Diagnosis of Syphilis
Diagnosis of syphilis is based on history, physical examination, and laboratory investigation. It is essential that the stage of syphilis be accurately assessed and documented in order to ensure appropriate management of cases and contacts.
Darkfield Microscopy & Direct or Indirect
1. Fluorescent Antibody Test (DFA/IFA):
Darkfield microscopy and DFA/IFA testing of lesion exudates or tissues are the definitive methods for diagnosing early syphilis when an active chancre, mucous patch, or condyloma latum is present. It is also useful for testing nasal discharge in a neonate with snuffles.
Darkfield microscopy is often not practical (it is not available in most labs including CPL) as it requires a skilled technician on-site. In addition, specimens must be appropriately collected and quickly examined within 5-20 minutes of collection. Positive tests on these materials for immunofluorescent (DFA) testing are diagnostic.
Samples collected from serous exudates from a chancre or secondary skin or mucous membrane lesions for DFA testing should be submitted on a slide and sent to CPL. CPL requests an additional dry Dacron swab be collected for nucleic acid amplification testing (NAAT), and transported in a dry sterile urine container. NAAT is used for syphilis subtyping and not for diagnosis. Prior arrangements are generally not required.
Serology:
Serologic tests for syphilis are essential for diagnosis of individuals, for following the efficacy of therapy, and for screening purposes. They detect antibodies formed during the course of syphilitic infection. A presumptive diagnosis is possible with the use of two types of serologic tests for syphilis; nonspecific nontreponemal antibody tests (VDRL and RPR) and specific treponemal antibody tests (FTA-ABS and TP-PA). To establish a diagnosis of syphilis, both types of serologic tests are usually necessary. It should be emphasized that serologic test results for syphilis on rare occasions may be negative in active cases, especially in older patients, or very early in primary infections. After hours testing is conducted for transplant and other emergent purposes. An appropriate sample is 5-10 ml of
blood collected in a red-stoppered tube which should be sent to CPL with a request for “serum for VDRL”. The CPL lab requisition should also provide information on the reason for testing (sexual contact to case, genital ulcer, clinical findings, etc). It is extremely important to include the relevant history on the lab requisition.
Routine screening of umbilical cord blood is NOT recommended for serological testing where a diagnosis of congenital syphilis is considered.
Testing of maternal serum is preferred to testing infant serum because infant serum can be
nonreactive if maternal serology is low titre or if the infection was late in pregnancy. Cord blood that is contaminated with maternal blood may lead to a
false positive test result.
2. Nontreponemal Tests (VDRL and RPR):
Syphilitic infection leads to the production of nonspecific antibodies (IgM and IgG) directed against a lipoidal antigen resulting from the interaction of host tissues with T. pallidum or from T. pallidum itself. This antibody-antigen reaction is the basis of nontreponemal tests such as the Venereal Disease Research Laboratory slide test (VDRL) and the rapid plasma reagin test (RPR).
The RPR test is more sensitive than the VDRL. CPL uses the rapid plasma reagin card test (RPR). After adequate treatment of syphilis, nontreponemal tests (NTT) eventually become nonreactive. However, even with sufficient treatment, patients sometimes have a persistent low-level positive nontreponemal test referred to as a serofast conversion. Nontreponemal test titres of persons who have been treated for latent or late stages of syphilis or who have become reinfected do not decrease as rapidly as do those of persons in the early stages of their first infection. In fact these persons may remain serofast for life.
VDRL and RPR become positive one to four weeks after the appearance of the primary chancre or six weeks after exposure. Biologic false positive reactions occur at a rate of 1-2% in the general population. Acute false positive tests lasting less than six months can occur following a febrile illness or immunization. As a rule, 90% of false positive titres are less than 1:8, but low titres are also seen in latent infection. False positive rates in pregnancy are similar to the general population. More than 10% of IDU may have false positive results (18). HIV infection has not been associated with increased false positive NTT in individuals at low risk of IDU.
Serial nontreponemal tests are useful to determine the stage of the disease; a four-fold rise in titre may indicate recent infection, reinfection in an adequately treated person, or relapse in an inadequately treated person. Adequate treatment of infectious syphilis is indicated by a four-fold or greater decline in titre within one year. Titres should generally become non-reactive or weakly reactive within one year following treatment of primary syphilis and within two years after treatment for secondary syphilis. Treatment of late latent or late syphilis usually has little or no effect on the titre and should not be used to gauge the adequacy of the treatment. Titres tend to become lower with time, but serum frequently remains reactive, usually in low titre. As with all quantitative serologic tests, only a four-fold or greater change in titre is meaningful.
3. Specific Treponemal Tests:
These tests measure antibodies against specific T. pallidum antigens and are used primarily to confirm the diagnosis of syphilis in patients with a reactive nontreponemal test. The principal specific treponemal antibody tests performed in most laboratories are the T. pallidum particle agglutination tests (TP-PA) and fluorescent treponemal antibody-absorption test (FTA-ABS).
Most patients who have reactive treponemal tests will have reactive tests for the remainder of their lives, regardless of treatment or disease activity. However, reversion to a nonreactive status may occur in up to 10% of patients, especially in those who are treated early (3). Treponemal test antibody titres correlate poorly with disease activity and should not be used to assess treatment response.
False positive results can occur especially when the FTA-ABS test is used in patients with Lyme disease, HIV, pregnancy, drug addiction, toxoplasmosis, H. pylori, autoimmune disorders like lupus and rheumatoid arthritis, and in persons with other treponemal diseases such as yaws, pinta or bejel.
Confirmatory Test:
The TP-PA test is a specific treponemal test for the serologic detection of antibodies to various species and subspecies of treponemes. Reports from CPL refer to the TP-PA as a confirmatory test result.
Reference Test:
The FTA-ABS test is an indirect immunofluorescent antibody test using T. pallidum from rabbit testis as the antigen. Its interpretation is subjective and requires great attention to detail. Its principal use is to verify the diagnosis of syphilis. Reports from CPL refer to this as the reference test.
4. Tests for Neurosyphilis:
No single test is diagnostic for neurosyphilis; the CSF-VDRL is highly specific but it is insensitive; as low as 30%. Most other tests are both insensitive and nonspecific and must be interpreted in relation to other test results and the clinical assessment.
Diagnosis of neurosyphilis usually depends on the combination of patient history, physical examination, reactive serologic test results, and abnormalities of CSF (cell count, protein, or a reactive CSF-VDRL). The CSF leukocyte count is usually elevated (>5 WBC/mm3) in patients with neurosyphilis. The CSF leukocyte count can also be used as a sensitive measure of the effectiveness of therapy. A positive CSF-VDRL result in the appropriate clinical setting establishes the diagnosis of neurosyphilis although serum antibody contamination is possible. A negative CSF-VDRL does not rule out the possibility of neurosyphilis. Normalization of CSF markers is affected by the stage of syphilis at which treatment is initiated and pretreatment levels of particular CSF markers. In patients without HIV infection treated with penicillin regimens, the CSF pleocytosis and VDRL titre normalize within one year. Reversion of pleocytosis is more likely when pretreatment CSF WBC counts are high. CSF-VDRL normalization
is less likely when pretreatment CSF-VDRL titres are high. In HIV-infected patients, CSF WBC count, protein, and VDRL may be slow to normalize. CSF-VDRL titres are less likely to normalize after treatment when CD4+ counts are <200 cells/μL compared to CD4+ counts >200 cells/μL. Therefore in HIV-infected patients, it is not possible to exclude treatment failure and more intensive regimens may be required.
Examination of CSF should be considered in the following circumstances:
1. Congenital syphilis;
2. Neurologic, ophthalmic, or otologic signs and symptoms;
3. Tertiary syphilis;
4. Previously treated patients who fail to achieve an adequate serologic response;
5. HIV coinfection with late latent or syphilis of unknown duration;
6. Where HIV coinfection exists, a lumbar puncture
(LP) is strongly recommended when neurological signs or symptoms are present, VDRL/RPR ≥1:32 dilutions, CD4+ counts <350 cells/μL or treated syphilis with suboptimal decline in VDRL/RPR titre. Some experts recommend a LP for all syphilis cases with HIV coinfection. A LP may be considered in other patients on a case by case basis.
Diagnosis of syphilis is based on history, physical examination, and laboratory investigation. It is essential that the stage of syphilis be accurately assessed and documented in order to ensure appropriate management of cases and contacts.
Darkfield Microscopy & Direct or Indirect
1. Fluorescent Antibody Test (DFA/IFA):
Darkfield microscopy and DFA/IFA testing of lesion exudates or tissues are the definitive methods for diagnosing early syphilis when an active chancre, mucous patch, or condyloma latum is present. It is also useful for testing nasal discharge in a neonate with snuffles.
Darkfield microscopy is often not practical (it is not available in most labs including CPL) as it requires a skilled technician on-site. In addition, specimens must be appropriately collected and quickly examined within 5-20 minutes of collection. Positive tests on these materials for immunofluorescent (DFA) testing are diagnostic.
Samples collected from serous exudates from a chancre or secondary skin or mucous membrane lesions for DFA testing should be submitted on a slide and sent to CPL. CPL requests an additional dry Dacron swab be collected for nucleic acid amplification testing (NAAT), and transported in a dry sterile urine container. NAAT is used for syphilis subtyping and not for diagnosis. Prior arrangements are generally not required.
Serology:
Serologic tests for syphilis are essential for diagnosis of individuals, for following the efficacy of therapy, and for screening purposes. They detect antibodies formed during the course of syphilitic infection. A presumptive diagnosis is possible with the use of two types of serologic tests for syphilis; nonspecific nontreponemal antibody tests (VDRL and RPR) and specific treponemal antibody tests (FTA-ABS and TP-PA). To establish a diagnosis of syphilis, both types of serologic tests are usually necessary. It should be emphasized that serologic test results for syphilis on rare occasions may be negative in active cases, especially in older patients, or very early in primary infections. After hours testing is conducted for transplant and other emergent purposes. An appropriate sample is 5-10 ml of
blood collected in a red-stoppered tube which should be sent to CPL with a request for “serum for VDRL”. The CPL lab requisition should also provide information on the reason for testing (sexual contact to case, genital ulcer, clinical findings, etc). It is extremely important to include the relevant history on the lab requisition.
Routine screening of umbilical cord blood is NOT recommended for serological testing where a diagnosis of congenital syphilis is considered.
Testing of maternal serum is preferred to testing infant serum because infant serum can be
nonreactive if maternal serology is low titre or if the infection was late in pregnancy. Cord blood that is contaminated with maternal blood may lead to a
false positive test result.
2. Nontreponemal Tests (VDRL and RPR):
Syphilitic infection leads to the production of nonspecific antibodies (IgM and IgG) directed against a lipoidal antigen resulting from the interaction of host tissues with T. pallidum or from T. pallidum itself. This antibody-antigen reaction is the basis of nontreponemal tests such as the Venereal Disease Research Laboratory slide test (VDRL) and the rapid plasma reagin test (RPR).
The RPR test is more sensitive than the VDRL. CPL uses the rapid plasma reagin card test (RPR). After adequate treatment of syphilis, nontreponemal tests (NTT) eventually become nonreactive. However, even with sufficient treatment, patients sometimes have a persistent low-level positive nontreponemal test referred to as a serofast conversion. Nontreponemal test titres of persons who have been treated for latent or late stages of syphilis or who have become reinfected do not decrease as rapidly as do those of persons in the early stages of their first infection. In fact these persons may remain serofast for life.
VDRL and RPR become positive one to four weeks after the appearance of the primary chancre or six weeks after exposure. Biologic false positive reactions occur at a rate of 1-2% in the general population. Acute false positive tests lasting less than six months can occur following a febrile illness or immunization. As a rule, 90% of false positive titres are less than 1:8, but low titres are also seen in latent infection. False positive rates in pregnancy are similar to the general population. More than 10% of IDU may have false positive results (18). HIV infection has not been associated with increased false positive NTT in individuals at low risk of IDU.
Serial nontreponemal tests are useful to determine the stage of the disease; a four-fold rise in titre may indicate recent infection, reinfection in an adequately treated person, or relapse in an inadequately treated person. Adequate treatment of infectious syphilis is indicated by a four-fold or greater decline in titre within one year. Titres should generally become non-reactive or weakly reactive within one year following treatment of primary syphilis and within two years after treatment for secondary syphilis. Treatment of late latent or late syphilis usually has little or no effect on the titre and should not be used to gauge the adequacy of the treatment. Titres tend to become lower with time, but serum frequently remains reactive, usually in low titre. As with all quantitative serologic tests, only a four-fold or greater change in titre is meaningful.
3. Specific Treponemal Tests:
These tests measure antibodies against specific T. pallidum antigens and are used primarily to confirm the diagnosis of syphilis in patients with a reactive nontreponemal test. The principal specific treponemal antibody tests performed in most laboratories are the T. pallidum particle agglutination tests (TP-PA) and fluorescent treponemal antibody-absorption test (FTA-ABS).
Most patients who have reactive treponemal tests will have reactive tests for the remainder of their lives, regardless of treatment or disease activity. However, reversion to a nonreactive status may occur in up to 10% of patients, especially in those who are treated early (3). Treponemal test antibody titres correlate poorly with disease activity and should not be used to assess treatment response.
False positive results can occur especially when the FTA-ABS test is used in patients with Lyme disease, HIV, pregnancy, drug addiction, toxoplasmosis, H. pylori, autoimmune disorders like lupus and rheumatoid arthritis, and in persons with other treponemal diseases such as yaws, pinta or bejel.
Confirmatory Test:
The TP-PA test is a specific treponemal test for the serologic detection of antibodies to various species and subspecies of treponemes. Reports from CPL refer to the TP-PA as a confirmatory test result.
Reference Test:
The FTA-ABS test is an indirect immunofluorescent antibody test using T. pallidum from rabbit testis as the antigen. Its interpretation is subjective and requires great attention to detail. Its principal use is to verify the diagnosis of syphilis. Reports from CPL refer to this as the reference test.
4. Tests for Neurosyphilis:
No single test is diagnostic for neurosyphilis; the CSF-VDRL is highly specific but it is insensitive; as low as 30%. Most other tests are both insensitive and nonspecific and must be interpreted in relation to other test results and the clinical assessment.
Diagnosis of neurosyphilis usually depends on the combination of patient history, physical examination, reactive serologic test results, and abnormalities of CSF (cell count, protein, or a reactive CSF-VDRL). The CSF leukocyte count is usually elevated (>5 WBC/mm3) in patients with neurosyphilis. The CSF leukocyte count can also be used as a sensitive measure of the effectiveness of therapy. A positive CSF-VDRL result in the appropriate clinical setting establishes the diagnosis of neurosyphilis although serum antibody contamination is possible. A negative CSF-VDRL does not rule out the possibility of neurosyphilis. Normalization of CSF markers is affected by the stage of syphilis at which treatment is initiated and pretreatment levels of particular CSF markers. In patients without HIV infection treated with penicillin regimens, the CSF pleocytosis and VDRL titre normalize within one year. Reversion of pleocytosis is more likely when pretreatment CSF WBC counts are high. CSF-VDRL normalization
is less likely when pretreatment CSF-VDRL titres are high. In HIV-infected patients, CSF WBC count, protein, and VDRL may be slow to normalize. CSF-VDRL titres are less likely to normalize after treatment when CD4+ counts are <200 cells/μL compared to CD4+ counts >200 cells/μL. Therefore in HIV-infected patients, it is not possible to exclude treatment failure and more intensive regimens may be required.
Examination of CSF should be considered in the following circumstances:
1. Congenital syphilis;
2. Neurologic, ophthalmic, or otologic signs and symptoms;
3. Tertiary syphilis;
4. Previously treated patients who fail to achieve an adequate serologic response;
5. HIV coinfection with late latent or syphilis of unknown duration;
6. Where HIV coinfection exists, a lumbar puncture
(LP) is strongly recommended when neurological signs or symptoms are present, VDRL/RPR ≥1:32 dilutions, CD4+ counts <350 cells/μL or treated syphilis with suboptimal decline in VDRL/RPR titre. Some experts recommend a LP for all syphilis cases with HIV coinfection. A LP may be considered in other patients on a case by case basis.
5. Tests for congenital syphilis:
Venous samples should be obtained from both mother and baby for serology (treponemal and nontreponemal tests). Cord blood is not suitable for testing. The interpretation of reactive antibodies in the neonate must take into consideration the maternal history, including stage of syphilis, history of treatment, and syphilis serology results.
Placenta, neonatal nasal discharge, or skin lesions may be examined by darkfield microscopy or DFA/IFA for T. pallidum. CSF examination should be performed on all infants with suspected congenital syphilis. Long bone x-rays should also be performed.
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