Investigations
1st investigations to order
dark-field microscopy of swab from lesion
Test
Performed to identify Treponema pallidum.
Can provide a definitive diagnosis of syphilis, but is not usually available outside specialist settings.[36]
The lesion is cleansed and abraded with a gauze pad until serous exudates appear, which are collected onto a glass slide for microscopic analysis.
A single negative result does not exclude infection; ideally 3 negative examinations on different days are required.
Primary syphilis: sensitivity of dark-field microscopy is 74% to 86%, specificity is 85% to 100%.[5][6][45]
Secondary syphilis: dark-field microscopy may be positive from ulcerative anogenital lesions.
Gummata in tertiary syphilis have few, if any, identifiable T pallidum organisms.
Congenital syphilis: test suspicious lesions (e.g., bullous rash or nasal discharge).[8]
Result
coiled spirochaete bacterium with a corkscrew appearance and motility
serum treponemal enzyme immunoassay (EIA)
Test
A treponemal serology test.
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active infection or past (treated) infection.
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a ‘reverse sequence screening algorithm’).[46]
False-positive results may occur with other non-sexually transmitted treponemal infection (e.g., yaws, pinta, bejel).
False-negative results may occur in incubating and early primary syphilis. It usually takes 3 weeks for an EIA IgG/IgM test to become positive after infection with Treponema pallidum.
EIA is the test generally used for screening.[4]
Primary syphilis: EIA sensitivity 82% to 100%, and specificity 97% to 100%.[73]
Secondary syphilis: EIA sensitivity is 100%.[73]
Late latent syphilis: EIA sensitivity is 98% to 100%.[73]
Result
positive
serum Treponema pallidum particle agglutination (TPPA)
Test
The TPPA test is the preferred treponemal test.[36]
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active or past (treated) infection.
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a "reverse sequence screening algorithm").[46]
Primary syphilis: TPPA sensitivity is 85% to 100%, and specificity is 98% to 100%.[74][75]
Secondary and late latent syphilis: TPPA sensitivity is 98% to 100%.[75]
Result
positive
serum Treponema pallidum haemagglutination (TPHA)
Test
A treponemal serology test.
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active or past (treated) infection.
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a 'reverse sequence screening algorithm').[46]
Result
positive
serum fluorescent treponemal antibody absorption (FTA-ABS) test
Test
A treponemal serology test.
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active infection or past (treated) infection.
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a 'reverse sequence screening algorithm').[46]
FTA-ABS is used less often than TPHA and TPPA because it is less specific.
Result
positive
immunocapture assay
Test
A treponemal serology test.
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active infection or past (treated) infection.
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a 'reverse sequence screening algorithm').[46]
Result
positive
line immunoassay (LIA) serological test
Test
A treponemal serology test.
A patient with a positive treponemal test result will remain positive for life. Therefore, a positive result alone cannot distinguish between an active or past (treated) infection.
LIA serological tests (e.g., INNO-LIA syphilis test) can be used to confirm syphilis infection following initial serological treponemal testing. A single LIA test can confirm infection, making it more convenient than traditional methods of serological confirmation (which usually require multiple assays). Studies evaluating the performance of LIA tests for syphilis infection have demonstrated higher sensitivity and specificity compared with FTA-ABS and TPHA serology tests.[54][55]
Result
positive
serum rapid plasma reagin (RPR) test
Test
A non-treponemal serology test.
Provides a quantitative measure of disease activity and can be used to monitor treatment response (RPR titers decrease or become non-reactive with effective treatment).
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a 'reverse sequence screening algorithm').[46]
Despite adequate treatment, some patients maintain a persisting low level positive antibody titre (known as a serofast reaction).[50]
False positives may occur due to the presence of a variety of medical conditions (e.g., pregnancy, autoimmune disorders, and infections).
A false-negative test may occasionally occur in an undiluted specimen (the prozone phenomenon).
Primary syphilis: RPR sensitivity is 70% to 73%.[5][76]
Secondary syphilis: RPR sensitivity is 100%.[76]
Preferred test over the serum Venereal Disease Research Laboratory test.
Congenital syphilis: include paired maternal and neonatal non-treponemal serological titres using the same test, preferably conducted at the same laboratory.[8]
The US Food and Drug Administration (FDA) has issued a warning of false-positive RPR results linked to COVID-19 vaccination. RPR false reactivity has been observed in some individuals for at least 5 months following the vaccine. The issue has been identified in the Bio-Rad BioPlex 2200 Syphilis Total & RPR test kit. It is not yet known if other RPR tests are affected similarly. For healthcare professionals who use the Bio-Rad BioPlex 2200 Syphilis Total & RPR test kit, the FDA recommends performing confirmatory testing for all reactive results. In patients previously treated for syphilis who received a COVID-19 vaccine, and whose clinical presentation and epidemiologic considerations do not support syphilis reinfection, reactive RPR results obtained from the BioPlex 2200 Syphilis Total & RPR test kit should be confirmed using an RPR test from a different manufacturer.[77]
Result
positive
serum Venereal Disease Research Laboratory (VDRL) test
Test
A non-treponemal serology test.
Provides a quantitative measure of disease activity and can be used to monitor treatment response (VDRL titres decrease or become non-reactive with effective treatment).
The most common approach is to use a treponemal test as the initial serological test, followed by a non-treponemal test to confirm diagnosis and provide evidence of active disease or re-infection (i.e., a 'reverse sequence screening algorithm').[46]
Despite adequate treatment, some patients maintain a persisting low level positive antibody titre (known as a serofast reaction).[50]
False positives may occur due to the presence of a variety of medical conditions (e.g., pregnancy, autoimmune disorders, and infections).
A false-negative test may occasionally occur in an undiluted specimen (the prozone phenomenon).
Primary syphilis: VDRL sensitivity is 44% to 76%.[5]
VDRL is positive in 77% of cases of late latent syphilis.[5]
VDRL sensitivity in secondary syphilis is 100%.[76]
Congenital syphilis: include paired maternal and neonatal non-treponemal serological titres using the same test, preferably conducted at the same laboratory.[8]
Result
positive
Investigations to consider
lumbar puncture, cerebrospinal fluid (CSF) analysis
Test
Indicated for any patient with clinical evidence of neurological involvement (e.g., cranial nerve dysfunction, meningitis, stroke, acute or chronic altered mental status, or loss of vibration sense).
Indicated if syphilis of unknown duration exists in the presence of HIV co-infection.
Indicated in any child with congenital syphilis and neurological symptoms or signs.[8]
An elevated CSF WBC count and positive CSF Venereal Disease Research Laboratory (VDRL) suggests neurological involvement.[37]
Some patients with neurosyphilis have an isolated elevated CSF WBC count and negative rapid plasma reagin/VDRL.
Neurosyphilis is unlikely at CSF Treponema pallidum haemagglutination assay (TPHA)/T pallidum particle agglutination assay (TPPA) titres <1:320.
A non-reactive CSF-TPHA test result usually excludes neurosyphilis.
How to perform a diagnostic lumbar puncture in adults. Includes a discussion of patient positioning, choice of needle, and measurement of opening and closing pressure.
Result
WBC count >10 cells/mm³; CSF protein >50 mg/dL (0.50 g/L); CSF VDRL positive; CSF TPHA/TPPA/FTA-ABS positive
chest x-ray
Test
May detect possible thoracic aortic aneurysm or aortic calcification.
Required in people with symptoms or signs of aortic regurgitation, heart failure, or aortic aneurysm.
Result
possible widened thoracic aorta, aortic calcification
echocardiogram
Test
Required if cardiovascular syphilis is strongly suspected (e.g., a patient has symptoms or signs of aortic regurgitation, heart failure, or aortic aneurysm).
Result
may show evidence of heart failure, aortic regurgitation, or thoracic aortic aneurysm
computed tomography brain
Test
Performed before lumbar puncture to exclude elevated intracranial pressure, to ensure that a lumbar puncture procedure will be safe.
Elevated intracranial pressure is rarely caused by syphilis itself.
Result
usually normal
magnetic resonance imaging brain
Test
Performed before lumbar puncture to exclude elevated intracranial pressure, to ensure that a lumbar puncture procedure will be safe.
Elevated intracranial pressure is rarely caused by syphilis itself.
Result
usually normal
HIV test
Test
All patients with syphilis should be tested for HIV.
In geographical areas in which the prevalence of HIV is high, patients who have syphilis should be re-tested for HIV after 3 months, even if the first HIV test result is negative, and be offered HIV pre-exposure prophylaxis (PrEP).[8]
All infants and children at risk for congenital syphilis should be tested for HIV.[8]
Result
positive or negative
fetal ultrasound scan
Test
Should be performed on all pregnant women with syphilis or suspected of having syphilis.
Presence of fetal or placental syphilis indicates a greater risk of treatment failure for congenital syphilis.[71]
Result
may show hepatomegaly, ascites, hydrops fetalis, intrauterine growth retardation
full blood count
Test
Performed in infants with possible congenital syphilis.
Result
may show anaemia, thrombocytopenia, leukopenia, possible neutrophilia
long-bone x-rays
Test
May be indicated in infants with suspected congenital syphilis.[8]
Performed if osteochondritis suspected.
Result
may demonstrate osteochondritis
liver enzymes (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase) and bilirubin
Test
May be indicated in infants with suspected congenital syphilis.[8]
Performed if clinical findings suggestive of liver involvement (e.g., hepatomegaly).
Result
aspartate aminotransferase and alanine aminotransferase may be elevated
auditory brainstem response
Test
May be indicated in infants with suspected congenital syphilis.[8]
Performed if clinically indicated.
Result
may detect deafness
audiometry
Test
Neurosyphilis may involve cranial nerves (particularly the 8th cranial nerve).
Result
may detect hearing deficit
fetal skeletal survey
Test
Might aid diagnosis of congenital syphilis in stillborn infants.[8]
Result
osseous lesions
Emerging tests
Treponema pallidum polymerase chain reaction (PCR) (sample taken directly from ulcerative lesions)
Test
T pallidum PCR has been shown to have moderate sensitivity (70% to 80%) and high specificity >90% in the diagnosis of primary or secondary syphilis, when compared with adequate reference tests (e.g., serology, dark-field microscopy).[56]
The US Centers for Disease Control and Prevention considers PCR testing a valid method for diagnosing primary, secondary, and congenital syphilis, and its use is likely to increase.[8][57]
Result
positive
point of care (POC) testing with either treponemal or combination treponemal/non-treponemal antibody
Test
POC syphilis testing has been assessed in the setting of high-risk regions, where rapid and early diagnosis may be more important than accuracy. Several clinical trials have shown promise and POC testing has been recommended as part of the Pan American Health Organization strategy to diagnose and treat syphilis.[58][59]
Result
positive; however, a positive result for treponemal antibodies alone does not distinguish between current, past, or treated infection
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