< > Home : FAQ : Professional : Chlamydiales : Immunobiology : Infections : Diagnosis & Treatment : Links : Contact Us

Classic diagnostics: Antigen detection enzyme immunoassays

The first enzyme immunoassay (EIA) for the detection of chlamydial antigen in clinical specimens was the Abbott Chlamydiazyme^® which appeared around the same time as the Syva Microtrak^® direct immunofluorescence test. Unlike the Syva test, it is understood the Chlamydiazyme was based on an adsorbed polyclonal antiserum, rather than monoclonal antibody. Early clinical trials indicated that the test, which could be completed in four hours, had a sensitivity against male and female genital tract specimens of around 60 to 90%, depending on the sample and the reference technique [Jones et al., 1984; Howard et al., 1986; Taylor-Robinson et al., 1987].  Against direct immunofluorescence Taylor Robinson et al., 1987 reported a sensitivity of 67% for endocervical samples,  with positive and negative predictive values against sexually transmitted infections clinic populations of 63% and 90% respectively, and a specificity of 89%.  With the advent of more sensitive reference procedures, it is now believed the sensitivity of most antigen detection assays is of the order of 60% [see: nucleic acid amplification figure 1]. 

It soon became clear that there were specificity problems [Saikku et al., 1986]. Lack of specificity was associated with the finding that the enzyme immunoassay antibody reacted with strains of Acinetobacter calcoaceticus, Escherichia coli, Gardnerella vaginalis, Neisseria gonorrhoeae and group B streptococci [Taylor-Robinson et al., 1987]. In women urinary tract infection could cause false positive results [Demaio et al., 1991]. Specificity was, however, improved by the introduction of a confirmation test using blocking antibody [Moncada et al., 1990; Newhall et al., 1999]. 

Subsequently three further chlamydial antigen detection EIAs were introduced to the market, the Syva Microtrak EIA, the Pathfinder^® EIA (Sanofi/Kallestad) and the Boots-Celltech [subsequently  Dako] IDEIA^® . The latter was a radically different immunoassay, being based on the improved specificity of monoclonal antibody to the chlamydia-specific lipopolysaccharide epitope plus an ingenious, redox-linked, signal amplification system [Stanley et al., 1985]. Early results suggested that the sensitivity and specificity of these assays for use on endocervical or endourethral specimens were broadly comparable [Mahony et al., 1989; Thomas et al., 1994]. The tests were of practical use for screening high risk, high prevalence populations but lacked sufficient specificity for screening low risk populations [Thomas et al., 1994; see: specificity]. As Schachter & Chow 1995 pointed out, reduced specificity in a diagnostic test results in an underestimation of a risk factor's association with an infection, which bias is particularly severe in low prevalence populations. Specificity, however, could be significantly improved by carrying out a confirmatory test with a different procedure on positive specimens. These tests were usually either a blocking antibody test, or direct immunofluorescence [Beebe et al., 1996; Moncada et al., 1990; Schwebke et al., 1991; Thomas et al., 1994].

A range of rapid, qualitative enzyme immunoassay-based diagnostic tests for chlamydial antigen were also developed, aimed at the physicians office / "bedside testing". Such tests include the Unipath Clearview^® , the Kodak Surecell^® and the Quidel QuickVue^® .  The general impression is that these qualitative tests had lower sensitivity than their less rapid but quantitative counterparts, but they have been infrequently investigated. Rani et al., 2002 conducted a pilot study to evaluate the sensitivity and specificity of the Quidel QuickVue Chlamydia test, in populations with a low and high prevalence of chlamydial genital tract infection, against the polymerase chain reaction, for endocervical samples. The sensitivity and specificity of the QuickVue test compared to PCR were 65% (95% confidence interval [CI] 42-87%) and 100% respectively for the high prevalence population and 25% (95% CI: 5-70%) and 100% respectively for the low prevalence population. The positive predictive value was 100% for both high and low prevalence populations because of the high specificity. The sensitivity of the QuickVue test for a high prevalence population was comparable to laboratory-based enzyme immunoassay techniques, but not to nucleic acid amplification based methods. However, for a low prevalence population, this test, due to low sensitivity, failed to identify one in 4 cases.  [Rani et al., 2002].

[Comment: QuickVue is a rapid chlamydial antigen capture assay based on genus specific murine monoclonal antibody. In many respects it resembles the Unipath Clearview. Quidel corporation claim an overall sensitivity of 92.0% and specificity 98.6% against culture (manufacturers package insert). It must be remembered that culture itself is considered to have a sensitivity of only around 60 - 70% against more sensitive nucleic acid amplification tests; see: Fig 1. This is reflected in the results of Rani et al., 2002 which give a sensitivity for QuickVue  (against PCR) of 65%].

Many laboratories still use chlamydial antigen detection immunoassays because of their lower cost and ease of use. Research interest in this methodology, however, waned with the introduction of superior methods based on nucleic acid hybridisation  [Gen-Probe Pace 2^®] or amplification [see nucleic acid amplification]. Thus, despite steady development and use over more than a decade, few recent studies have compared these tests. However, one such study, using culture as reference standard, compared five different tests for the detection of C. trachomatis in endocervical specimens from 4,980 women attending family planning clinics in the northwestern United States. These tests were the Chlamydiazyme (Abbott), the Syva MicroTrak direct fluorescent antibody and Syva enzyme immunoassays,  the Pathfinder^® enzyme immunoassay and the Gen-Probe Pace 2 hybridisation test. All positive results  were confirmed, either by testing the original specimens with a blocking antibody test (Chlamydiazyme), by cytospin direct immunofluorescence (MicroTrak & Pathfinder EIAs), or by probe competition assay (Pace 2). This was a low risk population with a prevalence of culture-proven chlamydial infection of 3.9%. The sensitivities of the various tests ranged from 62 to 75%, [an overestimate since culture itself is only about 70% sensitive]. The positive predictive value for each test was 85% or higher and, surprisingly, the specificities of the tests, even without confirmation assays was greater than 99%. Performing confirmatory tests eliminated virtually all false positives [Newhall et al., 1999]. In high prevalence populations, such tests might be acceptable for screening purposes. Nevertheless, despite  low cost,  an analysis of diagnostic methods for chlamydia screening to prevent pelvic inflammatory disease in the state of Maryland, USA, concluded that nucleic acid amplification-based methods were ultimately the most cost effective [Howell et al., 1998]. 

Improvements continue to be made in chlamydial antigen detection enzyme immunoassay. Dako have introduced a new generation IDEIA^®, called the IDEIA PCE^®.  The latter test incorporates a polymer conjugate consisting of multiple copies of antibody and enzyme molecules to provide signal amplification [Okadome et al., 1999].  When tested against an Abbott LCx^® LCR reference standard on paired endocervical swabs from each of 1,123 women, the sensitivity of the IDEIA PCE against LCR was 91.8% after confirmation and the specificity a creditable 99.8%  [Chernesky et al., 2001]. 

[Comment: Given that LCR is one of the most sensitive and specific tests, these are impressive figures; studies by other groups are indicated]. 

A Japanese study of the new test compared the performance of the original Dako IDEIA, the new IDEIA PCE and the Amplicor^® PCR (Roche). The two study groups were 1) first voided urine samples from 193 male patients with urethritis and 2) endocervical and vaginal swabs from 187 high risk commercial sex workers. Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. This indicates that the new IDEIA test is a significantly improved antigen detection test, [bearing in mind that no test can improve on the PCR chosen here as the reference standard]. In laboratory tests, the sensitivity of the IDEIA PCE for different serovars of C. trachomatis and for C. muridarum varied from 38 inclusion forming units (IFU) per ml for C. trachomatis serovar F to 237 IFU per ml for serovar D. The incorporation of the polymer conjugate resulted in a worthwhile 2-5 fold increase in analytical sensitivity without any increase in cross reactivity with protein A producing strains of Staphylococcus aureus  [Okadome et al., 1999].

[Comment: Many clinical laboratories are familiar with enzyme immunoassay rather than with the more demanding nucleic acid amplification based methods. In these circumstances, and for moderate to high prevalences of infection (say >5%), the Dako IDEIA PCE may be useful but studies are needed by other groups].

[MEW] May 2002

NEXT: Point of care testing

References

Beebe, J. L., Masters, H., Jungkind, D., Heltzel, D. M. & Weinberg, A. (1996). Confirmation of the Syva MicroTrak enzyme immunoassay for Chlamydia trachomatis by Syva Direct Fluorescent Antibody Test. Sexually Transmitted Diseases  23, 465 - 470.

Chernesky, M., Jang, D., Copes, D., Patel, J., Petrich A., Biers, K., Sproston, A. & Kapala, J. (2001). Comparison of a polymer conjugate-enhanced enzyme immunoassay to ligase chain reaction for diagnosis of Chlamydia trachomatis in endocervical swabs. J 39, 2306 - 2307. Full article   [Surprisingly good performance by the DAKO IDEIA PCE against LCR]

Demaio, J., Boyd, R. S., Rensi, R. & Clark, A. (1991). False-positive Chlamydiazyme results during urine sediment analysis due to bacterial urinary tract infections. J 29, 1436 - 1438. 

Howard, L. V., Coleman, P. F., England, B. J. & Herrmann, J. E. (1986). Evaluation of Chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis. Journal of Clinical Microbiology 23, 329 - 332.

Howell, M. R., Quinn, T. C., Brathwaite, W. & Gaydos, C. A. (1998). Screening women for Chlamydia trachomatis in family planning clinics: the cost-effectiveness of DNA amplification assays. Sexually Transmitted Diseases 25, 108 - 117. [LCR is cost effective in Maryland for preventing pelvic inflammatory disease].

Jones, M. F., Smith, T. F., Houglum, A. J. & Herrmann, J. E. (1984). Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test. Journal of Clinical Microbiology 20, 465 - 467. [One of the first studies of the Chlamydiazyme EIA]

Mahony, J., Castriciano, S., Sellors, J., Stewart, I., Cunningham, I., Landis, S., Seidelman, W., Grant, L., Devlin, C. & Chernesky, M. (1989). Diagnosis of Chlamydia trachomatis genital infections by cell culture and two enzyme immunoassays detecting different chlamydial antigens. Journal of Clinical Microbiology 27, 1934 - 1938.

Moncada, J., Schachter, J., Bolan, G., Engelman, J., Howard, L., Mushahwar, I., Ridgway, G., Mumtaz, G., Stamm, W. & Clark, A. (1990). Confirmatory assay increases specificity of the Chlamydiazyme test for Chlamydia trachomatis infection of the cervix. Journal of Clinical Microbiology 28, 1770 - 1773.

Newhall, W. J., Johnson, R. E., DeLisle, S., Fine, D., Hadgu, A., Matsuda, B., Osmond, D., Campbell, J. & Stamm, W. E. (1999). Head-to-head evaluation of five chlamydia tests relative to a quality-assured culture standard. Journal of Clinical Microbiology 37, 681 - 685. Full article [Large study. A rare head to head evaluation of the later antigen detection immunoassays].

Okadome, A., Notomi, T., Nomura, S. & Nagayama, A. (2000). Reactivity of a dual amplified chlamydia immunoassay with different serovars of Chlamydia trachomatis. International  Journal of STD and AIDS 10, 460 - 463.

Rani, R., Corbitt, G., Killough, R., Curless, E. (2002). Is there any role for rapid tests for Chlamydia trachomatis? International Journal of STD and AIDS 13, 22 - 24. [Interesting pilot study].

Saikku, P., Puolakkainen, M., Leinonen, M., Nurminen, M. & Nissinen, A. (1986). Cross-reactivity between Chlamydiazyme and Acinetobacter strains. New England Journal of Medicine 314, 922 - 923. [A letter that hit the mark]

Schachter, J. & Chow, J. M. (1995). The fallibility of diagnostic tests for sexually transmitted diseases: the impact of behavioral and epidemiologic studies. Sexually Transmitted Diseases 22, 191 - 196. [Some good epidemiological and statistical points that needed spelling out]

Schwebke, J. R., Stamm, W. E., Handsfield, H. H. (1991). Use of sequential enzyme immunoassay and direct fluorescent antibody tests for detection of Chlamydia trachomatis infections in women. Journal of Clinical Microbiology 28 2473 - 2476.

Stanley, C. J., Johannsson, A. & Self, C. H. (1985). Enzyme amplification can enhance both the speed and the sensitivity of immunoassays. Journal of Immunological Methods 83, 89 - 95.  [The basis of the original IDEIA signal amplification, now superseded, but innovative in its time].

Stary, A., Kopp, W., Zahel, B., Muller, I., Nerad, S., Storch, M. (1994). Rapid diagnosis of Chlamydia trachomatis with a nucleic acid probe in male and female patients. Dermatology 188, 300 - 804.

Tanaka, M., Nakayama, H., Sagiyama, K., Haraoka, M., Yoshida, H., Hagiwara, T., Akazawa, K. & Naito, S. (2000). Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens. Journal of Clinical Pathology 53, 350 - 354. [Interesting investigation of the DAKO polymer conjugate enhanced IDEIA]

Taylor-Robinson, D., Thomas, B. J. & Osborn, M. F. (1987). Evaluation of enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis in genital tract specimens. Journal of Clinical Pathology 40, 194 - 199.

Thomas, B. J., MacLeod, E. J., Hay, P. E., Horner, P. J. & Taylor-Robinson, D. (1994). Limited value of two widely used enzyme immunoassays for detection of Chlamydia trachomatis in women. European Journal of Clinical Microbiology and Infectious Disease 13, 651 - 655. [A good reflective paper as usual from this stable]

v(2001). Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests. Journal of Clinical Microbiology 39, 1751 - 1756. Full article   

Wylie, J. L., Moses, S., Babcock, R., Jolly, A., Giercke, S. & Hammond, G. (1998). Comparative evaluation of Chlamydiazyme, PACE 2, and AMP-CT assays for detection of Chlamydia trachomatis in endocervical specimens. Journal of Clinical Microbiology

NEXT: Classic diagnostics: Serology