Rare phenomenon delays diagnosis

An unusual laboratory phenomenon accounts for a false negative result in a suspicious case for syphilis
Dr Madhara Weerasinghe Dr Stephen Davies

A 60-year-old heterosexual male presents to his GP with a three-week history of fatigue, arthralgia and night sweats.

Blood tests reveal liver function derangement with AST 291U/L (normal <40), ALT 736U/L (normal <35), GGT 206U/L (normal <50), ALP 214U/L (normal 30-110).

An autoimmune illness is considered, prompting rheumatology assessment which is inconclusive. 

One month later, the patient experiences bilateral reduction in visual acuity and is referred to an ophthalmologist. Ocular examination demonstrates abnormalities in the posterior segment (choroid) with visual acuity of 6/24 in the right and 6/12 in the left. 

The ophthalmologist conducts additional blood tests which show reactive syphilis chemiluminescent microparticle immunoassay (CMIA) and Treponema pallidum particle agglutination (TPPA) but non-reactive rapid plasma reagin test (RPR). 

The patient is referred to the local sexual health clinic, and on further history, reports unprotected oral and vaginal sex with an anonymous casual female partner one month prior to the onset of his symptoms.

He denies any sexual contact with male partners or any recreational substance use.

He reports he has had no other sexual contact in the last five years. He has had no prior investigations for sexually transmitted infections (STIs) including syphilis.


Oral cavity examination reveals a small ulcer (<1cm) on the lower gum, which is mildly firm at the base. Neurological and genital examination is unremarkable. There is no rash on the trunk or limbs, and there is full and painless range of movement in all joints.

The community laboratory which conducted the syphilis serology tests is asked to re-do the RPR after diluting the original sample. This returns a revised result of RPR reactive, titre 1:512, confirming the prozone phenomenon and making the diagnosis of ocular syphilis highly likely. 

The ulcer on the lower gum is swabbed and is positive for Treponema pallidum DNA. RPR is requested from the hospital laboratory (which routinely performs a parallel RPR test on a diluted sample at 1:16), and is reported as reactive, titre 1:256. 

The patient screens negative for other STIs, including HIV, hepatitis B, hepatitis C, chlamydia and gonorrhoea. 


The prozone phenomenon occurs when high concentrations of an antibody in the serum interferes with antigen-antibody complex formation, which forms the basis of a positive flocculation test, such as the RPR.4

Although uncommon, this can lead to false negative results with subsequent missed or delayed diagnoses, incorrect staging and onward transmission. 

The prozone phenomenon is estimated to occur in 0.2-2% of syphilis cases and is associated with HIV-coinfection, early stage of infection, neurosyphilis and pregnancy.5 It can also occur with other tests such as hormonal assays (eg, prolactin, thyroglobulin, HCG) and tumour markers (eg, PSA, CEA, CA-125).6 

Clinical presentations of syphilis are highly variable and may not necessarily follow the classical stages.1,2 Therefore, diagnosis and disease staging rely heavily on clinical suspicion and serological tests. In Australia, most laboratories use a treponemal test to screen for syphilis (eg, syphilis CMIA, enzyme immunoassay [EIA]).1-3

If reactive, a second treponemal test and a non-treponemal test (eg, RPR) are performed.1,2 Treponemal tests usually become reactive 2-4 weeks post-infection and usually remain reactive for life, even after appropriate treatment. Non-treponemal tests usually become reactive 3-4 weeks post-infection and indicate disease activity.2,3

Both types of tests do not have 100% sensitivity and therefore may be negative in early infection (ie, incubating and primary syphilis).1,2 The sensitivity of RPR in secondary syphilis is virtually 100%, however false negatives can occur, as seen in this case, due to the prozone phenomenon. 

The prozone effect can be unmasked by diluting the serum to reduce the concentration of circulating antibodies.4,6

When clinical findings are strongly suggestive of syphilis despite negative or unexpectedly low non-treponemal test results, request that the investigating laboratory repeat the test on specimens at higher dilution to uncover a possible prozone effect. 


The patient declines lumbar puncture and is treated presumptively with a neurosyphilis regimen of benzylpenicillin 24 million units (14.4g) daily via continuous intravenous infusion for 15 days as an outpatient. He reports subjective improvement in his ocular symptoms a few days after commencing treatment. 

Follow-up ophthalmology assessment finds a persistent reduction in close vision with no other complications. The RPR is repeated three months after treatment and is reactive, titre 1:64, indicating successful serological response to treatment.

Box 1: Key practice points
– Syphilis diagnoses are increasing, especially in gay, bisexual and other men who have sex with men (GBMSM), Aboriginal and Torres Strait Islander people and women of reproductive age.
– Diagnosis and disease staging of syphilis relies heavily on clinical suspicion and serological tests. 
– Consider the possibility of the prozone effect in cases that are highly suspicious for syphilis but with non-reactive or unexpectedly low non-treponemal tests.

Dr Madhara Weerasinghe is a GP and an advanced trainee in sexual health medicine at the Northern Sydney Sexual Health Service, Sydney, NSW. 

Dr Stephen Davies is a senior staff specialist sexual health physician at the Northern Sydney Sexual Health Service, Sydney, NSW.

The authors wish to acknowledge Associate Professor Bernard Hudson, director of microbiology and senior staff specialist, Royal North Shore Hospital, Sydney, NSW.

References on request from kate.kelso@adg.com.au.