The prozone phenomenon in syphilis testing.

Syphilis, a genital ulcerative disease, remains an important public health concern in the United States. In addition to facilitating the transmission of human immunodeficiency virus (HIV), untreated syphilis may progress to cause neurologic, cardiovascular, and musculoskeletal complications and in pregnancy may lead to serious complications including stillbirth, neonatal death, prematurity, or congenital syphilis syndromes. The overall rate of primary and secondary syphilis reported in the United States declined during the 1990s and in 2000; however, the rates of primary and secondary syphilis in men began increasing in 2000, leading to an overall rate increase in 2001. (1) This resurgence in early syphilis was associated with outbreaks in men who have sex with men (MSM), particularly those who were HIV-infected and/or practicing high-risk sexual behavior. (1) The South continues to have a higher rate of primary and secondary syphilis than other regions of the country, with 56% of reported cases occurring in this region in 2001. (1)

Important measures in controlling the ongoing spread of syphilis include testing and treating those with clinical manifestations of early syphilis (primary and secondary syphilis) or those who are asymptomatic with early syphilis (early latent syphilis). A nontreponemal serologic test, such as the rapid plasma reagin test (RPR) or Venereal Disease Research Laboratory test, is widely accepted as an effective testing strategy for detecting syphilis, although false-positive and false-negative reactions can occur. These tests detect antibodies reactive to a stabilized cardiolipin antigen. When serum from a syphilis-infected individual is added to a suspension of cardiolipin antigen and charcoal, an antibody-antigen lattice forms, with the flocculation reaction visualized as clumping of the charcoal.

The interesting case report by Smith and Holman (2) in this issue demonstrates an important limitation of nontreponemal tests in the diagnosis of early syphilis. They describe a man with HIV coinfection and clinical features of secondary syphilis who has a false-negative reaction to a qualitative serum RPR. On dilution of his serum, the serum RPR became reactive and remained positive to a dilution of greater than 1:2,056. False-negative results in screening with a nontreponemal test may occur due to the prozone phenomenon, test misinterpretation, or laboratory error in performing the test. (3) The prozone phenomenon is a false-negative reaction to a serologic test that occurs when very high antibody titers in the serum prevent the formation of the antibody-antigen lattice needed to visualize a flocculation reaction. (4) Dilution of the serum, as was done in this case, reestablishes the proper concentration of antibody needed for optimal antibody-antigen lattice formation and reveals the presence of the prozone phenomenon.

The prozone phenomenon has been reported primarily in early syphilis (especially secondary syphilis), (3,4) but has also been reported in the setting of isolated neurosyphilis. (5) It is unknown whether a cerebrospinal fluid nontreponemal test displays the prozone phenomenon, although this is much less likely to occur than in serum because of lower cerebrospinal fluid nontreponemal assay antibody titers. The prozone phenomenon has been reported to occur in 0.3 to 2% of all cases of syphilis, (3) and may be more common in diseases associated with B-cell dysregulation and excess antibody, such as HIV. (4) Antibody titers reported after serum dilution in the prozone phenomenon have ranged from 1:64 to greater than 1:2,056. (2,6) The prozone phenomenon has been reported to occur with use of other serologic tests such as the HIV enzyme-linked immunosorbent assay. (7)

In their case report, Smith and Holman (2) hypothesize that there may be an increase in the incidence of the prozone phenomenon in the HIV-infected population because of the increased frequency of serologic testing for syphilis in this population, which in most clinical settings is probably performed at least annually or more often based on risk assessment. It is important to remember that it is the B-cell dysregulation in HIV-infected individuals that likely leads to the prozone effect, and such dysregulation will most likely be seen in HIV-infected individuals with acquired immunodeficiency syndrome or who have HIV viral loads that are not controlled on highly active antiretroviral therapy. HIV-infected individuals with CD4 counts above 200 and low or undetectable HIV viral loads may not have a higher incidence of the prozone phenomenon.

No matter what stage of syphilis is being treated and whether there is other laboratory evidence to establish the diagnosis (ie, treponemes seen on darkfield microscopy), a nontreponemal test should always be performed before initiating treatment when possible, as nontreponemal titers correlate with disease activity and provide an objective measure for monitoring response to therapy. However, treatment in any patient with suspected primary or secondary syphilis based on their clinical presentation should not be held while awaiting test results. Although cost-effectiveness of routinely performing quantitative nontreponemal tests on all patients has not been demonstrated, quantitation of serum titers should be considered in the following settings: 1) when clinical findings are suggestive of primary or secondary syphilis; 2) in MSM who are HIV-infected or have high-risk sexual behavior; and 3) in pregnant women in areas of high syphilis prevalence.

Control of syphilis spread and achieving eradication of this disease in the United States will depend on a multifaceted approach including screening, treatment of infected patients and their partners, and educational efforts addressing high-risk sexual behavior, particularly in populations such as MSM and in areas of high disease prevalence, such as the South. Awareness of the prozone phenomenon and addressing it in the previously mentioned settings is a key component of this multifaceted approach.

Accepted July 25, 2003.

Copyright [c] 2004 by The Southern Medical Association

0038-4348/04/9704-0327

Please see "The Prozone Phenomenon with Syphilis and HIV-1 Co-infection" on page 379 of this issue.

References

1. Centers for Disease Control and Prevention, Division of STD Prevention. Sexually Transmitted Disease Surveillance 2001 Supplement: Syphilis Surveillance Report. Atlanta, Centers for Disease Control and Prevention, Feb 2003. Available at: http://www.cdc.gov/std/Syphilis2001/2001SyphSuppText.pdf. Accessed August 12, 2003.

2. Smith G, Holman RP. The prozone phenomenon with syphilis and HIV-1 co-infection. South Med J 2004;97:379-382.

3. el-Zaatari MM, Martens MG, Anderson GD. Incidence of the prozone phenomenon in syphilis serology. Obstet Gynecol 1994;84:609-612.

4. Jurado RL, Campbell J, Martin PD. Prozone phenomenon in secondary syphilis: has its time arrived? Arch Intern Med 1993;153:2496-2498.

5. Uribe CS, Garcia FA. Neurosyphilis and the prozone effect [in Spanish]. Rev Neurol 1998;27:970-972.

6. Berkowitz K, Baxi L, Fox HE. False-negative syphilis screening: the prozone phenomenon, nonimmune hydrops, and diagnosis of syphilis during pregnancy. Am J Obstet Gynecol 1990;163:975-977.

7. Imrie A, Best S, Penny R, et al. False-negative ELISA result for HIV antibody due to an apparent prozone phenomenon. Med J Aust 1987;147:42 (letter).

William M. Geisler, MD, MPH

From the Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL.

William Geisler is supported by the Centers for Disease Control and Prevention Sexually Transmitted Disease Faculty Expansion Program Grant (R30CCR421113).

Reprint requests to William M. Geisler, MD, MPH, Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 703 19th Street S., 242 Zeigler Research Bldg., Birmingham, AL 35294-0007. Email: [e-mail]