Laboratory Recommendations for Syphilis Testing

Publication Date: February 8, 2024
Last Updated: February 13, 2024

Summary of Recommendations

Recommendation for endpoint titers

Endpoint titers (the highest dilution yielding a reactive result) should be determined and clearly reported when testing serum with nontreponemal (lipoidal antigen) assays that detect antibodies to lipoidal antigens (i.e., rapid plasma reagin and Venereal Disease Research Laboratory). Reports should not contain mathematical symbols such as > or < signs.
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Recommendation for syphilis serologic testing algorithm

Serologic tests that measure antibodies to both nontreponemal (lipoidal) and treponemal antigens related to syphilitic infections should be used in combination, when the primary test is reactive, to aid in the diagnosis of syphilis. Sole reliance on one reactive serologic test result can misclassify a patient’s syphilis status. Both the traditional syphilis screening algorithm (initial screening with nontreponemal [lipoidal antigen] assays) and the reverse syphilis screening algorithm (initial screening with treponemal immunoassays) are acceptable. The preferred algorithm should be based on laboratory resources, including staff, space and costs, test volume, and patient populations served.
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Recommendation for serologic syphilis testing

Nontreponemal (lipoidal antigen) tests (e.g., rapid plasma reagin or Venereal Disease Research Laboratory) are not interchangeable when used to determine antibody titers; testing on follow-up samples must be performed with the same type of test. The Treponema pallidum particle agglutination test is the preferred manual treponemal test.
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Recommendation for syphilis serologic testing in pregnant persons

Nontreponemal (lipoidal antigen) and treponemal tests should be interpreted in the same manner regardless of pregnancy status.
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Recommendation for syphilis serologic testing in persons living with HIV/AIDS

Nontreponemal (lipoidal antigen) and treponemal tests should be interpreted in the same manner regardless of HIV status.
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Recommendation for the direct detection of Treponema pallidum by darkfield microscopy

Darkfield microscopy should be maintained if already in use or established in sexually transmitted diseases clinics where a point-of-care test for primary or secondary syphilis diagnosis would be beneficial for timely patient treatment.
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Recommendation for direct detection of Treponema pallidum by immunohistochemistry and silver staining

Immunohistochemistry is preferred over silver staining for formalin-fixed, paraffin-embedded tissue sections regardless of anatomic site.
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Algorithms that can be applied to screening for syphilis with serologic tests — CDC laboratory recommendations for syphilis testing in the United States, 2024

Comparison of traditional and reverse algorithms for syphilis screening by serology — CDC laboratory recommendations for syphilis testing, United States, 2024

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Parameter Traditional algorithm with a nontreponemal (lipoidal antigen) test as the initial test Reverse algorithm with a treponemal test as the initial test
Reagent cost Rapid and inexpensive reagents Higher reagent cost per specimen. Automated treponemal tests widely available with high throughput and lower human labor costs
Specimen throughput Good for small-throughput laboratories. Less suitable for high-throughput laboratories because of labor and resources needed and occupational hazard of pipetting of individual specimens. Possible batching of samples that could delay test result turnaround time
Performance characteristics of the first test in the algorithm Results of nontreponemal (lipoidal antigen) tests can be subjective, and there is laboratory variability in titers
Possible prozone reaction that might be falsely interpreted as negative unless the serum sample is diluted
Biologic false-positive resulting from nonspecific reactivity resulting from conditions other than syphilis
Might be less sensitive for detecting early and late/latent syphilis		 Treponemal tests produce objective results
No prozone reaction
Detects antibodies against Treponema pallidum antigens
Might have increased detection of patients with early syphilis
Screening applications Good for populations with a high likelihood of previous syphilis If algorithm is used in populations with a high likelihood of previous syphilis, an increased number of primary screening tests could be false positives*

Specimen types, storage, and transport for direct detection tests for Treponema pallidum — CDC laboratory recommendations for syphilis testing, United States, 2024

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Direct detection test Specimen types Specimen storage and transport
Darkfield microscopy Serous exudate of moist lesions (except oral lesions) should be collected directly on a microscope slide or using a sterile bacteriological loop; avoid red blood cells Fresh, room temperature (20°C to 26°C; 68°F to78.8°F)
Immunofluorescent antibody test staining Smear from suspected lesion(s) Fresh, room temperature (20°C to 26°C; 68°F to 78.8°F)
Immunohistochemistry staining Formalin-fixed and paraffin-embedded tissue sections of brain, placenta, umbilical cord, or skin lesions from secondary or tertiary syphilis Room temperature (20°C to 26°C; 68°F to 78.8°F)
Nucleic acid amplification test Primary syphilis:
Serous exudate of moist lesions should be collected with a sterile Dacron swab and placed in a commercial transport medium
Secondary syphilis:
Mucous patches and condyloma lata specimens should be collected with a sterile Dacron swab and placed in a commercial transport medium
Fresh frozen tissue biopsy or formalin-fixed and paraffin-embedded tissue
Neonatal whole blood or serum; whole blood should be collected in an EDTA (purple top) tube		 Frozen (−20°C to −80°C; −4°F to −112°F), frozen ice packs or dry ice

Recommendation Grading

Disclaimer

The information in this patient summary should not be used as a substitute for professional medical care or advice. Contact a health care provider if you have questions about your health.

Overview

Title

Laboratory Recommendations for Syphilis Testing

Authoring Organization

Publication Month/Year

February 8, 2024

Last Updated Month/Year

February 15, 2024

Supplemental Implementation Tools

Document Type

Guideline

Country of Publication

US

Document Objectives

This report provides new CDC recommendations for tests that can support a diagnosis of syphilis, including serologic testing and methods for the identification of the causative agent Treponema pallidum. These comprehensive recommendations are the first published by CDC on laboratory testing for syphilis, which has traditionally been based on serologic algorithms to detect a humoral immune response to T. pallidum. These tests can be divided into nontreponemal and treponemal tests depending on whether they detect antibodies that are broadly reactive to lipoidal antigens shared by both host and T. pallidum or antibodies specific to T. pallidum, respectively. Both types of tests must be used in conjunction to help distinguish between an untreated infection or a past infection that has been successfully treated. Newer serologic tests allow for laboratory automation but must be used in an algorithm, which also can involve older manual serologic tests. Direct detection of T. pallidum continues to evolve from microscopic examination of material from lesions for visualization of T. pallidum to molecular detection of the organism. Limited point-of-care tests for syphilis are available in the United States; increased availability of point-of-care tests that are sensitive and specific could facilitate expansion of screening programs and reduce the time from test result to treatment. These recommendations are intended for use by clinical laboratory directors, laboratory staff, clinicians, and disease control personnel who must choose among the multiple available testing methods, establish standard operating procedures for collecting and processing specimens, interpret test results for laboratory reporting, and counsel and treat patients. Future revisions to these recommendations will be based on new research or technologic advancements for syphilis clinical laboratory science.

Inclusion Criteria

Male, Female, Adolescent, Adult, Older adult

Health Care Settings

Ambulatory, Laboratory services

Intended Users

Nurse, nurse practitioner, physician, physician assistant

Scope

Diagnosis, Assessment and screening

Diseases/Conditions (MeSH)

D013587 - Syphilis

Keywords

Syphilis, Syphilis Testing

Source Citation

Papp JR, Park IU, Fakile Y, Pereira L, Pillay A, Bolan GA. CDC Laboratory Recommendations for Syphilis Testing, United States, 2024. MMWR Recomm Rep 2024;73(No. RR-1):1–32. DOI: http://dx.doi.org/10.15585/mmwr.rr7301a1