concentrations quantified. By using T. pallidum-specific primers for the amplification process, detection of the organism in patient samples can be completed with increased sensitivities of less than or equal to ten organisms, with some studies showing sensitivities of one to five organisms per specimen (2,19,23,24). However, there are disadvantages to this approach. Sensitivities can vary depending on specimen type, which can include lesion swabs, lymph node aspirates, CSF, blood, amniotic fluid, and fixed or unfixed tissue samples (2,25). Specificity can be altered by primer selection and skill of the technician, as well as by sample type, quality, and handling (2). Additionally, PCR-based methods are difficult to standardize between laboratories and interlaboratory differences can include source of primers and method of DNA extraction from specimens (19). An additional shortcoming of PCR detection for syphilis is that it was presumed that this methodology could not differentiate between dead and live organisms, thus resulting in incorrect reporting. However, recent studies comparing diagnostic techniques for T. pallidum infections in untreated individuals have demonstrated that positive PCR results are in fact indicative of an active infection (19).
To date, there are no commercially available PCR diagnostic tests for syphilis available in the United States and many believe that this condition must be rectified (2). Therefore, this approach continues to hold the interest of many laboratory diagnosticians. Multiple variations of traditional PCR methodology have been developed, such as multiplex PCR (MPCR), which uses PCR technology to simultaneously detect T. pallidum, herpes simplex virus type 1 and 2, and Haemophilus ducreyi in patient samples; reverse transcriptase PCR (RT-PCR), which targets the 16S rRNA from T. pallidum in order to detect the organism; and real-time PCR techniques targeting a variety of T. pallidum genes (19,26-29). However, as with traditional PCR methods, these tests have yet to be made commercially available.
Serology Testing for T. pallidum
Serological tests have been the principle tool for diagnosis of syphilis since the 1930s. However, serological diagnostic techniques used for the screening and diagnosis of syphilis are inherently flawed in that they rely on the presence of antibodies, either treponemal or nontreponemal, which may or may not necessarily correlate with the appearance or disappearance of pathogen (2). Therefore, it is common practice to use a combination of multiple tests, each targeting a different antigen or employing a different technical approach, in order to provide the most effective diagnostic method. However, much discussion has arisen from deciding which tests are most effective and in which order these assays should be used for screening and diagnosis.
Nontreponemal tests have been used to screen patients that are suspected of having or are at-risk for syphilis (4). These tests, also referred to as phospholipid antibody tests, use various techniques, such as flocculation tests or enzyme-linked immunosorbent assays (ELISA), in order to detect antibodies that target reagin, which is a complex of cardiolipin, lecithin, and cholesterol. This antigen is not unique to T. pallidum and is instead thought to be derived from the host and incorporated into the pathogen's membrane during infection, thus making the complex antigenic. Due to the nonspecific nature of this antibody-antigen interaction, assays that target anti-reagin antibodies are referred to as "nontreponemal" tests, while assays that monitor treponeme-specific antibodies are called "treponemal" tests.
As with all syphilis diagnostic techniques, nontreponemal tests have a specific set of disadvantages or flaws that are unique to the methods used in these assays. Due to the non-specific nature of the anti-reagin antibodies, non-treponemal tests are plagued with high rates of false-positives. Other conditions, such as tissue damage from recent or concurrent infectious diseases, autoimmune disorders, chronic inflammatory conditions or acute immune stimulation, pregnancy, or the aging process can also produce auto-antibodies that recognize the cardiolipin antigen and therefore give a false-positive result (3). False-negatives are also possible with these tests and can be explained by either very low antibody levels, which is more common during early or late stage syphilis, or very high levels of antibody (3,5,22). These high levels of antibodies, occurring most often during secondary syphilis, disrupt the optimal antibody to antigen ratio required for the agglutination tests, called a prozone phonemenon, and therefore give a false-negative test result (22). Finally, as indicated by the possibility of false-negatives during early and late stage syphilis, the sensitivities of these tests depend on the stage of disease at the time of diagnosis (3). In most cases, anti-reagin antibodies become detectable one to two weeks after the appearance of initial symptoms and tests therefore may not be positive until secondary stage syphilis (22).
Venereal Disease Research Laboratory (VDRL): The VDRL uses the modified cardiolipin antigen as a target. During the VDRL test, patient serum is mixed with the reagin antigen solution. If reactive antibodies are present, they bind to the antigen and flocculates form, which are visible using low magnification microscopy (30). Results of such a test can be reported as reactive, meaning medium to large clumps of antigen-antibody complexes were formed, weakly reactive, characterized by small clumps, and nonreactive or no flocculation (22,30). Such a report can be quantified by using serially diluted patient serum and the titer is reported as the greatest serum dilution to produce a fully reactive result (22). Due to the quantitative nature of this test, it is possible to use this assay both to detect syphilis and to evaluation the effectiveness of disease treatment (19). The VDRL is the only nontreponemal assay that can be used to test cerebrospinal fluid (CSF), due to the lack of sufficient specificity and sensitivity of other tests (31).
In 1987, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of IgG and IgM to the cardiolipin, lecithin, and cholesterol antigen used in the VDRL, a test that is referred to as VDRL ELISA (32). The VISUWELL Reagin test was then introduced as an indirect enzyme immunoassay (EIA) that was based on the VDRL ELISA (31,33). The benefits of this test include high sensitivities and specificities, as well as the potential for automated screening with the capacity for high volume (31).
Rapid Plasma Reagin (RPR): The Rapid Plasma Reagin assay is a simplified version of the VDRL. A modified version of the cardiolipin antigen complex, with the addition of carbon/charcoal particles, is placed on a disposable plastic card. Patient serum, potentially containing the nontreponemal anti-cardiolipin antibodies, is incubated with the card. A positive reaction results in flocculation of the carbon/charcoal particles, which allows for macroscopic interpretation of the results (19,22,34). Again, as with the VDRL, the results of the RPR assay can be both qualitative and quantitivate, using serial dilutions to obtain titers (19,22). Advantages of this test include its simplicity and its potential for automation, the antigen stability, macroscopic observation, and a slightly higher sensitivity compared to VDRL. Disadvantages stem from the fact that this technique is often not available at primary health care settings and thus diminishes its usefulness for widespread screening (1).
[FIGURE 9 OMITTED]
Historically, treponemal tests, which monitor levels of treponeme-specific antibodies, have been used in order to confirm positive results of nontreponemal test screening (4). There is a wide variety of different treponemal tests that are used for this purpose, suggesting that no one test is ideal for all stages of syphilis (19). Similar to nontreponemal tests, it is likely that an individual will be negative for treponemal antibodies during the earlier stages of disease. However, once reactive, patients who have been infected with T. pallidum usually remain seropositive for treponemal antibodies and will therefore test positive using treponemal diagnostic tests for life, regardless of treatment. Consequently, unlike nontreponemal tests, treponemal assays cannot be used to monitor treatment or confirm recovery (35).
T. pallidum Immobilization (TPI): The TPI, introduced by Robert Nelson, Judith Diesendruck, and John Eagen in 1949, was the first treponemal-specific serological test to be developed. This assay was originally based on the observation that T. pallidum organisms were immobilized in the presence of antibodies and complement (19). Therefore, in order to make this phenomenon useful in the diagnostic field, these researchers used serum from syphilis-infected patients in order to inhibit treponemal mobility in the presence of active complement (3). This assay was used until the 1980s, but its use was hindered by its complexity, the difficult to consistently reproduce results, and the high costs associated with running the test (19).
Fluorescent Treponemal Antibody test (FTA)/Fluorescent Treponemal Antibody Absorption (FTAABS): Due to the disadvantages outlined above, the TPI was largely replaced by FTA-ABS testing (19). The FTA and the FTA-ABS, which is simply a modified form of the FTA due to the addition of an absorption step, both use anti-human Ig antibodies labeled with fluorescein in order to detect host antibodies bound to T. pallidum organisms on a glass slide (36,37). The target spirochete, which is to be used as the antigen, is harvested from rabbit testis and then killed (22). Patient serum is then filtered during the absorption step, in order to remove any anti-treponemal antibodies that are not specific for T. pallidum. Any remaining antibodies recognizing T. pallidum antigens bind the organism during incubation and can then be labeled using the fluorescently tagged antihuman Ig antibodies. The intensity of fluorescence, measured using ultraviolet microscopy, is then described on a scale of zero to four. No fluorescence, or a score of zero, indicates a specimen negative for T. pallidum; a score of 1+ fluorescence is an equivocal result and requires retesting; and a score of 2+ or greater signifies a positive, reactive test (22). Due to the subjective nature of this assay, the results are not quantitative and therefore cannot be used to monitor treatment (22). With this test, there is the potential for false negatives to occur, due to a lack of an antibody response during the early stages of syphilis. However, the FTA or FTA-ABS is rarely negative when a nontreponemal screening test has already reactive as positive (22). Conversely, it is also possible for FTA or FTA-ABS results to be false-positives. These false-positives often occur for the same reasons nontreponemal false-positives occur and can usually be explained by the presence of some other condition that is producing cross-reactive antibodies that recognize the T. pallidum nonspecifically. However, compared to nontreponemal tests, false-positives are much less common when using a treponemal-specific test (22). Like the TPI, the FTA-ABS test is relatively subjective and labor intensive and has largely been replaced by the particle agglutination assay.
[FIGURE 10 OMITTED]
T. pallidum Hemagglutination Assay (TPHA)/T. pallidum Particle Agglutination Assay (TPPA): With a TPHA, red blood cells are coated, or sensitized, using antigens derived from a research strain of T. pallidum. These sensitized erythrocytes are then exposed to patient serum; if reactive antibodies are present, the red blood cells agglutinate in a visible fashion (3,22). This technique is simpler than that of FTA and has a relatively high sensitivity and specificity. However, this test is not sensitive enough for the diagnosis of primary syphilis, during which levels of anti-treponeme antibodies are low (3, 38). The T. pallidum particle agglutination assay is similar to the TPHA, but instead of red blood cells, biologically inert gel or latex particles are used (3, 22). The purified treponemal antigen is immobilized on these particles and then exposed to a patient sample. If reactive antibodies are present, particle agglutination will occur and a smooth mat of particles will be visible covering the bottom of the well. If the patient is negative for antibodies, the gel particles will settle at the bottom of the well in a characteristic "button" fashion (39). This assay produces fewer equivocal reactions than the TPHA and takes less time to complete, requiring only ten minutes for the particle agglutination compared to the two hours needed to complete the TPHA (3).
Enzyme Immunoassay (EIA): Treponemal EIA tests can be used to indirectly detect both IgM and IgG antibodies against T. pallidum. Multiple treponemal EIA tests have been developed and are variations of the basic technique of using a secondary antibody, linked with an enzyme, to label a primary, host antibody that is specific to the target antigen (31,34,40). Upon addition of substrate, an enzyme-mediate reaction occurs, thus indirectly signaling the presence of primary antibody. The benefits of the EIA assay are many and include the potential for automation, which increases the testing capacity for higher throughput volumes and removes the subjectivity of human analysis, and equal or better sensitivities and specificities when compared to other commonly used confirmatory tests (31,40,41).
Future Directions for Laboratory Diagnosis of Syphilis
In January, 2009, representatives from the CDC, the APHL, and various experts such as public health laboratorians and sexually transmitted disease (STD) researchers, clinicians, and program directors met in order to discuss the current diagnostic methods for syphilis. After extensively reviewing the relevant literature and addressing numerous concerns related to current techniques, this group of experts outlined a set of recommendations and research topics that they felt should direct the future of laboratory diagnosis of syphilis. These discussed recommendations, many of which are outlined below, have been used to up-date the official CDC Sexually Transmitted Guidelines for 201042 and offer valuable insight for readers of current, comprehensive reviews of syphilis diagnosis and disease management (2).
One point of discussion regarding the future of direct detection methods was the development of FDA-approved point-of-care (POC) tests for syphilis diagnosis. Researchers believe that dark field microscopy still holds potential as a valuable POC diagnostic tool and therefore its use should be expanded in the United States (2). Worldwide, over 20 rapid treponemal tests are commercially available (43). The World Health Organization (WHO) has begun a two-phase assessment of the performance of these rapid syphilis tests, beginning with the laboratory evaluation of tests from six different participating manufactures. Compared to TPHA or TPPA assays, sensitivities and specificities of these rapid treponemal tests ranged from 85-98% and 93-98%, respectively, with excellent overall performance compared to the reference standard using a kappa statistic (43). Reports on how these tests perform in a field-setting and their effectiveness in a disease control program has yet to be released.
A large part of serologic detection of T. pallidum relies on proper selection of diagnostic test. After reviewing current literature and discussing advantages and disadvantages of each diagnostic test, experts at the consultation meeting recommended that selecting the appropriate serologic test requires consideration of multiple factors. Not only should laboratories consider the performance of the test, the requirements for skilled professionals, cost, level of labor-intensity, and technical requirements, but characteristics of the patient population should also be considered. For example, the prevalence of the disease within the population and the diagnostic need of the patient, either for screening, confirmation, or disease management, are central to the test selection process. Consequently, high or low risk populations may require different screening systems, while patients at various stages of diseases may also have unique testing needs (2).
Because of the variability introduced by such conditions, experts suggested the use of multiple algorithms to dictate syphilis diagnosis. The use of the traditional algorithm has historically been supported by the cost-effectiveness of using nontreponemal tests to screen. However, in settings with high testing volumes, the potential for automation makes newer treponemal diagnostic assays, such as EIAs, more economically efficient and effective for screening (44). Therefore, researchers have suggested reversing the procedure, so that screening is done using treponemal tests and nontreponemal tests are used only as confirmatory assays. In addition to the potential for higher testing volumes, researchers believe that such modifications to the traditional algorithm would increase the objectivity, the sensitivity, and specificity to the screening process (2).
This theory has been put into practice by four different laboratories in New York, each using a modified algorithm for syphilis diagnosis (45). In each laboratory, a treponemal EIA test was used to screen at-risk patients. If the assay was nonreactive, the testing process was usually halted and the patient was considered to be negative for syphilis. If the EIA was reactive, the test was confirmed with a nontreponemal RPR. As with traditional algorithms, when a sample was reactive for both the EIA and RPR, it indicated a case of untreated syphilis, unless a history of prior treatment was documented. However, reversing the order of tests introduced a new combination of results. A CDC review of the results from these four laboratories showed that this reversed sequence of testing identified 3,664 (3%) of 116,822 specimens that were in fact reactive for treponemal antibodies, but not for the RPR (45). The traditional screening algorithm, in which testing would have been halted after the negative RPR, would have failed to identify this patient population. Interpreting this previously missed category of results is somewhat more difficult because it could either represent a patient population previously infected with syphilis, which may or may not have been treated, or could indicate the presence of late latent cases of syphilis (45).
Due to the ambiguity of this interpretation, researchers admitted that using modified screening procedures could lead to over diagnosis and over treatment (45). Therefore, accurate patient histories regarding disease and treatment, as well as further laboratory testing using different treponemal tests before confirming diagnosis, become even more essential. Additionally, such changes in procedures must be implemented only when the specific demands of the patient population served by the laboratory necessitate a change in traditional screening approaches (2).
With rising rates of syphilis infections not only in the United States but also in the global population as well, it is imperative that public health professionals take action to both improve diagnosis and treatment of T. pallidum infections and to prevent further spread of the disease. An advanced understanding of the clinical presentations of the disease, which can vary by stage of disease and by individual, is a prerequisite for any proposed action. Recognition of the clinical disease, especially in at-risk patient populations is necessary in order to indicate the need for supplemental laboratory diagnosis. Laboratory professionals must be prepared to address each individual case critically, as unique combinations of confounding factors may call for a modification of traditional diagnostic approaches. Being aware of the advantages and disadvantages of various diagnostic techniques under different clinical and laboratory settings will allow laboratory professionals to improve the accuracy and efficiency of syphilis diagnosis and therefore help provide more effective patient care.
Questions for STEP Participants
1. Long, slender, helically curved, gram-negative bacilli that present with tight coils are identified as belonging to which species?
2. Which of the following statements is true for congenital syphilis infection?
A. The disease is virtually nonexistent in the United States.
B. It can be effectively prevented by proper screening of expectant mothers.
C. The disease does not result in any long-term effects past the first year of life.
D. It is never life-threatening.
3. Systemic symptoms such as fever, weight loss, malaise, and loss of appetite can be present along with a widespread rash can be seen in patients suffering from which if the following?
A. primary syphilis.
B. secondary syphilis.
C. tertiary syphilis.
D. terminal stage syphilis.
4. Which of the following is a widely used nontreponemal serologic test?
5. A stage of venereal syphilis characterized by tissue destruction, central nervous disease, cardiovascular abnormalities, eye disease, and granuloma-like lesions is known as which of the following?
A. primary syphilis.
B. secondary syphilis.
C. tertiary syphilis.
D. quaternary syphilis.
6. Which of the following is a nontreponemal serologic test in which soluble antigen particles are coalesced to gtform larger particles that are visible as clumps when they are aggregated by antibody?
A. NTF (nontreponemal flocculation).
B. FTA-ABS (fluorescent treponemal antibody absorption) test.
C. VDRL (Venereal Disease Research Laboratory) test.
D. TP-PA (T. pallidum particle agglutination) test.
7. A specific treponemal serologic test performed by overlaying whole treponemes that are fixed to a slide with serum from patients suspected of having syphilis and using fluorescein-conjugated antihuman antibody reagent to detect specific antitreponemal antibodies is called which of the following?
A. RPR (rapid plasma reagin) test.
B. FTA-ABS (fluorescent treponemal antibody absorption) test.
C. VDRL (Venereal Disease Research Laboratory) test.
D. TP-PA (T. pallidum particle agglutination) test.
8. Which of the following is a test that utilizes gelatin particles sensitized with T. pallidum antigens to detect specific antitreponemal antibodies in patient serum?
A. RPR (rapid plasma reagin) test.
B. FTA-ABS (fluorescent treponemal antibody absorption) test.
C. VDRL (Venereal Disease Research Laboratory) test.
D. TP-PA (T. pallidum particle agglutination) test.
9. Primary syphilis includes which of the following symptoms?
A. fever, weight loss, malaise, loss of appetite, and a widespread rash
B. the appearance of a chancre at the site of inoculation and dissemination of the organism.
C. tissue destruction, central nervous disease, cardiovascular abnormalities, eye disease, and granuloma-like lesions.
D. subclinical but not necessarily dormant, at which time diagnosis can be made only by serologic tests.
10. Proper selection of the diagnostic test used for serologic detection of T. pallidum requires consideration of which of the following?
A. the performance of the test
B. technical requirements for the test
C. prevalence of disease within the population
D. all of the above
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Margaret Powers-Fletcher, PhD Candidate, Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio
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|Title Annotation:||Article 380: 2 Clock Hours|
|Publication:||Journal of Continuing Education Topics & Issues|
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