Types Of Serological Tests Pdf

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Aug 3, 2024, 4:58:15 PM8/3/24

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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.

These recommendations were developed by CDC staff members on the basis of evidence published in peer-reviewed scientific journals. Data available in Food and Drug Administration (FDA)-cleared syphilis diagnostic test inserts were reviewed and assessed for consistency with published findings. In 2017, the Association of Public Health Laboratories (APHL) assisted with the literature review through an independent work group formed to evaluate the scientific literature for CDC to consider in the development of evidence-based recommendations for syphilis testing in the United States. APHL work group members were selected based on expertise in the field of syphilis and represented public health and commercial laboratory directors, public- and private-sector providers, and academic researchers. The work group leads were experienced in conducting systematic reviews of the literature. Potential conflicts of interest were disclosed to APHL and are listed at the end of the work group (Supplementary Appendix 1, ). APHL staff members reviewed potential conflicts and concluded that no work group members had a financial interest or ongoing relationships that might bias the literature review and subsequent discussions. The APHL work group did not rank the evidence and did not make any recommendations based on the scientific literature review. CDC staff members involved in ranking the evidence and drafting recommendations based on the scientific literature certified that they did not have a perceived or actual competing interest with respect to this activity.

Draft recommendations were peer reviewed as defined by the Office of Management and Budget for influential scientific information ( -review.htm). In February 2022, draft recommendations were peer reviewed by four experts in the field of syphilis who were not U.S Federal employees, were not funded by CDC for syphilis research, and were not involved in the development of these recommendations (Supplementary Appendix 3, ). Comments submitted during the external peer review were addressed, and the document was available for a 60-day public comment period beginning April 5, 2023. Draft recommendations were reviewed by subject matter experts and stakeholders, including APHL, the American Society for Microbiology, the Centers for Medicare & Medicaid Services (CMS), and FDA. After the public comment and stakeholder review, CDC considered all comments in the development of final testing recommendations for syphilis.

Syphilis serologic tests were developed at the beginning of the 20th century and used by medical personnel to diagnose syphilis. The first test, known as the Wassermann test, was a complement fixation test that used liver extracts, initially from fetuses and subsequently from the heart tissue of patients with syphilis (24). The assay was further standardized to improve reproducibility by laboratories after the publication of a method to isolate cardiolipin and lecithin (phosphorylcholine) from beef heart and combine them with cholesterol as the antigens for these tests (25). Subsequent tests involving immobilization of T. pallidum, agglutination, or flocculation were based on the same principle of detecting serum that reacted to T. pallidum (T. pallidum immobilization [TPI] test) or to antigens found in the membranes of T. pallidum (cardiolipin [diphosphatidylglycerol], phosphorylcholine, and cholesterol) used in the rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests. In 1954, the World Health Organization convened an expert committee on treponematoses and made recommendations regarding antigen preparation, standardization of tests, and terminology (26). The terminology was based on the understanding of the contemporaneous scientific findings and became the basis for which to describe the serologic testing concepts for syphilis that are still used today (27). Over time, the use of the terms nontreponemal tests, treponemal tests, and nonspecific antibodies should be revisited and updated to be consistent with the scientific evidence related to the immunobiology of T. pallidum.

After entry through the mucosa or microabrasions in the skin, T. pallidum replicates locally and quickly spreads throughout the body, including the CNS, through the cardiovascular and lymphatic systems (41). The dearth of pathogen-associated molecular patterns on the cell surface of T. pallidum contributes to the inability of the innate immune system to clear the organism during primary infection and subsequent dissemination (42). Activation of the innate immune system might be downregulated by a treponemal phospholipid found in the outer membrane (43). However, dendritic cells phagocytize T. pallidum early during infection, and most migrate to draining lymph nodes where they present processed treponemal antigens (mostly protein antigens) to B- and T-cells to initiate adaptive immune responses (44).

Antigens that are processed and presented by phagocytic cells during T. pallidum infection are either unique to the organism or common to the organism, host cells, or both. Cardiolipin, a diphosphatidylglycerol, is an integral mitochondrial cell membrane phospholipid required for proper mitochondrial function (45). B1 cells, a subset of B-cells, secrete antibodies of low to moderate affinity in the absence of activation by previous infection (46). The B1-secreted antibodies are referred to as natural antibodies, and they can bind to cardiolipin and other phospholipids (e.g., cholesterol and phosphatidylcholine). However, other infections or conditions, in addition to syphilis and autoimmune diseases, can cause a transient increase in natural antibodies against cardiolipin (47). The cytoplasmic membrane of T. pallidum contains cardiolipin and other phospholipids that can contribute to immune stimulation during infection (48,49). Cholesterol and phosphatidylcholine are host phospholipids that are also constituent macromolecules in the T. pallidum cytoplasmic membrane (48). Phosphorylcholine can be a target for protective immunity, as demonstrated by the bactericidal effect of a monoclonal antibody binding to this antigen on the surface of T. pallidum (50). Antibodies to both cholesterol and phosphatidylcholine are elevated during certain stages of infection with T. pallidum (51) and are detected by RPR and VDRL tests.

Antibodies that reacted to the lipoidal antigens used in the Wassermann and subsequent agglutination or flocculation tests were either an indication of a concomitant T. pallidum infection or another condition related to host tissue damage and release of lipoidal antigens. The term nontreponemal test was first used in the literature in 1960 to differentiate tests based on antigens specific to T. pallidum (TPI, fluorescent treponemal antibody-absorption [FTA-ABS], microhemaggluntination assay for antibodies to T. pallidum [MHA-TP], T. pallidum hemagglutination assay [TPHA], and T. pallidum particle agglutination [TPPA]) from tests based on antigens (i.e., cardiolipin, phosphatidylcholine, and cholesterol) found in healthy animal tissues and other organisms in addition to T. pallidum and used in VDRL and RPR tests. The lipid composition of T. pallidum was first described in 1979 when it was reported that the organism contained all the phospholipids used in nontreponemal tests (48). Genomic analysis of T. pallidum further revealed the lack of certain enzymes for biosynthetic pathways necessary for these cytoplasmic and outer membrane phospholipids, indicating an inherent requirement for phospholipids from the host (52).

The increase in antibodies to cardiolipin, phosphatidylcholine, and cholesterol during T. pallidum infection is likely the result of a combination of antigens from both the bacteria and the host, not just from host tissue damage. In a rabbit model, T. pallidum cardiolipin induced a high antibody titer during active infection (49). Inoculating rabbits with inactivated T. pallidum resulted in a lower anticardiolipin titer, suggesting the increased response observed during active infection was attributable to immune stimulation from a combination of cardiolipin released from T. pallidum and damaged host cells (49). Because the antigens used in nontreponemal tests are found in T. pallidum membranes and host membranes, referring to these tests as nontreponemal is a misnomer. A 2019 study demonstrated that 11% of 526,540 reactive nontreponemal tests were not associated with syphilis, and in those cases, the tests were detecting antibodies to nontreponemal antigens generated by host tissue damage from other diseases (53). However, 89% of the reactive tests were associated with syphilis, implying that most nontreponemal tests detect antibodies triggered by T. pallidum phospholipid antigens during infection. Purported nontreponemal tests could more accurately be called lipoidal antigen tests. Hereafter in this report, these tests will be referred to as nontreponemal (lipoidal antigen) tests.