JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1990, P. 1647-1648 0095-1137/90/071647-02$02.00/0
Vol. 28, No. 7
Copyright © 1990, American Society for Microbiology
Ortho Enzyme Immunoassay versus McCoy Cell Monolayers Stained by iodine or Fluorescent Antibody for Detection of Chlamydia trachomatis L. E. PHILLIPS,t* P. B. SMITH, G. D. RIDDLE, S. FARO, H. K. GOODRICH, AND M. G. MARTENS Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030 Received 27 October 1989/Accepted 3 April 1990
We compared a new enzyme immunoassay (Ortho Diagnostics Systems, Inc., Raritan, N.J.) (EIA) with iodine and fluorescent-antibody staining of inoculated McCoy cell monolayers for detection of Chlamydia trachomatis in our female outpatient populations. EIA was more sensitive than iodine at a statistically significant level, but there were no statistically significant differences between EIA results and those for fluorescent-antibody staining.
Isolation in tissue cultures is considered the standard detection method for Chlamydia trachomatis. This method, however, is labor intensive and expensive and is often not available in settings where large-scale screening for sexually transmitted diseases is performed. Hence, diagnostic alternatives to cultivation in tissue culture have been sought. Current available methods include direct antigen detection in specimens with fluorescein-labeled antibodies, DNA probes, and enzyme immunoassay (EIA). Ortho Diagnostics Systems, Inc. (Raritan, N.J.), currently markets a 3-h EIA suitable for screening large numbers of specimens. This assay utilizes a treated microwell surface to immobilize chlamydia antigens extracted from clinical specimens. The sequential addition of a rabbit anti-chlamydia antibody, a horseradish-peroxidase-labeled anti-rabbit antibody, and substrate results in specific color development, which is read spectrophotometrically. We evaluated the Ortho EIA procedure for antigen detection of C. trachomatis to determine whether that procedure offers advantages over cultivation of the organism in our setting. Two female populations were sampled; 197 patients between the ages of 13 and 19 years old were attending a Teen Health Clinic at Jefferson Davis Hospital in Houston, Tex., and 50 patients over the age of 20 were pregnant and attending the prenatal obstetrical clinic at Jefferson Davis Hospital. Three endocervical specimens were collected with sterile Dacron swabs. The first two swabs were randomized; one was placed in chlamydia culture transport medium (Bartels Immunodiagnostics, Bellevue, Wash.), and the other was placed in an Ortho EIA transport tube according to instructions on the collection package. The third swab was used to prepare a direct smear by rolling the swab over a 6- to 8-mm circular area on a glass slide. Samples for chlamydia culture were stored at 2 to 8°C for up to 24 h or at -70°C for up to 72 h. Samples for analysis by EIA were stored at -70°C and tested within 7 days of collection. The smears were air dried, fixed in acetone, and stored at -70°C until stained. Specimens for cultivation of chlamydia were treated as described previously (5). Of the 247 monolayers, 149 were Corresponding author. t Present address: Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48105. *
stained with Jones iodine (Scott Laboratories, Inc., Fiskeville, R.I.) and 98 were stained with a fluorescent-antibody (FA) stain (Ortho Diagnostics). The portion of specimen not used for cultivation was frozen at -70°C. The EIA procedure was performed according to the package insert. Discrepant results between culture and the EIA procedure were further analyzed as follows: (i) the EIA was repeated with retained extract; (ii) the direct smear was stained with Ortho Chlamydia Direct FA reagent; (iii) the original culture inoculum and EIA tube were centrifuged at 3,000 x g for 1 h, and the sediments were dropped on a slide, air dried, acetone fixed, and stained with Ortho Chlamydia Direct FA test stain. Test results were compared by using standard formulas (2). The data were first analyzed by comparing EIA and culture results. Specimens that were negative by culture and positive by the direct FA stain of smear or centrifuged transport sediments were considered culture misses and counted as true positives. Statistical analysis of the differences between the sensitivities and specificities of EIA results, iodine-stained monolayers, and FA-stained monolayers was performed by using a two-tailed two-sample hypothesis test of percentages (6). Significant differences were detected if the critical ratio was greater than 2.58 with a 99% confidence limit. It was determined that a sample size of 19 or more was necessary in order to detect statistically significant differences between the methods. This calculation was made by assuming that the rate of disease (p) is 0.23 and expanding the binomial distribution (1 - p)n to attain a significance level of 0.01. Of the 197 adolescents, 44 (22%) were culture positive for C. trachomatis. The incidence of positive cultures by reason for clinical visit was 18 of 64 (28%) for postpartum examination, 21 of 96 (22%) for family planning, 4 of 32 (13%) for pregnancy test or sexually transmitted disease screen, and 1 of 5 (20%) for other medical reasons. Only those patients presenting for a sexually transmitted disease screen complained of symptoms. Of the 50 pregnant adults, 6 (12%) were culture positive for C. trachomatis. A comparison between iodine-stained monolayers and EIA results revealed 20 false-positive and 2 false-negative results by EIA (Table 1). However, discrepant-results analysis revealed that 11 specimens negative by iodine staining were positive (8 direct smears, 1 culture transport tube sediment, and 2 EIA tube sediments) after FA staining. A 1647
1648
J. CLIN. MICROBIOL.
NOTES
TABLE 2. Comparisons of Ortho EIA and FA-stained cultures with confirmed positive results
TABLE 1. Comparisons of Ortho EIA and initial iodine-stained cultures with confirmed positive results
No. of specimens with result by: Initial culture Confirmation
No. of specimens with result by: Test and
Initial
Test and result
Confirmation
culture
re s u lt_
+
-
19 2
20 108
+
+_
Ortho EIA
Ortho EIA + -
30
9
2
108
21
0 117
18 5
+
5 70
20 4
3 71
23 1
0 74
Initial culture
Initial culture +
-+_
il
a Sensitivity and specificity, respectively, for each comparison were as follows: EIA versus initial culture, 90.5 and 84.4%; EIA versus confirmation, 93.8 and 92.3%; initial culture versus confirmation, 65.6 and 100%.
comparison among EIA, culture, and confirmation results is also presented in Table 1. The EIA results were statistically more sensitive than results of the iodine-stained culture (critical ratio, 2.95). A comparison between FA-stained monolayers and EIA results demonstrated five false-negative and five false-positive results by EIA. Discrepant-results analysis revealed that one EIA-positive, culture-negative specimen was positive after FA staining of the EIA tube sediment, that one EIAnegative, culture-positive specimen was EIA positive on repeat testing, and that one EIA-positive, culture-negative specimen was EIA negative upon repeat testing. A comparison between EIA and culture with confirmed positive results is presented in Table 2. The sensitivity and specificity values for the EIA procedure were not statistically different from those for FA-stained monolayers (critical ratios, 1.41 and 1.77, respectively). In conclusion, we found that the sensitivity and specificity of Ortho EIA were similar to those obtained by culture and staining with FA reagents but that Ortho EIA was more sensitive than culture with iodine staining in the intermediate- to high-prevalence female populations studied. Furthermore, our results were similar to those obtained by other investigators who compared other EIAs with iodine-stained cultures (3, 4) or FA-stained cultures (1, 7, 8). The Ortho EIA was easier to perform than cultivation. Thus, the Ortho EIA procedure may be an acceptable alternative to culture for detection of C. trachomatis in female genital tract specimens. Further studies are necessary to more closely define the usefulness of the procedure in clinical laboratories.
+
a Sensitivity and specificity, respectively, for each comparison were as follows: EIA versus initial culture, 78.3 and 93.3%; EIA versus confirmation, 83.3 and 95.9%o; initial culture versus confirmation, 95.8 and 100%.
1.
2. 3. 4. 5.
6.
7.
8.
LITERATURE CITED Baselski, V. S., G. McNeeley, G. Ryan, and M. Robinson. 1987. A comparison of nonculture-dependent methods for detection of Chlamydia trachomatis infections in pregnant women. Obstet. Gynecol. 70:47-52. Galen, A. S. 1979. Predictive value theory. Diagn. Med. 1: 23-31. Jones, M. F., T. F. Smith, A. J. Houglum, and J. E. Herrmann. 1984. Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test. J. Clin. Microbiol. 20:465-467. Levy, R. A., and A. L. Warford. 1986. Evaluation of the modified ChlamydiazymeR immunoassay for detection of chlamydial antigen. Am. J. Clin. Pathol 86:330-335. Phillips, L. E., S. Faro, P. B. Smith, G. D. Riddle, K. H. Goodrich, and M. G. Martens. 1988. Premarket evaluation of monofluor reagent for detection of Chlamydia trachomatis in adolescent out-patients. Genitourin. Med. 64:164-168. Sanders, D. H., R. J. Eng, and A. F. Murph. 1985. Testing hypotheses and making decisions: two-sample procedures, p. 256-276. In C. L. Mehalik and E. Henson (ed.), Statistics: a fresh approach. McGraw Hill, New York. Smith, J. W., R. E. Rogers, B. P. Katz, J. F. Brickler, P. L. Lineback, B. Van Der Pol, and R. B. Jones. 1987. Diagnosis of chlamydial infection in women attending antenatal and gynecologic clinics. J. Clin. Microbiol. 25:868-872. Tjiam, K. H., B. Y. M. van HeiJst, A. van Zuuren, J. H. T. Wagenvoort, T. van Joost, E. Stolz, and M. F. Michel. 1986. Evaluation of an enzyme immunoassay for the diagnosis of chlamydial infections in urogenital specimens. J. Clin. Microbiol. 23:752-754.
Vol. 28, No. 7
Copyright © 1990, American Society for Microbiology
Ortho Enzyme Immunoassay versus McCoy Cell Monolayers Stained by iodine or Fluorescent Antibody for Detection of Chlamydia trachomatis L. E. PHILLIPS,t* P. B. SMITH, G. D. RIDDLE, S. FARO, H. K. GOODRICH, AND M. G. MARTENS Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030 Received 27 October 1989/Accepted 3 April 1990
We compared a new enzyme immunoassay (Ortho Diagnostics Systems, Inc., Raritan, N.J.) (EIA) with iodine and fluorescent-antibody staining of inoculated McCoy cell monolayers for detection of Chlamydia trachomatis in our female outpatient populations. EIA was more sensitive than iodine at a statistically significant level, but there were no statistically significant differences between EIA results and those for fluorescent-antibody staining.
Isolation in tissue cultures is considered the standard detection method for Chlamydia trachomatis. This method, however, is labor intensive and expensive and is often not available in settings where large-scale screening for sexually transmitted diseases is performed. Hence, diagnostic alternatives to cultivation in tissue culture have been sought. Current available methods include direct antigen detection in specimens with fluorescein-labeled antibodies, DNA probes, and enzyme immunoassay (EIA). Ortho Diagnostics Systems, Inc. (Raritan, N.J.), currently markets a 3-h EIA suitable for screening large numbers of specimens. This assay utilizes a treated microwell surface to immobilize chlamydia antigens extracted from clinical specimens. The sequential addition of a rabbit anti-chlamydia antibody, a horseradish-peroxidase-labeled anti-rabbit antibody, and substrate results in specific color development, which is read spectrophotometrically. We evaluated the Ortho EIA procedure for antigen detection of C. trachomatis to determine whether that procedure offers advantages over cultivation of the organism in our setting. Two female populations were sampled; 197 patients between the ages of 13 and 19 years old were attending a Teen Health Clinic at Jefferson Davis Hospital in Houston, Tex., and 50 patients over the age of 20 were pregnant and attending the prenatal obstetrical clinic at Jefferson Davis Hospital. Three endocervical specimens were collected with sterile Dacron swabs. The first two swabs were randomized; one was placed in chlamydia culture transport medium (Bartels Immunodiagnostics, Bellevue, Wash.), and the other was placed in an Ortho EIA transport tube according to instructions on the collection package. The third swab was used to prepare a direct smear by rolling the swab over a 6- to 8-mm circular area on a glass slide. Samples for chlamydia culture were stored at 2 to 8°C for up to 24 h or at -70°C for up to 72 h. Samples for analysis by EIA were stored at -70°C and tested within 7 days of collection. The smears were air dried, fixed in acetone, and stored at -70°C until stained. Specimens for cultivation of chlamydia were treated as described previously (5). Of the 247 monolayers, 149 were Corresponding author. t Present address: Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48105. *
stained with Jones iodine (Scott Laboratories, Inc., Fiskeville, R.I.) and 98 were stained with a fluorescent-antibody (FA) stain (Ortho Diagnostics). The portion of specimen not used for cultivation was frozen at -70°C. The EIA procedure was performed according to the package insert. Discrepant results between culture and the EIA procedure were further analyzed as follows: (i) the EIA was repeated with retained extract; (ii) the direct smear was stained with Ortho Chlamydia Direct FA reagent; (iii) the original culture inoculum and EIA tube were centrifuged at 3,000 x g for 1 h, and the sediments were dropped on a slide, air dried, acetone fixed, and stained with Ortho Chlamydia Direct FA test stain. Test results were compared by using standard formulas (2). The data were first analyzed by comparing EIA and culture results. Specimens that were negative by culture and positive by the direct FA stain of smear or centrifuged transport sediments were considered culture misses and counted as true positives. Statistical analysis of the differences between the sensitivities and specificities of EIA results, iodine-stained monolayers, and FA-stained monolayers was performed by using a two-tailed two-sample hypothesis test of percentages (6). Significant differences were detected if the critical ratio was greater than 2.58 with a 99% confidence limit. It was determined that a sample size of 19 or more was necessary in order to detect statistically significant differences between the methods. This calculation was made by assuming that the rate of disease (p) is 0.23 and expanding the binomial distribution (1 - p)n to attain a significance level of 0.01. Of the 197 adolescents, 44 (22%) were culture positive for C. trachomatis. The incidence of positive cultures by reason for clinical visit was 18 of 64 (28%) for postpartum examination, 21 of 96 (22%) for family planning, 4 of 32 (13%) for pregnancy test or sexually transmitted disease screen, and 1 of 5 (20%) for other medical reasons. Only those patients presenting for a sexually transmitted disease screen complained of symptoms. Of the 50 pregnant adults, 6 (12%) were culture positive for C. trachomatis. A comparison between iodine-stained monolayers and EIA results revealed 20 false-positive and 2 false-negative results by EIA (Table 1). However, discrepant-results analysis revealed that 11 specimens negative by iodine staining were positive (8 direct smears, 1 culture transport tube sediment, and 2 EIA tube sediments) after FA staining. A 1647
1648
J. CLIN. MICROBIOL.
NOTES
TABLE 2. Comparisons of Ortho EIA and FA-stained cultures with confirmed positive results
TABLE 1. Comparisons of Ortho EIA and initial iodine-stained cultures with confirmed positive results
No. of specimens with result by: Initial culture Confirmation
No. of specimens with result by: Test and
Initial
Test and result
Confirmation
culture
re s u lt_
+
-
19 2
20 108
+
+_
Ortho EIA
Ortho EIA + -
30
9
2
108
21
0 117
18 5
+
5 70
20 4
3 71
23 1
0 74
Initial culture
Initial culture +
-+_
il
a Sensitivity and specificity, respectively, for each comparison were as follows: EIA versus initial culture, 90.5 and 84.4%; EIA versus confirmation, 93.8 and 92.3%; initial culture versus confirmation, 65.6 and 100%.
comparison among EIA, culture, and confirmation results is also presented in Table 1. The EIA results were statistically more sensitive than results of the iodine-stained culture (critical ratio, 2.95). A comparison between FA-stained monolayers and EIA results demonstrated five false-negative and five false-positive results by EIA. Discrepant-results analysis revealed that one EIA-positive, culture-negative specimen was positive after FA staining of the EIA tube sediment, that one EIAnegative, culture-positive specimen was EIA positive on repeat testing, and that one EIA-positive, culture-negative specimen was EIA negative upon repeat testing. A comparison between EIA and culture with confirmed positive results is presented in Table 2. The sensitivity and specificity values for the EIA procedure were not statistically different from those for FA-stained monolayers (critical ratios, 1.41 and 1.77, respectively). In conclusion, we found that the sensitivity and specificity of Ortho EIA were similar to those obtained by culture and staining with FA reagents but that Ortho EIA was more sensitive than culture with iodine staining in the intermediate- to high-prevalence female populations studied. Furthermore, our results were similar to those obtained by other investigators who compared other EIAs with iodine-stained cultures (3, 4) or FA-stained cultures (1, 7, 8). The Ortho EIA was easier to perform than cultivation. Thus, the Ortho EIA procedure may be an acceptable alternative to culture for detection of C. trachomatis in female genital tract specimens. Further studies are necessary to more closely define the usefulness of the procedure in clinical laboratories.
+
a Sensitivity and specificity, respectively, for each comparison were as follows: EIA versus initial culture, 78.3 and 93.3%; EIA versus confirmation, 83.3 and 95.9%o; initial culture versus confirmation, 95.8 and 100%.
1.
2. 3. 4. 5.
6.
7.
8.
LITERATURE CITED Baselski, V. S., G. McNeeley, G. Ryan, and M. Robinson. 1987. A comparison of nonculture-dependent methods for detection of Chlamydia trachomatis infections in pregnant women. Obstet. Gynecol. 70:47-52. Galen, A. S. 1979. Predictive value theory. Diagn. Med. 1: 23-31. Jones, M. F., T. F. Smith, A. J. Houglum, and J. E. Herrmann. 1984. Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test. J. Clin. Microbiol. 20:465-467. Levy, R. A., and A. L. Warford. 1986. Evaluation of the modified ChlamydiazymeR immunoassay for detection of chlamydial antigen. Am. J. Clin. Pathol 86:330-335. Phillips, L. E., S. Faro, P. B. Smith, G. D. Riddle, K. H. Goodrich, and M. G. Martens. 1988. Premarket evaluation of monofluor reagent for detection of Chlamydia trachomatis in adolescent out-patients. Genitourin. Med. 64:164-168. Sanders, D. H., R. J. Eng, and A. F. Murph. 1985. Testing hypotheses and making decisions: two-sample procedures, p. 256-276. In C. L. Mehalik and E. Henson (ed.), Statistics: a fresh approach. McGraw Hill, New York. Smith, J. W., R. E. Rogers, B. P. Katz, J. F. Brickler, P. L. Lineback, B. Van Der Pol, and R. B. Jones. 1987. Diagnosis of chlamydial infection in women attending antenatal and gynecologic clinics. J. Clin. Microbiol. 25:868-872. Tjiam, K. H., B. Y. M. van HeiJst, A. van Zuuren, J. H. T. Wagenvoort, T. van Joost, E. Stolz, and M. F. Michel. 1986. Evaluation of an enzyme immunoassay for the diagnosis of chlamydial infections in urogenital specimens. J. Clin. Microbiol. 23:752-754.
