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Fig 1. Transcription mediated amplification in pictures (double click on slide; approx 100 kb)

The detection system is the Hybridization Protection Assay (HPA) previously illustrated, utilising acridinium ester technology.

In 1997 two studies compared the performance of the Gen-Probe AMP-CT, either against the Abbott LCx^® LCR, the Roche COBAS AMPLICOR^® PCR [Goessens et al., 1997] and cell culture [Mouton et al., 1997]. In the first of these studies, urine, urethral and cervical specimens were obtained from 544 men and 456 women visiting an outpatient sexually transmitted disease clinic. The sensitivities of LCx, COBAS AMPLICOR and AMP-CT against cell culture were 79, 86, and 78%, respectively. After discrepant analysis using an in-house PCR, the sensitivities of LCx, COBAS AMPLICOR, and AMP-CT were 84, 93, and 85%, respectively. Specificity exceeded 99% for all three of these amplification assays while sensitivity for all three methods was consistently lower for urines  from females compared to males  [Goessens et al., 1997]. The second study in that year compared AMP-CT with cell culture using urine samples collected from 1000 patients attending an STD outpatient clinic in Rotterdam [Mouton et al., 1997]. After discrepant analysis, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, for samples from women and 100, 99.2, 93.1, and 100%, respectively, for samples from men. To place this in context,  for cell culture the corresponding values were 72.5, 99.2, 92.5, and 98%, for women  and 57.4, 99.0, 86.1, and 95.4%, respectively, for men. It was concluded that the AMP-CT test was a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males [Mouton et al., 1997]. 

Crotchfelt et al., 1998 also compared the performance of AMP-CT with cell culture on endocervical and urine specimens collected in Baltimore, USA. Direct immunofluorescence on sedimented transport medium was used to resolve discrepant samples. After discrepant analysis for 480 female urine specimens, AMP CT had a sensitivity of 93.8% and a specificity of 100% while for 464 male urine specimens, the corresponding results  were 95.6 and 98.7%, respectively. On 479 endocervical swab specimens, the sensitivity of AMP-CT was 100% and the specificity was 99.5%. Again it was concluded that AMP CT was a highly sensitive test for the detection of C. trachomatis in both endocervical specimens and in urine from both sexes [Crotchfelt et al.,1998].

At a dermato-venereology clinic in Vienna, Stary et al., 1998 compared the performance of the Abbott LCx and the AMP-CT with endocervical, urine, and vulval specimens from women and urethral and urine specimens from men. Chlamydial culture was also performed on endocervical, vulval, and urethral swabs. Of the 308 women and 240 men tested, 25 (8.1%) and 44 (18.3%), respectively, were shown to be infected. Using the infected individual as an expanded "gold standard", the TMA and LCR assays gave similar sensitivity results, with sensitivity of 93.2%on male urethral samples and 88.6 and 86.4% on male urine samples. In women, the sensitivities of the TMA assay for endocervical and vulval samples were 88 and 92%, respectively, compared to values of 92% for the LCR on both sample types and 52 and 8%, respectively, for culture. Using first-void urine for chlamydial diagnosis in women, LCR detected 96% and TMA detected 76% of infected individuals, showing a significantly lower sensitivity for urine in women (P = 0.0253). The results indicated a high overall agreement for both amplification techniques for all specimens, except female urine. Importantly it confirmed the previous observation that vulval swabs are an effective alternative noninvasive sample type to urine for the detection of C. trachomatis infection in women by nucleic acid-based amplification technologies [Stary et al., 1998].   

Pasternack et al., 1999 compared the performance of the AMP-CT TMA with the Roche COBAS AMPLICOR CT/NG PCR  for the detection of C. trachomatis in urine. Urine specimens were collected from 658 patients: 320 men and 338 women. The results were concordant for 98.8% of these patients. In particular, the tests  were uniformly positive for 39 male and 33 female patients. Discrepant specimens were retested by an alternative TMA assay and a major outer membrane protein gene-based PCR. All positive results were correctly identified by the TMA assay, whereas PCR gave two false-negative and six false-positive results [Pasternack et al., 1999]. [Caveat lector: Retesting only discrepant specimens introduces bias, see discrepant analysis].

Amplification assays are potentially susceptible to inhibitors in urine, which can lead to false negative results. In an interesting and relevant study, the effect of Inhibitors in the urine of pregnant or non pregnant women on various amplification-based tests was assessed [Mahoney et al., 1998]. The prevalence of inhibitors causing complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. Logistic regression analysis revealed that inhibition was associated with: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR; nitrites for LCR (OR, 14.4); and haemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Fortunately, most inhibition was removed by storage overnight at 4º or -70º degrees C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. Thus, the prevalence of nucleic acid amplification inhibitors in female urine was different for each technology, though storage and dilution remove most of the inhibitors [Mahoney et al., 1998]. [Caveat lector again: When assessing laboratory studies of the performance of nucleic acid amplification-based tests on urine, be aware that batch testing of stored urine may overestimate the test's performance with fresh urine]. 

A complementary study by a different group found that the overall prevalence of C. trachomatis increased with time after the last menstrual bleeding until three weeks later, when the sensitivities of the nucleic acid amplification based tests all decreased because of the secretion of inhibitors. Vaginal flush samples were found to be superior to urine for detecting C. trachomatis infection in women. When screening young asymptomatic females, samples should ideally be obtained in the latter part of the menstrual cycle [Moller et al., 1999]. [Self collected vaginal fluid on tampons or a vaginal introitus specimen is an important alternative and non invasive specimen to urine; see  Stary et al., 1998; also non-invasive testing].

Lauderdale et al., 1999 collected one urine and four endocervical swabs from 787 women attending one of four obstetrics / gynaecology clinics. The tests compared were the Clearview^® rapid EIA (Wampole Laboratories),  the Gen-Probe PACE 2 and AMP CT assays and the Abbott LCx^®  assay. They used an expanded reference standard, considering that patients were true positives if three or more assays (swab and/or urine) were positive. Discrepant results from the EIA or PACE 2^® assays were confirmed by alternate amplified assays.  After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Overall prevalence of C. trachomatis was 8.4%. As might be expected, the amplification technologies were the most sensitive methods with either swab (AMP CT assay best) or urine (LCx assay best) specimens [Lauderdale et al., 1999].  

An excellent study looked at the performance of several nucleic acid amplification-based tests for screening symptomatic and asymptomatic patients. In symptomatic patients, 618 specimens from 140 men (140 urethral swabs and 140 urines) and 202 women (202 endocervical swabs and 136 urines) were tested by cell culture, AMP-CT and Roche Amplicor C. trachomatis micro well plate (MWP) PCR on genital samples, and AMP-CT and Amplicor on urine. A clinical specimen was considered to be truly positive if either cell culture and/or both AMP-CT and Amplicor were positive. In the asymptomatic patients, a total of 300 urines (136 women and 164 men) were tested by four amplification methods, AMP-CT, Abbott LCx, the Roche Amplicor MWP and Cobas PCRs. In this group a subject was considered infected when two or more amplification methods were positive. In the symptomatic population (prevalence 13%), concordant results were observed in 320/342 cases (93.5%). After analysis of discrepant results, the sensitivity of AMP-CT, Amplicor, and culture was 100%, 95.5%, 68.8%, and the specificity was 98.3%, 99.3%, 100%, respectively. The number of false negative results by AMP-CT in urine, probably due to labile inhibitors, was 3/276 (1%). In the asymptomatic population, the results were concordant in 298/300 (99.3%), with seven positive and 291 negative specimens. The authors concluded that, compared to the other amplification methods tested, AMP CT is at least as sensitive for the identification of chlamydial infection in symptomatic and asymptomatic men and women on genital or urine specimens [de Barbeyrac et al., 2000].

Gomes et al., 2002 looked at the effect on AMP-CT sensitivity of pooling either four or eight urine samples together  versus testing each sample individually.  Thirty-three known polymerase chain reaction positive urine specimens were combined with 231 fresh first-catch urine samples in 33 groups of four or in 33 groups of eight urine samples. Gen-Probe AMP-CT assay was performed on the pools and on individual samples at the same time. Where discrepancy occurred, the known positive samples were diluted 1:4 and 1:8 using the manufacturer's dilution buffer and retested. Additional positive specimens found among fresh first catch urine samples were also tested by the Roche AMPLICOR assay to confirm their positivity. The sensitivities of 8X pooling, 4X pooling and individual testing were 86.5%, 94.3% and 91.9%, respectively. It was concluded that the Gen-Probe AMP-CT assay applied to a 4X urine pools was a sensitive and useful method of screening large numbers of urines.

[MEW] September 2002

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References

de Barbeyrac, B., Geniaux, M., Hocke, C., Dupon, M. & Bebear, C. (2000). Detection of Chlamydia trachomatis in symptomatic and asymptomatic populations with urogenital specimens by AMP CT (Gen-probe incorporated) compared to other commercially available amplification assays. Diagnostic Microbiology and Infectious Diseases 37, 181 - 185.

Beebe, J. L., Gershman, K. A., Kelley, J. K., Hagner, D. & Creede, P. (1999). How adequate is adequate for the collection of endocervical specimens for Chlamydia trachomatis testing? Sexually Transmitted Disease 26, 579 - 583.

Crotchfelt, K. A., Pare, B., Gaydos, C. & Quinn, T. C. (1998). 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. Journal of Clinical Microbiology 36, 391 - 394. Full article.

Ferrero, D V., Meyers, H. N., Schultz, D. E. & Willis, S. A. (1998). Performance of the Gen-Probe AMPLIFIED Chlamydia trachomatis assay in detecting Chlamydia trachomatis in endocervical and urine specimens from women and urethral and urine specimens from men attending sexually transmitted disease and family planning clinics. Journal of Clinical Microbiology 36, 3230 - 3233. Full article.  

Goessens, W. H., Mouton, J. W., van der Meijden, W. I., Deelen, S., van Rijsoort-Vos, T. H., Lemmens-den Toom, N., Verbrugh, H. A. & Verkooyen, R. P. (1997). Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine. Journal of Clinical Microbiology 35, 2628 - 2633. Full article

Gomes, J. P., Viegas, S., Paulino, A. & Catry, M. A. (2002). Sensitivity evaluation of the Gen-Probe AMP-CT assay by pooling urine samples for the screening of Chlamydia trachomatis urogenital infection. International Journal of STD and AIDS. 13, 540 - 542.

Hill, C. S. (2001) Molecular diagnostic testing for infectious diseases using TMA technology. Expert Reviews of Molecular Diagnostics 1, 445 - 455.

Lauderdale, T. L., Landers, L., Thorneycroft, I. & Chapin, K. (1999). Comparison of the PACE 2 assay, two amplification assays, and Clearview EIA for detection of Chlamydia trachomatis in female endocervical and urine specimens. Journal of Clinical Microbiology 37, 2223 - 2229. Full article

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

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

Mouton, J. W., Verkooyen, R., van der Meijden, W. I., van Rijsoort-Vos, T. H., Goessens, W. H., Kluytmans, J. A., Deelen, S. D., Luijendijk, A. & Verbrugh, H. A. (1997). Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test. Journal of Clinical Microbiology 35, 1369 - 1372. Full article 

Pasternack, R., Vuorinen, P. & Miettinen, A. (1999). Comparison of a transcription-mediated amplification assay and polymerase chain reaction for detection of Chlamydia trachomatis in first-void urine. European Journal of Clinical Microbiology and Infectious Disease 18, 142 - 144.

Semeniuk, H., Zentner, A., Read, R. & Church, D. (2002). Evaluation of sequential testing strategies using non-amplified and amplified methods for detection of Chlamydia trachomatis in endocervical and urine specimens from women. Diagnostic Microbiology and Infectious Disease 42, 43 - 51.

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   

NEXT: Second generation TMA