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All nucleic acid-based chlamydial diagnostic assays depend on hybridization. In direct hybridisation probe tests, signal is generated without any intermediary target amplification. 

The Gen-Probe PACE 2 test was first introduced in 1988. Its sensitivity is founded on the fact that it is targeted against high copy number chlamydial rRNA which effectively serves as a natural pre-amplification. As rRNA is mostly single stranded, no thermal denaturation for strand separation is necessary prior to hybridisation, which takes 60 minutes at 60C. The test is completed in approximately two hours (Fig 1).  The test is popular because of its relative  simplicity to perform and its scalability using conventional laboratory equipment. An acridinium ester probe  generates a signal in the LEADER^®  luminometer following addition of hydrogen peroxide.

Fig 1.  The Gen-Probe PACE 2 System for diagnosing C. trachomatis (also N. gonorrhoeae).

Fig 1A. Simple diagram of the PACE 2 procedure.  Double click on thumb nail to view. Note that relatively little special equipment is required, other than a simple magnetic separator, a water bath and a luminometer.  Download pdf of figure .  [1.2mb]	Fig 1B. Protocol for the probe competition confirmatory assay. These figures reproduced here by courtesy of Gen-Probe Incorporated. Download pdf of figure . [1.2mb]

Fig 2. The Gen-Probe PACE 2 Hybridisation protection assay (HPA) in pictures (each slide approx 100 kb).

Click HERE for a brief audio video presentation from Gen-Probe on the HPA (235 kb; Windows media player; broadband connection best).

PACE 2 tests are available for C. trachomatis or N. gonorrhoeae alone, or for the two tests combined in a single test tube (the PACE 2C Assay). Subsequently a probe competition assay was introduced to help resolve any border line results, or to verify positive results. Early results indicated that the Gen-Probe PACE 2 was highly specific, with a sensitivity roughly equivalent to the IDEIA^® III antigen detection test [LeBar et al., 1989] and better than the Chlamydiazyme^® test [Stary et al., 1994]. However many comparisons are of limited value today because they were made against a culture reference standard, which we now know is itself only about 70% sensitive [see: nucleic acid amplification] and because there have also been steady interim improvements in the test. These included halving the volume of the transport fluid to increase sensitivity and the introduction of the probe competition assay for verifying positive results. In the cervix since chlamydiae primarily infect columnar endothelial cells of the endocervical canal it is important to ensure that the sample contains adequate numbers of endocervical cells [see sampling]. The number of endocervical cells in a sample for the PACE 2 CT increase sensitivity in a mathematically predictable manner [Beebe et al., 1999]. The need for the probe competition assay has also been analyzed. It was concluded that the probe competition assay should be a routine supplement to the Gen-Probe PACE 2 for specimens with an initial signal by PACE 2 CT of less than 1,500 relative light units [Woods & Garza 1996].

The Pace 2 test has the advantage of being relatively simple to perform but at reduced sensitivity compared to the leading nucleic acid amplification based techniques. Wylie et al., 1998 compared Chlamydiazyme^® antigen detection assay (Abbott Laboratories), PACE 2 and the AMP CT (Gen-Probe) amplification assay for their ability to detect C. trachomatis in endocervical samples from 787 women. Taking AMP CT as the reference standard, the corresponding sensitivities of the   PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP CT assays were 79.3 and 63.4%, respectively. Both tests had a specificity of 100% and the overall prevalence of C. trachomatis was 10.4%. Some specimens in the intermediate zone by the PACE 2 assay were confirmed by subsequent probe competition assay. Interestingly, amplification testing these borderline specimens did not significantly increase yield. The majority of specimens which tested positive by the AMP-CT assay were not borderline in the PACE 2 assay [Wylie et al., 1998]. Methods of purifying DNA for nucleic acid based tests after they have been processed for the PACE 2, i.e. using amplification confirmation, have been detailed by Gossack & Beeb, 1998. 

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^® LCR 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 TMA assays targeting different sequences.  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. However the PACE 2 assay was considered to have acceptable sensitivity while being a simpler and less expensive assay. The Clearview^® CT EIA, although yielding a rapid physician-office result, had unacceptably low sensitivity [Lauderdale et al., 1999].  

Another study of endocervical samples from 493 women found no difference between the Abbot LCx or the PACE 2, despite the former being a nucleic acid amplification-based test [Doing et al., 1999].  

A major virtue of the Pace 2 is that it is relatively robust. In an interesting study, duplicate specimens collected from 1,017 women in South Carolina were either shipped by courier (faster transit time) or through the U.S. mail (longer transit, elevated temperatures). There was a 99% concurrence of results, showing that the transport method was immaterial under these conditions [Parker et al., 1999]. This is obviously relevant the emphasis in STD control is shifting to the return of samples  collected by patients through the mail [see non invasive sampling].

[Conclusion: The Gen-Probe PACE 2 is a relatively simple and very robust test. Although its sensitivity is less than the leading amplification-based tests, the combination of simplicity and low cost may make it attractive in many settings. The Pace 2 Combo's ability to test for two pathogens on one swab makes it particularly attractive in the US, where gonorrhoea is generally more prevalent than in Europe. In Europe, the preference is for the Pace 2 CT test.] 

The Digene Hybrid Capture II Test

In the late 90s, a new hybridization assay was introduced by the Digene Corporation of Gaithersburg, Maryland, USA. This test, the Digene Hybrid Capture^® II (HC II) CT/GC test, is a nucleic acid, signal amplification-based test designed for detecting C. trachomatis or N. gonorrhoeae genital tract infections. It uses a special conical shaped brush for cervical specimen collection from nonpregnant women and swabs from pregnant women.  The test uses a microwell format. The sample is lysed with alkali to release target nucleic acid, at the same time both destroying the RNA and denaturing the DNA. An RNA probe for a specific DNA target is added, hybridization occurs, and the hybrid is captured onto a solid phase with a universal capture antibody. A mixture of alkaline phosphatase labelled antibodies bind to the bound hybrid. After washing, a dioxetane substrate is added and a chemiluminescent signal is generated. Since the test uses an EIA-type format, it is potentially readily scaled up [see Digene corporation web site]. In a head-to-head study by the Digene corporation on 1,746 patients from two centres, the new test, which could be performed using the PACE 2 specimen transport buffer,  was substantially more sensitive than the PACE 2 test [Modarress et al., 1998]. Another study considered the test comparable to PCR for sensitivity and specificity [Girdner et al., 1999]. 

A multi-centre study on cervical specimens from 1,370 women found the Digene assay had a sensitivity of 93% and specificity of 98.5% against culture for N. gonorrhoeae and a sensitivity of 97.7% and specificity of 98.2% against culture for C. trachomatis. After allowing for discrepant analysis using PCR, it was concluded the test is more sensitive than C. trachomatis culture and is at least as sensitive as culture for gonococci [Schachter et al., 1999]. A more recent study after discrepant analysis by direct immunofluorescence, PCR and culture, found that the Digene Hybrid Capture II (HC II) CT/GC test had a relative specificity and sensitivity of 94.8% and 99.8% overall against cervical specimens, whereas the sensitivity of culture was 83.6%. 

[This is a new direct probe test to the market that, on the data reported, looks sensitive. Moreover it uses an enzyme immunoassay format with which many laboratories are familiar. However, there are relatively few studies. More studies against good nucleic acid amplification tests and against the DAKO IDEIA^® PCE are indicated]. 

[MEW] May 2002

NEXT: Nucleic acid amplification

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