Received 23 September 1997/Returned for modification 16 January 1998/Accepted 19 February 1998

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1998, p Vol. 36, No /98/$ Copyright 1998, American Society for Microbiology Comparison of Performances of Two Commercially Available Tests, a PCR Assay and a Ligase Chain Reaction Test, in Detection of Urogenital Chlamydia trachomatis Infection MIRJA PUOLAKKAINEN, 1 * EIJA HILTUNEN-BACK, 2 TIMO REUNALA, 2 SATU SUHONEN, 3 PEKKA LÄHTEENMÄKI, 3 MATTI LEHTINEN, 4 AND JORMA PAAVONEN 5 Haartman Institute, Department of Virology, 1 and Departments of Dermatology and Venereology 2 and Obstetrics and Gynecology, 5 University of Helsinki, Family Federation of Finland, 3 and Department of Infectious Disease Epidemiology, National Public Health Institute, 4 Helsinki, Finland Received 23 September 1997/Returned for modification 16 January 1998/Accepted 19 February 1998 The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections. Chlamydia trachomatis is one of the most common causes of treatable sexually transmitted diseases (28). Serious sequelae, including pelvic inflammatory disease, tubal factor infertility, ectopic pregnancy, chronic abdominal pain, and reactive arthritis are associated with C. trachomatis infection (4, 14). Nonspecific clinical signs and symptoms of C. trachomatis infection and the frequent occurrence of asymptomatic infections render the clinical diagnosis of C. trachomatis infections difficult (24). Since asymptomatic individuals serve as a reservoir of infection and since complications also occur after silent infection, detection of infected individuals is a major challenge to the preventive health care system. With advances in DNA technology, laboratory methods for the amplification and detection of the multicopy plasmid DNA present in all C. trachomatis serovars have been introduced for the diagnosis of C. trachomatis infection (19). Theoretically, this approach offers high sensitivity (multicopy target) and high specificity (the target is present only in C. trachomatis). The PCR or ligase chain reaction (LCR) technology is demanding with respect to laboratory facilities and equipment, but standardized easy-to-use kits are available. Furthermore, partially automated assays (e.g., the LCx Chlamydia trachomatis assay; Abbott Laboratories, North Chicago, Ill.) and fully automated assays (Cobas Amplicor Chlamydia trachomatis [CT/NG] test; Roche Diagnostic Systems Inc., Branchburg, N.J.) have been developed and are already available for routine use. Assays * Corresponding author. Mailing address: Haartman Institute, Department of Virology, P.O. Box 21, FIN University of Helsinki, Helsinki, Finland. Phone: Fax: Mirja.Puolakkainen@Helsinki.fi. based on PCR (Cobas Amplicor) and LCR (LCx) tests have been applied in the detection of C. trachomatis infection worldwide. Testing of first-void urine (FVU) specimens has been a major breakthrough in the detection of chlamydial infection in both symptomatic and asymptomatic males and females (1 3, 5, 11, 12, 18, 20 22, 26). Both PCR and LCR tests have proven to be superior in the diagnosis of C. trachomatis infection when compared to the previously used methods in terms of both sensitivity and specificity. However, reports of few studies that have compared the two most widely used amplification methods have been published (6, 17). Our objective was to compare the performance of an automated PCR test (Roche Cobas Amplicor Chlamydia trachomatis [CT/NG] Test) and an LCR test (Abbott LCx Chlamydia trachomatis assay) with endocervical and urethral swab specimen culture as a reference test. We also wanted to assess the rate of inhibition of the amplification reaction in our material using the PCR test, which includes an internal control (IC). The effect of freezing on the performance of PCR and LCR testing of urine was also studied. In addition, the clinical utility of testing urine samples instead of swab specimens was studied. MATERIALS AND METHODS Patients. From May 1996 through October 1996, a total of 1,030 patients were recruited into the study. Informed consent was obtained from all patients, and a questionnaire regarding symptoms, sexual behavior, and contraception was completed. Altogether, 912 patients attending a sexually transmitted disease (STD) clinic and 118 females attending a clinic for adolescents in Helsinki were included in the study. Both symptomatic and asymptomatic patients were included. None of the patients had received antibiotic treatment 1 week prior to the visit. Specimen collection and aliquoting. One endocervical swab specimen obtained from each woman or a urethral swab specimen obtained from each man was placed into C. trachomatis transport medium (2SP). Another endocervical or 1489

2 1490 PUOLAKKAINEN ET AL. J. CLIN. MICROBIOL. urethral swab (LCx STD swab; Abbott) specimen was placed into an LCx STD swab Transport Tube. The order of collection of the swabs was alternated weekly. FVU (10 to 50 ml) was collected after swabbing of the urethra for males and before swabbing of the endocervix for females and was transferred to a 15-ml polypropylene tube. The 2SP specimens were stored at 4 C, transported to the laboratory, and processed within 24 h; the specimens collected on Friday afternoons, however, were stored at 4 C over the weekend and were processed on the following Monday morning. This is a standard practice in our laboratory. The specimens in 2SP were aliquoted for culture, PCR, and direct fluorescent-antibody (DFA) testing. The FVU specimens were stored at 4 C and transported to the laboratory within 24 h (except the specimens collected on Friday afternoons, which were stored at 4 C over the weekend and transported on the following Monday morning) and were then aliquoted to be tested fresh (stored at 4 C for up to 4 [LCR] or 7 [PCR] days) and frozen ( 20 C) and to be used in discrepant analyses. Specimen testing. Culture for C. trachomatis was performed with an aliquot from the 2SP medium. PCR was performed with one aliquot of fresh urine and one aliquot of frozen urine, as well as with an aliquot of 2SP medium. LCR was performed with one aliquot of fresh urine, one aliquot of frozen urine, and an aliquot of the LCx swab transport medium. Culture of C. trachomatis. Specimens in 2SP medium were vortexed vigorously. Aliquots of 250 l were inoculated onto McCoy cell monolayers growing on glass coverslips in 24-well plates. After the plates were centrifuged at 3,400 g for1h at 32 C, the inoculum was removed and was replaced with culture medium (BHK-21 with 10% fetal calf serum, 2 mm glutamine, gentamicin, vancomycin, and nystatin) containing 0.5 g of cycloheximide per ml. The cultures were incubated at 37 C in a 5% CO 2 atmosphere. After 48 h, the cultures were fixed with methanol, stained with fluorescein isothiocyanate-labeled MikroTrak Chlamydia trachomatis Culture Confirmation Reagent (Syva, Palo Alto, Calif.), and examined microscopically for the presence of chlamydial inclusions. DFA testing. An aliquot of 2SP medium (100 l) was centrifuged at 10,500 g for 10 min, and the pellet was suspended in phosphate-buffered saline (PBS) and smeared on a glass slide. The slide was fixed in methanol at 20 C for 10 min and stained with fluorescein isothiocyanate-labeled MikroTrak Chlamydia trachomatis Direct Specimen Reagent (Syva) and examined microscopically for the presence of C. trachomatis elementary bodies (EBs). Cobas Amplicor CT/NG test. The Cobas Amplicor CT/NG PCR test was performed according to the manufacturer s instructions. Fresh urine samples were vortexed vigorously. Frozen urine samples were completely thawed and were vortexed vigorously. A 500- l aliquot of urine was transferred to a polypropylene tube containing 500 l of CT/NG Urine Wash Buffer, and the contents were mixed well by vortexing. The tubes were incubated at 37 C for 15 min and centrifuged at 12,500 g for 5 min. The supernatant was discarded and the remnants of the supernatant in each tube were blotted on a separate sheet of absorbent paper. A total of 250 l of CT/NG Lysis Buffer was added to each tube, and the contents were mixed well by vortexing. After incubation for 15 min at room temperature, 250 l of CT/NG Specimen Diluent was added to each tube and the contents were mixed by vortexing. The tubes were centrifuged at 12,500 g for 10 min, and a 50- l aliquot of each supernatant was used for testing. The endocervical or urethral swab specimens collected in 2SP medium were processed as follows: 100 l of CT/NG Lysis Buffer was mixed with 100 l ofthe 2SP medium and the mixture was incubated at room temperature for 10 min. A total of 200 l of CT/NG Specimen Diluent was added the each tube, and the tubes were incubated at room temperature for 10 min. Aerosol-barrier pipette tips were used throughout the procedure. Of the processed specimens, an aliquot of 50 l was transferred to A-tubes containing the Working Master Mix, and the tubes were placed into the thermal cycler segments of the Cobas Amplicor. Amplification and detection of C. trachomatis and IC DNA were automatically performed by the Cobas Amplicor system. Results were expressed as A 660 and were regarded as positive (C. trachomatis A 660, 0.8), as negative (C. trachomatis A 660, 0.2; IC A 660, 0.2), or in a gray zone (C. trachomatis A 660, 0.2 but 0.8). For inhibitory specimens the A 660 of the IC was 0.2 and that of C. trachomatis was 0.2. For specimens containing inhibitory substances, another aliquot was tested at full strength and following 1:10 dilution in CT/NG Specimen Diluent. Abbott LCx Chlamydia trachomatis assay. The LCR test was performed according to the manufacturer s instructions. A 1-ml aliquot of the FVU was centrifuged at 13,000 g for 15 min and the supernatant was discarded. The pellet was resuspended in 1 ml of LCx Urine Specimen Resuspension Buffer. The treated FVU samples and swab specimens in the transport tubes were heated at 97 C for 15 min. For amplification of Chlamydia by LCx, 100 l of the treated specimens was used. An LCx Analyzer (microparticle enzyme immunoassay) was used to detect the amplicons. The LCx Analyzer calculated the cutoff value (0.45 mean of LCx Chlamydia Calibrator Values), and specimens with an assay value equal to or greater than the cutoff value were considered positive. Discrepant analysis. The results for any set of patient specimens without unanimous results for both specimen types in all tests were considered discrepant. The specimens were retested as follows. To check for the presence of nonviable C. trachomatis EBs in culture-negative samples with discrepant results, the DFA assay was performed with the 2SP medium sediment. If either the FVU or the swab tested in the LCx assay had discrepant results, the specimen was retested both undiluted and following 1:10 dilution in LCx Urine Specimen Buffer. The LCx assay was repeated with the processed swab specimen. One of the frozen aliquots of urine was also processed and tested. Retesting of the specimens with discrepant results by the Cobas Amplicor CT/NG test and confirmatory PCR for the major outer membrane protein (MOMP) were done blindly at Roche Diagnostic Systems Inc., Basel, Switzerland. Samples (aliquots of unprocessed 2SP and frozen urine) were sent there coded, and the set included known positive and known negative samples. However, the results obtained by retesting were not used to create an expanded reference standard to avoid bias in the analyses (10). Statistical analysis. The diagnostic consistency of different tests for different specimen types was calculated as described by Cohen (7). The diagnostic accuracy of different tests for different specimen types was calculated according to current guidelines for evaluation of C. trachomatis diagnostic tests (9). RESULTS Of the 1,030 patients, 15 patients had an incomplete set of specimens and were excluded, leaving 1,015 patients for evaluation. By culture, C. trachomatis infection was detected in 7.4% (42 of 565) of the males and 4.2% (19 of 449) of the females (one culture could not be evaluated due to toxicity). Higher prevalence was obtained by testing swabs or FVUs by PCR (males, 8.8 to 9.2%; females, 6.0 to 6.3%) or LCR (males, 8.0 to 8.1%; females, 4.9 to 5.6%) (Table 1). It was noteworthy that the gender-specific difference seen by culture was much smaller than that seen by PCR or LCR. The results of all five tests were identical for 945 (93%) of the 1,015 patients. All of the available test results were identical for an additional 39 patients. Altogether, all available test results were in agreement for 984 (97%) of the 1,015 patients. The patient group with an incomplete set of results includes 1 patient with a toxic chlamydial culture, 10 patients whose specimens were inhibitory by using an IC in the PCR, 3 patients whose specimens did not give a result in the LCR (repeatedly error code 166), and 25 patients for whom LCR results were not available. Statistical testing of the diagnostic consistency of the PCR and LCR methods gave kappa coefficients of 0.93 for swab specimens and 0.95 for FVU specimens. In order to calculate test performance, a patient was considered to have a proven C. trachomatis infection if the culture result was positive (61 patients) or if both PCR and LCR performed with either specimen type gave a positive result (16 patients). Thus, 77 patients had true-positive results and 938 patients had true-negative results. Culture detected 79.2% of the positive patients. The sensitivities of testing of FVU by PCR and LCR were 96.1 and 93.3%, respectively (Table 1). The specificities of all the tests used were high (100% for culture and LCR and greater than 99% for PCR). The discrepant results for 31 patients are presented in Table 2. For 10 patients (three females and seven males), C. trachomatis culture failed to detect an infection, although the same swab specimen was positive by PCR and another swab specimen was positive by LCR (eight patients; results of LCR with swab specimens were not available for two patients). Also, testing of FVU by both PCR and LCR gave a positive result for eight patients, but the result remained negative for two patients. When an aliquot of the 2SP was studied by the DFA assay, six of the eight samples tested showed typical C. trachomatis EBs. Discrepant analysis detected C. trachomatis plasmid DNA or MOMP DNA in specimens from all these patients. Also, culture failed for five patients (all females), for whom only PCR and LCR testing of FVU gave a positive result. No C. trachomatis MOMP DNA could be found in aliquots of 2SP or processed LCx swab specimens, whereas FVU aliquots from two of the four patients tested were also positive for C. trachomatis MOMP DNA. Testing of FVU by PCR failed to detect C. trachomatis in one male patient, and testing of FVU by LCR failed to detect

3 VOL. 36, 1998 PCR AND LCR IN DETECTION OF C. TRACHOMATIS INFECTION 1491 Patient sex and test (specimen) TABLE 1. Diagnostic performance of different samples in detection of urogenital C. trachomatis infection No. of specimens positive/ no. of specimens tested (%) Sensitivity (%) Specificity (%) PPV (%) a NPV (%) b Women Culture (swab c ) 19/449 (4.2) 67.9 (19/28) d 100 (421/421) 100 (19/19) 97.9 (421/430) PCR (swab) 27/447 (6.0) 78.6 (22/28) 98.8 (414/419) 81.5 (22/27) 98.6 (414/420) LCR (swab) 22/448 (4.9) 81.5 (22/27) 100 (421/421) 100 (22/22) 98.8 (421/426) PCR (FVU) 28/442 (6.3) 96.4 (27/28) 99.8 (413/414) 96.4 (27/28) 99.8 (413/414) LCR (FVU) 25/443 (5.6) 92.6 (25/27) 100 (406/406) 100 (25/25) 99.5 (406/408) Men Culture (swab) 42/565 (7.4) 85.7 (42/49) 100 (516/516) 100 (42/42) 98.7 (516/523) PCR (swab) 52/565 (9.2) 100 (49/49) 99.4 (513/516) 94.2 (49/52) 100 (513/513) LCR (swab) 45/557 (8.1) 100 (45/45) 100 (512/512) 100 (45/45) 100 (512/512) PCR (FVU) 50/565 (8.8) 95.9 (47/49) 99.4 (513/516) 94.0 (47/50) 99.6 (513/515) LCR (FVU) 45/560 (8.0) 93.8 (45/48) 100 (512/512) 100 (45/45) 99.4 (512/515) Combined Culture (swab) 61/1,014 (6.0) 79.2 (61/77) 100 (937/937) 100 (61/61) 98.3 (937/953) PCR (swab) 79/1,012 (7.8) 92.2 (71/77) 99.1 (927/935) 89.9 (71/79) 99.4 (927/933) LCR (swab) 67/1,005 (6.7) 93.1 (67/72) 100 (933/933) 100 (67/67) 99.5 (933/938) PCR (FVU) 78/1,007 (7.7) 96.1 (74/77) 99.6 (926/930) 94.9 (74/78) 99.7 (926/929) LCR (FVU) 70/993 (7.0) 93.3 (70/75) 100 (918/918) 100 (70/70) 99.5 (918/923) a PPV, positive predictive value. b NPV, negative predictive value. c Swab, endocervical swab from females and urethral swab from males. d Values in parentheses are numbers used to calculate sensitivity, specificity, PPV, and NPV (9). C. trachomatis in one female and two males. However, discrepant analysis revealed C. trachomatis DNA in another aliquot of FVU from these three patients. Eleven patients had positive results only by PCR. FVU gave a positive signal for four patients and 2SP gave a positive signal for eight patients (both the swab and the urine were positive or one patient). Two of these 11 patients were confirmed to be positive by PCR for MOMP and plasmid-based PCR with another aliquot of FVU, 2SP, or a processed LCx swab specimen. These patients were not counted as true positives when calculating the performance of the tests, since an expanded gold standard was not created. However, 9 of these 11 patients most likely initially had false-positive PCR results because they remained negative when retested by PCR for No. of patients Culture with swab PCR with swab TABLE 2. Analysis of discrepant test results by culture, PCR, and LCR a Result LCR with swab PCR with FVU LCR with FVU MOMP and plasmid-based PCR. For one female, only tests performed with the 2SP medium (culture, PCR, and the DFA assay) remained negative, although PCR of FVU and LCR of swabs as well as LCR of FVU gave positive results, suggesting technical failure in swabbing. Detection of the IC by PCR. Specimens from 10 (1%) patients were found to be inhibitory when tested for the IC in the Cobas Amplicor Chlamydia trachomatis (CT/NG) Test. These were eight urine samples and three endocervical swab specimens (from one female, both the FVU and the swab specimen turned out to be inhibitory). All samples were from females (mean age, 34 years; age range, 20 to 51 years). Six were symptomatic and four were asymptomatic. One used birth control pills, and none was taking antibiotics. In addition to these No. of M/ no. of F Comment 6 5/1 FN by culture b 2 NA 1/1 FN by culture 2 1/1 FN by culture; no C. trachomatis DNA in FVU 5 0/5 No C. trachomatis in swabs 3 2/1 FN by LCR with FVU 1 NA 1/0 FN by PCR with FVU 3 2/1 FP by PCR with FVU c 7 0/7 FP by PCR with swab (n 6) d ;TPbyPCR with swab (n 1) e 1 1/0 TP by PCR e 1 0/1 Obvious swabbing failure a Abbreviations: FN, false negative; FP, false positive; TP, true positive; NA, not available; M, male; F, female. b The DFA assay showed C. trachomatis particles in the culture specimen; both the culture specimen and FVU contained MOMP DNA by PCR. c The initial positive results by PCR of FVU could not be confirmed by any other test, and also reanalysis of another (frozen) aliquot of FVU remained negative by PCR for plasmid DNA and PCR for MOMP DNA. d The initial positive results by PCR with swabs could not be confirmed by any other test. e C. trachomatis MOMP DNA was found in the culture specimen and in FVU by PCR.

4 1492 PUOLAKKAINEN ET AL. J. CLIN. MICROBIOL. 11 specimens, the amplification of the IC was negative for another 11 specimens that were positive for C. trachomatis. Testing of frozen FVU. Aliquots of FVU were stored at 20 C for 2 to 6 months before retesting by PCR and LCR. Fewer positive results were obtained by both PCR and LCR when frozen aliquots of FVU were thawed and analyzed. Ten of the 78 initially PCR-positive FVU specimens became negative. Six of these were from patients with other positive markers for C. trachomatis infection, and four were most likely initially false positive. Four of the 10 aliquots of FVU were also initially LCR positive and all also became negative by LCR. Seven of the 70 initially LCR-positive FVU specimens became negative by LCR. Six of these seven were from patients with true C. trachomatis infection, and one was most likely an initially false-positive result. Four of the seven aliquots of FVU were also initially PCR positive and all also became negative by PCR. DISCUSSION Most urogenital C. trachomatis infections are initially asymptomatic but may subsequently cause considerable long-term morbidity. Consequently, accurate diagnosis of C. trachomatis infection requires the use of specific laboratory techniques. Performance of these methods varies considerably. Culture was earlier considered the gold standard, but PCR and LCR studies suggests that the sensitivity of the culture even in expert laboratories is as low as 75 to 85% (5, 21). It is universally accepted that culture can no longer serve as a reference method in the evaluation of diagnostic tests for C. trachomatis. Recently introduced PCR and LCR techniques have been shown to be very sensitive in the diagnosis of chlamydial infection of the male urethra and in detecting chlamydial DNA in male urine specimens. Lower sensitivity has usually been observed when female specimens have been tested, possibly due to the more frequent occurrence of inhibitors in the specimens. For the first time, we compared the diagnostic performance of commercially available PCR and LCR with both swabs and FVU using cell culture as the reference. In earlier studies evaluating PCR and LCR, FVU had been tested by the amplification methods and swabs had been tested by culture (17) or by culture and the DFA assay (6). In our hands, testing of FVU from females and urethral swabs from males by nucleic acid amplification methods detected most C. trachomatis-positive patients. Culture was less sensitive for female endocervical swabs than for male urethral swabs (67.9 versus 85.7%). Thus, culture missed every third infected female and 14% of the infected males. The site of infection might, however, well explain why swab PCR and LCR failed to detect C. trachomatis infection in six patients, because all these women had negative cervical specimen culture results and chlamydial DNA was found in the FVU only. This study showed that PCR and LCR showed excellent consistency by kappa coefficients and performed better than culture when compared to our standard (positive culture result or positive PCR and LCR results with either specimen type). Use of discrepant analysis is one attempt to improve the performance characteristics of the reference method. Discrepant analysis aims to identify by an alternative (confirmatory) method true-positive specimens originally missed by the reference method. However, if only samples with discrepant results are tested by confirmatory tests, which is usually the case, a bias is introduced (10). The selective testing includes only specimens initially giving discrepant results and could theoretically favor the performance of the tests being evaluated. In our material, the criteria for proven infection were culture positivity or both PCR and LCR positivity. Discrepant analysis was done, but the results were used only to clarify the initial results. An expanded reference standard was not created, although this was recommended recently by the STD Diagnostic Initiative (9). Our retesting showed that 60% of the swab specimens that were obviously culture failures did contain C. trachomatis EBs when the specimens were analyzed by DFA staining. This is as expected, since culture and DFA assay are considered less sensitive than PCR. Clinical specimens are known to contain several factors that inhibit DNA polymerase. Roche has developed a system that permits the identification of such polymerase reaction-inhibiting factors: detection of amplification of an IC in included in the Cobas Amplicor test, thus ensuring the integrity of the result. In our material, the occurrence of such inhibitors was low (less than 1%). Others have noted higher inhibition rates detectable with the IC system with clinical specimens (16) or spiked clinical specimens (27). It is possible that in this study obvious false-negative LCR results were due to the presence of inhibitors of the LCR, but this could not be confirmed in our study, because the LCx does not incorporate an internal control. Our earlier experience with the PCR, LCR, and transcription-mediated amplification (TMA) techniques in routine clinical microbiology laboratory use suggests that some specimens contain factors that inhibit the amplification reactions, but the factors are not identical for different techniques. Specificity is another concern with the DNA amplification techniques. Theoretically, the tests are highly specific, but because the potential of the amplification reaction is enormous, even a slight contamination can result in erroneous results. In published studies, PCR is usually found to be more sensitive and LCR is found to be more specific when compared to the sensitivity and specificity of a created standard (6, 17). This was also the case in our study when FVU specimens were analyzed: The sensitivities of PCR and LCR were 96.1 versus 93.3%, respectively, and the specificities of the tests were 99.6 versus 100%, respectively. To increase the specificities of the amplification techniques, retesting of the initially positive specimens or confirmation of the results by another amplification method can be done. However, because freezing and thawing of the specimen can have an effect on test performance, the results may not be comparable. Freezing and thawing can eliminate inhibitory factors (25), but as our results suggest, the DNA in the specimens can also be degraded. The most attractive aspects of DNA amplification methods are their excellent sensitivities and good performance with FVU samples. In our study, 96 and 93% of the infections could be detected by testing FVU by PCR or LCR, respectively, when the results for symptomatic and asymptomatic patients were analyzed together. In the detection of both symptomatic and asymptomatic infections, testing of FVU by PCR or LCR is thus a preferred alternative to testing of swab specimens for both females and males. We have shown earlier that screening for chlamydial infections by the PCR test with FVU is costbeneficial in a low-prevalence population if the prevalence of infection exceeds 3.9% (15). Because recent studies suggest that C. trachomatis infection is also a risk factor for adverse pregnancy outcomes other than ectopic pregnancy (8) and is possibly also a risk factor for the development of cervical neoplasia (13), the cost-effectiveness of screening of the sexually active population by DNA amplification methods needs to be reevaluated. ACKNOWLEDGMENTS We thank Marja Pietilä, Maarit Ohranen, and Maaria Vainio for skillful technical assistance. The skillful personnel of the STD clinic,

5 VOL. 36, 1998 PCR AND LCR IN DETECTION OF C. TRACHOMATIS INFECTION 1493 Helsinki University Central Hospital, as well as Maritta Slotte and Katriina Bildjuschkin, Sexual Health Clinic of Väestöliitto, Counseling and Services for Adolescents, the Family Federation of Finland, Helsinki, are gratefully acknowledged for collecting all the samples. This study was supported by a grant from the Helsinki University Central Hospital Research Funds and by Roche Diagnostic Systems Inc., Basel, Switzerland. REFERENCES 1. Ånestad, G., B. P. Berdal, O. Scheel, R. Mundal, O. Odinsen, K. Skaug, O. S. Khalil, P. Plier, and H. Lee Screening urine samples by leukocyte esterase test and ligase chain reaction for chlamydial infections among asymptomatic men. J. Clin. Microbiol. 33: Bauwens, J. E., A. M. Clark, M. J. Loeffelholz, S. A. Herman, and W. E. Stamm Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine. J. Clin. Microbiol. 31: Bauwens, J. E., A. M. Clark, and W. E. Stamm Diagnosis of Chlamydia trachomatis endocervical infections by a commercial polymerase chain reaction assay. J. Clin. Microbiol. 31: Cates, W., and J. E. Wasserheit Genital chlamydial infections: epidemiology and reproductive sequelae. Am. J. Obstet. Gynecol. 164: Chernesky, M. A., D. Jang, H. Lee, J. D. Burczak, H. Hu, J. Sellors, S. J. Tomazic-Allen, and J. B. Mahony Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction. J. Clin. Microbiol. 32: Chernesky, M., S. Chong, D. Jang, K. Luinstra, J. Sellors, and J. B. Mahony Ability of commercial ligase chain reaction and PCR assays to diagnose Chlamydia trachomatis infections in men by testing first-void urine. J. Clin. Microbiol. 35: Cohen, J A coefficient of agreement for nominal scales. Educ. Psychol. 20: Gencay, M., M. Puolakkainen, T. Wahlström, P. Ämmälä, L. Mannonen, A. Vaheri, and M. L. Koskiniemi Chlamydia trachomatis detected in human placenta. J. Clin. Pathol. 50: Global Access to STD Diagnostics Guidelines for evaluating Chlamydia trachomatis Diagnostic Tests, p STD Initiative Program for Appropriate Technology in Health, vol. 4. Program for Appropriate Technology in Health (PATH), Seattle, Wash. 10. Hadgu, A The discrepancy in discrepant analysis. Lancet 348: Jaschek, G., C. A. Gaydos, L. E. Welsh, and T. C. Quinn Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. J. Clin. Microbiol. 31: Lee, H., M. A. Chernesky, J. Schachter, J. D. Burczak, W. W. Andrews, S. Muldoon, G. Leckie, and W. E. Stamm Diagnosis of Chlamydia trachomatis geniotourinary infection in women by ligase chain reaction assay of urine. Lancet 345: Lehtinen, M., J. Dillner, T. Luostarinen, A. Aromaa, M. H. Hibma, R. Kirnbauer, A. Miettinen, J. Paavonen, J. T. Schiller, and M. Hakama Nested case-control study of the role of human papillomavirus type 16 and Chlamydia trachomatis in cervical carcinoma: nested case-control study. Br. Med. J. 312: Paavonen, J Chlamydia trachomatis a major threat to reproduction. Eur. J. Obstet. Gynecol. Reprod. Biol. 49: Paavonen, J., M. Puolakkainen, M. Paukku, and H. Sintonen. Cost-benefit analysis of universal first void urine Chlamydia trachomatis screening program. Obstet. Gynecol., in press. 16. Pasternack, R., P. Vuorinen, A. Kuukankorpi, T. Pitkäjärvi, and A. Miettinen Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens. J. Clin. Microbiol. 34: Pasternack, R., P. Vuorinen, T. Pitkäjärvi, M. Koskela, and A. Miettinen Comparison of manual amplicor PCR, Cobas Amplicor PCR, and LCx assays for detection of Chlamydia trachomatis infection in women by using urine specimens. J. Clin. Microbiol. 35: Paukku, M., M. Puolakkainen, D. Apter, S. Hirvonen, and J. Paavonen First-void urine testing for Chlamydia trachomatis by polymerase chain reaction in women in a low prevalence population. Sex. Transm. Dis. 24: Quinn, T Advances in the molecular diagnosis of Chlamydia trachomatis, p In A. Stary (ed.), Proceedings of the Third Meeting of the European Society for Chlamydia Research. Societa Editrice Esculapio, Bologna, Italy. 20. Quinn, T. C., L. Welsh, A. Lentz, K. Crotchfelt, J. Zenilman, J. Newhall, and C. Gaydos Diagnosis by AMPLICOR PCR of Chlamydia trachomatis infection in urine from women and men attending sexually transmitted disease clinic. J. Clin. Microbiol. 34: Ridgway, G. L., G. Mumtaz, A. J. Robinson, M. Franschini, C. Carder, J. Burczak, and H. Lee Comparison of the ligase chain reaction with cell culture for the diagnosis of Chlamydia trachomatis infection in women. J. Clin. Pathol. 49: Schachter, J., J. Moncada, R. Whidden, H. Swah, G. Bolan, J. D. Burczak, and H. Lee Noninvasive tests for diagnosis of Chlamydia trachomatis infection: application of ligase chain reaction to first-catch urine specimens of women. J. Infect. Dis. 172: Schachter, J Evolution of diagnostic tests for Chlamydia trachomatis infections, p In A. Stary (ed.), Proceedings of the Third Meeting of the European Society for Chlamydia Research. Societa Editrice Esculapio, Bologna, Italy. 24. Stamm, W. E., L. A. Koutsky, J. K. Benedetti, J. L. Jourden, R. C. Brunham, and K. K. Holmes Chlamydia trachomatis urethral infections in men: prevalence, risk factors, and clinical manifestations. Ann. Intern. Med. 100: Stary, A., B. Choueiri, I. Hörting-Müller, P. Halisch, and L. Teodorowich Detection of Chlamydia trachomatis in urethral and urine samples from symptomatic and asymptomatic male patients by the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 15: Toye, B., R. W. Peeling, P. Jessamine, P. Claman, and I. Gemmill Diagnosis of Chlamydia trachomatis infections in asymptomatic men and women by PCR assay. J. Clin. Microbiol. 34: Verkooyen, R. P., A. Luijendijk, W. M. Huisman, W. H. F. Goessens, J. A. J. W. Kluytmans, J. H. van Rijsoort-Vos, and H. A. Verbrugh Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis Assay. J. Clin. Microbiol. 34: World Health Organization An overview of selected curable sexually transmitted diseases. Global Programme on AIDS, World Health Organization, Geneva, Switzerland.