Abbott Laboratories, Abbott Park, Illinois4. Received 28 December 1994/Returned for modification 21 March 1995/Accepted 1 June 1995. Urine samples from ...
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1995, p. 2483–2484 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 9
Screening Urine Samples by Leukocyte Esterase Test and Ligase Chain Reaction for Chlamydial Infections among Asymptomatic Men G. ÅNESTAD,1* B. P. BERDAL,2 O. SCHEEL,2 R. MUNDAL,3 O. ODINSEN,4 K. SKAUG,1 O. S. KHALIL,4 P. PLIER,4 AND H. LEE4 Department of Virology, National Institute of Public Health,1 Norwegian Defence Microbiological Laboratory,2 and Defence Command Norway, Joint Medical Services,3 Oslo, Norway, and Abbott Laboratories, Abbott Park, Illinois4 Received 28 December 1994/Returned for modification 21 March 1995/Accepted 1 June 1995
Urine samples from 358 asymptomatic males were screened for urethral inflammation by the leukocyte esterase (LE) test and for Chlamydia trachomatis by the ligase chain reaction (LCR). LE and LCR positivity rates were 7.5% (27 of 358 samples) and 2.8% (10 of 358 samples), respectively. Eight of the 10 LCR-positive samples were detected by the LE screening test. The urine LE prescreening test in combination with the LCR assay may be a reasonable approach for genitourinary chlamydial disease control. toms of urethritis. The urethral swabs were taken by using a fine cotton-tipped metal holder introduced 3 to 5 cm into the urethra. After it was removed, the cotton tip was cut off aseptically and placed into a vial containing 1 ml of 0.2 M sucrosephosphate (2-SP) medium. LE test. Urine samples were tested immediately after collection with urine LE test strips (Ecur4-Test; Boehringer Mannheim GmbH, Mannheim, Germany) according to the manufacturer’s recommendations. The test strips were dipped into the urine for 1 s, and excess urine was drained off on the rim of the sample tube. After 2 min, the color of the test strip was compared with a color scale on the label of the package. The test was counted as negative or positive ($11). LCR. The urine LCR assay (Abbott Laboratories, Chicago, Ill.) was performed as described previously (3). Within 24 h of collection, urine samples were briefly vortexed and 1 ml was transferred and centrifuged at 13,000 3 g for 10 min. The supernatant was removed, and the pellet was resuspended in 1 ml of urine resuspension buffer and heated to 958C for 20 min. After cooling, 100 ml of the suspension was transferred to a unit-dose amplification vial. Each vial contained four C. trachomatis plasmid probes, thermostable DNA ligase, DNA polymerase, deoxynucleoside triphosphates, and cofactors in a buffer (pH 7.8). Amplification was effected for 40 cycles at a denaturation temperature of 978C and an annealing and ligation temperature of 628C. The amplified products were finally processed and assessed with an LCX analyzer (Abbott). The LCR results were expressed as counts per second, with positive results defined as a value equal to or greater than the product of the mean of the two positive calibrator values multiplied by 0.45. Cell culture from urethral swabs. The urethral swabs were kept on melting ice for up to 8 h before inoculation onto monolayers of cycloheximide-treated McCoy cells, as described previously (10). Briefly, 200 ml of the sample in 2-SP medium was inoculated into each cell culture tube. After centrifugation at 3,000 3 g for 60 min, the cells were incubated at 378C for 48 h, washed, fixed in methanol, stained with fluorescein isothiocyanate-labelled monoclonal antibodies to C. trachomatis (Tissue Culture Confirmation Test; Syva Company, Palo Alto, Calif.), and examined by UV microscopy at 3100 magnification.
Males serve as an important reservoir for Chlamydia trachomatis. Like women, they may be asymptomatic carriers (6, 12). Because of the discomfort associated with specimen collection from the male urethra, laboratory diagnosis of C. trachomatis infection has been more difficult for men than for women. Recently, various tests for detecting C. trachomatis in urine samples have been developed, of which enzyme immunoassay (EIA) is the most frequently used. However, both the sensitivity and the specificity characteristics of the EIAs on urine samples limit the utility of this approach (2, 4, 10). The simple, dipstick-mounted urinary leukocyte esterase (LE) test indicates mucosal inflammation, which may be induced by, for example, urethral chlamydial infection (7, 8, 11, 13). The nonspecific nature of the LE test necessitates the need to confirm all LE-positive test results with a C. trachomatis-specific test. A newly developed test, the ligase chain reaction (LCR), amplifies chlamydial plasmid DNA (1, 5). This test has been shown to have greater sensitivity than either EIA of urine samples or culture of urethral swabs (3, 9). The purpose of the present study was to evaluate the reliability of LCR testing of urine samples as well as the diagnostic suitability of the LE test as a nonspecific, prescreening test for C. trachomatis infection. Samples. Fifteen milliliters of first-void morning urine was collected from 358 Norwegian male military conscripts aged 19 to 21 years. All subjects answered a questionnaire concerning urethral symptoms and use of antimicrobial agents. LE and LCR testing were performed on all of the urine samples. All LCR- or LE-positive samples, together with a similar number of negative control samples, were retested by LCR. A second urine sample and an urethral swab were collected from nine LCR-positive and nine LE-positive and LCR-negative subjects. Similar samples were also collected from 26 LCR- and LE-negative controls. This second round of samples was collected 12 days after the first urine specimen was obtained. This group of 44 subjects also answered a second questionnaire that requested information on previously overlooked clinical symp* Corresponding author. Mailing address: Department of Virology, National Institute of Public Health, Geitmyrsveien 75, 0462 Oslo, Norway. 2483
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TABLE 1. Comparison of LE test and LCR test results for urine samples from 358 Norwegian military conscripts No. of samplesa LE result LCR positive
LCR negative
Total
8 2 10
19 329 348
27 331 358
Positive Negative Total
a For the LE test compared with the LCR assay, the sensitivity was 80% (8 of 10 samples), the specificity was 94.5% (329 of 348), the positive predictive value was 29.6% (8 of 27), and the negative predictive value was 99.4% (329 of 331).
Discordant results. In cases of discrepancies between LCR and cell culture results, the discrepant samples, together with a similar number of negative control samples, were tested by a second LCR assay with probes for the gene of the major outer membrane protein (MOMP). Results and discussion. Twenty-seven urine samples (7.5%) were LE positive, and 10 urine samples (2.8%) were LCR positive (Table 1). Eight of the 10 LCR-positive samples were also LE positive. Thus, the overall positivity of LCR among the LE-positive samples was 29.6% (8 of 27 samples). Altogether, 58 urine samples were retested by the LCR assay. The results were identical to those obtained in the first test. The test results for the 44 second urine samples and the urethral swabs are presented in Table 2. The second urine sample from one of the two initially LCR-positive and LEnegative subjects was LCR negative. Urethral swab culture was also negative. MOMP-LCR assays confirmed the plasmid LCR test results. The conscript from whom this sample was obtained had been treated for otitis media with both erythromycin and tetracycline immediately before the first urine sample was collected. It is conceivable that the first LCR test was a true positive, detecting chlamydial plasmid DNA from nonviable bacteria. Workowski et al. (14) found that gene amplification assays could remain positive 1 to 2 weeks after the initiation of therapy. If so, the negative LE test result may be more informative because the urethral inflammation will diminish during the course of therapy. A second urine sample from another conscript whose initial urine sample tested positive by both LCR and LE was also LCR positive, but the urethral swab was culture negative. The second urine sample was MOMP-LCR
TABLE 2. Test results for second urine samples and urethral swabs from 44 military conscripts tested for C. trachomatis by LCR and cell culture No. of conscripts
6 1b 1 1c 9 26
Result for first urine samplea
LCR1, LCR1, LCR1, LCR1, LCR2, LCR2,
LE1 LE1 LE2 LE2 LE1 LE2
Result for second sample LCR of urine
Urethral culture
1 1 1 2 2 2
1 2 1 2 2 2
a LCR1, LCR positive; LCR2, LCR negative; LE1, LE positive; LE2, LE negative. b Positive by MOMP-LCR for the second urine sample. c Positive by MOMP-LCR for the first urine sample and negative for the second urine sample.
positive, indicating that the discrepancy may be explained by swab or culture failure. Chernesky et al. (3), using an ‘‘expanded gold standard,’’ found that LCR testing of urine samples had a sensitivity of 96%. Compared with culture and EIA, testing of urine samples by LCR was the most sensitive technique. This is in agreement with our findings (Table 2). Sixteen of the 358 conscripts reported previous symptoms consistent with genital infection. At the time of collection of the urine samples, all but two of the conscripts were symptomfree. One of these two subjects had an LCR-positive urine sample. In the second round of questioning, after the laboratory results had been communicated to the conscripts, another LCR-positive conscript admitted slight urethral symptoms. Thus, 8 of the 10 LCR-positive subjects were truly asymptomatic. Although only 8 of the 10 LCR-positive urine samples were detected by the LE screening test, we agree with Genc et al. (7) that in asymptomatic male populations, screening of urine by the LE test and then confirmation by a specific test for C. trachomatis is a reasonable strategy for urogenital chlamydial disease control. From a cost-benefit point of view, the alternative is likely to be no testing at all. REFERENCES 1. Birkenmeyer, L. G., and I. K. Mushahwar. 1991. DNA probe amplification methods. J. Virol. Methods 35:117–126. 2. Chernesky, M., S. Castriciano, J. Sellors, I. Stewart, I. Cunningham, S. Landis, W. Seidelman, L. Grant, C. Devlin, and J. Mahony. 1990. Detection of Chlamydia trachomatis antigens in urine as an alternative to swabs and cultures. J. Infect. Dis. 161:124–126. 3. Chernesky, M. A., D. Jang, H. Lee, J. D. Burczak, H. Hu, J. Sellors, S. J. Tomazic-Allen, and J. B. Mahony. 1994. Diagnosis of Chlamydia trachomatis infections in men and women by testing first-void urine by ligase chain reaction. J. Clin. Microbiol. 32:2682–2685. 4. Demaio, J., R. S. Boyd, R. Rensi, and A. Clark. 1991. False-positive Chlamydiazyme results during urine sediment analysis due to bacterial urinary tract infections. J. Clin. Microbiol. 29:1436–1438. 5. Dille, B. J., C. C. Butzen, and L. G. Birkenmeyer. 1993. Amplification of Chlamydia trachomatis DNA by ligase chain reaction. J. Clin. Microbiol. 31:729–731. 6. Fraser, J. J., P. T. Rettig, and D. W. Kaplan. 1983. Prevalence of cervical Chlamydia trachomatis and Neisseria gonorrhoeae in female adolescents. Pediatrics 71:333–336. 7. Genc, M., L. Ruusuvaara, and P.-A. Mårdh. 1993. An economic evaluation of screening for Chlamydia trachomatis in adolescent males. JAMA 270: 2057–2064. 8. McNagny, S. E., R. M. Parker, J. M. Zenilman, and J. S. Lewis. 1992. Urinary leukocyte esterase test: a screening method for the detection of asymptomatic chlamydial and gonococcal infections in men. J. Infect. Dis. 165:573–576. 9. Schachter, J., W. E. Stamm, T. C. Quinn, W. W. Andrews, J. D. Burczak, and H. H. Lee. 1994. Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix. J. Clin. Microbiol. 32:2540–2543. 10. Scheel, O., G. Ånestad, R. Mundal, and B. P. Berdal. 1993. Detection of Chlamydia trachomatis in the urine of young Norwegian males by enzyme immunoassay. Eur. J. Clin. Microbiol. Infect. Dis. 12:746–749. 11. Sellors, J. W., J. B. Mahony, L. Pickard, D. Jang, D. Groves, K. E. Luinstra, and M. A. Chernesky. 1993. Screening urine with a leukocyte esterase strip and subsequent chlamydial testing of asymptomatic men attending primary care practitioners. Sex. Transm. Dis. 20:152–157. 12. Shafer, M.-A., V. Prager, J. Shlawitz, 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. 13. Shafer, M.-A., J. Schachter, A. B. Moscicki, A. Weiss, J. Shalwitz, E. Vaughan, and S. G. Millstein. 1989. Urinary leukocyte esterase screening test for asymptomatic chlamydial and gonococcal infection in males. JAMA 262:2562–2566. 14. Workowski, K. A., M. F. Lampe, K. G. Wong, M. B. Watts, and W. E. Stamm. 1993. Long-term eradication of Chlamydia trachomatis genital infection after antimicrobial therapy: evidence against persistent infection. JAMA 270: 2071–2075.
