Introduction

Syphilis is a sexually transmitted infection (STI) caused by the spirochete bacterium Treponema pallidum. There are different stages of syphilis: primary, secondary, and tertiary, which are defined by clinical manifestations of the disease. Those who are infected but are asymptomatic have latent syphilis. Without adequate treatment, neurological and cardiovascular anomalies may develop, and infection in pregnancy can lead to congenital syphilis. Thus, detecting syphilis promptly is crucial in preventing the progression and fatal consequences of the disease. Traditionally, rapid plasma reagin (RPR) and venereal disease research laboratory (VDRL) tests are the non-treponemal serologic tests used to screen for active syphilis infection and monitor response to treatment. Due to their lack of specificity, positive results are confirmed by tests that are treponemal-specific, such as fluorescent treponemal antibody absorption (FTA) test, T. pallidum enzyme-immunoassay (TP-EIA) test, T. pallidum particle agglutination (TPPA) test, and the automated chemiluminescent immunoassay (CLIA) test.[1]

Etiology and Epidemiology

Syphilis is a prevalent, sexually transmitted infection worldwide. It is caused by Treponema pallidum, a motile spirochete bacterium, with humans as its only natural host.[2] There were 5.6 million cases of syphilis globally in 2012, according to the World Health Organization (WHO).[3] There are an estimated 10 to 12 million new cases of syphilis annually. The risk of acquiring syphilis escalates with an increasing number of sexual partners. Since the late 1990s, an epidemic of syphilis has been seen in homosexual males.[2] There is also an increasing number of people who are co-infected with other sexually transmitted infections, such as the human immunodeficiency virus (HIV).[4] Additionally, early stages of syphilis have been recognized more often amongst heterosexual men and women in the United Kingdom (U.K.). In regards to congenital syphilis, there are an estimated 500,000 to 1 million new cases globally each year.[2]

Pathophysiology

T. pallidum enters the body through areas that have undergone “microtrauma,” most commonly mucous membranes. As such, sexual intercourse remains the most common route of transmission, including oral sex and oral-anal intercourse. The disease is most likely to spread while individuals have primary and secondary syphilitic lesions.[2] Less commonly, it can be spread through blood transfusions or organ donations from patients with active syphilis.[4]

Syphilis is classified by its clinical manifestations into primary, secondary, latent, and tertiary syphilis.

Primary syphilis is associated with the highest probability of transmission and is characterized by a single painless chancre, although multiple lesions may present in a minority of patients.[5] A macule develops at the site of inoculation and eventually transforms into a papule, which ulcerates. The incubation period between the time of inoculation and the development of a lesion is two to three weeks. At this stage, the lesion will heal spontaneously in approximately five weeks if left untreated.[2]

Secondary syphilis is defined by a maculopapular rash on the palms and soles bilaterally. Patients may also have bacteremia with fevers and fatigue. If secondary syphilis goes untreated, individuals improve within three to six weeks. Secondary syphilis recurs in approximately 25% of infected patients within 1 year.

Patients then progress to latent syphilis, in which they are asymptomatic but have positive serology. Latent syphilis is further classified into early latent and late latent syphilis. According to WHO, patients with early latent syphilis have been infected for less than two years, and patients with late latent syphilis have been infected for over two years.

Approximately 35% of patients with late latent syphilis will progress to tertiary syphilis, which is characterized by chronic end-organ damage several years after inoculation. This includes neurosyphilis, gummatous syphilis, and cardiovascular syphilis. In those with neurosyphilis, T. pallidum has traveled to the brain and spinal cord 5 to 20 years after primary syphilis. The dorsal columns are primarily affected. In tabes dorsalis or spinal cord syndrome, individuals exhibit ataxia, loss of vibration and proprioception, urinary and fecal incontinence, and absent limb reflexes. They may also show psychiatric disease and dementia. Gummatous syphilis manifests as granulomatous lesions of the bone or skin 3 to 12 years after primary syphilis. Cardiovascular syphilis is characterized by aortitis of the proximal aorta approximately 15 to 30 years after primary syphilis.

Approximately one-third of infants born to infected mothers are diagnosed with congenital syphilis. Syphilis can be transmitted to the fetus by a transplacental transmission during pregnancy if the mother is infected. This is more likely to occur within the first two years of the mother becoming infected. Spontaneous abortions and stillbirths occur in one-third of syphilitic pregnancies. Children with congenital syphilis commonly exhibit hepatosplenomegaly, abnormal dental development, nasal chondritis (“snuffles”), deafness, and craniofacial abnormalities.[2] Congenital syphilis can be prevented with antenatal screening and treatment during pregnancy.[5] The treatment for syphilis of any stage is parenteral penicillin.[2]

Diagnostic Tests

Serological testing is currently the primary method of diagnosing syphilis. The two types of serological tests are non-treponemal (non-specific) and treponemal (specific). Non-treponemal tests include RPR, VDRL, and toluidine red unheated serum test (TRUST). RPR and VDRL are most commonly used. Treponemal tests include TPPA, CLIA, and TP-EIA. Syphilis can also be diagnosed using dark ground microscopy of samples of genital lesions in those with primary and secondary syphilis.[2]

The Centers for Disease Control and Prevention (CDC) recommends using non-treponemal reagin tests as the first-line for screening for syphilis infection. The two main non-treponemal tests used are RPR and VDRL.[6] The RPR assay is 86% sensitive in primary syphilis, 73% sensitive in latent syphilis, and 100% sensitive in secondary syphilis.[3]

Testing Procedures

RPR card tests are non-treponemal flocculation assays that work by detecting reagin antibodies in plasma and serum. RPR antigen mixture is prepared with cholesterol, nonspecific cardiolipin, and lecithin. A 0.05 mL drop of serum is placed on a circle within a testing card for each assay. The serum is spread throughout the circle. One drop of prepared antigen suspension is then added to each specimen. The testing cards are rotated in a humidified chamber at 100 rpm for 8 minutes. The cards are then rotated by hand and read under a light source macroscopically. If IgM or IgG antibodies to lipoidal antigens are present, agglutination is visualized on the card.[3] The titers of samples with reactive results are first determined at 1:1. The titers are diluted in series in phosphate-buffered saline (PBS) to achieve an endpoint titer at a maximum of 1:16 dilution. Alternatively, for dilutions greater than 1:16, a 1:50 non-reactive serum-PBS solution is used. The endpoint titer is the maximum dilution at which aggregation is visible. Patients are at greater risk of neurosyphilis with RPR titers of greater than or equal to 1:32.[7]

Interfering Factors

False-negative results in non-treponemal testing can be attributed to several factors. The primary cause of false-negative results is testing during the early stages of primary infection.[7] False-negative results are also associated with HIV infection and testing during the late syphilis stages in patients who have gone untreated.[2][7] This may be explained by the “prozone effect,” in which the efficacy of antibodies to form immune complexes is impaired when extremely high concentrations of antibodies or antigens are present. Lastly, the manual RPR test is susceptible to subjective human error. Because testing depends upon the person reading the test, the results may be misinterpreted, and interpretation may vary from person to person.[7] False-positive results are associated with IV drug use, HIV infection, hepatitis B infection, and autoimmune diseases. In these circumstances, it is not unusual for titers to be 1:8 or lower.[7] This may lead to invasive testing and unnecessary treatments, including unwarranted lumbar punctures and the adverse consequences that come with it.[8] Additionally, since the antigens in RPR testing are also natural constituents of human cells, it is impossible to determine if the RPR test is measuring components released by bacteria, damaged host cells, or a combination of the two.[3] A false-positive result in non-treponemal testing occurs in 1% to 2% of patients in the United States.[7] Consequently, a specific treponemal test is used with RPR to minimize false-positive diagnoses.[3]

Results, Reporting, and Critical Findings

There are only two classes of RPR interpretation: reactive or nonreactive. Occasionally, it is necessary to repeat testing or report an “indeterminate” result, which requires further evaluation.[3] Reactive results of non-treponemal tests are confirmed with treponemal tests. Those with concurrent HIV infection are recommended to follow up with serologic testing for syphilis every 3 months for 1 year and at 2 years after treatment. Those without concurrent HIV are recommended to follow up with serologic testing for syphilis at 6 and 12 months.[7]

Syphilis is a reportable STI in the United States. The demographics, sex, ethnicity, and age of each patient are recorded. The CDC receives data on reportable STIs through the National Electronic Telecommunications System for Surveillance. Certain cases may be further investigated by the local health department, and additional risk factors such as men who have sex with men (MSM) and HIV infection may be reported. In the United States, the rate of reported cases of primary and secondary syphilis has significantly increased within the past 15 years. In 2001, there was a historic decline in reported cases with 2 cases per 100,000; however, the reported rate tripled in 2016 with 8.7 cases per 100,000.[9]

Clinical Significance

Because RPR is utilized as a first-line test to screen for syphilis infection, its use is significant in preventing the progression and sequelae of syphilis in its early stages. These sequelae include cardiac and neurologic conditions that may be fatal as a consequence of this disease. Additionally, the use of RPR in pregnant women is important in detecting and treating syphilis before transmission to the fetus occurs, resulting in congenital syphilis.[1] Although RPR testing is primarily used for diagnosing syphilis, it can also be performed for monitoring syphilis, as the reactivity of RPR tests usually declines after treatment. RPR testing can also be used to assess for reinfection. Positive serology in RPR testing usually occurs within three to six weeks of exposure to syphilis.[3]

There is a high prevalence of coinfection with syphilis in those infected with HIV in certain populations. Additionally, syphilis infection may facilitate the transmission of HIV. Thus, using RPR to screen for syphilis, especially in the primary and secondary stages, allows for public health officials and clinical providers to protect those who are HIV negative from acquiring HIV and to also identify and help those already infected with HIV living without medical care.[9]

Quality Control and Lab Safety

RPR testing is considered to be a relatively inexpensive and simple procedure. However, there are reports documenting a wide range of variability between laboratories. One report by Gupta et al. in 2009 found a 58.7% variation in RPR testing amongst 138 serology panels between 26 laboratories. This variability was attributed to several factors, including delayed testing, insufficient laboratory temperatures, use of different RPR card tests in various labs, and skewed interpretation of results. RPR testing requires the provision of standard resources and strict adherence to proper guidelines. This includes refrigeration to store antigens, monitoring laboratory temperatures and humidity, a light source, and sufficient electricity supply. There have been cautions regarding antigen susceptibility to sunlight, freezing, and thawing. Additionally, it is important to have properly trained personnel to interpret RPR results correctly.[3]

Of note, automated RPR testing is also available and has been noted to be able to handle a larger sample size and requires less reader subjectivity.[6] However, while automated non-treponemal testing has been suggested to obliterate problems with manual RPR testing, inconsistent results have been documented with automated testing in monitoring response to treatment.[3]

Enhancing Healthcare Team Outcomes

In addition to physicians, pharmacists have become more involved in screening patients for syphilis, thus allowing for more patients to receive treatment when necessary. This also prevents further transmission and complications of infection. Dried blood spot (DBS) testing is available for purchase in select pharmacies in the U.K. and the United States. Patients use a lancet on their fingertips to draw blood themselves and place the sample on a testing cassette.[10] Although patients self-collect their blood, they do not self-test.[11] Results are available within 15 minutes. The sensitivity and specificity of the kit are reportedly over 99%. In one study performed in England, pharmacists were trained to counsel patients and collect samples. They were advised to talk about testing to patients who were at high risk of syphilis infection, including those participating in needle exchange programs or undergoing substitution therapy for opiate abuse. While this method of syphilis screening is easily accessible to patients, it involves training and commitment from pharmacists. If not properly trained to handle testing, pharmacists are at risk of blood-borne infections. There is potential for an increase in DBS testing, but more evidence is needed to determine the success of syphilis screening in pharmacies.[10] [Level 2]

Other interventions to improve the number of conducted diagnostic tests include “task shifting,” in which specific clinical tasks are transferred to healthcare providers other than physicians. For instance, nurses are responsible for conducting syphilis screening in various health clinics in the United States, the Netherlands, and Australia.[11] [Level 5]