CLINICAL IMMUNOLOGY AND MICROBIOLOGY Original Article

Chlamydiazyme Plus Blocking Assay to Detect Chlamydia trachomatis in Endocervical Specimens RUSSELL D. MILLS, M.T. (B.S.),1 ANITA YOUNG, B.A.,1 KAREN CAIN, M.T. (B.S.),1 TINA M.H. BLAIR, M.D.,2 MICHAEL A. SITORIUS, M.D.,3 AND GAIL L. WOODS, M.D.1

Chlamydia trachomatis is the most common cause of sexually transmitted disease in the United States and is responsible for more than 4 million infections each year.1,2 Tissue culture is considered the reference method to detect C. trachomatis in clinical specimens; however, this approach to diagnosis is time consuming, with a minimum turn-around time of 48 hours, labor-intensive, and relatively costly. The development of antigen detection methods—direct immunofluorescence using monoclonal antibodies to the species-specific outer membrane protein and enzyme immunoassays (EIA)—has allowed rapid and cost-effective screening for C. trachomatis. Compared with culture, the sensitivity rate of direct immunofluorescence assay (DFA) in women has ranged

100%, and 99%, respectively, for culture and the enzyme-linked immunoabsorbent assay plus blocking assay and 74%, 98%, 83%, and 96%, respectively, for the direct immunofluorescence assay. In this population of women, using the enzyme-linked immunoabsorbent assay with the confirmatory assay is a rapid, reliable, and cost-effective alternative to culture for diagnosing infection with C. trachomatis. (Key words: Chlamydia trachomatis; Enzyme-linked immunoassay; Chlamydiazyme; Blocking assay) Am J Clin Pathol 1992;97:209-212

from 70 to 100%, depending on the type of population studied and the cutoff used for a positive result, and the specificity rate has been about 99%.3~8 Of the commercially available EIA kits, Chlamydiazyme (Abbott Laboratories, North Chicago, IL) has been evaluated most extensively; and in women, its sensitivity rat, compared with culture, has ranged from about 70 to 90%, depending on the population evaluated, and the specificity rate has varied from about 89 to 98%.9"13 Because of the lower specificity rate of Chlamydiazyme, using this test primarily in populations with a high prevalence of infection with C. trachomatis and not in low-risk populations, in which the predictive value is lower, has been recommended. However, the sensitivity of tissue culture for detecting C. trachomatis is less than 100%. Therefore, when culture is used as the reference to which other tests, such as Chlamydiazyme, are compared, a true-positive result could be From the Departments of ' Pathology and Microbiology, 2SurgeryEmergency Medicine, and ^Family Practice, University of Nebraska incorrectly labeled as a false-positive result because the Medical Center, Omaha, Nebraska. culture was negative.514 To distinguish between true-positive EIA results missed by culture and actual false-positive Received March 20, 1991; received revised manuscript and accepted for publication June 10, 1991. EIA results caused by nonspecific binding of test reagents Supported in part by Abbott Laboratories. or by the presence of high concentrations of bacteria with Gail Woods is associated with the Department of Pathology and Laboratory Medicine, Medical College of Pennsylvania, Philadelphia, Penn- cross-reacting antigens (such as Streptococcus, Acinetosylvania. bacter, Klebsiella, and Gardnerella) in the clinical speciAddress reprint requests to Mr. Mills: Department of Pathology and men, a confirmatory assay in which a monoclonal antiMicrobiology. University of Nebraska Medical Center, 600 South 42nd body reacts with the chlamydia-specific lipopolysaccharide Street, Omaha, Nebraska 68198-3135

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Three methods to detect Chlamydia trachomatis in endocervical swab specimens collected from 502 women with genitourinary or abdominopelvic symptoms were evaluated: (1) a direct immunofluorescence assay, (2) an enzyme-linked immunoabsorbent assay, confirming positive samples with a blocking assay, and (3) conventional tissue cell culture. C. trachomatis was detected by at least one method in 72 specimens, of which 56 (11%) were determined to be true-positive results by repeated testing and by performing a confirmatory assay. The sensitivity, specificity, and positive and negative predictive values were 91%, 100%,

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Article epitope, blocking the specific reactivity of the Chlamydiazyme antibody but not the binding of cross-reactive bacteria, has been developed.15 To evaluate this confirmatory assay, endocervical swab samples collected from women visiting the Emergency Department and attending the Family Health Clinic at the University of Nebraska Medical Center were tested by tissue culture; DFA, the method used routinely to screen for C. trachomatis at this institution; and Chlamydiazyme, confirming EIA-positive, culture-negative specimens by repeating the test with the blocking antibody. MATERIALS AND METHODS Study

Population

plate containing McCoy cell monolayers on 12-mm glass coverslips was inoculated with 0.5 mL of that mixture. A positive and negative control were included with each specimen run. Plates were centrifuged at 2,500g for 1 hour at 33 °C. After incubation at 35 °C in 5% C 0 2 for 2 hours, 1 mL of chlamydial culture medium with cycloheximide was added, and plates were incubated for 48 hours. Medium was removed and coverslips were fixed in 95% ethanol for 10 minutes, washed twice, stained with monoclonal antibodies (MicroTrak, Syva Co.), mounted on glass slides, and examined at 100X and 400X magnification using a Nikon epifluorescent microscope (Nikon, Garden City, NY). The presence of one or more positively fluorescing inclusion(s) was considered positive. DFA

Specimen Collection The exocervix was cleaned to remove excess mucous and exudate, and three swab specimens were collected from each individual. For tissue culture, one swab was placed into 1.5 mL of 2-sucrose phosphate (2-SP) transport medium, transported to the laboratory, and stored at —70 °C until processed for culture (not longer than 1 week). For DFA, one swab was processed according to manufacturer (Syva Co., Palo Alto, CA) directions and stored at 4 °C until stained and examined (no more than 24 hours). For Chlamydiazyme, the swab provided in the collection kit was placed in the Abbott transport tube and held at 4 °C for no more than 5 days. To prevent sampling bias, the order of specimen collection was rotated as follows: the laboratory technologists prepared packages consisting of the 2-SP medium and the DFA and Chlamydiazyme collection kits, each of which was labeled 1, 2, or 3 to indicate order of collection. Numbering was alternated to insure that the collection order was equally distributed. All physicians in the two departments participated in the study. Culture Specimens were thawed, vortexed, and sonicated for 3 minutes in a Mettler ultrasonic cleaner (Mettler Electronics, Anaheim, CA). To each specimen, 1 mL of Eagles minimum essential medium (Gibco, Grand Island, NY) containing 3% chlamydia-free fetal bovine serum (Wittacker M.A. Bioproducts, Walkersville, MD), gentamicin, and fungizone was added. Each of two wells of a 24-well A.J.C.P. •

Methanol-fixed smears were stained with the MicroTrak (Syva Co.) fluorescein-conjugated monoclonal antibodies according to the manufacturer directions. A positive and negative control slide (Syva Co.) were included with each specimen run. Using a Nikon Labophot epifluorescent microscope, which has a mercury light source, slides were scanned at 400X magnification and then examined at 1000X magnification to confirm the morphologic findings. The presence of three or more characteristic elementary bodies was considered positive.16 Samples containing fewer than 10 columnar epithelial cells or 1 or 2 elementary bodies and those with excessive amounts of mucus were considered inconclusive, and collection of a new specimen was recommended. Chlamydiazyme The Chlamydiazyme test was performed according to manufacturer directions. Briefly, samples, controls, and reagents were warmed to room temperature and 1 mL of specimen dilution buffer was added to each sample tube and allowed to stand for 10 to 15 minutes. Each sample was vortexed for 15 seconds three times, the fluid was expressed from the swab and discarded, and 150 pL of each vortexed control and sample was placed into one well of the reaction tray, to which one bead was added. Trays were sealed and incubated at 45 °C for 40 minutes in the Dynamic Incubator used in conjunction with the Abbott Commander Parallel Processing Center, which included a bead washer, reagent pipettor, spectophotometer, and data management system (all by Abbott Laboratories, Chicago, IL). Cover seals were removed and trays were processed through the Commander: beads were washed automatically and 150 fiL of rabbit antibody to C. trachomatis was added. Trays were sealed and incubated as previously described. Seals were removed, and trays were processed: beads were washed and 150 /ih of horseradish peroxidase-conjugated rabbit immunoglob-

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Endocervical swab specimens were collected from 502 women who came to the Emergency Department or Family Health Clinic at the University of Nebraska Medical Center with genitourinary or abdominopelvic complaints that warranted pelvic examination.

MILLS ET AL. Chlamydiazytne and Blocking Assay to Detect C. trachomatis ulin G was added. Trays were resealed, incubated as described above, and processed: beads were washed and 300 mL of o-phenylenediamine was added, and incubated in the dark at room temperature for 30 minutes. Cover seals were removed and trays were processed: sulfuric acid was added to stop the reaction and the absorbance of each well was read and calculated automatically. Positive samples were saved and tested in the blocking assay. Chlamydiazytne Blocking Assay

RESULTS On initial testing, C. trachomatis was detected by at least one method in 72 specimens; 51 were positive by culture, 64 were positive by Chlamydiazyme, 47 were positive by DFA (5 of which had between 3 and 10 elementary bodies), and 36 were positive by all three methods (Table 1). Fifty-seven specimens (11.3%) yielded incon-

TABLE 1. COMPARISON OF CELL CULTURE, DFA, AND CHLAMYDIAZYME FOR DETECTING C. TRACHOMATIS IN CERVICAL SPECIMENS Cell Culture

_ + + + + + -

Chlamydiazyme

DFA

_ -

IC

_

+ +

IC

+

+ -

+ -

+ -

IC

+

+

No. of Specimens 376 54 36 2 8 3 12(4)

KD

2 8

IC = inconclusive (see text); + = positive; - = negative. Numbers in parentheses refer to the number that were confirmed to be true-positive results by the blocking assay.

elusive DFA results, of which about one half were due to excessive amounts of mucus and about one half were due to inadequate numbers of columnar epithelial cells. Of the 13 samples testing positive by Chlamydiazyme alone, 5 were shown to be true-positive results by the confirmatory assay. The 8 specimens shown to be positive only by DFA, of which three had between 3 and 10 elementary bodies, were considered false-positive results because repeated culture and EIA testing yielded negative results. The slides were no longer available for review. Therefore, of the 72 initially positive specimens, 56 (11%) were considered true-positive results. The sensitivity, specificity, and positive and negative predictive values were 91%, 100%, 100%, and 99%, respectively, for culture and 91%, 98%, 86%, and 99%, respectively, for Chlamydiazyme. By identifying false-positive EIA results with the confirmatory assay, the sensitivity, specificity, and positive and negative predictive values of Chlamydiazyme equalled those of culture. The sensitivity, specificity, and positive and negative predictive values for DFA, based only on conclusive test results, were 74%, 98%, 83%, and 96%, respectively. DISCUSSION As with most evaluations of tests for detection of C. trachomatis, there were inherent problems in our study design. First, when multiple samples are obtained from one site, the order of specimen collection could bias the results. To limit this bias, the order of sample collection was rotated. In only some cases the false-negative culture or Chlamydiazyme results could be explained by the order of specimen collection. For example, of the five cases in which culture yielded false-negative results, the swab for culture was collected first once and was collected second and third in two cases each. Of the five culture-positive, Chlamydiazyme-negative samples, the swab for culture was collected first once, was collected third three times, and for one the order of collection was unknown. Second, freezing samples submitted for culture at - 7 0 °C for shortterm storage may have adversely affected the sensitivity of the test. Based on the results of a study by Jaqua-Stewart and associates,16 we elected to use three elementary bodies as the cut-off for a positive DFA test. If we had used 10 elementary bodies as the cut-off for a positive test, the number recommended by the manufacturer, the sensitivity, specificity, and positive and negative predictive values would be 70%, 99%, 88%, and 96%, respectively. In summary, in this population of women with genitourinary or abdominopelvic symptoms in whom the prevalence of C. trachomatis was about 11%, DFA was the least reliable of the tests evaluated for detecting infec-

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The blocking assay was performed manually according to manufacturer directions and reactions were measured using the Abbott Quantum II spectophotometer. Briefly, controls and positive specimens were repeated in duplicate as described above for the Chlamydiazyme assay, except that to one of the wells for each specimen the blocking antibody preparation (consisting of 10 ;uL of a murine monoclonal antibody raised against the lipopolysaccharide of C. trachomatis 434 [an L2 serovar] plus 250 nL of the anti-C trachomatis reagent) was added in place of the unmodified anti-C. trachomatis solution. A reduction of 50% or more in the absorbance value of the confirmatory assay compared with the absorbance value of the Chlamydiazyme retest verified the presence of chlamydial antigens, whereas a reduction of less than 50% indicated a false-positive result.

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CLINICAL IMMUNOLOGY AND MICROBIOLOGY Original Article

tion with C. trachomatis, although the test provides rapid results and allows evaluation of specimen quality. The 74% sensitivity of DFA was similar to what we and others reported in the past.1317 Chlamydiazyme used in conjunction with the confirmatory assay had sensitivity, specificity, and positive and negative predictive values equal to those of culture. Therefore, in this type of population, using Chlamydiazyme and confirming positive results with the blocking assay appears to be a rapid, reliable, and cost-effective alternative to culture. Acknowledgments. The authors thank the nursing staff in the Emergency Room and Family Health Clinic for organizing the collection of specimens and Val Rementer for secretarial assistance.

6.

7.

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9.

10.

11.

1. Bell TA, Grayston JT. Center or Disease Control guidelines for prevention and control of Chlamydia trachomatis infections. Ann Intern Med 1986;104:524-526. 2. Centers for Disease Control. Chlamydia trachomatis infections. Policy guidelines for prevention and control. MMWR 1985;34(Suppl):535-545. 3. Baselski VS, McNeeley SG, Ryan G, Robinson SA. A comparison of nonculture-dependent methods for detection of Chlamydia trachomatis infections in pregnant women. Obstet Gynecol 1987;70:47-52. 4. Chernesky MA, Mahoney JB, Castriciano S, et al. Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women. J Infect Dis 1986; 154:141-148. 5. Lefebvre J, Laperiere H, Rousseau H, Masse R. Comparison of three techniques for detection of Chlamydia trachomatis in endocervical

13. 14. 15.

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specimens from asymptomatic women. J Clin Microbiol 1988; 26: 726-731. Lipkins ES, Moncada JV, Shafer M-A, et al. Comparison of monoclonal antibody staining and culture in diagnosing cervical chlamydial infection. J Clin Microbiol I986;23:l 14-117. Phillips RS, HanffPA, Kaufman RS, Aronson MD. Use of a direct fluorescent antibody test for detecting Chlamydia trachomatis cervical infection in women seeking routine gynecologic care. J Infect Dis 1987;156:575-581. Quinn TC, Warfield P, Kappus E, et al. Screening for Chlamydia trachomatis infection in an inner-city population: A comparison of diagnostic methods. Infect Dis 1985; 152:419-423. Amortegui AJ, Meyer MP. Enzyme immunoassay for detection of Chlamydia trachomatis from the cervix. Obstet Gynecol 1985;65: 523-526. Howard LV, Coleman PF, England BJ, Herrmann JE. Evaluation of Chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis. J Clin Microbiol 1986;23:329-332. Smith JW, Rogers RE, Katz BP, et al. Diagnosis of chlamydial infection in women attending antenatal and gynecologic clinics. J Clin Microbiol 1987;25:868-872. Kellogg JA, Seiple JW, Murray CL, Levisky JS. Effect of endocervical specimen quality on detection of Chlamydia trachomatis and on the incidence of false-positive results with the Chlamydiazyme method. J Clin Microbiol 1990;28:1108-1113. Barnes RC. Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 1989; 2:119-136. Schachter J. Rapid diagnosis of sexually transmitted disease. Yale J Biol Med 1986;58:443-452. Moncada J, Schachter J, Bolan G, et al. Confirmatory assay increases specificity of the Chlamydiazyme test for Chlamydia trachomatis infection of the cervix. J Clin Microbiol 1990;28:1770-1773. Jaqua-Stewart MJ, Tichota-Lee J, Amundson LH, et al. Defining the threshold of positivity for MicroTrak direct fluorescent Chlamydia detection. In: Abstracts of the Annual Meeting of the American Society for Microbiology. Washington, DC, 1988, p 369. Woods GL, Young A, Scot JC, et al. Evaluation of a nonisotopic probe for detection of Chlamydia trachomatis in endocervical specimens. J Clin Microbiol 1990;28:370-372.

Chlamydiazyme plus blocking assay to detect Chlamydia trachomatis in endocervical specimens.

Three methods to detect Chlamydia trachomatis in endocervical swab specimens collected from 502 women with genitourinary or abdominopelvic symptoms we...
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