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A survey of general practitioners in England and Wales by the Public Health Laboratory Service indicated that, among infectious diseases, the greatest opportunity for improving public health was seen to be the better management of chlamydial genital tract infections. Improved availability of diagnostic tests for chlamydial infections was also a major priority [McNulty et al., 2001]. Nucleic acid amplification-based methods are now of prime importance for the diagnosis of chlamydial infections [Lisby et al., 1999]. Indeed, the development of chlamydial tests based on nucleic acid amplification technology (NAAT) has been considered the most important advance for the detection of chlamydial infections since cell culture [Stary, 2000]. A number of commercial products using nucleic acid amplification technology are now available [Chernesky  1999; Chernesky et al., 1999]. These tests amplify either a) the target nucleic acid, DNA or RNA ; or b) the probe after it has annealed to target nucleic acid. Such tests  are generally more sensitive than liquid or solid phase hybridization tests which do not embody an amplification process [Chernesky 1999] and are considerably more sensitive than culture or antigen detection methods [Ostergaard, 1999]. 

The major target for amplification based tests against C. trachomatis are generally multiple-copy gene products, such as the cryptic chlamydial plasmid or ribosomal   RNA . Starting with a multiple copy gene offers a clear starting advantage with respect to sensitivity. Initial nucleic acid amplification based tests were based on the detection of chlamydial DNA in clinical samples. The well known polymerase chain reaction, e.g. the Roche Amplicor^® PCR , uses selected primers, nucleotide triphosphates and taq polymerase to amplify chlamydial DNA sequences, which are captured by hybridization on oligonucleotide-coated microplates. The Roche Amplicor^® PCR was the first commercial nucleic acid amplification test to be made available for the diagnosis of human chlamydial infections. The ligase chain reaction, commercialised as the Abbott LCx^®  also detects chlamydial DNA and is based on the ligation of chlamydia-specific oligonucleotide probes which serve as a copy of the original target sequence and which are adjacent to each other. Allowing for the problems of discrepant analysis, the true sensitivity of PCR and LCR is of the order of 90 to 97% [Cheng et al., 2001]. The more recent BDProbeTec^®ET (Becton Dickinson, USA) Chlamydia trachomatis and Neisseria gonorrhoeae amplified DNA assay uses a novel isothermal strand displacement amplification (SDA) method coupled with fluorescent energy transfer (ET) measurement to detect amplified product as it is produced [Stary 2000; van der Pol et al., 2001]. 

 mRNA is much more labile than DNA and is not commercially used for chlamydial diagnosis. For research, the presence of chlamydial mRNA is indicative of ongoing bacterial transcriptional activity, and often approximated to microbial viability, probably naively [Birch et al., 2001]. Ribosomal RNA is much more stable and is particularly useful for diagnosis as there may be several thousand copies per bacterial cell. 

The first isothermal and one step transcription based amplification system was described by Guatelli et al., 1990 [see: excellent review of Chan & Fox, 1999]. The process was modelled on the enzymes essential for retrovirus replication, including ribonuclease H, reverse transcriptase  and a DNA-dependent RNA polymerase to produce first cDNA and then copies of the original target. These products then functioned as a template for a series of transcription and reverse transcription reactions that were self sustained, amplifying the RNA target some ten million fold [Chan & Fox, 1990].Two commercial variants of this basic methodology are the  Transcription Mediated Amplification assay of Gen-Probe Incorporated, [the Gen-Probe AMP-CT], and the Organon Teknika Nuclisens^® Nucleic Acid Sequence Based Amplification (NASBA).  Unlike reverse transcriptase   PCR, both NASBA and TMA are isothermal ; there is no need for thermal cycling. In NASBA, the product is an antisense single stranded RNA, so the high temperature denaturation of double stranded DNA characteristic of RT-PCR is not required. NASBA differs from TMA in using Ribonuclease H as well as AMV reverse transcriptase and T7 RNA polymerase .  In NASBA and TMA compared with RT-PCR, there is an exponential rather than binary increase in amplified product, resulting in much greater amplification [Chan & Fox, 1999]. 

Fig 1.

Advantages and disadvantages

The main advantage of the nucleic acid amplification-based diagnosis for chlamydiae is that such methods combine unsurpassed sensitivity with good specificity. Thus, In a five-city study of 3,551 women, which compared the results of commercial nucleic acid amplification tests (PCR and LCR)  with the non-amplified PACE 2 nucleic acid hybridization test, the sensitivities of PACE 2, LCR, and PCR tests with cervical specimens were 78.1, 96.9, and 89.9%, respectively. The specificity of all three tests was high at  99.3, 97.5, and 98.2%, respectively. Sensitivities obtained with cervical swabs exceeded those obtained with urine specimens by small amounts. In this study, nucleic acid amplification based tests improved the detection of infected women by 17 to 38% compared to the PACE 2 hybridization test [Black et al., 2002]. Such tests enable a high detection rate for C. trachomatis in symptomatic individuals and an adequate detection rate in asymptomatic individuals (where there are often fewer chlamydiae present). In women, PCR and other amplification based tests can be usefully combined with routine cervical smear tests for cervical cancer [Bianchi et al., 2002; see: cervical cytology screening]. They also permit the use of non invasive clinical samples such as urine [Ostergaard, 1999; Stary et al., 1998; Stary, 2000].  Performance of some of the major commercial tests on urine based samples in a major quality control study is covered elsewhere on this site [see urine QC]. In general specificity is now excellent but there are still issues concerning sensitivity.

However the greater sensitivity of these assays means that accidental contamination with amplified product (amplicon) is a  problem of major importance for kit design,  laboratory workflow and personnel. The tests require carefully trained laboratory technicians, which tends to remove them from the clinic where they are needed to specialised laboratories. Statistical methods of coping with the problem of contamination have been suggested [Shapiro, 1999]. Specimen throughput can also be a problem, although the tests are increasingly becoming automated. Nucleic acid amplification tests tend to be more expensive (but more effective) than other laboratory methods of testing for chlamydial infection.  The clinic or laboratory contemplating adopting such tests therefore needs to consider not just sensitivity, specificity and the clinical requirement, but also the suitability of the test for the facilities and human resources available. Fortunately, for high throughput testing, kit manufacturers can provide instrumentation to achieve at least partial automation. Alternatively, with a little ingenuity it may be possible to adapt other programmable laboratory dispensing / assay equipment [see: robotics].

Cost effectiveness of nucleic acid amplification assays

The reproducibility and performance of the better tests, and their suitability for automation, means that it is now feasible to perform epidemiological studies on target populations having a relatively low level of prevalence, so as to inform and develop infection control strategies. While cost is undoubtedly a limiting factor, for screening purposes the pooling of clinical specimens offers an effective way of substantially reducing costs. A number of studies have purported to demonstrate the cost effectiveness for health services of using nucleic acid amplification tests for screening for chlamydial genital tract infection in order to prevent sequelae, even though such tests are more expensive than antigen detection or hybridization assays. One paper will suffice to make the point here. In a cohort of 18,000 women in Maryland it was expected that there would be 497 cases of pelvic inflammatory disease in the absence of screening, which  would cost an estimated  $2.2 million in  medical costs. The use of antigen detection EIAs would prevent 240 cases and save a calculated $887,000 while the use of nucleic acid amplification on urine specimens would save a further $287,100. It was concluded that, compared with EIA screening, (the strategy with the lowest program costs), a screening strategy that combined the use of NAAT on cervical specimens in women receiving pelvic examinations, and NAAT of urine in women with no medical indications that necessitated a pelvic examination, would prevent the most cases of pelvic inflammatory disease and thus provide the highest cost savings  [Howell et al., 1998]. 

For Africa, Sahin-Hodoglugil et al., 2003 compared the cost effectiveness of three different strategies for the diagnosis and treatment of gonococcal and chlamydial infections in Africa. These strategies were "gold standard" care based on microbiological diagnosis of infection, syndromic management or mass treatment. A decision tree model was constructed and Monte-Carlo simulations were run to test the robustness of the cost effectiveness estimates to changes in the inevitable underlying assumptions. In the African context it was concluded that mass treatment with doxycycline for chlamydia was the most cost-effective protocol in terms of cost per cure for chlamydial infection whereas syndromic management had the lowest costs. However No single protocol was optimal. The treatment-seeking behaviour, STD prevalence, and coverage of each locale must be evaluated to determine the most cost-effective and highest impact program.

For a recent review of nucleic acid amplification tests see Chernesky, 2002.

Caveat emptor

Which may be translated as "let the purchaser beware". As Verhoeven et al., 2003 point out, a number of problems can be expected if  diagnostic tests are implemented in large scale routine clinical practice. These are:

• Multiple testing may be needed for accurate results, but is seldom possible because of cost and inconvenience
• In low prevalence populations the positive predictive value of a test is inevitably fairly low
• In practice reproducibility problems do occur with diagnostic tests and they vary over time. Where the problem is caused by low target numbers in the specimen, a repeat sample may not include the target. Clinicians need to be made aware of this issue where results are borderline. Occasionally chronic reproducibility problems lead to the withdrawal of a test, as in the case of the Abbott LCx.
• Diagnostic accuracy may be affected by contamination in the laboratory
• Detection of minute amounts of chlamydial DNA may not be clinically significant

Finally it is important to remember that a diagnosis of C. trachomatis genital tract infection may have important psychosocial impact on the patient and his or her partner. Consequently information provided by the clinician should normalise and destigmatise chlamydial genital tract infection [Duncan et al., 2001]. Provided that these factors are kept in mind, the current generation of nucleic acid amplification based tests generally do an excellent job.

[MEW] September 2003

NEXT: PCR

Sahin-Hodoglugil, N. N., Woods, R., Pettifor, A. & Walsh, J. (2003). A comparison of cost-effectiveness of three protocols for diagnosis and treatment of gonococcal and chlamydial infections in women in Africa. Sexually transmitted diseases 30, 455 - 469.

van der Pol, B., Quinn, T. C., Gaydos, C. A., Crotchfelt, K., Schachter, J., Moncada, J., Jungkind, D., Martin, D. H., Turner, B., Peyton, C. & Jones, R. B. (2000). Multicenter evaluation of the AMPLICOR and automated COBAS AMPLICOR CT/NG tests for detection of Chlamydia trachomatis. Journal of Clinical Microbiology 38, 1105-1112. Full article   [Good multicentre study]

Verhoeven, V., Ieven, M., Meheus, A., Avonts, D. & Goosens, H. (2003). First, do not harm: also an issue in NAA assay diagnostics for chlamydial infection. Sexually Transmitted Infections 79, 76 - 77. [A letter making some good points].