Serological tests for syphilis.

INTRODUCTION

T. pallidum subsp. pallidum is the causative organism for syphilis. Three morphologically identical subspecies of T. pallidum have been classified as follows:

T. pallidum subsp. pallidum (syphilis)

T. pallidum subsp. pertenue (yaws), and

T. pallidum subsp. endemicum (bejel).

The immune response pattern in syphilis and the nonvenereal treponematoses is similar. This complicates the interpretation of reactive serologic tests for syphilis (both nontreponemal, e.g., Venereal Disease Research Laboratory [VDRL] and treponemal tests, e.g., T. pallidum hemagglutination assay [TPHA]) in patients who have lived in areas where endemic treponematoses are still prevalent

Syphilis has diverse clinical manifestations and shares many clinical features with other treponemal and nontreponemal diseases. Therefore, it is mandatory that the clinical diagnosis is always supported by appropriate laboratory tests and that the test results are interpreted with reference to the patient's history and physical examination findings.

HISTORY

Wassermann test or Wassermann reaction

It was the First blood test for syphilis and the first test in the nontreponemal tests (NTT) category developed by Wassermann, Julius Citron, and Albert Neisser in 1906

It is based on Complement fixation principle in which a sample of blood or CSF was taken and introduced to the antigen which was cardiolipin extracted from bovine muscle or heart. Treponemal nonspecific antibodies react with the lipid – the Wassermann reaction (WR) of antiphospholipid antibodies (APAs)

The intensity of the reaction (classed 1, 2, 3, or 4) indicates the severity of the condition

Newer NTTs, such as the rapid plasma reagin (RPR) and VDRL tests, have mostly replaced it.

INVESTIGATIONS FOR SYPHILIS

Although Treponema pallidum cannot be grown in culture, there are many tests for the direct and indirect diagnosis of syphilis [Table 1].[1]

Table 1

Diagnostic tests for syphilis

Direct diagnostic methods	Serological tests (indirect diagnostic method)
Dark-field microscopic examination of fluid or smears from lesions	Nontreponemal tests
Disadvantages:	For screening
Requires a trained, experienced microscopist	VDRL test
Success is dependent	Variants of VDRL test:
Various factors, including	RPR test
Too little or too much fluid on the slide	TRUST
The presence of refractile elements in the specimen	USR test
Improper thickness of the slide or cover slip, etc.	RST
Treatment with antibiotics may result in a false-negative finding	ART
	Treponemal tests
	For confirmation
	TPI test
	TPHA
	FTA-Abs test
	EIA
	Western blot technique
Histological examination of tissue sections with T pallidum antibody-based nonfluorescent stain such as the IHC stain
Direct fluorescent antibody test for T pallidum
Nucleic acid amplification methods such as PCR

PCR=Polymerase chain reaction; RPR=Rapid plasma regain; TRUST=Toludine red unheated serum test; USR=Unheated serum reagin; RST=Reagin screen test; ART=Automated reagin test; TPI=Treponema pallidum immobilization; TPHA=Treponema pallidum hemagglutination assay; FTA-Abs=Fluorescent treponemal antibody absorption; EIA=Treponemal enzyme immunoassay; IHC=Immunohistochemistry; VDRL=Venereal Disease Research Laboratory

NONTREPONEMAL TESTS

Venereal Disease Research Laboratory test

Mechanism: This detects both immunoglobulin (Ig) G and IgM APA (Ab against cardiolipin) formed by the host in response to lipoidal material released by damaged host cells early in infection and lipid from the cell surfaces of the treponeme itself

Seroconversion occurs from 21 days of exposure till about up to 6 weeks after infection.

Variants of Venereal Disease Research Laboratory test

RPR test: Results are available within 5 min

Conventional RPR card test[2] Automated RPR test

Uses cardiolipin Ag with carbon Immunoassay technique using latex

Particle to detect regain Ab Particles coated with lecithin and cardiolipin

Toludine red unheated serum test

Unheated serum reagin test

Reagin screen test

Automated reagin test.

Interpretation

Qualitative test for screening syphilis

Results are reported as nonreactive, reactive, and weakly reactive

In reactive NTT:

A fourfold increase in titer indicates infection, reinfection, or treatment failure

A fourfold decrease in titer indicates effective therapy.

Quantitative VDRL: The VDRL test can be quantitated by examining serial dilutions of serum and can be used to follow the course of illness, including the response to therapy

Patients with early syphilis who have been treated with appropriate doses and preparations of benzathine penicillin should be evaluated clinically and serologically, using a NTT, after 3 months to assess the results of therapy

A second evaluation should be performed after 6 months and, if indicated by the results at this point, again after 12 months to reassess the condition of the patient and detect possible reinfection.[3]

When a nontreponemal serologic test shows persistent reactivity with no signs of decline of titer at 6 months after adequate therapy or if it fails to show a fourfold decrease of initial high titer within 1 year, it is considered a seroresistance or serofast state.

Advantages

Inexpensive and simple

Suitable for mass screening

The baseline titer can be used to follow-up the treatment response.

Limitations

Reduced sensitivity in primary syphilis and late latent syphilis

False-positive results due to technical error and variations in normal population, due to antiphospholipid autoantibodies (biologic false positivity) [Table 2]

Table 2

Causes of biologic false-positive serological test

Infectious causes			Noninfectious causes Advanced cancer
Bacterial	Viral	Parasitic
Bacterial endocarditis	HIV	Malaria	Chronic liver diseases
Chancroid	Chickenpox	Trypanosomiasis	Connective tissue disease
Leprosy	Infectious mononucleosis		Pregnancy

False-negative results: In VDRL test, the optimal ratio of the antigen antibody yields an insoluble precipitate that is visible, thus rendering the test positive. The zone of equivalence defines this optimal ratio

However, undiluted serum specimens have a high quantity of reagin antibody which will give false-negative reaction. This is known as PROZONE phenomenon.[4] Further, on serial dilutions, the test becomes positive.

It is clinically significant in certain populations at risk like:

Those who are on continuous immunosuppressive drugs

Those who are HIV seropositive

Antigen excess can also result in false-negative result which is known as POSTZONE phenomenon Figure 1.

Figure 1

Pro and post zone phenomenon

Thus, confirmation by a treponemal test is required.

TREPONEMAL TESTS

Treponema pallidum immobilization test

This test uses the virulent T. pallidum (Nichol's strain) obtained from rabbits

It detects antibody which inhibits the normal movements of T. pallidum in the presence of complement (specific treponemal immobilizing antibody). The reaction of treponemes in the presence of patient's serum is observed by dark field microscopy

The test is positive if 50% or more of the treponemes are immobilized

It becomes positive few days to a week later than the VDRL test

Specificity is 100%, but as the test is time consuming, expensive, and hazardous, it is not performed nowadays.

Currently used treponemal tests are as follows:

Treponema pallidum hemagglutination assay

Microhemagglutination assay for IgM and IgG antibodies in which sensitized sheep erythrocytes are coated with T. pallidum (Nichol's strain)

Results are reported as reactive if agglutination occurs in a dilution of 1:80 or more

Reactivity can be expected around the 4th to 5th week of infection

Fluorescent treponemal antibody absorption test

Indirect Immunofluorescence antibody test

Presence of antibody in patient's serum is indicated by fluorescence. Intensity of fluorescence is reported as nonreactive, borderline, or reactive

Reactivity begins in the 3rd week of infection

It is the most sensitive serological test in the early stage of syphilis.

Fluorescent treponemal antibody absorption double-staining test

A fluorochrome-labeled counterstain for T. pallidum and antihuman IgG conjugate labeled with tetramethylrhodamine isothiocyanate to detect antibody in patient's serum are employed in this test

False positivity can occur in 1% of sera.[5]

Treponemal enzyme immunoassay

In this test, antigen is fixed to wells in microtiter plates, and then serum is added and rinsed off after 30–60 min

Antibody to T. pallidum binds to antigen and reacts in the 2nd incubation with an enzyme labeled antihuman globulin

Result is available in 3–4 h.

Advantages

Automated (or semi-automated) processing[6]

Objective reading of results

Interfacing with the laboratory computer system to allow electronic report generation

New algorithm suggests screening with treponemal enzyme immunoassay alone followed by a NTT.

Western blot technique

Based on the whole T. pallidum lysate antigen

The presence of antibodies to the immunodeterminants with molecular weights 15.5, 17, 44.5, and 47 kDa appears to be diagnostic for acquired syphilis

When an IgM-specific conjugate is used, the test has value in the diagnosis of congenital syphilis.

Treponemal tests may remain reactive for years with or without treatment, and treponemal test antibody titers correlate poorly with disease activity

Therefore, treponemal tests should not be used to evaluate response to therapy, relapse, or reinfection in previously treated patients

Treponemal tests do not differentiate venereal syphilis from endemic syphilis (yaws and pinta).

Sensitivity and specificity of commonly used serologic tests

Sensitivity: It is defined as the proportion of people who test positive for the disease among those who have the disease[7]

Specificity: It is defined as the proportion of healthy patients known not to have the disease who will test negative for it [Tables 3, 4 and Figure 2].

Table 3

Sensitivity and specificity of various serological tests

	Sensitivity (%)				Specificity (%)
	Primary syphilis	Secondary syphilis	Latent syphilis	Tertiary syphilis
VDRL	78	100	95	71	98
RPR	86	100	98	73	98
FTA-Abs	84	100	100	96	97
TPHA	76	100	97	94	99
EIA	93	100	100

VDRL=Venereal Disease Research Laboratory; FTA-Abs=Fluorescent treponemal antibody absorption; RPR=Rapid plasma regain; EIA=Treponemal enzyme immunoassay; TPHA=Treponema pallidum hemagglutination assay

Table 4

Window period of various serological tests

Serological test	Window period
FTA-Abs	3rd week
VDRL	2-6 weeks
TPHA	4-5th week

VDRL=Venereal Disease Research Laboratory; FTA-Abs=Fluorescent treponemal antibody absorption; TPHA=Treponema pallidum hemagglutination assay

Figure 2

Sensitivity and specificity of various serological tests

Other tests

Oral fluid test for syphilis

Time-resolved fluorescence immunoassay to detect antibodies to T. pallidum recombinant antigens in the oral fluid specimens collected using “Oracol” swab

In early syphilis – Sensitivity: 100%

Specificity: 97.9%.

Patients with positive syphilis serology – Sensitivity: 76.5%

Specificity: 96.9%.

Potentially useful when collection of blood is not practicable.

CEREBROSPINAL FLUID EXAMINATION IN SYPHILIS

Indications

Neurological, ophthalmic, or auditory symptoms and signs

Other clinical evidence of active infection – aortitis, gumma, or iritis

Treatment failure

HIV infection

A nontreponemal serum titer of >32 if the duration of syphilis is over 1 year

A nonpenicillin-based treatment regimen is planned

All infants suspected of prenatal syphilis.

The typical CSF findings of neurosyphilis are:

Moderate mononuclear pleiocytosis (10–400 cells/mL)

Elevated total protein (0.46–2.0 g/L)

Positive CSF VDRL criteria.

The CSF VDRL test is highly specific, and false-positive results are rare in the absence of blood contamination

Most venerologists consider an examination of CSF unnecessary in early syphilis and prefer to examine CSF after 1–2 years of posttreatment follow-up

In untreated asymptomatic late syphilis, a CSF examination should always be done

A normal CSF is by definition an essential prerequisite for a diagnosis of latent syphilis.

TESTING POLICY

NTTs such as RPR test detect almost all cases of early syphilis, but false positives are possible

Treponemal tests, such as TPHA and FTA-Ab, are used to confirm NTT results[8]

Quantitative RPR titers can help evaluate the response to treatment [Table 5]

Table 5

Interpretation of rapid plasma regain test results

	RPR	RPR titer	TPHA
Active infection	+	>1:8	+
Latent syphilis	+	Often <1:4	+
False positive	+	Usually <1:4	−
Successful treatment	+ or −	2 titers decrease (e.g., from 1:16 to 1:4)	+

TPHA=Treponema pallidum hemagglutination assay; RPR=Rapid plasma regain

When additional tests are not available, all clients with reactive RPR should be treated

The Centers for Disease Control and Prevention (CDC) has proposed an algorithm for evaluating syphilis. Patients who have a reactive treponemal test on two different tests with a negative NTT are candidates for treatment if they have not been treated in the past [Figure 3].

Figure 3

Algorithm for evaluation of syphilis

GUIDELINES FOR SCREENING OF SYPHILIS IN PREGNANCY AND NEWBORNS

The WHO STI guideline recommends screening all pregnant women for syphilis during the first antenatal care (ANC) visit and to be repeated early in the 3rd trimester[9]

In areas with a high prevalence of syphilis, the CDC has recommended re-screening in the 3rd trimester and again at the time of delivery to detect new infections during pregnancy

For all syphilis-positive women detected during ANC, their newborns should be tested by RPR using infant serum and not cord blood as it can yield false-positive result

All newborns showing fourfold rise in titer compared to that of their mothers' titer need to be hospitalized to initiate penicillin treatment for 10 days.

Serological tests in HIV

Syphilis is common among patients with HIV infection and vice versa. The course of clinical disease and serologic markers may be altered or more aggressive in co-infected patients

Serologic tests for syphilis are still the cornerstone of diagnosing untreated syphilis infection, independent of the HIV status

Unusual serologic responses in co-infected patients can be seen such as:

Increased biological false positivity

Higher-than-expected serologic VDRL titers

Seronegative cases of syphilis.

Delayed titer responses after treatment

Return of titers to nonreactivity as immunosuppression advances

Currently, routine periodic screening (at least annually and 2–4 times yearly among high-risk groups, such as Men who have sex with Men (MSM) is strongly recommended

Irrespective of signs and symptoms, all HIV-positive patients should have baseline VDRL screening and follow-up at 3 months to rule out the possibility of false-negative results, as seroconversion generally takes about 4–6 weeks after infection

In the course of reactive VDRL, the diagnosis of syphilis should be confirmed by the specific treponemal test

According to the current CDC guidelines, indications of lumbar puncture in HIV and syphilis co-infection are as follows:[10]

Patients with neurologic symptoms should undergo immediate lumbar puncture to rule out neurosyphilis

HIV-infected patients who present with late, latent syphilis or syphilis of unknown duration

Patients with evidence of serologic failure after receiving syphilis therapy

Risk of neurosyphilis is increased substantially – three- to fivefold – in HIV-infected patients with RPR titers 1:32 or in whom CD4 counts are <350

HIV testing is critical for all patients with a new diagnosis of syphilis.

Syphilis screening using rapid point-of-care tests

Operational requirements for RPR/VDRL testing are not available at most primary care. Delay in obtaining test results through referrals to offsite laboratories can delay or result in missed opportunities for treatment. Very often, patients may not return for follow-up, particularly if they are asymptomatic

Aim: To integrate rapid, simple, and technologically appropriate syphilis testing at venues with limited resources

Six test kits validated by the WHO.

CONCLUSION

Every patient suspected of syphilis should be subjected to a NTT (i.e., qualitative VDRL) followed by a treponemal test (i.e., TPHA). VDRL test indicates recent infection and is also useful in follow-up to monitor response of therapy. While treponemal tests are more specific tests, due to the limitation that they remain positive lifelong, they are not indicative of recent infection.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.