Vol. 30, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2762-2764
0095-1137/92/112762-03$02.00/0 Copyright © 1992, American Society for Microbiology
Multicenter Evaluation of the AntigEnz Chlamydia Enzyme Immunoassay for Diagnosis of Chlamydia trachomatis Genital Infection A.
CLARK,`* W. E. STAMM,' C. GAYDOS,2 L. WELSH,2 T. C. QUINN,2
J. SCHACHITER,3 AND J. MONCADA3 Department of Medicine, University of Washington, Seattle, Washington, 981041; Johns Hopkins University, Baltimore, Maryland, 21205; and Department of Laboratoty Medicine, University of California, San Francisco, California 941433 Received 22 May 1992/Accepted 30 July 1992
To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for ETA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9%S/ in Baltimore (n = 532), 11% in Seattle (n = 500), and 9%to in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100o among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99%o in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.
MATERTALS AND METHODS
Chlamydial infection is recognized as the most prevalent bacterial sexually transmitted disease in the United States, causing more than 4,000,000 infections each year (3). Although most of these cases are minimally symptomatic initially, many progress to produce serious sequelae, including pelvic inflammatory disease, perihepatitis, infertility, and ectopic pregnancy (4, 8, 13, 17-19, 21). Cervical infection caused by chlamydia may also increase the risk of acquiring or transmitting human immunodeficiency virus (1, 9, 10). Finally, chlamydial infection during pregnancy places the newborn at risk for infant pneumonia and neonatal conjunctivitis (2, 12). For all of these reasons, screening of young women to ensure early diagnosis and treatment of chlamydial infection is important. More recently, asymptomatic young men with unrecognized chlamydial urethritis have also been identified as a target for screening (15, 16, 20). Because cultures for chlamydia are not widely available, noncultural tests such as enzyme immunoassay (EIA) directed against various chlamydial antigens have been introduced to facilitate laboratory screening and diagnosis of chlamydial infection. The advantages of EIA include the fact that microscopy is not required, viable organisms are not necessary, and the test itself is rapid and technically simple. We evaluated the performance of a new chlamydial EIA, AntigEnz Chlamydia (Baxter/Bartels), which employs a microplate format to detect soluble chlamydial antigen.
*
Patient populations and specimen collection. All men and women attending public clinics for sexually transmitted diseases at the three evaluation sites (San Francisco, Seattle, and Baltimore) were asked to participate in the study. Men were eligible only if they had not urinated in the past hour. Duplicate endocervical or urethral swabs were collected from participating patients. For culture, swabs were placed in a sucrose-phosphate buffer solution containing fetal bovine serum and appropriate antibiotics and transported to the laboratory. For AntigEnz Chlamydia testing, swabs were placed in a transport medium consisting of Eagle minimum essential medium containing fetal bovine serum, sugars, and appropriate antibiotics supplied by the manufacturer
(Northumbria Biologicals Ltd., Northumberland, United Kingdom). Swab order was alternated, with the first specimen being used for the AntigEnz Chlamydia for one-half of the samples and for culture in one-half of the specimens. Laboratory methods. Specimens for chlamydial isolation were cultured on cycloheximide-treated McCoy cells within 24 to 48 h of collection. Specimens were held in transport medium at 4°C until processed. In Baltimore, isolates were inoculated onto monolayers grown in microtiter plates, while in San Francisco, monolayers were grown on coverslips in glass vials as described by Ripa and Mardh (11). Workers at both sites performed one blind passage. At the Seattle site, McCoy cell monolayers were grown in 96-well microtiter plates, and passages were not done. Chlamydial inclusions were detected by using fluoresceinated anti-major outer membrane protein species-specific monoclonal antibody
Corresponding author. 2762
AntigEnz CHLAMYDIA EIA
VOL. 30, 1992
2763
TABLE 1. Comparison of AntigEnz Chlamydia EIA with culture Site and population
Baltimore Women (n = 430) Men (n = 102) San Francisco Women (n = 401) Men (n = 96)
Revised
Revised
Sensitivity (%)
9.1 8.8
92.3 (39) 100 (9)
94.6 (391) 88.2 (93)
100
8.2 10.4
84.8 (33) 100 (10)
97.0 (368) 96.5 (86)
86.5 (37) 100 (12)
98.1 (364) 98.8 (84)
9 16.4
79.4 (34) 75 (20)
98.5 (342) 98 (101)
79.4 (34) 76.2 (21)
98.5 (344)
8.8 12.2 9.5
85.8 (106) 87.2 (39) 86.5 (145)
96.6 (1,101) 94.3 (280) 95.5 (1,318)
88.3 (128) 90.5 (53) 89.4 (181)
98.6 (1,081) 99.3 (267) 99 (1,348)
Seattle
Women (n = 378) Men (n = 122) All sites Women (n = 1,209) Men (n = 320) Total (n = 1,529)
Specificity (%)b
Prevalence (%)
94.7 (57)
(20)
99.2 (373) 100
99
(82)
(101)
a Numbers in parentheses are total positives in agreement. b Numbers in parentheses are total negatives in agreement. c Revised parameters include those reevaluated discrepant samples also positive by DFA.
preparations at all sites. For the EIA, test specimens and controls were vortexed and then placed in a heated block at 100°C (+5°C) for 10 min, cooled, and revortexed. An aliquot of patient sample was placed in the microwell along with a murine monoclonal antibody specific for chlamydial lipopolysaccharide and a horseradish peroxidase-conjugated anti-murine immunoglobulin G antibody. Following a 60-min incubation at 37°C and subsequent rinsing, the chromogenic substrate 3,3',5,5'-tetramethyl benzidine (TMB) was added. At the end of substrate incubation (20 min), a stopping solution of 2 N H2SO4 was added to each well. Readings were performed on a spectrophotometer able to measure 250-,ul volumes and record the A450 of microwells. A colored enzyme substrate product above the upper threshold level (calculated by adding 0.15 absorbance units to the average absorbance of the negative control) indicated the presence of chlamydia in the patient specimen. All specimens giving readings between the lower threshold (adding 0.1 absorbance units to the average absorbance of the negative control) and the upper threshold (described above) fell into the designated grey zone and were retested along with the original controls. All specimens giving repeat absorbances greater than the threshold were then recorded as positive, while those with absorbances equivalent to or lower than the threshold were recorded as negative. Analysis of discrepant results. Where differing results were obtained by the tissue culture method and AntigEnz Chlamydia, a 200-,ul aliquot of the original EIA transport buffer was centrifuged at a minimum of 10,000 x g for 15 min, suspended in phosphate-buffered saline, and respun. The resulting pellet was spotted to a slide, fixed with methanol, and then stained with Syva Microtrak Chlamydia trachomatis Direct Specimen Test Reagent (Syva Co., Palo Alto, Calif.) to detect elementary bodies (EBs). A positive slide was defined as having two or more chlamydial EBs. RESULTS A total of 1,529 patients were studied by both the AntigEnz Chlamydia EIA and conventional cell culture. The prevalences of infection as judged by culture in Baltimore, San Francisco, and Seattle were, respectively, 9% (39 of 430), 8% (33 of 401), and 9% (34 of 378) in women and 9% (9 of 102), 10% (10 of 96), and 16% (20 of 122) in men. The sensitivities of EIA and culture differed by study site.
Among women, the sensitivity was 92% in Baltimore, 85% in San Francisco, and 79% in Seattle. In men, similar variations in sensitivity of the EIA by study site location were observed. Overall sensitivity of the EIA for all sites combined was 86% in women and 87% in men. Specificity of the EIA ranged from 88 to 90% for men and from 95 to 99% among women at each site. Positive predictive values in women were 63 to 84%, and negative predictive values ranged from 98 to 99%. In men, positive and negative predictive values were 77 to 88 and 95 to 100%, respectively. On further analysis of specimens that originally fell in the grey zone, 6 of 15 specimens were EIA positive upon repeat assay at the Baltimore site, and 3 of the 6 showed EBs in direct immunofluorescense assay (DFA; 3, 7, and >20 EBs per slide). All nine that were EIA negative upon repeat assay were negative by culture and DFA. Two of five grey-zone specimens were positive upon repeat EIA at San Francisco, and neither showed EBs in a DFA. The three specimens that were negative upon repeat were also neative by DFA and culture. In Seattle, one of three grey-zone specimens was positive upon repeat. This specimen was also positive by DFA. Of two grey-zone specimens that were negative upon repeat, both were negative by DFA and culture. Thus, combining all of the sites, 9 of 23 borderline results were positive on repeat (of which 4 were confirmed by DFA), and 14 were negative. All specimens with discrepant results were reevaluated to determine whether EBs were present by DFA analysis. In all, AntigEnz Chlamydia detected 161 of the 181 culturepositive specimens. Of the 20 culture-positive patients not detected by EIA, 7 had chlamydial EBs seen by DFA. Of the 53 specimens positive by EIA alone, 17 were positive by DFA. Defining true positives as any specimen positive by routine culture or any specimen positive by DFA, we recalculated the sensitivity and specificity of the AntigEnz Chlamydia (Table 1). The revised sensitivity of the EIA in women ranged from 79% in Seattle to 95% in Baltimore. The revised specificity was 98 to 99%. In men, the revised sensitivity ranged from 76 to 100% (Table 1), while revised specificity was 99 to 100%. Revised positive and negative predictive values were 82 to 95 and 99%, respectively, for women and 92 to 100 and 98 to 100%, respectively, for men.
2764
J. CLIN. MICROBIOL.
CLARK ET AL.
DISCUSSION
Many different forms of EIA that detect chlamydial antigen in genital specimens are now available. Most of these assays are well suited to testing large numbers of specimens, and if they were sufficiently sensitive and specific, they would be useful either for screening or for diagnostic testing in large clinics or laboratories. Hands-on time to perform these assays is short compared with tissue culture, and results are available the same day. The reading of these assays is also objective. An instrument measures the color intensity of each reaction, thus eliminating variation in interpretation of positives, as may occur with DFA. However, comparison of the performance of one EIA evaluated in one laboratory with the same assay or another EIA tested in a different laboratory is problematic (19). Comprehensive reviews of the performances of these various assays have demonstrated a wide range of reported sensitivities and even a surprisingly broad range of specificities. Unless two tests are directly compared in the same patient population and laboratory, variables such as specimen collection quality, transport time, type of population tested, and sensitivity of comparative culture systems all influence the observed sensitivity and specificity of the EIA. The sensitivity of an EIA, for example, is directly proportional to the number of inclusion-forming units in chlamydial culture (14). Thus, in symptomatic populations with higher median inclusion counts, EIAs usually perform better than in asymptomatic populations with lower mean inclusion counts. Similarly, the sensitivity of an EIA is usually greater in women than in men because the median number of inclusion-forming units in cervical specimens is typically 10-fold that found in urethral specimens. The factors outlined above may explain the variation in EIA sensitivity that we observed from site to site in our study. In conclusion, as judged by reports in the literature and our previous experience with antigen assays (5-7, 14), we found that the sensitivity and specificity of AntigEnz Chlamydia EIA compared favorably with those of other available EIAs. We recommend this assay as a diagnostic or screening test in moderate- to high-risk populations. Further testing is needed to determine its performance in screening lower-
prevalence populations. ACKNOWLEDGMENT This work was supported in part by a grant from the Northumbria
Corp. REFERENCES 1. Aral, S. O., and K. K. Holmes. 1991. Sexually transmitted diseases in the AIDS era. Sci. Am. 264(2):62-69. 2. Beem, M. O., and E. M. Saxon. 1977. Respiratory-tract colonization and a distinctive pneumonia syndrome in infants infected with Chiamydia trachomatis. N. Engl. J. Med. 296:306-310. 3. Centers for Disease Control. 1985. Chlamydia trachomatis infections. Policy guidelines for prevention and control. Morbid. Mortal. Weekly Rep. 34:53S-54S. 4. Department of Health and Human Services, Sexually Transmitted Disease Branch, National Institutes of Health. 1991. Pelvic in-
5.
6.
7.
8.
9.
10. 11. 12.
13.
14.
flammatory disease: research directions in the 1990s. Sex. Transm. Dis. 18:46-64. Gaydos, C., C. Reichart, J. Long, L. Welsh, T. Neumann, E. Hook III, and T. Quinn. 1990. Evaluation of Syva enzyme immunoassay for detection of Chlamydia trachomatis in genital specimens. J. Clin. Microbiol. 28:1541-1544. Moncada, J., J. Schachter, G. Bolan, J. Engelman, L. Howard, I. Mushahwar, G. Ridgway, G. Mumtaz, W. Stamm, and A. Clark. 1990. Confirmatory assay increases specificity of the Chlamydiazyme test for Chlamydia trachomatis infection of the cervix. J. Clin. Microbiol. 28:1770-1773. Moncada, J., J. Schachter, G. Bolan, J. Nathan, M.-A. Shafer, G. Clark, J. Schwebke, W. Stamm, T. Mroczkowski, Z. Selibroska, and D. H. Martin. Evaluation of Syva's enzyme immunoassay for the detection of Chlamydia trachomatis in urogenital specimens. J. Diagn. Microbiol. Infect. Dis., in press. Paavonen, J., P. Saikku, and J. von Knorring. 1981. Association of infection with Chiamydia trachomatis with Fitz-Hugh-Curtis syndrome. J. Infect. Dis. 144:176. Plummer, F., J. Simonsen, D. Cameron, J. Ndinya-Achola, J. Kreiss, M. Gakinya, P. Waiyaki, M. Cheang, P. Piot, A. Ronald, and E. Ngugi. 1991. Cofactors in male-female sexual transmission of human immunodeficiency virus type 1. J. Infect. Dis. 163:233-239. Quinn, T., J. Mann, J. Curran, and P. Piot. 1986. AIDS in Africa: an epidemiologic paradigm. Science 234:955-963. Ripa, K. T., and P. A. Mardh. 1977. Cultivation of Chlamydia trachomatis in cycloheximide-treated McCoy cells. J. Clin. Microbiol. 6:328-331. Sandstrom, K. I., T. A. Bell, J. W. Chandler, C. C. Kuo, S. P. Wang, J. T. Grayston, H. M. Foy, W. E. Stamm, M. K. Cooney, A. L. Smith, and K. K. Holmes. 1984. Microbial causes of neonatal conjunctivitis. J. Pediatr. 5:706-711. Schachter, J. 1991. Chlamydiae, p. 1045-1053. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. Schwebke, J. R., W. E. Stamm, and H. H. Handsfield. 1990. Use of sequential enzyme immunoassay and direct fluorescent antibody tests for detection of Chlamydia trachomatis infections in women. J. Clin. Microbiol. 28:2473-2476.
15. Shafer, M. A., V. Prager, J. Shalwitz, E. Vaughan, B. Moscicki, R. Brown, C. Wibbelsman, and J. Schachter. 1987. Prevalence of urethral Chlamydia trachomatis and Neisseria gonorrhoeae among asymptomatic, sexually active adolescent boys. J. Infect. Dis. 156:223-224. 16. Shafer, M. A., J. Schachter, A. B. Moscicki, et al. 1989. Urinary leukocyte esterase screening test for asymptomatic chlamydial and gonococcal infections in males. JAMA 262:2562-2566. 17. Stamm, W. E. 1986. Diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infections using antigen detection methods. Diagn. Microbiol. Infect. Dis. 4:93S-99S. 18. Stamm, W. E. 1988. Diagnosis of Chlamydia trachomatis genitourinary infections. Ann. Intern. Med. 108:710-717. 19. Stamm, W. E. 1990. Laboratory diagnosis of chlamydial infection, p. 459-470. In W. R. Bowie, H. D. Caldwell, R. 0. Jones, P.-A. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.), Chlamydial infections: proceedings of the 7th International Symposium on Human Chlamydial Infections. Cambridge University Press, Cambridge. 20. Stamm, W. E., and B. Cole. 1986. Asymptomatic Chlamydia trachomatis urethritis in men. Sex. Transm. Dis. 13:163-165. 21. Wasserheit, J. N., T. A. Bell, N. B. Kiviat, et al. 1986. Microbial causes of proven pelvic inflammatory disease and efficacy of clindamycin and tobramycin. Ann. Intern. Med. 104:187-193.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2762-2764
0095-1137/92/112762-03$02.00/0 Copyright © 1992, American Society for Microbiology
Multicenter Evaluation of the AntigEnz Chlamydia Enzyme Immunoassay for Diagnosis of Chlamydia trachomatis Genital Infection A.
CLARK,`* W. E. STAMM,' C. GAYDOS,2 L. WELSH,2 T. C. QUINN,2
J. SCHACHITER,3 AND J. MONCADA3 Department of Medicine, University of Washington, Seattle, Washington, 981041; Johns Hopkins University, Baltimore, Maryland, 21205; and Department of Laboratoty Medicine, University of California, San Francisco, California 941433 Received 22 May 1992/Accepted 30 July 1992
To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for ETA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9%S/ in Baltimore (n = 532), 11% in Seattle (n = 500), and 9%to in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100o among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99%o in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.
MATERTALS AND METHODS
Chlamydial infection is recognized as the most prevalent bacterial sexually transmitted disease in the United States, causing more than 4,000,000 infections each year (3). Although most of these cases are minimally symptomatic initially, many progress to produce serious sequelae, including pelvic inflammatory disease, perihepatitis, infertility, and ectopic pregnancy (4, 8, 13, 17-19, 21). Cervical infection caused by chlamydia may also increase the risk of acquiring or transmitting human immunodeficiency virus (1, 9, 10). Finally, chlamydial infection during pregnancy places the newborn at risk for infant pneumonia and neonatal conjunctivitis (2, 12). For all of these reasons, screening of young women to ensure early diagnosis and treatment of chlamydial infection is important. More recently, asymptomatic young men with unrecognized chlamydial urethritis have also been identified as a target for screening (15, 16, 20). Because cultures for chlamydia are not widely available, noncultural tests such as enzyme immunoassay (EIA) directed against various chlamydial antigens have been introduced to facilitate laboratory screening and diagnosis of chlamydial infection. The advantages of EIA include the fact that microscopy is not required, viable organisms are not necessary, and the test itself is rapid and technically simple. We evaluated the performance of a new chlamydial EIA, AntigEnz Chlamydia (Baxter/Bartels), which employs a microplate format to detect soluble chlamydial antigen.
*
Patient populations and specimen collection. All men and women attending public clinics for sexually transmitted diseases at the three evaluation sites (San Francisco, Seattle, and Baltimore) were asked to participate in the study. Men were eligible only if they had not urinated in the past hour. Duplicate endocervical or urethral swabs were collected from participating patients. For culture, swabs were placed in a sucrose-phosphate buffer solution containing fetal bovine serum and appropriate antibiotics and transported to the laboratory. For AntigEnz Chlamydia testing, swabs were placed in a transport medium consisting of Eagle minimum essential medium containing fetal bovine serum, sugars, and appropriate antibiotics supplied by the manufacturer
(Northumbria Biologicals Ltd., Northumberland, United Kingdom). Swab order was alternated, with the first specimen being used for the AntigEnz Chlamydia for one-half of the samples and for culture in one-half of the specimens. Laboratory methods. Specimens for chlamydial isolation were cultured on cycloheximide-treated McCoy cells within 24 to 48 h of collection. Specimens were held in transport medium at 4°C until processed. In Baltimore, isolates were inoculated onto monolayers grown in microtiter plates, while in San Francisco, monolayers were grown on coverslips in glass vials as described by Ripa and Mardh (11). Workers at both sites performed one blind passage. At the Seattle site, McCoy cell monolayers were grown in 96-well microtiter plates, and passages were not done. Chlamydial inclusions were detected by using fluoresceinated anti-major outer membrane protein species-specific monoclonal antibody
Corresponding author. 2762
AntigEnz CHLAMYDIA EIA
VOL. 30, 1992
2763
TABLE 1. Comparison of AntigEnz Chlamydia EIA with culture Site and population
Baltimore Women (n = 430) Men (n = 102) San Francisco Women (n = 401) Men (n = 96)
Revised
Revised
Sensitivity (%)
9.1 8.8
92.3 (39) 100 (9)
94.6 (391) 88.2 (93)
100
8.2 10.4
84.8 (33) 100 (10)
97.0 (368) 96.5 (86)
86.5 (37) 100 (12)
98.1 (364) 98.8 (84)
9 16.4
79.4 (34) 75 (20)
98.5 (342) 98 (101)
79.4 (34) 76.2 (21)
98.5 (344)
8.8 12.2 9.5
85.8 (106) 87.2 (39) 86.5 (145)
96.6 (1,101) 94.3 (280) 95.5 (1,318)
88.3 (128) 90.5 (53) 89.4 (181)
98.6 (1,081) 99.3 (267) 99 (1,348)
Seattle
Women (n = 378) Men (n = 122) All sites Women (n = 1,209) Men (n = 320) Total (n = 1,529)
Specificity (%)b
Prevalence (%)
94.7 (57)
(20)
99.2 (373) 100
99
(82)
(101)
a Numbers in parentheses are total positives in agreement. b Numbers in parentheses are total negatives in agreement. c Revised parameters include those reevaluated discrepant samples also positive by DFA.
preparations at all sites. For the EIA, test specimens and controls were vortexed and then placed in a heated block at 100°C (+5°C) for 10 min, cooled, and revortexed. An aliquot of patient sample was placed in the microwell along with a murine monoclonal antibody specific for chlamydial lipopolysaccharide and a horseradish peroxidase-conjugated anti-murine immunoglobulin G antibody. Following a 60-min incubation at 37°C and subsequent rinsing, the chromogenic substrate 3,3',5,5'-tetramethyl benzidine (TMB) was added. At the end of substrate incubation (20 min), a stopping solution of 2 N H2SO4 was added to each well. Readings were performed on a spectrophotometer able to measure 250-,ul volumes and record the A450 of microwells. A colored enzyme substrate product above the upper threshold level (calculated by adding 0.15 absorbance units to the average absorbance of the negative control) indicated the presence of chlamydia in the patient specimen. All specimens giving readings between the lower threshold (adding 0.1 absorbance units to the average absorbance of the negative control) and the upper threshold (described above) fell into the designated grey zone and were retested along with the original controls. All specimens giving repeat absorbances greater than the threshold were then recorded as positive, while those with absorbances equivalent to or lower than the threshold were recorded as negative. Analysis of discrepant results. Where differing results were obtained by the tissue culture method and AntigEnz Chlamydia, a 200-,ul aliquot of the original EIA transport buffer was centrifuged at a minimum of 10,000 x g for 15 min, suspended in phosphate-buffered saline, and respun. The resulting pellet was spotted to a slide, fixed with methanol, and then stained with Syva Microtrak Chlamydia trachomatis Direct Specimen Test Reagent (Syva Co., Palo Alto, Calif.) to detect elementary bodies (EBs). A positive slide was defined as having two or more chlamydial EBs. RESULTS A total of 1,529 patients were studied by both the AntigEnz Chlamydia EIA and conventional cell culture. The prevalences of infection as judged by culture in Baltimore, San Francisco, and Seattle were, respectively, 9% (39 of 430), 8% (33 of 401), and 9% (34 of 378) in women and 9% (9 of 102), 10% (10 of 96), and 16% (20 of 122) in men. The sensitivities of EIA and culture differed by study site.
Among women, the sensitivity was 92% in Baltimore, 85% in San Francisco, and 79% in Seattle. In men, similar variations in sensitivity of the EIA by study site location were observed. Overall sensitivity of the EIA for all sites combined was 86% in women and 87% in men. Specificity of the EIA ranged from 88 to 90% for men and from 95 to 99% among women at each site. Positive predictive values in women were 63 to 84%, and negative predictive values ranged from 98 to 99%. In men, positive and negative predictive values were 77 to 88 and 95 to 100%, respectively. On further analysis of specimens that originally fell in the grey zone, 6 of 15 specimens were EIA positive upon repeat assay at the Baltimore site, and 3 of the 6 showed EBs in direct immunofluorescense assay (DFA; 3, 7, and >20 EBs per slide). All nine that were EIA negative upon repeat assay were negative by culture and DFA. Two of five grey-zone specimens were positive upon repeat EIA at San Francisco, and neither showed EBs in a DFA. The three specimens that were negative upon repeat were also neative by DFA and culture. In Seattle, one of three grey-zone specimens was positive upon repeat. This specimen was also positive by DFA. Of two grey-zone specimens that were negative upon repeat, both were negative by DFA and culture. Thus, combining all of the sites, 9 of 23 borderline results were positive on repeat (of which 4 were confirmed by DFA), and 14 were negative. All specimens with discrepant results were reevaluated to determine whether EBs were present by DFA analysis. In all, AntigEnz Chlamydia detected 161 of the 181 culturepositive specimens. Of the 20 culture-positive patients not detected by EIA, 7 had chlamydial EBs seen by DFA. Of the 53 specimens positive by EIA alone, 17 were positive by DFA. Defining true positives as any specimen positive by routine culture or any specimen positive by DFA, we recalculated the sensitivity and specificity of the AntigEnz Chlamydia (Table 1). The revised sensitivity of the EIA in women ranged from 79% in Seattle to 95% in Baltimore. The revised specificity was 98 to 99%. In men, the revised sensitivity ranged from 76 to 100% (Table 1), while revised specificity was 99 to 100%. Revised positive and negative predictive values were 82 to 95 and 99%, respectively, for women and 92 to 100 and 98 to 100%, respectively, for men.
2764
J. CLIN. MICROBIOL.
CLARK ET AL.
DISCUSSION
Many different forms of EIA that detect chlamydial antigen in genital specimens are now available. Most of these assays are well suited to testing large numbers of specimens, and if they were sufficiently sensitive and specific, they would be useful either for screening or for diagnostic testing in large clinics or laboratories. Hands-on time to perform these assays is short compared with tissue culture, and results are available the same day. The reading of these assays is also objective. An instrument measures the color intensity of each reaction, thus eliminating variation in interpretation of positives, as may occur with DFA. However, comparison of the performance of one EIA evaluated in one laboratory with the same assay or another EIA tested in a different laboratory is problematic (19). Comprehensive reviews of the performances of these various assays have demonstrated a wide range of reported sensitivities and even a surprisingly broad range of specificities. Unless two tests are directly compared in the same patient population and laboratory, variables such as specimen collection quality, transport time, type of population tested, and sensitivity of comparative culture systems all influence the observed sensitivity and specificity of the EIA. The sensitivity of an EIA, for example, is directly proportional to the number of inclusion-forming units in chlamydial culture (14). Thus, in symptomatic populations with higher median inclusion counts, EIAs usually perform better than in asymptomatic populations with lower mean inclusion counts. Similarly, the sensitivity of an EIA is usually greater in women than in men because the median number of inclusion-forming units in cervical specimens is typically 10-fold that found in urethral specimens. The factors outlined above may explain the variation in EIA sensitivity that we observed from site to site in our study. In conclusion, as judged by reports in the literature and our previous experience with antigen assays (5-7, 14), we found that the sensitivity and specificity of AntigEnz Chlamydia EIA compared favorably with those of other available EIAs. We recommend this assay as a diagnostic or screening test in moderate- to high-risk populations. Further testing is needed to determine its performance in screening lower-
prevalence populations. ACKNOWLEDGMENT This work was supported in part by a grant from the Northumbria
Corp. REFERENCES 1. Aral, S. O., and K. K. Holmes. 1991. Sexually transmitted diseases in the AIDS era. Sci. Am. 264(2):62-69. 2. Beem, M. O., and E. M. Saxon. 1977. Respiratory-tract colonization and a distinctive pneumonia syndrome in infants infected with Chiamydia trachomatis. N. Engl. J. Med. 296:306-310. 3. Centers for Disease Control. 1985. Chlamydia trachomatis infections. Policy guidelines for prevention and control. Morbid. Mortal. Weekly Rep. 34:53S-54S. 4. Department of Health and Human Services, Sexually Transmitted Disease Branch, National Institutes of Health. 1991. Pelvic in-
5.
6.
7.
8.
9.
10. 11. 12.
13.
14.
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