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Molecular diagnostics: Non-invasive sampling

The sensitivity and specificity of modern molecular diagnostics for chlamydial infection plus the fact that it is no longer necessary to maintain chlamydial viability has opened up the use of alternative specimen sources. Traditionally, the main specimens for the laboratory diagnosis of chlamydial genital tract infection have been the urethra in men and women and the endocervix in women [see: specimen collection]. All of these sites involve  genital tract examination with attendant discomfort and embarrassment for patients. Moreover a significant proportion of endocervical or urethral samples submitted for laboratory diagnosis by PCR were found to contain insufficient cellular material to be a valid specimen [Coutlee et al., 2000], a problem long recognized with specimens submitted for direct diagnostic immunofluorescence. Clearly there is a need to explore alternative approaches.

Urine testing

The key first step was made (in 1988) by Owen Caul and colleagues. Using relatively insensitive, commercial chlamydial antigen immunoassays in comparison with 'gold standard' direct detection by fluorescent monoclonal antibody, these workers demonstrated that good results could be obtained for the detection of chlamydial antigen in genital tract samples or in urine [Caul et al., 1988; Paul & Caul, 1990]. Subsequently (and at the time surprisingly) it was found that female urine was almost as good as an endocervical swab for the detection of female genital tract infection [Lee et al., 1995]. 

Urine tests for Chlamydia trachomatis using molecular diagnostic methods permit the expansion of screening beyond traditional clinic environments [Carder et al., 1999; Marrazo et al., 1997]. Urine specimens in both men and women are now used to assess the prevalence of chlamydial infection in large numbers of individuals in low risk populations as a preliminary to improved  control strategies [for example: Fenton et al., 2001]. The disadvantage of urine testing, particularly female urine, is that inhibitors in the urine such as nitrites, crystals or hormones may inhibit amplification reactions [Chenersky et al., 1998; Mahony et al., 1998]. This problem is reduced by storage of specimen overnight or longer in the refrigerator or freezer [Chenersky et al., 1998], and by the careful and complete removal of urine off centrifuged deposit. Any residual inhibition can be monitored using  the inhibition controls that are available for some but not all of the nucleic acid amplification-based tests. Where residual inhibition is experienced, dilution of the sample 1:10 often helps, though at the potential cost of sensitivity. With care, non invasive screening of female as well as male urine has adequate sensitivity and specificity for most purposes to obviate the need for cervical or endourethral swabs. It is particularly useful for screening for asymptomatic infection where there is usually no easy justification for genital examination and sampling. Sugunendran et al., 2001 found that, in males, LCR testing of male urine gave better results than the traditional second endourethral swab [where the first swab is used for gonococcal diagnosis].

Performance of different tests on urine samples.

Verkooyen et al., 2003 reported the results of a major European Quality Control Concerted Action to assess the ability of laboratories to detect C. trachomatis in a panel of urine samples by various nucleic acid amplification tests. The test panel consisted of three negative, two strongly positive, and five weakly positive lyophilised urine samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples. A further 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 43% of data sets, the Abbott LCx assay [now withdrawn] in 30%, the Roche Amplicor manual assay in 9%, an in-house PCR was performed in 9%, the Becton Dickinson ProbeTec ET assay was performed in 4.9% and the GenProbe TMA assay [superseded by the improved Aptima test] in  3.9%. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively, though the number of data sets for these assays was small. Overall the results, particularly for specificity, were better than reported for most quality control studies. However sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the performance of these assays [Verkooyen et al., 2003].

Self-collected 'at home' samples 

Vulval swabs are also  a convenient and useful alternative to urine or endocervical swabs for the detection of C. trachomatis genital tract infection in women by ligase chain reaction [Stary et al., 1997] or transcription mediated amplification [Stary et al., 1998]. Vaginal tampons are also a convenient alternative [Alary et al., 2001; Tabrizi et al., 2000] . Vaginal flush samples are reported to be superior to urine for detection of chlamydial infections in women because  they are less affected by inhibitors [Moller et al., 1999]. One of the advantages of urine, vulval, tampon or penile glans samples is that they may be self-collected at home by the subject, then mailed to the testing laboratory. In a pilot study on 104 women, using the Abbott LCx^® LCR as the diagnostic nucleic acid amplification based test, the sensitivity of the cervical sample was 96.3%; the vulval-introital sample in LCR buffer 92.6%; the vulval-introital swab collected dry 88.9%; first catch urine stored at +2-8 degrees C 81.5%; first catch urine stored at room temperature 77.8% and first catch urine stored with 2% w/v boric acid at room temperature 87.0%. This indicated that self-collected vulval samples or first catch urine stored in boric acid as preservative gave good results for 'at home' sampling [Carder et al., 1999]. 

There have been few studies of the effect of the post on samples, although one group reported that samples for the GenProbe Pace 2 hybridization test were little affected by posting through the US mail [Parker et al., 2000]. Swabs may also be mailed dry. Thus,  Gaydos et al., 2002 found that the sensitivity of dry swabs versus true positives was 91.3% among female military recruits and the specificity was 99.3%. This is attractive in non clinic settings, particularly where there are obstacles to accessing medical care [Gaydos et al., 2002 ]. [Comment: It is also valuable where the diagnostic laboratory, either for work scheduling purposes or reagent economy, wishes to retain flexibility as to which test will be used on receipt of the specimen].

Home screening

The previous section indicates the feasibility of patient collected specimens being used to shift the emphasis of control strategies to the collection of specimens at home. Studies which have utilised this approach report that home screening forms a potentially useful and acceptable component of a chlamydial screening programme [Carder et al., 1999; Stephenson et al., 2000]. Ostergaard et al., 2000 compared the efficacy of a home screening program for urogenital C. trachomatis infection with conventional swab sampling performed at a physician's office. Female students at 17 high schools in the county of Aarhus, Denmark, were divided into a study group (tested by home sampling) and a control group (tested in a physician's office). The number of new infections and the number of subjects who reported being treated for pelvic inflammatory disease were assessed after one year.  51.1% of 867 women in the intervention group and 58.5% of 833 women in the control group were available for follow-up. Thirteen (2.9%) and 32 (6.6%) new infections were identified in the intervention and control groups, respectively.  Nine women in the intervention group (2.1%) and 20 (4.2%) in the control group reported being treated for pelvic inflammatory disease (P=.045), indicating that a screening strategy involving home sampling was associated with a lower prevalence of C. trachomatis infection and a lower proportion of reported cases of pelvic inflammatory disease [Ostergaard et al., 2000]. All persons aged 21 to 23 years in Aarhus county (30,439 people) were offered a mail home sampling test as part of a structured 14 week public information campaign on chlamydia. Materials for home sampling could be requested via the internet or on an answering machine. However, during the campaign, only 0.8% of the potential females and 0.4% of the potential males took up the offer. It was concluded there was a need to research more effective outreach programs for home based population screening [Andersen et al., 2001].

Undaunted, a different strategy was then applied. All persons aged 21 to 23 years in Aarhus county (30,439 people) were divided into three groups. Group 1 (4,500) had a home sampling kit directly mailed to their centrally registered home address with which a home sampling kit could be ordered; group 2 (4,500) had a reply card mailed to their home address enabling them to order a kit; group 3 (21,439) received usual care. For women in groups 1 and 2 the relative risks of being tested were 4.1 and 3.5. For men they were an astonishing 19.1 and 11.8. Thus both screening strategies were highly effective, particularly for men [Andersen et al., 2002]. This is a very important finding which points the way, in conjunction with sensitive modern tests, for new screening strategies to control chlamydial infection. The cost effectiveness of this approach will no doubt be evaluated by the same enterprising and innovative group. A study based on Maryland, USA on women attending family planning clinics indicated that the use of 'expensive' nucleic acid amplification based diagnostic tests rather than 'cheaper' antigen detection tests was cost effective [Howell et al., 1998; see: cost effectiveness].  

Home collected samples also have considerable potential for epidemiological research. In Aarhus, Kjaer et al., 2000 conducted an interesting survey over 24 weeks on patient-collected and mailed urine or vaginal specimens of recurring infection in women who had been treated for chlamydial infection. Two weeks after treatment 89% of patients tested negative, but there was a cumulative incidence of recurrent infection of 29% over the 24 weeks of follow up [Kjaer et al., 2000].

Bloomfield et al., 2003 assessed the feasibility of repeat chlamydia screening using self collected, mailed specimens, in the San Francisco area. A letter offering home re-screening was mailed to 399 adults who had previously tested positive for genital chlamydial infection. Kits were mailed to anyone who did not actively decline. The kits contained instructions on how to collect a urine specimen and return it  by mail. A short survey asked individuals their level of concern about confidentiality, safety, and privacy of mail screening. The specimens were tested with strand displacement amplification. 22.4% of kit recipients responded, with response rates highest among homosexual and bisexual men (38.6%), people 35 years or older (34.3%), and white people (34.6%). The overall positivity rate was 3.2% (2/63). In women 18-25 years old, the positivity was much higher at 13.3% (2/15). It was concluded that home testing with mailed urine collection kits is a feasible and acceptable method to screen for recurrent chlamydial infection, with young women being most likely to benefit.

Primary care setting

In the UK and other countries there are moves to make diagnostic tests for chlamydial infection available in primary care. Kufeji et al., 2003 investigated current patterns of testing for chlamydial genital tract infection in primary care in the Nottingham area of the UK. They found that most tests (63.1%) were being performed on women over the age of 25, even though the prevalence of infection is greatest in sexually active women under the age of 25. In this study the proportion of positive tests was highest in 15-19 year olds (13.3%) followed by 20-24 year olds (8.2%). A higher ratio of GP principals to female practice population was associated with higher testing rates and more chlamydia identified, but the ratio of female GP principals to patients was associated with higher testing rates only in 20 - 24 year olds. Diagnosed incidence was greatest in more socioeconomically deprived practice populations, and this was not explained by higher testing rates. Men were rarely tested. It was concluded that current testing practice in primary care did not reflect the known epidemiology of genital chlamydial infection. Practices with lower doctor-patient ratios do less testing, and measures to enable their active participation in any organised screening programme will be important in order to reach all at-risk groups [Kufeji et al., 2003].

Resource poor areas

[Comment: Since the studies of Caul et al., there has been an enormous interest in the use of noninvasive sampling strategies for the screening and diagnosis of C. trachomatis genital tract infections. These methods generally seem to be patient acceptable and to work well, though there are some pitfalls, such as hormonally influenced inhibitors of nucleic acid amplification reactions in the urine of some women. The apparent efficacy of non invasive samples is a tribute to the excellent sensitivity and specificity of modern, commercial, nucleic acid-based amplification tests for chlamydial infection. A number of studies, most notably the excellent studies from Denmark, indicate the exciting possibilities for controlling genital chlamydial infection of using a home based sampling strategy].

[MEW] updated July 2003

NEXT: Screening for chlamydiae during cervical cytology

References

Alary, M., Poulin, C., Bouchard, C., Fortier, M., Murray, G., Gingras, S., Aube, M. & Morin, C. (2001). Evaluation of a modified sanitary napkin as a sample self-collection device for the detection of genital chlamydial infection in women. Journal of Clinical Microbiology 39, 2508 - 2512.

Andersen, B., Olesen, F., Moller, J. K. & Ostergaard, L. (2002). Population-based strategies for outreach screening of urogenital Chlamydia trachomatis infections: a randomized, controlled trial. Journal of Infectious Diseases 185, 252 - 258.

Andersen, B., Ostergaard, L., Moller, J. K., Olesen, F. (2001). Effectiveness of a mass media campaign to recruit young adults for testing of Chlamydia trachomatis by use of home obtained and mailed samples. Sexually Transmitted Infections 77, 416 - 418.

Bloomfield, P. J., Steiner, K. C., Kent, C. K. & Klausner, J. D. (2003). Repeat chlamydia screening by mail, San Francisco. Sexually Transmitted Infections 79, 28 - 30. Full article

Carder, C., Robinson, A. J., Broughton, C., Stephenson, J. M. & Ridgway, G. L. (1999). Evaluation of self-taken samples for the presence of genital Chlamydia trachomatis infection in women using the ligase chain reaction assay. International Journal of STD and AIDS 10, 776 - 779.

Caul, E. O., Paul, I. D., Milne, J. D., Crowley, T. (1988). Non-invasive sampling method for detecting Chlamydia trachomatis. Lancet 2 (no 8622), 1246 - 1247.

Chernesky M, Chong S, Jang D, Luinstra K, Faught M, Mahony J. (1998). Inhibition of amplification of Chlamydia trachomatis plasmid DNA by the ligase chain reaction associated with female urines. Clinical Microbiology and Infection 4, 397 - 404.

Clark, A. M., Steece, R., Crouse, K., Campbell, J., Zanto, S., Kartchner, D., Mottice, S. & Pettit, D. (2001). Multisite pooling study using ligase chain reaction in screening for genital Chlamydia trachomatis infections. Sexually Transmitted Diseases 28, 565 - 568.

Coutlee, F., de Ladurantaye, M., Tremblay, C., Vincelette, J., Labrecque, L. & Roger, M. (2000). An important proportion of genital samples submitted for Chlamydia trachomatis detection by PCR contain small amounts of cellular DNA as measured by beta-globin gene amplification. Journal of Clinical Microbiology 38, 2512 - 2515. Full article.

Fenton, K. A., Korovessis, C., Johnson, A. M., McCadden, A., McManus, S., Wellings, K., Mercer, C. H., Carder C., Copas, A. J., Nanchahal, K., Macdowall, W., Ridgway, G., Field, J. & Erens, B. (2001). Sexual behaviour in Britain: reported sexually transmitted infections and prevalent genital Chlamydia trachomatis infection. Lancet 358, 1851 - 1854.

Gaydos, C. A, Crotchfelt, K. A., Shah, N., Tennant, M., Quinn, T. C., Gaydos, J. C., McKee, K. T. Jr. & Rompalo, A. M. (2002). Evaluation of dry and wet transported intravaginal swabs in detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections in female soldiers by PCR. Journal of Clinical Microbiology 40, 758 - 761. [Interesting study. Can mail dry as well as wet swabs, with ~10% loss of sensitivity].

Hedin, G., Abrahamsson, G. & Dahlberg, E. (2001). Urethritis associated with Chlamydia trachomatis: comparison of leukocyte esterase dipstick test of first-voided urine and methylene blue-stained urethral smear as predictors of chlamydial infection. APMIS. 109, 595 - 600.

Howell, M. R., Quinn, T. C., Brathwaite, W. & Gaydos, C. A. (1998). Screening women for Chlamydia trachomatis in family planning clinics: the cost-effectiveness of DNA amplification assays. Sexually Transmitted Diseases 25, 108 - 117.

Kjaer, H. O., Dimcevski, G., Hoff, G., Olesen, F. & Ostergaard, L. (2000). Recurrence of urogenital Chlamydia trachomatis infection evaluated by mailed samples obtained at home: 24 weeks' prospective follow up study. Sexually Transmitted Infections 76, 169 - 172. [Good study from a group that has done much to evaluate alternative approaches to screening and control]

Kufeji, O., Slack, R., Cassell, J. A., Pugh, S. & Hayward, A. (2003). Who is being tested for genital chlamydia in primary care? Sexually transmitted infections 79, 234 - 236.

Lee, H. H., Chernesky, M. A., Schachter, J., Burczak, J. D., Andrews, W. W., Muldoon, S., Leckie, G. & Stamm, W. E. (1995). Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet 345, (no 8944) 213 - 216.

Mahony J, Chong S, Jang D, Luinstra K, Faught M, Dalby D, Sellors J, Chernesky M. (1998) Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. Journal of Clinical Microbiology 36, 3122 - 3126. Full article

Marrazzo, J. M., White, C. L., Krekeler, B., Celum, C. L,. Lafferty, W. E,. Stamm, W. E. & Handsfield, H. H. (1997). Community-based urine screening for Chlamydia trachomatis with a ligase chain reaction assay. Annals of Internal Medicine 127, 796 - 803.

Moller, J. K., Andersen, B., Olesen, F., Lignell, T. & Ostergaard, L. (1999). Impact of menstrual cycle on the diagnostic performance of LCR, TMA, and PCE for detection of Chlamydia trachomatis in home obtained and mailed vaginal flush and urine samples.  Sexually Transmitted Infections 75, 228 - 230

Morre, S. A., van Valkengoed, I. G., de Jong, A., Boeke, A. J., van Eijk, J. T., Meijer, C. J. & van den Brule, A. J. (1999). Mailed, home-obtained urine specimens: a reliable screening approach for detecting asymptomatic Chlamydia trachomatis infections. Journal of Clinical Microbiology 37, 976 - 980.   Full article

Ostergaard, L., Andersen, B., Moller, J. K. & Olesen, F. (2000). Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: a cluster-randomized 1-year follow-up study. Clinical Infectious Disease 31, 951 - 957.

Parker, E. K., Wozniak, A., White, S. D., Beckham, C. & Roberts, D. (1999). Stability study on specimens mailed to a state laboratory and tested with the Gen-Probe PACE 2 assay for chlamydia. Sexually Transmitted Diseases 26, 213 - 215.

Shafer, M. A., Pantell, R. H. & Schachter J. (1999). Is the routine pelvic examination needed with the advent of urine-based screening for sexually transmitted diseases? Archives of Pediatric and Adolescent Medicine 153, 119 - 125.

Stary A, Najim, B. & Lee, H. H. (1997). Vulval swabs as alternative specimens for ligase chain reaction detection of genital chlamydial infection in women. Journal of Clinical Microbiology 35 836 - 838. Full article

Stary, A., Schuh, E., Kerschbaumer, M., Gotz, B. & Lee, H. (1998). Performance of transcription-mediated amplification and ligase chain reaction assays for detection of chlamydial infection in urogenital samples obtained by invasive and noninvasive methods. Journal of Clinical Microbiology 36, 2666 - 2670. Full article

Stephenson, J., Carder, C., Copas, A., Robinson, A., Ridgway, G. & Haines, A. (2000). Home screening for chlamydial genital infection: is it acceptable to young men and women? Sexually Transmitted Infections 76, 25 - 27.

Sugunendran, H., Birley, H. D., Mallinson, H., Abbott, M. & Tong, C. Y. (2001). Comparison of urine, first and second endourethral swabs for PCR based detection of genital Chlamydia trachomatis infection in male patients. Sexually Transmitted Infections 77, 423 - 426.

Tabrizi, S. N., Fairley, C. K., Cehn, S., Giouzeppos, O., Paterson, B., Bowden, F. J. & Garland, S. M. (2000). Evaluation of patient-administered tampon specimens for Chlamydia trachomatis and Neisseria gonorrhoeae. Sexually Transmitted Diseases 27, 133 - 137.

Verkooyen, R. P., Noordhoek, G. T., Klapper, P. E., Reid, J., Schirm, J., Cleator, G. M., Ieven, M. & Hoddevik G. (2003). Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories. Journal of Clinical Microbiology 41, 3013 - 3016.