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Frederick Mitchell
MSc FIBMS
Biomedical Scientist 3 (BMS3)
Regional Virus Laboratory
Kelvin Belfast Health and Social Care Trust – RVH
Belfast, UK
Infection by the bacterium Chlamydia trachomatis is the commonest sexually transmitted infection diagnosed in the UK and one that has shown a dramatic rise over the last decade. In some women, complications of chlamydial infection can cause pelvic inflammatory disease (PID) and infertility. In men, long-term complications of infection are less serious, but untreated males are able to transmit the infection. Presentation of the infection is often asymptomatic in both women and men, so early diagnosis and treatment are very important. In an uncomplicated infection, treatment with antibiotics is usually very effective.
For many years, culture of the organism in living cells was the routine diagnostic method. The culture technique was highly specific but had relatively poor sensitivity. Culture was later replaced by direct detection of chlamydial elementary bodies by fluorescein-labelled monoclonal antibodies and detection of chlamydial antigen by use of enzyme immunoassays. Both methods were an improvement over culture due to their ease of use in the laboratory and their ability to utilise nonviable patient specimens, although endocervical swabs in women and male urethral swabs were still the specimens of choice.
NAAT test
Recently, a major advance in the laboratory diagnosis of chlamydial infections has been the availability of commercial nucleic acid amplification tests (NAATs). These tests present superior sensitivity (and high specificity) compared with previous methods, although they also have a greater cost. They also allow the testing of noninvasive specimens, and a first-catch urine sample from a female patient is now an acceptable alternative specimen to the traditional endocervical swab (as well as self-taken vaginal swabs). Urine is also a satisfactory sample for male patients, so a urethral swab, with its obvious discomfort in the taking, is no longer necessary.
The NAAT consists of three basic steps: specimen preparation, amplification and detection. Preparation of specimens for amplification may simply consist of heating the specimen in lysis buffer or detergent to release the organism or employing a method of extraction such as purification of nucleic acid including chlamydial DNA. The latter protocol will reduce inhibitors in the specimen that may affect amplification. Commercial NAATs for chlamydia detection currently utilise a variety of amplification methods, including the polymerase chain reaction (PCR), strand displacement amplification (SDA) and transcription-mediated amplification (TMA). Detection may be “real time” (ie, collection of signal during amplification) or may occur as a post-amplification step. NAATs can be susceptible to false negatives due to inhibitors in the specimen, and all current assays incorporate an inhibition check by parallel amplification of an internal control.
What is available on the market?
In the UK, Becton Dickinson’s ProbeTec™ ET assay (SDA) and the Roche Cobas(®) Amplicor™ assay (PCR) currently dominate the marketplace for chlamydia testing, with 48% and 27% market share, respectively. Both assays utilise basic methods of sample preparation, including heating, use of lysis buffer and detergent, and also require an initial step of urine centrifugation and concentration. The ProbeTec system is available in a semiautomated format, requiring a number of manual steps, or, with the Viper instrumentation, a floor-standing platform using robotic technology. The Viper allows automation of prepared sample transfer for the amplification and detection steps (in one unit) and claims to process 920 samples in a shift. The Roche test uses the ubiquitous Cobas Amplicor analyser, a benchtop instrument that, like the Viper, combines amplification and detection in one instrument. Specimen preparation of both urine samples and swabs is aided by the use of the Tecan Evo75, a liquid-handling platform that automates a number of specimen preparation steps. Recently Roche, for chlamydia testing, has employed the Cobas(®) TaqMan(®) 48 Analyzer, a compact instrument that allows realtime PCR amplification and detection.
Gen-Probe is a company with a much smaller share of the market that offers a family of automated systems for low-, medium- and high-volume throughput (up to 1,600 specimens in eight hours) using the Aptima(®) CT assay (TMA). This instrument group comprising the Tigris(®) DTS(®) systems, allows streamlined specimen preparation, amplification and detection in the one unit and also incorporates a unique pierceable cap on the specimen collection tube. This innovation means the user is saved the time-consuming job of removing and replacing the caps before and after testing. The Gen-Probe assay uses a sophisticated method of specimen preparation with extraction of chlamydial ribosomal RNA by its capture onto magnetic particles. These allow separation from the sample by a magnet and permit subsequent washing to purify the rRNA prior to amplification and detection.
Abbott Laboratories was, for many years, a major worldwide player in NAATs for chlamydial diagnosis. It distributed the Abbott LCx(®) assay, which utilised the ligase chain reaction (LCR) for amplification. The assay was withdrawn in 2003 due to ongoing problems with test performance. Recently Abbott, in the form of Abbott Molecular, has re-entered the market with the new m2000 system, consisting of the m2000sp nucleic acid extraction platform and the m2000rt real-time PCR instrument for amplification and detection. It is this system that we are currently using in the Regional Virus Laboratory in Belfast.
Previously, for many years, we used an enzyme immunoassay for chlamydia diagnosis, and then two years ago we switched to a NAAT with amplification by the Artus PCR assay and detection by the ABI 7000 real-time PCR instrument. Sample preparation required urine centrifugation, mechanical shaking of swabs and then specimen purification in the ABI 6100 Nucleic Acid PrepStation. These preparation steps were tedious and labour-intensive, and in May 2006 we moved to our current NAAT with the new Abbott m2000 system. The move has proved successful as it has streamlined the specimen preparation process with the m2000sp automated platform for nucleic acid extraction and reduced hands-on time for staff.
New system for testing
A unique aspect of the new system is the Abbott multi-Collect specimen tube containing lysis buffer. This tube can collect either urine or swab samples and be stored at room temperature up to 14 days before testing. On reception at the laboratory, it can be placed directly on the m2000sp extraction platform without any further sample preparation. The nucleic acid extraction, as with the Gen-Probe system, utilises magnetic particles in the purification procedure. The magnetic particles are coated with silica, which, on mixing with the sample and lysis buffer, allows adsorption of nucleic acid.
Activation of a magnet attracts the magnetic particles to one side of the reaction tube to permit aspiration of the sample. Switching the magnet on and off appropriately allows washing of the particles in buffer, and finally, addition of an elution buffer decouples the nucleic acid from the particles. This eluate (purified nucleic acid) is added to the mastermix (PCR reagents) in a microwell plate, in a separate step on the automated m2000sp platform. The plate is then placed in the m2000rt real-time PCR instrument for amplification and detection. Each test run has 45 specimens, and we generally perform two runs per day, although three runs are possible, with a total of about 20,000 specimens tested in a year. The magnetic particle extraction method employed in the m2000 system has significantly reduced inhibitory specimens, from approximately 2% in our first NAAT, the Artus assay, to 0.09% in the Abbott test. This translates to about 400 patients being recalled each year for repeat testing, as opposed to 18 with our current assay.
Conclusion
The majority of NAATs for chlamydia, including those described here, permit tracking of specimens from barcode labelling at reception to the downloading of results to the laboratory information system (LIS). Most laboratories, before reporting positives, will confirm the result, either by retesting in the same assay or by using a different NAAT.
Some companies, including Becton Dickinson and Abbott, also offer a combined assay for Chlamydia trachomatis and Neisseria gonorrhoea, allowing simultaneous detection of both pathogens.Chlamydia testing by NAATs has allowed “opportunistic” screening of susceptible groups in the population, as urine or self-collected specimens are now viable as samples.
A National Chlamydia Screening Programme (NCSP) is currently being rolled out in the UK to detect genital chlamydia infection in young men and women to help reduce pelvic inflammatory disease and the occurrence of ectopic pregnancies. Chlamydia testing by NAATs is a requirement for this programme because of their high sensitivity (>90%) and specificity (approaching 100%), as well as their ability to test noninvasive specimens. The use of NAATs for chlamydia testing in the UK will soon be the routine means of diagnosis of infection. Laboratories will always require different solutions in their choice of NAAT due to factors such as assay capacity, equipment footprint, price and the constraints of staff numbers. In general, the trend in development of commercial assays is for extraction of nucleic acid in specimens to reduce inhibition, more automation in sample handling and a greater choice in assay capacity to meet individual laboratory needs.
