Abstract
The serovars of Chlamydia trachomatis-positive urine specimens (n = 81; as detected by PCR and ligase chain reaction) were successfully analyzed in 94% of cases by omp1 PCR-based RFLP analysis. The use of urine specimens and this simple and sensitive typing method will greatly facilitate epidemiological studies of C. trachomatis serovar distribution in asymptomatic C. trachomatis infections in both females and males.
The species Chlamydia trachomatis comprises 15 serovars, serovars A to L and additional variants. Serotyping of these C. trachomatis isolates is performed on cell cultures of cervical and urethral specimens by using polyclonal and monoclonal antibodies (13). Recent studies showed the feasibility of typing clinical isolates (5, 14) and direct typing of cervical specimens (8, 9) by a nested omp1 PCR-based restriction fragment length polymorphism (RFLP) assay for the differentiation of isolates into serovars and genovariants (12). By a nested PCR assay, 0.01 inclusion-forming unit (IFU) of serovar L2 DNA could be typed in reconstruction experiments (8). Nowadays, urine specimens are more frequently used for the detection of C. trachomatis in both males and females by nucleic acid amplification assays (LCx, COBAS, NASBA, and AMP-CT [2, 6, 7, 10, 11]). This noninvasive technique has a sensitivity higher than that of cell culture of tissue from the urethra and the cervix (6) and a sensitivity comparable to that of C. trachomatis detection in cervical scrape specimens by amplification assays. The use of urine specimens facilitates the screening of asymptomatically infected males and females. Typing asymptomatic C. trachomatis infections in urine specimens would enable the performance of large epidemiological typing studies. However, urine specimens cannot be used for serotyping purposes since cell culture using urine specimens is impossible. Also PCR-based genotyping directly on urine (e.g., 10 or 20 μl) will result in the introduction of urinary inhibitors which negatively influence C. trachomatis amplification. Furthermore, commercial assays (LCx [Abbott Laboratories, Chicago, Ill.] and COBAS Amplicor [Roche Diagnostic Systems, Inc., Branchburg, N.J.]) are more frequently used in routine laboratories for detection of C. trachomatis in clinical specimens and generate buffer sample solutions of which the composition, which is not exactly known, inhibits the PCR in the PCR-based RFLP genotyping. Therefore, the aim of this study was to develop a simple and sensitive method for the typing of C. trachomatis serovars in urine specimens. Firstly, the DNA isolation procedure for urine specimens had to be optimized, and secondly, C. trachomatis typing using the purified DNA had to be evaluated.
To optimize the genotyping of C. trachomatis from urine specimens, different DNA isolation methods were compared. For this, a dilution series of serovar L2 (range, 101 to 10−3 IFU) in the background of a C. trachomatis-negative urine specimen (as determined by PCR and ligase chain reaction) was used to determine the sensitivities of a silica-based DNA isolation method (1) and a filter tube-based isolation method (High Pure PCR Template Preparation [HPPTP] kit; Boehringer Mannheim B.V., Almere, The Netherlands) by amplifying the plasmid target (11) and the omp1 gene (9, 12). In addition, pellets from 1, 3, and 5 ml of five C. trachomatis-positive urine specimens (as determined by PCR and ligase chain reaction) were analyzed in the two different DNA isolations. Purified DNAs were subjected to a nested omp1 PCR (1.1 kb) as previously described (9), after which the PCR product was used for RFLP analysis (12). In the comparison of the silica-based DNA isolation method with the HPPTP method, the latter not only was 10 times more sensitive (0.1 versus 0.01 IFU) for both the C. trachomatis plasmid target and the omp1 target but also proved to be faster (30 versus 60 min) and easier to perform as well. For the five C. trachomatis-positive urine specimens, an input of 5 ml of urine gave the strongest amplification signals.
Subsequently, C. trachomatis-positive urine specimens (n = 81), obtained from asymptomatically infected men and women during a screening program in Amsterdam, The Netherlands, were used for genotyping. Urine pellets were used to isolate DNA by the HPPTP method. Serovars could be determined for 76 samples by a PCR-based RFLP method. The serovar distribution is shown in Table 1. Serovars F, E, and D (D and D−) were the most prevalent C. trachomatis serovars in asymptomatic infected females and males. Furthermore, two C. trachomatis variants were found to have unknown RFLP patterns which were not due to double infections. Variants have been found in other studies, but in those studies cervical specimens were used (12). The obtained typing data are in agreement with those of other typing studies using cervical and urethral swabs (15, 16). For the urine specimens in which omp1 amplification and typing were not possible (n = 5), inhibition in three eluates (urine specimens from two females and one male) was shown by means of spiking of serovar L2 DNA in the HPPTP eluates and subsequent plasmid PCR. In the two remaining urine specimens, partial inhibition could be the reason for the inability to amplify the longer omp1 fragment. This is still a subject of further investigation.
TABLE 1.
Serovar distribution as found for C. trachomatis-positive urine specimens from asymptomatic infected patients
| Serovar | No. of patients
|
Total no. (%) | |
|---|---|---|---|
| Female | Male | ||
| D | 2 | 3 | 5 (7) |
| D− | 2 | 3 | 5 (7) |
| E | 20 | 13 | 33 (43) |
| F | 6 | 6 | 12 (16) |
| G | 2 | 5 | 7 (10) |
| H | 1 | 1 | 2 (3) |
| I | 1 | 2 | 3 (4) |
| J | 3 | 1 | 4 (5) |
| K | 1 | 2 | 3 (4) |
| Var.a | 2 | 0 | 2 (3) |
Var., variant with aberrant PCR-based RFLP pattern.
Since nowadays commercial assays (LCx and COBAS Amplicor) are increasingly used in routine laboratories for C. trachomatis detection in cervical or urethral and urine specimens, the HPPTP kit was also optimized for DNA isolation from the buffer sample solutions since PCR could not directly be applied to buffer sample solutions. From the LCx and COBAS Amplicor sample buffer solutions, 300 and 500 μl, respectively, were mixed with 1 volume of isopropanol and centrifuged for 10 min at 14,000 rpm. The supernatant was removed, and the obtained pellet was resuspended in 200 μl of phosphate-buffered saline. Subsequently, the HPPTP protocol was followed for DNA isolation and C. trachomatis serovars were determined by the PCR-based RFLP method. From the C. trachomatis-positive urine specimens, 80 corresponding sample buffer solutions (40 each from the LCx and Amplicor assays) were used for typing. Serovars identical to those found by DNA isolation directly from the urine specimens were found. For one LCx mixture and three Amplicor mixtures obtained from different urine specimens the subsequent omp1 amplification was not successful and the mixtures could therefore not be typed. All samples (urines and sample buffer solutions) in which omp1 amplification and subsequent RFLP typing were not successful were noncorresponding samples.
In this study it was shown that C. trachomatis typing, despite inhibition in a small number of the samples, could be successfully performed for 94% of the urines. C. trachomatis typing of 95% (4 of 80) of the buffer-urine solutions from the commercial assays was also possible. Preliminary results showed that identical serovars were found in urines and the corresponding cervical scrapes (n = 24), supporting the feasibility of urine for C. trachomatis typing. The typing of C. trachomatis serovars in routine collected material (urine specimens and buffer-resuspended clinical material) provides the opportunity to study the epidemiology of C. trachomatis serovars and genovariants in an asymptomatically infected population (3, 4, 15).
In conclusion, the use of urine specimens in combination with this simple and sensitive C. trachomatis typing method will greatly facilitate epidemiological studies of C. trachomatis serovars in both asymptomatically and symptomatically infected females and males.
REFERENCES
- 1.Boom R, Sol C L M, Salimans M M M, Jansen C L, Wertheim-van Dillen P M E, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Crotchfelt K A, Pare B, Gaydos C, Quinn T C. Detection of Chlamydia trachomatis by the Gen-Probe AMPLIFIED Chlamydia Trachomatis Assay (AMP CT) in urine specimens from men and women and endocervical specimens from women. J Clin Microbiol. 1998;36:391–394. doi: 10.1128/jcm.36.2.391-394.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Dean D, Millman K. Molecular and mutation trends analyses of omp1 alleles for serovar E of Chlamydia trachomatis. J Clin Investig. 1997;99:475–483. doi: 10.1172/JCI119182. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dean D, Oudens E, Bolan G, Padian N, Schachter J. Major membrane variants of Chlamydia trachomatis are associated with severe upper genital tract infections and histopathology in San Francisco. J Infect Dis. 1995;172:1013–1022. doi: 10.1093/infdis/172.4.1013. [DOI] [PubMed] [Google Scholar]
- 5.Frost E H, Deslandes S, Veilleux S, Bourgaux-Ramoisy D. Typing Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein. J Infect Dis. 1991;163:1103–1107. doi: 10.1093/infdis/163.5.1103. [DOI] [PubMed] [Google Scholar]
- 6.Goessens W H F, Mouton J W, van der Meijden W J, Deelen S, van Rijsoort-Vos T H, Lemmens-den Toom N, Verbrugh H A, Verkooyen R P. Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for the detection of Chlamydia trachomatis infections in first-void urine. J Clin Microbiol. 1997;35:2628–2633. doi: 10.1128/jcm.35.10.2628-2633.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Jaschek G, Gaydos C A, Welsh L E, Quinn T. Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. J Clin Microbiol. 1993;31:1209–1212. doi: 10.1128/jcm.31.5.1209-1212.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Lan J, Walboomers J M M, Roosendaal R, van Doornum G J J, MacLaren D M, Meijer C J L M, van den Brule A J C. Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis. J Clin Microbiol. 1993;31:1060–1065. doi: 10.1128/jcm.31.5.1060-1065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lan J, Ossewaarde J M, Walboomers J M M, Meijer C J L M, van den Brule A J C. Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR. J Clin Microbiol. 1994;32:528–530. doi: 10.1128/jcm.32.2.528-530.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Lee H H, Chernesky M A, Schachter J, Burczak J D, Andrews W W, Muldoon S, Leckie G, Stamm W E. Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet. 1995;345:213–216. doi: 10.1016/s0140-6736(95)90221-x. [DOI] [PubMed] [Google Scholar]
- 11.Morré S A, Sillekens P, Jacobs M V, van Aarle P, de Blok S, van Gemen B, Walboomers J M M, Meijer C J L M, van den Brule A J C. RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples. J Clin Microbiol. 1996;34:3108–3114. doi: 10.1128/jcm.34.12.3108-3114.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Morré S A, Ossewaarde J M, Lan J, van Doornum G J J, Walboomers J M M, MacLaren D M, Meijer C J L M, van den Brule A J C. Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as confirmed by omp1 nucleotide sequence analysis. J Clin Microbiol. 1998;36:345–351. doi: 10.1128/jcm.36.2.345-351.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Ossewaarde J M, Rieffe M, de Vries A, Derksen-Nawrocki R P, Hooft H J, van Doornum G J J, van Loon A M. Comparison of two panels of monoclonal antibodies for serovar determination of Chlamydia trachomatis. J Clin Microbiol. 1994;32:2968–2974. doi: 10.1128/jcm.32.12.2968-2974.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Sayada C, Danamur E, Orfila J, Catalan F, Elion J. Rapid genotyping of Chlamydia trachomatis major outer membrane protein by the polymerase chain reaction. FEMS Microbiol Lett. 1991;83:73–78. doi: 10.1016/0378-1097(91)90447-i. [DOI] [PubMed] [Google Scholar]
- 15.Van de Laar M J W, Lan J, van Duynhoven Y T H H, Fennema J S A, Ossewaarde J M, van den Brule A J C, van Doornum G J J, Coutinho R A, van den Hoek J A R. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med. 1996;72:261–265. [PMC free article] [PubMed] [Google Scholar]
- 16.Workowski K A, Suchland R J, Pettinger M B, Stamm W E. Association of genital infections with specific chlamydia serovars and race. J Infect Dis. 1992;166:1445–1449. doi: 10.1093/infdis/166.6.1445. [DOI] [PubMed] [Google Scholar]