704
Article
Vol. 11. No. 8
Eur. J. Clin. Microbiol. Infect. Dis., August 1992, p. 704-708 0934-9723/92/08 0704-05 $ 3.00/0
Evaluation of a Chemiluminometric Immunoassay for Detection of Chlamydia trachomatis in the Urine of Male and Female Patients
C. S c i e u x 1., A . B i a n c h i 1, S. H e n r y I , N. B r u n a t 1, S. A b d e n n a d e r 2, D . V e x i a u 2, M. J a n i e r 2, E M o r e l 2, E H . L a g r a n g e 1
A chemiluminometric immunoassay (Magic Lite Chlamydia) for detection of Chlamydia trachomatis antigens in first-void urine samples was compared with cell culture using urogenital swabs from 221 men and 242 women. The rate of isolation of Chlamydia trachomatis was 23.5 % in men, nearly 80 % of whom had symptoms of urethritis, and 8.3 % in women, in whom both cervix and urethra samples were tested. In urine sediments from men and women respectively the chemiluminometric assay showed a sensitivity of 80.8 % and 70 %, a specificity of 97 % and 95 %, a positive predictive value of 89.4 % and 58.3 %, and a negative predictive value of 94.3 % and 97.2 %. Discrepanciesbetween results obtained with the chemiluminometric assay and cell culture were resolved using two polymerase chain reaction techniques to test urogenital samples. The detection of Chlamydia trachomatis in urine samples with the chemiluminometric assay was confirmed to be superior for screening symptomatic men with urogenital infections than women as a lower prevalence population.
Chlamydia trachomatis is now recognized as a m a j o r cause of sexually transmitted disease t h r o u g h o u t the world. T h e detection of chlamydial genital infections still poses a challenge for b o t h clinicians and laboratory workers (1). R e c o v e r y of Chlamydia trachomatis in cell cultures of urogenital specimens is considered the most sensitive and specific m e t h o d but has known drawbacks and is unavailable in m a n y clinical settings. Alternative m e t h o d s have been developed for detecting chlamydial antigen and chlamydial D N A (1-12). I m m u n o a s s a y s for testing first-void urine sediment h a v e b e e n evaluated in the diagnosis of chlamydial urethritis in m e n and to a lesser extent in w o m e n (13-22). We evaluated the p e r f o r m a n c e of a new chemiluminometric sandwich i m m u n o a s s a y for detection of Chlamydia trachomatis in urine samples f r o m m e n and w o m e n c o m p a r e d with cell culture of urethral and cervical swabs. Discrepancies b e t w e e n the results of culture and the i m m u n o a s s a y were resolved by using two p o l y m e r a s e chain reaction techniques to test urogenital samples. 1Laboratoire de Bact6riologie-Virologie and 2Centre Clinique et Biologique des Maladies Sexuellement Transmissibles, H6pital Saint-Louis, Universit6 Paris VII, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
Materials and Methods
Patients. From January to July 1991, 463 sexually active patients (242 women and 221 men) attending the Sexual Transmitted Diseases (STD) Clinic at St-Louis Hospital, Paris, were entered in the study after giving verbal consent. Patients were excluded from the study if they had received antibiotics within the preceding two weeks. Specimens. Specimens for culture consisted of a total of 705 urogenital swabs collected from the endocervix (n = 242), female urethra (n = 242) and male urethra (n = 221) using plastic swabs (Bactopick, Labomoderne, France). Swabs were placed in buffered sucrose phosphate (2-SP) transport medium kept at 4 *C and always inoculated onto culture cells within 6h. First-void urine specimens (approximately 15 to 50 ml) for the immunoassay were collected in large sterile vials and centrifuged at 3,500 xg for 20 rain at 4 *C. The pellets were suspended in 0.2 ml of specimen dilution buffer and 0.75 ml of reagent (0.34 N sodium hydroxide, chenodeoxycholate salt and EDTA) according to the instructions of the manufacturer. After vortexing twice for 15 see urine specimens were stored at 4 °C for less than a week or at -70"C until tested. The order of collection was urine specimens followed by swabs in 103 men and 116 women, and swabs followed by urine specimens in 118 men and 126 women. The time of last voiding ranged from 1 h 15 min to 15 h, and exceeded 3 h in 48.4 % of the men and 41.3 % of the women.
Chemiluminometric bnmunoassay. The chemiluminometric sandwich immunoassay (Magic Lite Chlamydia,
Vol. 11, 1992
Ciba Corning, USA) was performed according to the manufacturer's instructions. A volume of 0.2 ml of treated specimen was transferred to assay vials to which reagents (0.11 M citric acid, 0.18 M hydrogen peroxide and 0.5 M TAPS buffer), MOMP-speeiflc anti-Chlamydia trachomatis monoclonal antibody conjugated with aeridinium ester and antichlamydial polyclonal antibody immobilized on magnetic particles as solid phase were subsequently added. After incubation for 90 rain the solid phase particles were separated magnetically, washed and resuspended for counting. After addition of chemiluminescence-stimulating reagents, the emission of light was recorded as relative light units (RLU) in the Magic Lite Analyser. A specimen was classified as positive when the ratio of the specimen R L U to the negative control RLU was ~ 2 in duplicate tests.
Cell Culture. After vortex mixing, 0.2 mI of the transport medium containing the swab was inoculated onto freshly trypsinized Hela 229 cells in suspension in two to four wells of a mieroeulture plate, centrifuged (3,500 x g) at 36 °C for 40 rain, and incubated at 37 °C in 5 % CO2 for 48-72 h in maintenance medium containing cycloheximide (1 rag/l). Following fixation with cold absolute methanol for 10 rain, plates were stained using a genusspecific antichlamydial monoclonal antibody with a peroxidase-antiperoxidase detection system (Orthodiagnostics, USA). Chlamydial inclusion bodies were detected by bright field microscopy under magnification (200 times) with an inverted microscope. Each assay was scored independently and the result recorded without knowledge of the result of the other test. Sensitivity, specificity and predictive values were calculated by the usual methods (23). The p values were based on the Pearson chi-square test and are stated if the value was < 0.1. Specimens with discordant results were submitted to serial passage in cell culture after storage at -70 °C. Briefly, the samples were inoculated onto Hela 229 cells, and after 48 h of incubation the cell monolayer of one well was diluted in 0.3 ml of transport medium, disrupted by ultrasonic vibrator (Bioblock Scientific, France) and inoculated onto three wells of fresh Hela 229 cells. Two serial passages were performed.
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(10 mM "iris, 50 mM KCI, 1.5 mM MgCI2, 0.1 g/l gelatin, 200 ~M of each nucleotide, 2.5 units T a q - D N A potymerase [Cetus Corporation, USA], 0.5 /aM of each primer). For the chromosome PCR, the samples were submitted to 30 cycles of denaturation for 30 sec at 92 °C, annealing for 30 sec at 54 °C and extension for 1 rain at 72 °C in a D N A thermocycler (11). For the plasmid PCR, the samples were submitted to 40 cycles of denaturation for 2 rain at 94 °C, annealing for 2 rain at 57 °C and extension for 4 rain at 70 °C in a D N A thermocycler (12). Amplified D N A products were fractioned by 3 % or 1.2 % agarose gel eleetrophoresis. The specific fragment of 129bp (located in a conserved region of the major outer membrane protein gene) detected in the chromosome PCR, and the specific fragment of 473bp detected in the plasmid PCR, were stained with ethydium bromide visualized by UV fluorescence (11, 12). After Southern blotting onto Hybond N membranes (Amersham, UK), hybridization was performed using the 32p_ labelled specific internal probe (11, 12). The B-globin gene segment PCR was used to eliminate the possibility of false negative results due to the presence of PCR inhibitors. All specimens negative in the PCR were accordingly tested using the primer set PCO3 and PCO4 yielding amplified D N A of the l l 0 b p fragment (24).
Results
PCR Techniques. In 47 clinical specimens (16 endocervix,
T h e a v e r a g e a g e o f t h e 463 p a t i e n t s t e s t e d w a s 30 y e a r s f o r w o m e n ( r a n g e 15 t o 68 y e a r s ) a n d 33 y e a r s for m e n ( r a n g e 17 to 74 y e a r s ) . L e u c o r r h e a was t h e p r i m a r y r e a s o n for a t t e n d i n g t h e clinic in 83.3 % o f t h e w o m e n . O f t h e m e n , 3 7 . 1 % att e n d e d d u e to clinical e v i d e n c e o f u r e t h r a l disc h a r g e a n d 40.3 % d u e to u r e t h r a l d i s c o m f o r t o r dysuria. The reasons of the remaining patients for a t t e n d i n g t h e S T D clinic i n c l u d e d a s y m p t o m a t i c check-up, symptomatic partner, previous genital i n f e c t i o n a n d infertility.
16 female urethra, 15 male urethra specimens) from 31 patients in which results were discordant, specimens in 2-SP transport medium were tested using two PCR techniques to detect chromosomal and plasmid DNA respectively. Negative clinical specimens (n = 25) and positive clinical specimens (n = 20) were included as controls. An amount of 0.5 ml of vortexed transport medium was collected using a positive-deptacement pipette (Microman R, Gilson, USA) and centrifuged at 11,000 x g for 20 rain at 4 °C. The pellet was digested in 100 pl of lysis buffer containing 20 pg of proteinase K in 10 mM Tris 1 mM EDTA for 30 min at 37 °C and boiled for 10 min at 100 °C. The primer sets CT1, CT2 and the internal probe CT3 were used for the chromosome PCR as described by Duthil et al. (11). The primer set IIa, IIB and internal probe PIA were used for the plasmid PCR as described by Oostergaard et al. (12). All the oligonucleotides were synthesized by the phosphoramidite method (Applied Biosystems, USA). Each PCR was performed with 30 13I of treated specimen in 100 ~1 in a final reaction mixture
O f t h e 242 w o m e n t e s t e d , 20 (8.3 % ) w e r e p o s i t i v e f o r Chlamydia trachomatis o n c u l t u r e o f u r o g e n i tal swabs. T h e results o f t h e c h e m i l u m i n o m e t r i c assay a n d c u l t u r e a c c o r d i n g to s a m p l e o r i g i n a r e s h o w n in T a b l e 1. T h e p e r f o r m a n c e o f t h e c h e m i l u m i n o m e t r i c a s s a y in u r i n e s e d i m e n t s f r o m w o m e n is s h o w n in T a b l e 2. A l l w o m e n with a p o s i t i v e c u l t u r e in b o t h c e r v i x a n d u r e t h r a l s p e c i m e n s h a d d e t e c t a b l e c h l a m y d i a l a n t i g e n in urine sediment. The sensitivity of the chemil u m i n o m e t r i c a s s a y r a n g e d f r o m 73.3 % t o 83.3 % a c c o r d i n g to t h e o r i g i n o f a c u l t u r e p o s i t i v e specimen (cervix or urethral). Higher sensitivity o f t h e a s s a y w a s f o u n d in s p e c i m e n s y i e l d i n g m o r e t h a n 10 i n c l u s i o n s p e r w e l l on c u l t u r e ( a d j u s t e d :(2 = 4.63, p < 0.05 in c e r v i x s p e c i m e n s ; a d j u s t e d :(2 = 3.01, p < 0.1 in u r e t h r a s p e c i m e n s ) .
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Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: Results in the detection of Chlamydia trachomatisusing the chemiluminometric assay in firstvoid urine specimens compared with culture of urogenital specimens. Culture
Chemiluminometrie assay Positive
Negative
Cervix and urethra positive (n = 7) Cervix positive and urethra negative (n = 8) Cervix negative and urethra positive (n = 5) Cervix and urethra negative (n = 222)
7 4 3 10
0 4 2 212
Total (n = 242)
24
218
Urethra positive (n = 52) Urethra negative (n = 169)
42 5
10 164
Total (n = 221)
47
174
Women
Men
Table 2: Performance in the detection of Chlaraydia trachomatis of the chemituminometric assay in first-void urine specimens compared with cell culture and/or PCR in urogenital specimens. Chemiluminometric assay Sensitivity (%)
Specificity (%)
Culture Women (n = 242) Men (n = 221)
70 80.8
95.5 97
58.3 89.4
97.2 94.3
Culture and/or PCR Women (n = 242) Men (n = 221)
73.9 81.8
96.8 98.8
70.8 95.7
97.2 94.3
O f the 221 m e n tested, 52 (23.5 % ) were positive for Chlamydia trachomatis on culture of urogential swabs. T h e rate of a positive culture ranged f r o m 16.8 % to 3 4 . 1 % in m e n presenting with dysuria or urethral discharge. In 47 patients antigen was detected in urine sediment with the c h e m i l u m i n o m e t r i c assay (Table 1). T h e perform a n c e of the c h e m i l u m i n o m e t r i c assay is shown in Table 2. T h e sensitivity of the chemiluminometric assay was higher in patients yielding m o r e than ten inclusion bodies per well on culture of urethral specimens (adjusted %2 = 4.26, p < 0.05), but was not significantly different according to w h e t h e r the patient had urethral discharge or dysuria. In male and f e m a l e patients, no significant difference in sensitivity of the chemiluminometric assay in the testing of urine samples was noted ac-
Positive predictive Negative predictive value (%) value (%)
cording to the time of the last voiding, or the order of collection of samples. On serial passage of specimens with discordant results, no further culture-positive specimens were found in men or w o m e n . In the P C R the s a m e results w e r e o b t a i n e d using the plasmid P C R and the c h r o m o s o m e P C R . All 16 patients with positive culture and negative assay results were confirmed as positive in the PCR. O f the w o m e n in w h o m only the cervix or urethra specimen was positive on culture, P C R confirmed the result in all cases and the specimen f r o m the second site was also positive in three cases. O f the four w o m e n in w h o m only the cervix specimen was positive on culture and the assay result positive, the urethra specimen was positive on P C R in two cases. Of the 15 patients with negative culture and positive assay results, six were positive and
Vo1.11,1992
nine negative in the PCR so that the assay results in urine samples could be considered false positive. The presence of inhibitors of PCR was not demonstrated in any PCR negative specimen using the B-globin gene segment PCR. The performance of the chemiluminometric assay compared with culture and/or PCR is reported in Table 2.
Discussion
The findings of the present study in patients attending a STD clinic suggest that detection of Chlamydia trachomatis antigen in first-void urine sediments with the chemiluminometric assay can yield an accurate diagnosis of urethritis in men. However, in the screening of first-void urine in women the performance of the assay was inferior and did not parallel results achieved by culture of cervical swabs possibly because not all women have an infected urethra (13, 14, 18, 20). In addition, the disparity could be due to lower isolation rates in women compared to men. Previous evaluations of screening for Chlamydia trachomatis in first-void urine samples revealed a sensitivity of 72.6 % to 87 % in men and 54 % to 76.9 % in women using an enzyme immunoassay or fluorescence assay compared with culture of cervical and urethral specimens (13, 15, 17, 21). We obtained similar results which are superior to the low sensitivity of about 40 % reported in the testing of first-void urine sediment in men half of whom had no symptoms of urethritis or in women attending a family planning clinic (20, 22). Non-invasive collection of first-void urine samples during a patient's visit to the STD clinic was practical, and analysis of data according to time of the last voiding and order of collection of urine and urogenital specimens showed no impact on positivity rates in the chemiluminometric assay, as previously described (13, 16, 21, 22, 25). In both male and female patients, the sensitivity of the chemiluminometric assay strongly correlated with the number of chlamydial inclusion bodies in cell culture, as previously reported for antigen detection tests in urogenital and urine specimens (1-3, 6, 22). As recent studies have shown an excellent performance of PCR compared with cell culture, PCR could be considered the reference method to resolve discrepancies between results of cell culture and antigen detection methods (10-12). True false-positive results in the chemiluminometric
707
assay were uncommon in both men and women (9 of 463 patients). The chemiluminometric assay which uses species-specific monoclonal antibody and immobilized antichlamydial polyclonal antibody, has high specificity, limiting problems associated with urinary tract infection and bacterial contamination of urine sediment (26). In conclusion, the chemiluminometric assay, which has been tested previously in urogential specimens (6, 7), can be used to test urine specimens, giving results similar to those obtained with other widely used antigen detection tests for the diagnosis of chlamydial genital infection (13-21). The test is easy to perform and rapid, results being available within three hours. A direct comparison of this method with other existing methods in the testing of first-void urine specimens could be made in further studies. Detection of Chlamydia trachomatis in urine sediments using the chemiluminometric assay could be useful in a high-risk population of men when cell culture cannot be performed. However, screening of first-void urine in a lower prevalence population of women does not suffice to establish a diagnosis of genital chlamydial infection.
References
1. BarnesRC-'Laboratorydiagnosisof human chlamydial infections.ClinicalMicrobiologyReviews1989,2:119136. 2. Hipp SS, Han Y, Murphy D: Assessment of enzyme immunoassayand immunofluorescencetests for detection of Chlamydia trachomatis. Journal of Clinical Microbiology 1987, 25: 1938--1943. 3. B~ickman M, Ruden AKM, Ringertz O, Sandstrom EG: Evaluationof a commercialenzymeimmunoassay
versus culture for the detection of Chlamydia trachomat&. European Journal of Clinical Microbiology & Infectious Diseases 1989, 8: 778-782. 4. Coleman P, Varilek V, Mushahwar IK, Marchlewicz B, Safford J, Hansen J, Kurpiewski G, Grief T: TestPack Chlamydia,a new rapid assayfor the direct detec-
tion of Chlamydia trachomatis. Journal of Clinical Microbiology 1989, 27: 2811-2814. 5. Phillips LE, Smith PB, Riddle GD, Faro S, Goodrich HK, Martens MG: Ortho enzymeimmunoassayversus
McCoy cell monolayers stained by iodine or fluorescent antibody for detection of Chlamydia trachomatis. Journal of Clinical Microbiology1990, 28: 1647-1648. 6. Scieux C, Bianchi A, Vassias I, Meouchy R, Felten
A, Morel P, P~rol Y:Evaluationof a new chemiluminometric immunoassay, Magic Lite Chlamydia, for detecting Chlamydia trachomatis antigen from uro* genital specimens.SexuallyTransmittedDiseases 1992, 19: 161-164.
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7. Neman-Simha V, Delmas-Beauvieux MG, Geniaux M, B~b~ar C: Evaluation of a chemiluminometric immunoassay and a direct immunofluorescence test for detecting Chlamydia trachomatis in urogenital specimens. European Journal of Clinical Microbiology & Infectious Diseases 1991, 10: 662--665. 8. Lebar W, Herschman B, Jemai C, Pierzchala J: Comparison of DNA probe, monoclonal antibody enzyme immunoassay and cell culture for the detection of Chlamydia trachornatis. Journal of Clinical Microbiology 1989, 27: 826--828. 9. Woods GL, Young A, Scott JC, Blair TMH, Johnson AM: Evaluation of a nonisotopic probe for detection of Chlamydia trachornatis in endocervical specimens. Journal of Clinical Microbiology 1990, 28: 370-372.
17. Moncada J, Schachter J, Bolan G, Chale I: Detection of Chlamydia trachomatis in urine samples collected from males attending an STD clinic. In: Bowie WR, Caldwell HD, Jones RP, Mardh PA, Ridgway GL, Schachter J, Stamm WE, Ward ME (ed): Chlamydial infections. Cambridge University Press, 1990, p. 475478.
18. Patel R, Kinghorn GR, Kudesia G, VanHegzan R: 19.
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10. Claas HCJ, Wagenvoort JHT, Niesters HGM, Tio TT, van Rijsoort-Vos JH, Quint WGV: Diagnostic value of polymerase chain reaction for Chlamydia detection as determined in a follow-up study. Journal of Clinical Microbiology 1991, 29: 42-45.
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11. Dntilh B, B~b~ar C, Rodriguez P, Vekris A, Bonnet J, Garret M: Specific amplification o[ DNA sequence common to all Chlamydia trachomatis serovars using the polymerase chain reaction. Research in Microbiology 1989. 140: 7-16. 12. Ooslergaard L, Birkelund S, Chrisliansen G: Use of polymerase chain reaction for detection of Chlamydia trachomatis. Journal of Clinical Microbiology 1990, 28: 1254-1260. 13. Chernesky M, Castriciano S, Sellors J, Stewart I, Cunningham I, Landis S, Seidelman W, Grant L, Devlin C, Mahony J: Detection of Chlamydia trachomatis antigen in urine as an alternative to swabs and cultures. Journal of Infectious Diseases 1990, 161: 124-126. 14. Gem; M, Slary A, Bergman S, Mardh PA: Detection of Chlamydia trachomatis in first-void urine co]letted from men and women attending a venereal clinic. Acta Pathologica Microbiologica et Immunologica Scandinavica 1991, 99: 455--459. 15. Jawad A J, Manuel G, Matlhews R, Wise R, Clay JCG: Evaluation of a genus-specific monoclonal antibody in an amplified enzyme-linked immunoassay in the detection of Chlamydia in urine samples from men. Sexually Transmitted Diseases 1990, 17: 87-89. 16. While DJ, Malel RM, BigneU CJ: Non-invasive sampiing method for detecting Chlamydia trachomatis. Lancet 1989, i: 96-97.
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Chlamydia trachomatis detection and non-invasive sampling methods. Lancet 1991, 337: 1169. Paul ID, Caul EO: Evaluation of three Chlamydia trachomatis immunoassays with an unbiased, non invasive clinical sample. Journal of Clinical Microbiology 1990, 28: 220-222. SeUors JW, Mahony JB, Pickard L, Goldsmith CH, Gafni A, Chernesky M: Comparison of cervical, urethral, and urine specimens for the detection of Chlamydia trachomatis in women. Journal of Infectious Diseases 1991, 64: 205-208. SeUors J, Mahony J, Janf D, Pickard L, Castrieiano S, Landis S, Stewart I, Seidelman W, Cunninghanl I, Chernesky M: Rapid, on-site diagnosis of chlamydial urethritis in men detection of antigens in urethral swabs and urine. Journal of Clinical Microbiology 1991, 29: 407-409. Schwebke JR, Clark AM, Pellinger MB, Nsubga P, Stature WE: Use of a urine enzyme immunoassay as a diagnostic tool for Chlamydia trachomatis urethritis in men. Journal of Clinical Microbiology 1991, 29: 2446-2449. Schachler J: Immunodiagnosis of sexually transmitted disease. Yale Journal of Biology and Medicine 1985, 58: 443-452.
24. Saiki RK, Scharf S, Falcoona F, Mullis KB, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of B-globin genomic sequences restriction site analysis for diagnosis of sickle cell anemia. Science 1985, 230: 1350-1354. 25. Thomas BJ, Giichrisl C, Hay PE, Taylor-Robinson D: Simplification of procedures used to test urine samples for Chlamydia trachomatis. Journal of Clinical Pathology 1991, 44: 374-375. 26. Demaio J, Boyd RS, Rensi R, Clark A: False-positive Chlamydiazyme results during urine sediment analysis due to bacterial urinary tract infections. Journal of Clinical Microbiology 1991, 29: 1436-1438.
Article
Vol. 11. No. 8
Eur. J. Clin. Microbiol. Infect. Dis., August 1992, p. 704-708 0934-9723/92/08 0704-05 $ 3.00/0
Evaluation of a Chemiluminometric Immunoassay for Detection of Chlamydia trachomatis in the Urine of Male and Female Patients
C. S c i e u x 1., A . B i a n c h i 1, S. H e n r y I , N. B r u n a t 1, S. A b d e n n a d e r 2, D . V e x i a u 2, M. J a n i e r 2, E M o r e l 2, E H . L a g r a n g e 1
A chemiluminometric immunoassay (Magic Lite Chlamydia) for detection of Chlamydia trachomatis antigens in first-void urine samples was compared with cell culture using urogenital swabs from 221 men and 242 women. The rate of isolation of Chlamydia trachomatis was 23.5 % in men, nearly 80 % of whom had symptoms of urethritis, and 8.3 % in women, in whom both cervix and urethra samples were tested. In urine sediments from men and women respectively the chemiluminometric assay showed a sensitivity of 80.8 % and 70 %, a specificity of 97 % and 95 %, a positive predictive value of 89.4 % and 58.3 %, and a negative predictive value of 94.3 % and 97.2 %. Discrepanciesbetween results obtained with the chemiluminometric assay and cell culture were resolved using two polymerase chain reaction techniques to test urogenital samples. The detection of Chlamydia trachomatis in urine samples with the chemiluminometric assay was confirmed to be superior for screening symptomatic men with urogenital infections than women as a lower prevalence population.
Chlamydia trachomatis is now recognized as a m a j o r cause of sexually transmitted disease t h r o u g h o u t the world. T h e detection of chlamydial genital infections still poses a challenge for b o t h clinicians and laboratory workers (1). R e c o v e r y of Chlamydia trachomatis in cell cultures of urogenital specimens is considered the most sensitive and specific m e t h o d but has known drawbacks and is unavailable in m a n y clinical settings. Alternative m e t h o d s have been developed for detecting chlamydial antigen and chlamydial D N A (1-12). I m m u n o a s s a y s for testing first-void urine sediment h a v e b e e n evaluated in the diagnosis of chlamydial urethritis in m e n and to a lesser extent in w o m e n (13-22). We evaluated the p e r f o r m a n c e of a new chemiluminometric sandwich i m m u n o a s s a y for detection of Chlamydia trachomatis in urine samples f r o m m e n and w o m e n c o m p a r e d with cell culture of urethral and cervical swabs. Discrepancies b e t w e e n the results of culture and the i m m u n o a s s a y were resolved by using two p o l y m e r a s e chain reaction techniques to test urogenital samples. 1Laboratoire de Bact6riologie-Virologie and 2Centre Clinique et Biologique des Maladies Sexuellement Transmissibles, H6pital Saint-Louis, Universit6 Paris VII, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
Materials and Methods
Patients. From January to July 1991, 463 sexually active patients (242 women and 221 men) attending the Sexual Transmitted Diseases (STD) Clinic at St-Louis Hospital, Paris, were entered in the study after giving verbal consent. Patients were excluded from the study if they had received antibiotics within the preceding two weeks. Specimens. Specimens for culture consisted of a total of 705 urogenital swabs collected from the endocervix (n = 242), female urethra (n = 242) and male urethra (n = 221) using plastic swabs (Bactopick, Labomoderne, France). Swabs were placed in buffered sucrose phosphate (2-SP) transport medium kept at 4 *C and always inoculated onto culture cells within 6h. First-void urine specimens (approximately 15 to 50 ml) for the immunoassay were collected in large sterile vials and centrifuged at 3,500 xg for 20 rain at 4 *C. The pellets were suspended in 0.2 ml of specimen dilution buffer and 0.75 ml of reagent (0.34 N sodium hydroxide, chenodeoxycholate salt and EDTA) according to the instructions of the manufacturer. After vortexing twice for 15 see urine specimens were stored at 4 °C for less than a week or at -70"C until tested. The order of collection was urine specimens followed by swabs in 103 men and 116 women, and swabs followed by urine specimens in 118 men and 126 women. The time of last voiding ranged from 1 h 15 min to 15 h, and exceeded 3 h in 48.4 % of the men and 41.3 % of the women.
Chemiluminometric bnmunoassay. The chemiluminometric sandwich immunoassay (Magic Lite Chlamydia,
Vol. 11, 1992
Ciba Corning, USA) was performed according to the manufacturer's instructions. A volume of 0.2 ml of treated specimen was transferred to assay vials to which reagents (0.11 M citric acid, 0.18 M hydrogen peroxide and 0.5 M TAPS buffer), MOMP-speeiflc anti-Chlamydia trachomatis monoclonal antibody conjugated with aeridinium ester and antichlamydial polyclonal antibody immobilized on magnetic particles as solid phase were subsequently added. After incubation for 90 rain the solid phase particles were separated magnetically, washed and resuspended for counting. After addition of chemiluminescence-stimulating reagents, the emission of light was recorded as relative light units (RLU) in the Magic Lite Analyser. A specimen was classified as positive when the ratio of the specimen R L U to the negative control RLU was ~ 2 in duplicate tests.
Cell Culture. After vortex mixing, 0.2 mI of the transport medium containing the swab was inoculated onto freshly trypsinized Hela 229 cells in suspension in two to four wells of a mieroeulture plate, centrifuged (3,500 x g) at 36 °C for 40 rain, and incubated at 37 °C in 5 % CO2 for 48-72 h in maintenance medium containing cycloheximide (1 rag/l). Following fixation with cold absolute methanol for 10 rain, plates were stained using a genusspecific antichlamydial monoclonal antibody with a peroxidase-antiperoxidase detection system (Orthodiagnostics, USA). Chlamydial inclusion bodies were detected by bright field microscopy under magnification (200 times) with an inverted microscope. Each assay was scored independently and the result recorded without knowledge of the result of the other test. Sensitivity, specificity and predictive values were calculated by the usual methods (23). The p values were based on the Pearson chi-square test and are stated if the value was < 0.1. Specimens with discordant results were submitted to serial passage in cell culture after storage at -70 °C. Briefly, the samples were inoculated onto Hela 229 cells, and after 48 h of incubation the cell monolayer of one well was diluted in 0.3 ml of transport medium, disrupted by ultrasonic vibrator (Bioblock Scientific, France) and inoculated onto three wells of fresh Hela 229 cells. Two serial passages were performed.
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(10 mM "iris, 50 mM KCI, 1.5 mM MgCI2, 0.1 g/l gelatin, 200 ~M of each nucleotide, 2.5 units T a q - D N A potymerase [Cetus Corporation, USA], 0.5 /aM of each primer). For the chromosome PCR, the samples were submitted to 30 cycles of denaturation for 30 sec at 92 °C, annealing for 30 sec at 54 °C and extension for 1 rain at 72 °C in a D N A thermocycler (11). For the plasmid PCR, the samples were submitted to 40 cycles of denaturation for 2 rain at 94 °C, annealing for 2 rain at 57 °C and extension for 4 rain at 70 °C in a D N A thermocycler (12). Amplified D N A products were fractioned by 3 % or 1.2 % agarose gel eleetrophoresis. The specific fragment of 129bp (located in a conserved region of the major outer membrane protein gene) detected in the chromosome PCR, and the specific fragment of 473bp detected in the plasmid PCR, were stained with ethydium bromide visualized by UV fluorescence (11, 12). After Southern blotting onto Hybond N membranes (Amersham, UK), hybridization was performed using the 32p_ labelled specific internal probe (11, 12). The B-globin gene segment PCR was used to eliminate the possibility of false negative results due to the presence of PCR inhibitors. All specimens negative in the PCR were accordingly tested using the primer set PCO3 and PCO4 yielding amplified D N A of the l l 0 b p fragment (24).
Results
PCR Techniques. In 47 clinical specimens (16 endocervix,
T h e a v e r a g e a g e o f t h e 463 p a t i e n t s t e s t e d w a s 30 y e a r s f o r w o m e n ( r a n g e 15 t o 68 y e a r s ) a n d 33 y e a r s for m e n ( r a n g e 17 to 74 y e a r s ) . L e u c o r r h e a was t h e p r i m a r y r e a s o n for a t t e n d i n g t h e clinic in 83.3 % o f t h e w o m e n . O f t h e m e n , 3 7 . 1 % att e n d e d d u e to clinical e v i d e n c e o f u r e t h r a l disc h a r g e a n d 40.3 % d u e to u r e t h r a l d i s c o m f o r t o r dysuria. The reasons of the remaining patients for a t t e n d i n g t h e S T D clinic i n c l u d e d a s y m p t o m a t i c check-up, symptomatic partner, previous genital i n f e c t i o n a n d infertility.
16 female urethra, 15 male urethra specimens) from 31 patients in which results were discordant, specimens in 2-SP transport medium were tested using two PCR techniques to detect chromosomal and plasmid DNA respectively. Negative clinical specimens (n = 25) and positive clinical specimens (n = 20) were included as controls. An amount of 0.5 ml of vortexed transport medium was collected using a positive-deptacement pipette (Microman R, Gilson, USA) and centrifuged at 11,000 x g for 20 rain at 4 °C. The pellet was digested in 100 pl of lysis buffer containing 20 pg of proteinase K in 10 mM Tris 1 mM EDTA for 30 min at 37 °C and boiled for 10 min at 100 °C. The primer sets CT1, CT2 and the internal probe CT3 were used for the chromosome PCR as described by Duthil et al. (11). The primer set IIa, IIB and internal probe PIA were used for the plasmid PCR as described by Oostergaard et al. (12). All the oligonucleotides were synthesized by the phosphoramidite method (Applied Biosystems, USA). Each PCR was performed with 30 13I of treated specimen in 100 ~1 in a final reaction mixture
O f t h e 242 w o m e n t e s t e d , 20 (8.3 % ) w e r e p o s i t i v e f o r Chlamydia trachomatis o n c u l t u r e o f u r o g e n i tal swabs. T h e results o f t h e c h e m i l u m i n o m e t r i c assay a n d c u l t u r e a c c o r d i n g to s a m p l e o r i g i n a r e s h o w n in T a b l e 1. T h e p e r f o r m a n c e o f t h e c h e m i l u m i n o m e t r i c a s s a y in u r i n e s e d i m e n t s f r o m w o m e n is s h o w n in T a b l e 2. A l l w o m e n with a p o s i t i v e c u l t u r e in b o t h c e r v i x a n d u r e t h r a l s p e c i m e n s h a d d e t e c t a b l e c h l a m y d i a l a n t i g e n in urine sediment. The sensitivity of the chemil u m i n o m e t r i c a s s a y r a n g e d f r o m 73.3 % t o 83.3 % a c c o r d i n g to t h e o r i g i n o f a c u l t u r e p o s i t i v e specimen (cervix or urethral). Higher sensitivity o f t h e a s s a y w a s f o u n d in s p e c i m e n s y i e l d i n g m o r e t h a n 10 i n c l u s i o n s p e r w e l l on c u l t u r e ( a d j u s t e d :(2 = 4.63, p < 0.05 in c e r v i x s p e c i m e n s ; a d j u s t e d :(2 = 3.01, p < 0.1 in u r e t h r a s p e c i m e n s ) .
706
Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: Results in the detection of Chlamydia trachomatisusing the chemiluminometric assay in firstvoid urine specimens compared with culture of urogenital specimens. Culture
Chemiluminometrie assay Positive
Negative
Cervix and urethra positive (n = 7) Cervix positive and urethra negative (n = 8) Cervix negative and urethra positive (n = 5) Cervix and urethra negative (n = 222)
7 4 3 10
0 4 2 212
Total (n = 242)
24
218
Urethra positive (n = 52) Urethra negative (n = 169)
42 5
10 164
Total (n = 221)
47
174
Women
Men
Table 2: Performance in the detection of Chlaraydia trachomatis of the chemituminometric assay in first-void urine specimens compared with cell culture and/or PCR in urogenital specimens. Chemiluminometric assay Sensitivity (%)
Specificity (%)
Culture Women (n = 242) Men (n = 221)
70 80.8
95.5 97
58.3 89.4
97.2 94.3
Culture and/or PCR Women (n = 242) Men (n = 221)
73.9 81.8
96.8 98.8
70.8 95.7
97.2 94.3
O f the 221 m e n tested, 52 (23.5 % ) were positive for Chlamydia trachomatis on culture of urogential swabs. T h e rate of a positive culture ranged f r o m 16.8 % to 3 4 . 1 % in m e n presenting with dysuria or urethral discharge. In 47 patients antigen was detected in urine sediment with the c h e m i l u m i n o m e t r i c assay (Table 1). T h e perform a n c e of the c h e m i l u m i n o m e t r i c assay is shown in Table 2. T h e sensitivity of the chemiluminometric assay was higher in patients yielding m o r e than ten inclusion bodies per well on culture of urethral specimens (adjusted %2 = 4.26, p < 0.05), but was not significantly different according to w h e t h e r the patient had urethral discharge or dysuria. In male and f e m a l e patients, no significant difference in sensitivity of the chemiluminometric assay in the testing of urine samples was noted ac-
Positive predictive Negative predictive value (%) value (%)
cording to the time of the last voiding, or the order of collection of samples. On serial passage of specimens with discordant results, no further culture-positive specimens were found in men or w o m e n . In the P C R the s a m e results w e r e o b t a i n e d using the plasmid P C R and the c h r o m o s o m e P C R . All 16 patients with positive culture and negative assay results were confirmed as positive in the PCR. O f the w o m e n in w h o m only the cervix or urethra specimen was positive on culture, P C R confirmed the result in all cases and the specimen f r o m the second site was also positive in three cases. O f the four w o m e n in w h o m only the cervix specimen was positive on culture and the assay result positive, the urethra specimen was positive on P C R in two cases. Of the 15 patients with negative culture and positive assay results, six were positive and
Vo1.11,1992
nine negative in the PCR so that the assay results in urine samples could be considered false positive. The presence of inhibitors of PCR was not demonstrated in any PCR negative specimen using the B-globin gene segment PCR. The performance of the chemiluminometric assay compared with culture and/or PCR is reported in Table 2.
Discussion
The findings of the present study in patients attending a STD clinic suggest that detection of Chlamydia trachomatis antigen in first-void urine sediments with the chemiluminometric assay can yield an accurate diagnosis of urethritis in men. However, in the screening of first-void urine in women the performance of the assay was inferior and did not parallel results achieved by culture of cervical swabs possibly because not all women have an infected urethra (13, 14, 18, 20). In addition, the disparity could be due to lower isolation rates in women compared to men. Previous evaluations of screening for Chlamydia trachomatis in first-void urine samples revealed a sensitivity of 72.6 % to 87 % in men and 54 % to 76.9 % in women using an enzyme immunoassay or fluorescence assay compared with culture of cervical and urethral specimens (13, 15, 17, 21). We obtained similar results which are superior to the low sensitivity of about 40 % reported in the testing of first-void urine sediment in men half of whom had no symptoms of urethritis or in women attending a family planning clinic (20, 22). Non-invasive collection of first-void urine samples during a patient's visit to the STD clinic was practical, and analysis of data according to time of the last voiding and order of collection of urine and urogenital specimens showed no impact on positivity rates in the chemiluminometric assay, as previously described (13, 16, 21, 22, 25). In both male and female patients, the sensitivity of the chemiluminometric assay strongly correlated with the number of chlamydial inclusion bodies in cell culture, as previously reported for antigen detection tests in urogenital and urine specimens (1-3, 6, 22). As recent studies have shown an excellent performance of PCR compared with cell culture, PCR could be considered the reference method to resolve discrepancies between results of cell culture and antigen detection methods (10-12). True false-positive results in the chemiluminometric
707
assay were uncommon in both men and women (9 of 463 patients). The chemiluminometric assay which uses species-specific monoclonal antibody and immobilized antichlamydial polyclonal antibody, has high specificity, limiting problems associated with urinary tract infection and bacterial contamination of urine sediment (26). In conclusion, the chemiluminometric assay, which has been tested previously in urogential specimens (6, 7), can be used to test urine specimens, giving results similar to those obtained with other widely used antigen detection tests for the diagnosis of chlamydial genital infection (13-21). The test is easy to perform and rapid, results being available within three hours. A direct comparison of this method with other existing methods in the testing of first-void urine specimens could be made in further studies. Detection of Chlamydia trachomatis in urine sediments using the chemiluminometric assay could be useful in a high-risk population of men when cell culture cannot be performed. However, screening of first-void urine in a lower prevalence population of women does not suffice to establish a diagnosis of genital chlamydial infection.
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