Clinical Articles Detection of cervical Chlamydia trachoma tis and Neisseria gonorrhoeae with deoxyribonucleic acid probe assays in obstetric patients Ian K. Hosein, MD: Andrew M. Kaunitz, MD,. and Susan J. Craft, MTa

Jacksonville, Florida OBJECTIVE: The Gen-Probe PACE 2 deoxyribonucleic acid probe assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae are targeted against the ribosomal ribonucleic acid of each pathogen. Our study compared the performance of the probe assays with culture for Chlamydia trachomatis (246 patients) and Neisseria gonorrhoeae (310 patients) while screening obstetric patients. STUDY DESIGN: Using culture as a gold standard, we assessed the sensitivity, specificity, positive predictive value, and negative predictive value of the chlamydia and gonorrhea probes. RESULTS: The prevalence of chlamydia by culture was 13.4% and gonorrhea 4.8%. Against culture, the chlamydia probe assay performed as follows: sensitivity 93.9%, specificity 99.1 %, positive predictive value 93.9%, and negative predictive value 99.1 %. Values for the gonorrhea probe assay were 93.3%, 99.0%, 82.4%, and 99.7%, respectively. Additional molecular analysis of probe-positive-culture-negative specimens suggests that the gonorrhea probe-positive predictive value may be even higher. CONCLUSION: The Gen-Probe PACE 2 deoxyribonucleic acid probe assays for chlamydia and gonorrhea appear to be promising as convenient, reliable, and cost-effective alternatives to conventional cultures in screening obstetric patients. (AM J OSSTET GVNECOL 1992;167:588-91.)

Key words: Gonorrhea, chlamydia, deoxyribonucleic acid probes Chlamydia and gonorrhea are highly prevalent sexually transmitted diseases with a worldwide distribution. In the United States some 4 million cases of chlamydia and 700,000 cases of gonorrhea occur annually.1. 2 Although often asymptomatic, perinatal transmission of these organisms frequently cause neonatal ocular and, in the case of chlamydia, pulmonary infections. Although classified as a bacterium, Chlamydia trachomatis is an obligate intracellular pathogen; conventional method of detection involves cell culture with McCoy cells.' This approach is both time consuming and labor intensive, with delayed reporting. Conventional laboratory diagnosis of gonorrhea is based on culture with selective media such as Thayer-Martin or Martin-Lewis. However, Neisseria gonorrhoeae is a fastidious organism and may fail to grow because of delay From the Departments of Clinical Microbiology' and Obstetrics and Gynecology,' University of Florida Health Science Center. Received for publication December 17, 1991; revised March 18, 1992; accepted March 31, 1992. Reprints not available. 611138304


in specimen transport and incubation. Also, certain strains are sensitive to vancomycin in the standard selective media" To address problems associated with cultures, tests for chlamydia and gonorrhea not based on culture have emerged. Enzyme immunoassay tests for detection of chlamydial and gonococcal antigens include Chlamydiazyme and Gonozyme, respectively (Abbott Laboratories, Chicago). Chlamydiazyme has a sensitivity ranging from 70% to 90% of culture and a specificity ranging from 92% to 99%, depending on prevalence.' The Gonozyme test is specific and sensitive for detecting gonococcal urethral infection in men, but it is less sensitive (87%) for gonococcal infections in women. 6 The specificity of the test has also been questioned, with Neisseria lactamica and Neisseria cinerea giving false-positive reactions. The chlamydia direct fluorescent antibody stain has a similar performance profile, with sensitivities from 70% to 90% and specificities >95%.7 However, the direct fluorescent antibody procedure requires a fluorescent microscope and a high level of skill in interpretation. Nucleic acid hybridization offers the potential for

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rapid, sensitive, specific, and cost-effective noncultural detection of both organisms. s The Cen-Probe PACE 2 (Cen-Probe, Inc., San Diego) deoxyribonucleic acid (DNA) probes are complementary strands of DNA (cDNA) directed against the ribosomal ribonucleic acid of C. trachomatis and N. gonorrhoeae, respectively, and have been marketed in the United States since 1989. With this system DNA probe assays for both pathogens can be performed with one cervical swab. Our study compares the performance of these problem assays with standard culture for chlamydia and gonorrhea from cervical specimens in a population of low-income obstetric patients. Material and methods

Patient population. Our study was approved by the Institutional Review Board of University Medical Center,Jacksonville, Florida. From October 1989 to August 1990 endocervical specimens were collected from 322 consecutive patients during their first prenatal examinations at University Medical Center. The specimens were obtained during vaginal speculum examination for cervical cytology and sexually transmitted disease screening, which is routinely performed during the initial prenatal examination. University Medical Center is a teaching hospital serving a predominantly low-income population with approximately 5000 annual deliveries. Among obstetric patients screened in this study the racial distribution was 79% black and 21 % white. At the time of screening the median age, parity, and gestational age ofthese patients were 21 years, para 1, and 22 weeks, respectively. All of the original culture and DNA probe assays were performed in the clinical microbiology laboratory at University Medical Center. Technologists performing either culture or probe assays were blinded to the other corresponding results. Patients with positive results by culture or DNA probe were treated with standard antimicrobial regimens. Laboratory methods. After the exocervix was cleaned, three swabs were taken for cultures and DNA probe assays. One Dacron swab was used for chlamydia culture, one cotton swab for gonococcal culture, and one Dacron swab for both gonococcal and chlamydial probe assays. Clinicians were told not to follow any given order of specimen collection. The swab for chlamydia culture was placed in 1.5 ml of chlamydial transport medium with 0.2 mollL sucrose and 10% fetal bovine serum (Bartels/Baxter, Sacramento, Calif.) and was sent to the laboratory immediately. Samples were processed on receipt or stored at 4° C for processing the next day. After the samples were vortexed, they were centrifuged onto cycloheximide-treated McCoy cells on coverslips in a 2-vial shell vial procedure (Bartels). After incubation at 35° C for 48 to 72 hours, both coverslips were stained with a

fluorescein-labeled monoclonal antibody for inclusions. 9 The presence of one or more fluorescing inclusions was considered a positive result. The swab for gonococcal culture was planted on to Jembec plates (Martin-Lewis agar) at the bedside and transported to the laboratory immediately in a carbon dioxide pouch. The plates were incubated in 5% carbon dioxide at 35° C, for 72 hours. Colonies suspicious by Cram stain and oxidase for N. gonorrhoeae were confirmed by conventional sugar fermentation and coagglutination (Meritek, Meridian Diagnostics, Cincinnati).'o Techniques for chlamydial and gonococcal probe assays have been previously described. I I. 12 Results

Overall, this study involved 322 obstetric patients. Among these, 234 had probe assay and culture results for both pathogens. An additional 76 had gonococcal culture and probe results, and 12 had chlamydia culture and probe results. Performance of chlamydia probe assay. Of 246 cultures, 33 (13.4%) were positive for chlamydia. With culture as the gold standard the chlamydia probe assay had a sensitivity of 93.9%, specificity of 99.1 %, positive predictive value of93.9%, and negative predictive value of 99.1 %. Two chlamydia probe-positive-culture-negative results were false positive by definition. A further competitive probe binding assayl2 was performed by the Cen-Probe staff on a frozen aliquot (-70° C) of one of the original samples for probe assay (the other was not available), and it was positive for chlamydia. If this specimen is considered truly positive, the adjusted performance of the chlamydia probe becomes sensitivity 94.1 %, specificity 99.5%, positive predictive value 97.0%, and negative predictive value 99.1 %. Performance of gonorrhea probe assay. Of 310 cultures, 15 (4.8%) were positive for gonorrhea. The probe assay for gonorrhea had a sensitivity of 93.3%, specificity of 99.0%, positive predictive value of 82.4%, and negative predictive value of 99.7%. Among three gonococcal probe-positive-culture-negative results, probe competition assay was positive in two for gonococcal nucleic acid. If these two probe competition assay positives are considered to be truly positive, the adjusted performance of the gonococcal probe becomes sensitivity 94.1 %, specificity 99.7%, positive predictive value 94.1 %, and negative predictive value 99.7%. Comment

Both chlamydia and gonorrhea are highly prevalent sexually transmitted diseases. Although frequently asymptomatic, infection with either organism in pregnant women may lead to a variety of perinatal sequelae. Unfortunately, significant problems are associated with


Hosein, Kaunitz, and Craft

both chlamydia and gonorrhea cultures. Because of the prevalence of these pathogens and their serious health consequences, there has been a search for rapid, sensitive, specific, and cost-effective tests. DNA probe technology based on the specific binding of complementary strands of nucleic acid may offer each of these benefits. The Gen-Probe PACE 2 probe assays for C. trachomatis and N. gonorrhoeae are the newest versions of prototype chemiluminescent DNA probes for the direct detection of these two sexually transmitted organisms. Iwen et al. l ' evaluated the PACE 2 chlamydia probe versus culture with endocervical swabs from emergency room patients with a chlamydia prevalence of 9.4%. Sensitivity, specificity, and positive and negative predictive values were 93%, 98%, 85%, and 99%, respectively. Panke et al. II evaluated the PACE 2 gonococcal probe in a mixed population of symptomatic and asymptomatic (prevalence 2%) women and found the probe to be equivalent to culture in both populations. They did recommend, however, that borderline results be repeated to obtain consistent negative values. Because both probe assays can be performed with a single swab, our study evaluated the performance of the probe assays for chlamydia and gonorrhea against culture in a population of low-income obstetric patients. To our knowledge this is the first report evaluating these probe assays for both organisms at the same time. The prevalence by culture of chlamydia was 13.4% and gonorrhea 4.8%. Among the 234 patients in whom cultures for both pathogens were performed, 31 were positive for chlamydia, 12 were positive for gonorrhea, and 5 were positive for both. Our failure to specify the order in which swabs were collected for the cultures and probe assays may represent a limitation of this study. However, a large number of clinicians were involved in the collection process, and they were told not to use any given order. Because we believe that randomization was approached, if not achieved, bias introduced into our results as a result of swab order should be minor, if present at all. The occurrence of two chlamydia probe-positiveculture-negative results could be explained by storing the culture specimens at 4 0 C for an excessive period of time. Refrigeration of specimens for >24 hours is known to reduce recovery of chlamydia. 13 In this series, however, specimens for chlamydia were processed in :=;24 hours. Therefore delayed processing should not have been a factor in explaining these two apparent false-negative chlamydia culture results. Even without the probe competition assay data on probe-positive-culture-negative samples, performance features of the chlamydia probe assay and all but one (positive predictive value) for gonorrhea are >90%. If the probe competition assay results of probe-positive-

September 1992 Am J Obstet Gyneco!

culture-negative samples are considered to be accurate, the positive predictive value for gonorrhea is increased from 82.4% to 94.1 %. Sample collection for the DNA probe is convenient: one swab is used for both chlamydia and gonorrhea. The shelf-life of the collection kits (swab and transport tube) is 6 months at room temperature; after collection, the sample in the transport tube is stable for 7 days at room temperature. Both of these features facilitate use by clinics distant from the laboratory. In the laboratory the procedure for probe binding, washing, and detection takes place in a single test tube; necessary equipment, luminometer, and magnetic separation rack are supplied by the manufacturer. Because the separation step (bound from unbound probe) uses a magnetic field, it is much faster than centrifugation. The detection system uses chemiluminescence, thereby eliminating the safety problems and short shelf-life characteristic of radioactive labels. Because one specimen can be analyzed for both chlamydia and gonorrhea in 2 hours, results can be reported the day of specimen receipt. At University Medical Center, Jacksonville, a cost analysis indicates considerable savings with probe assays over culture for chlamydia and gonorrhea. Charges for the equipment needed to perform the DNA probe assays are incorporated into reagent costs. The higher the volume of assays performed, the lower the overall costs of assays become. Our hospital laboratory processes between 1000 and 2000 samples for probe analysis each month. Our combined cost for performing chlamydial and gonococcal probe assays is approximately $12. In contrast, our combined cost for performing chlamydia and gonorrhea cultures is $27. In summary, this study demonstrates that PACE 2 DNA probe assays for chlamydia and gonorrhea offer a convenient and, for busy laboratories, cost-effective alternative to cultures. Experience in our prenatal clinics indicates that probe reliability is high when used in obstetric populations with infection prevalence comparable with those we have reported. REFERENCES 1. Washington E, johnson RE, Sanders L. Chlamydia trachomatis infections in the United States: what are they costing us? JAM A 1987;257:2070-2. 2. Dallabetta G, Hook EW III. Gonococcal infections. Infect Dis Clin North Am 1987;1:25-54. 3. Ripa KT, Mardh PA. Cultivation of Chlamydia trachomatis in cycloheximide-treated McCoy cells. j Clin Microbiol 1977;6:328-31. 4. Mirret S, Reller LB, Knapp jS. Neisseria gonorrhoeae strains inhibited by vancomycin in selective media and correlation with auxotype. j Clin Microbiol 1981;14:94-6. 5. Grillner L, Beckman S, Hammar H. Comparison of two enzyme immunoassays and an immunofluorescence test for detection of C. trachomatis. J Clin Microbiol 1986;5: 559-62.

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6. Schacter], McCormack WM, Smith RF, Parks RM, Bailey R, Ohlin AC. Enzyme immunoassay for diagnosis of gonorrhea.] Clin Microbiol 1984;19:57-9. 7. Chernesky MA, Mahony ]B, Castriciano S, et al. Detection of Chlamydia trachomatis antigens by enzyme immunoassay and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women.] Infect Dis 1986;154:141-8. 8. Highfield PE, Dougan G. DNA probes for microbial diagnosis. Med Lab Sci 1985;42:352-60. 9. Stamm WE, Tam M, Koester M, Cles L. Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluorescein-conjugated monoclonal antibodies. ] Clin Microbiol 1983;17:666-8. 10. Morello ]A, Janda WM, Doern GV. Neisseria and Bran-

DNA probes for chlamydia and gonorrhea

hamella. In: Balows A, ed. Manual of clinical microbiology. 5th ed. Washington, D.C.: American Society for Microbiology, 1991:258-76. 11. Panke ES, Yang LI, Leist PA, et al. Comparison of Gen Probe DNA probe test and culture for the detection of Neisseria gonorrhoeae in endocervical specimens.] Clin Microbiol 1991 ;29:883-8. 12. Iwen PC, Blair TM, Woods GL. Comparison of the GenProbe PACE 2 system, direct fluorescent antibody and cell culture for detecting Chlamydia trachomatis in cervical specimens. Am] Clin PathoI1991;95:578-82. 13. Mahony]B, Chernesky MA. Effect of swab type and storage temperature on the isolation of Chlamydia trachomatis from clinical specimens.] Clin Microbiol 1985;22:865-7.

Three-group metaphase as a morphologic criterion of progressive cervical intraepithelial neoplasia Marian J.E. Mourits, MD," Wim J.L.M. Pieters, MD, PhD: Harry Hollema, MD, PhD,b and Matthe P.M. Burger, MD, PhD" Groningen and Winschoten, The Netherlands OBJECTIVE: The purpose of our study was to investigate the presence of three-group metaphase in progressive cervical intraepithelial neoplasia. STUDY DESIGN: This was a retrospective histologic study on the conization specimens of 41 women with microinvasive cervical carcinoma, 28 of whom were enrolled in the study. Three-group metaphase was scored in the invasive part of the lesion and in the adjacent cervical intraepithelial neoplasia. RESULTS: Three-group metaphase was found in 93% of cervical intraepithelial neoplasia adjacent to the invasive part of the lesion. However, three-group metaphase was found in 11 % of the microinvasive cervical carcinoma cases with an infiltration depth of

Detection of cervical Chlamydia trachomatis and Neisseria gonorrhoeae with deoxyribonucleic acid probe assays in obstetric patients.

The Gen-Probe PACE 2 deoxyribonucleic acid probe assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae are targeted against the ...
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