Objectives To investigate the performance of the fully automated cobas 4800 CT/NG test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.
Methods The study was conducted using 900 clinical specimens (496 urine and 404 swab specimens) for C trachomatis testing, of which 498 specimens (318 urine and 180 swab specimens) were also tested for N gonorrhoeae. The results of the cobas 4800 CT/NG test were compared with those obtained from the Roche COBAS AMPLICOR CT/NG and COBAS TaqMan CT assays. N gonorrhoeae-positive specimens were further tested using in-house, real-time PCR assays. A panel of 223 Neisseria isolates was used to further investigate the performance of the cobas 4800 N gonorrhoeae assay.
Results For urine specimens, the sensitivity, specificity and negative and positive predictive values of the cobas 4800 CT/NG test were 94.5%, 99.5%, 98.8% and 97.7%, respectively, for C trachomatis, and 92.9%, 100%, 99.7% and 100%, respectively, for N gonorrhoeae. For swab specimens, the sensitivity, specificity and negative and positive predictive values were 92.0%, 100%, 99.5% and 100%, respectively, for C trachomatis, and 100%, 99.4%, 100% and 90.0%, respectively, for N gonorrhoeae. All N gonorrhoeae isolates were positive and all non-gonococcal Neisseria strains were negative by the cobas 4800 N gonorrhoeae assay.
Conclusions The cobas 4800 CT/NG test is suitable for high through-put identification of C trachomatis and N gonorrhoeae infections.
- Chlamydia trachomatis
- DNA amplification
- Neisseria gonorrhoea
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Nucleic acid amplification tests (NAATs) are widely used for routine detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The many advantages of NAATs, including optimal sensitivity and the option of non-invasively collected specimens such as urine, make them ideal for high through-put identification. However, notable problems have been observed with both N gonorrhoeae and C trachomatis NAATs. First, there have been continuing problems with the specificity of certain NAATs for N gonorrhoeae.1 For example, the current Roche COBAS AMPLICOR N gonorrhoeae assay (Roche Diagnostics, Australia) is known to cross-react with strains of several commensal Neisseria species, including Neisseria subflava, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica and Neisseria sicca.2 3 Consequently, supplementary confirmatory testing for N gonorrhoeae NAATs is now widely used.1 4 Second, there have been problems of false-negative results caused by sequence variation. A variant of C trachomatis with a 377 bp deletion in the cryptic plasmid was reported in Sweden in 2007. The deleted sequence comprised the sequence targets for commercial C trachomatis NAATs producing false-negative results in both the Roche COBAS AMPLICOR and Abbott LCx C trachomatis assays. As a result, there was unchecked spread of the new variant throughout Sweden.5 6 This experience has led to calls for NAATs to target at least two genetic sequences to reduce the potential for false-negative results.6 7 Lower sensitivity or poorly specific diagnostic assays can have significant public health impacts.
In this study, we investigated the performance of the new cobas 4800 CT/NG test for detection of C trachomatis and N gonorrhoeae. The cobas × 480 performs automated DNA extraction, and PCR setup, while the cobas z 480 is a real-time PCR amplification platform. The cobas 4800 CT/NG assay is a multiplex PCR method that comprises reactions for C trachomatis, N gonorrhoeae and internal controls. Notably, the assay comprises two target sequences for detecting C trachomatis, including the C trachomatis cryptic plasmid and major outer membrane protein gene. The newly designed N gonorrhoeae assay targets a direct repeat region called DR-9. This target region is repeated three times on the N gonorrhoeae genome and has two highly conserved sequence variations. The cobas 4800 CT/NG test uses two sets of NG primers and probes to detect any combination of both target variations.
A total of 900 clinical specimens submitted for C trachomatis and/or N gonorrhoeae testing and including specimens from both symptomatic and asymptomatic patients were used in the study. These comprised 496 urine specimens submitted to Pathology Queensland (Path-Qld) located in Brisbane, Queensland, and 404 swab specimens (168 cervical, 7 eye, 2 mouth, 2 penile, 21 rectal, 1 throat, 10 urethral, 142 vaginal, and 51 swabs for which the site was not recorded) submitted to the Institute of Medical and Veterinary Science, South Australian Pathology (IMVS-SA) located in Adelaide, South Australia. The specimens were collected over a 4-month period from January 2009 to May 2009. In the latter part of the study, specimens positive in the initial round of routine diagnostic testing were oversampled to provide sufficient numbers for a valid assessment of sensitivity of the cobas 4800 platform. Specimens were stored at 4°C for up to 5 days before being tested by the reference methods, and at 4°C for up to 7 days before being prepared for cobas 4800 CT/NG testing, at which point a 1.0 ml aliquot of each urine specimen was also stored at −20°C.
cobas 4800 CT/NG testing
cobas 4800 CT/NG testing was conducted at the Queensland Paediatric Infectious Diseases Laboratory, Brisbane, Queensland. Urine specimens were prepared for cobas 4800 CT/NG testing by adding 750 μl of each urine specimen to 750 μl of cobas PCR media. Swab specimens were initially resuspended in 1.0 ml of phosphate-buffered saline (PBS), 500 μl of which was then added to 500 μl of cobas PCR media. Specimens were then tested in the cobas 4800 system. Briefly, samples (either 22 or 94 per run), controls and reagents were loaded onto the cobas × 480 analyser (extraction instrument), which performed the DNA extraction using magnet bead technology. Internal controls for C trachomatis and N gonorrhoeae were added to each sample and control during extraction, controlling for extraction, amplification, inhibition and competition. This instrument then loaded the extracted DNA and controls and amplification reagents into a 96-well amplification plate. The plate was then covered and placed into the cobas z 480 real-time PCR instrument. Results were described as positive, negative or invalid by an algorithm within the cobas 4800 software.
The C trachomatis and N gonorrhoeae results obtained from Path-Qld and IMVS-SA were used as the reference standard for the cobas 4800 investigation.
C trachomatis results were available for all 900 specimens used in the study. At Path-Qld, specimens were tested for C trachomatis DNA using the COBAS TaqMan; 200 μl of urine was extracted on the Corbett X-tractor Gene (Corbett, Australia) and eluted in 100 μl, of which 50 μl was added the COBAS TaqMan CT master mix prepared in accordance with the manufacturer's guidelines and amplified and detected on the TaqMan 48 using the Roche test definition file. The results generated by the software were positive, negative or invalid (Roche Diagnostics, Australia). At IMVS-SA C trachomatis testing was performed on the COBAS TaqMan 48 or if both C trachomatis and N gonorrhoeae were requested the CT test was performed on the COBAS AMPLICOR (Roche Diagnostics, Australia). Swab samples were tested using similar methods to Path-Qld, with the exception that 200 μl of swab sample was extracted on the MagNA Pure LC (Roche, Mannheim) using the DNA I Isolation kit (Roche, Mannheim) and DNA I FastProtocol with the addition of poly A, and eluted in 100 μl. The master mix preparation, amplification and detection on the COBAS TaqMan 48 were preformed as described by the manufacturer. For tests performed on the COBAS AMPLICOR the manufacturer's guidelines were followed with the exception that 147 μl of MgCl2 was added directly to the CT/NG master mix. This was done because an alternative DNA extraction protocol was used; in the original COBAS AMPLICOR protocol the CT/NG master mix obtains its MgCl2 from the elution buffer used in the COBAS AMPLICOR manual extraction method.
N gonorrhoeae results were available for 498 specimens, comprising 318 urine and 180 swab specimens. The smaller number of N gonorrhoeae results was due to a subset of specimens (402) being submitted for C trachomatis testing only and were therefore excluded from the N gonorrhoeae analyses. At Path-Qld and IMVS-SA, specimens were screened for N gonorrhoeae DNA using the Roche COBAS AMPLICOR method. Specimens providing positive results in the COBAS AMPLICOR N gonorrhoeae assay were re-extracted by MagNA Pure LC and further tested by supplementary real-time PCR assays. At Path-Qld, a N gonorrhoeae duplex in-house real-time PCR method8 targeting the gonococcal porA pseudogene and opa genes was used, and at IMVS-SA a LightCycler-based, in-house, real-time PCR targeting the gonococcal porA pseudogene was used as previously described.9 Specimens providing positive results in both the COBAS AMPLICOR and supplementary assays were considered positive for N gonorrhoeae according to the Australian Public Health Laboratory Network guidelines for N gonorrhoeae NAAT testing.4
Neisseria species panel
A panel of Neisseria species (223 isolates) was used to further investigate the specificity and conservation of the target sequences used by the cobas 4800 N gonorrhoeae assay. These included one N canis, 10 N cinerea, one N elongata, two N flavescens, 76 N gonorrhoeae, 20 N lactamica, 39 N meningitidis, nine N mucosa, five N polysacchareae, eight N sicca, 37 N subflava, one N weaveri and 14 Moraxella (Branhamella) catarrhalis isolates. For each isolate, three to six colonies from a 24 h single colony subculture of each isolate were suspended in 1.0 ml of distilled water. Of this, 20 μl was added to 980 μl of cobas PCR media and tested in the cobas 4800 CT/NG assay as described above.
C trachomatis results
Of the 496 urine specimens (Path-Qld) tested for C trachomatis DNA, eight specimens were excluded from further calculations because they were flagged as either failed or invalid by the cobas 4800 system. Of the remaining 488 urine specimens, 86 provided positive results (53 men and 33 women; cobas 4800 cycle threshold values ranging from 25 to 40 cycles, mean and median 33 cycles) and 395 specimens provided negative results in both cobas 4800 and reference C trachomatis PCR assays. Seven urine specimens provided discrepant results; five specimens provided positive results in the reference C trachomatis assay but were negative in the cobas 4800 C trachomatis method, whereas two specimens were positive by the cobas 4800 assay only (samples A–G; table 2).
Of the 404 swab specimens (IMVS-SA) tested for C trachomatis DNA, one cervical swab specimen provided an invalid result by the cobas 4800 and was excluded from further calculations. Of the remaining 403 swab specimens, 23 provided positive results (10 cervical, one eye, two rectal, nine vaginal and one for which the site was not recorded; cobas 4800 cycle threshold values ranging from 26 to 37 cycles, mean 31 cycles, median 30 cycles) and 378 specimens provided negative results in both cobas 4800 and reference C trachomatis PCR assays. Two vaginal swabs (samples H and I; table 2) provided positive results in the reference C trachomatis assay but were negative in the cobas 4800 C trachomatis method.
N gonorrhoeae results
Of the 318 urine specimens tested for N gonorrhoeae DNA, six specimens were excluded from further calculations because they were flagged as either failed or invalid by the cobas 4800. Of the remaining 312, 13 specimens provided positive results (eight men and five women; cobas 4800 cycle threshold (Ct) values ranging from 24 to 36 cycles, mean 28 cycles, median 27 cycles) and 298 specimens provided negative results in both the cobas 4800 N gonorrhoeae assay and the reference N gonorrhoeae testing algorithm. One urine specimen was negative by the cobas 4800 N gonorrhoeae assay and positive in the reference testing algorithm (sample J; table 2). This specimen was positive in the opa component (39 cycles) but negative in the porA pseudogene component of the NG duplex supplementary assay used by Path-Qld.
Of the 180 swab specimens tested for N gonorrhoeae DNA, nine specimens provided positive results (seven vaginal and two for which the site was not recorded; cobas 4800 Ct values ranging from 28 to 39 cycles, mean and median 32 cycles) and 170 specimens provided negative results in both the cobas 4800 N gonorrhoeae assay and the reference N gonorrhoeae testing algorithm. One vaginal swab specimen was positive in the cobas 4800 N gonorrhoeae assay (Ct=39 cycles) but was considered negative in the reference algorithm because it was negative in the supplementary test (sample K; table 2).
Neisseria species panel
Upon testing the Neisseria species panel in the cobas 4800 N gonorrhoeae assay, all 147 non-gonococcal isolates provided negative results and all 76 gonococci provided positive results (Ct values ranging from 18 to 36 cycles, mean 26 cycles, median 24 cycles).
Specimens labelled ‘failed’ or ‘invalid’
In total, five specimens were flagged by the cobas 4800 as ‘failed’, indicating a failure to extract the specimen, and four specimens flagged as ‘invalid’, indicating that the internal control results were negative. This provided an overall extraction failure rate of 0.6% and an inhibition rate of 0.4% for the 900 clinical specimens tested. One urine specimen was positive for N gonorrhoeae by both the cobas 4800 (Ct=24 cycles) and reference assays but provided an ‘invalid’ result for the cobas 4800 C trachomatis reaction because the internal control was negative. Two urine specimens that failed to undergo DNA extraction in the cobas × 480 and therefore did not provide valid results for either C trachomatis or N gonorrhoeae in the cobas 4800 were positive for N gonorrhoeae DNA by the reference methods.
Retesting in both the cobas 4800 and TaqMan 48 assays was performed to further investigate the nine specimens providing discrepant C trachomatis results. Original aliquots of urine specimens stored at −20°C were retested. The original PBS suspensions for the two swab specimens that provided discrepant C trachomatis results were not available for retesting, therefore the remaining cobas PCR media suspensions of these specimens were split and tested in both assays. Upon retesting, all specimens provided agreement in both assays (table 2). The performance characteristics of the cobas 4800 were not re-calculated on the basis of these results. The two specimens providing discrepant N gonorrhoeae results were not retested.
Using the cobas 4800, a CT/NG test run could be performed in approximately 3.5 h for 96 samples or 2.75 h for 24 samples, once reagents were equilibrated at room temperature. This included daily and weekly maintenance (approximately 7 min each), new run set up (20–30 min), extraction (60–100 min) and PCR (approximately 90 min). New extraction runs could be initiated on the cobas × 480 while PCR on the cobas z 480 was still underway from a previous run.
The cobas 4800 CT/NG assay proved to be highly suitable for high through-put identification of C trachomatis and N gonorrhoeae nucleic acid. The system automation enables three to four 96-sample runs to be performed within a standard working day by a single staff member. Further, the results of the cobas 4800 CT/NG assay gave good agreement with established methods. A number of discrepant results were observed, particularly for C trachomatis, but all these samples provided cycle threshold (Ct) values at or above cycle 36 and were among the highest seen in the study. Ct values are inversely proportional to DNA load, indicating that these samples represent the lowest DNA loads. Thus, sampling problems associated with low DNA load may account for some of these discrepancies. Specimen handling difficulties may also have contributed to discrepant results, including the fact that after reference assay testing, specimens were stored at 4°C for up to 7 days before being added to the cobas PCR media for cobas 4800 testing, and therefore some DNA degradation may have occurred. This is supported by the fact that discrepant C trachomatis specimens later provided agreement upon retesting in both the cobas 4800 and reference tests. It should also be noted that the preparation of specimens in this evaluation is not consistent with manufacturer instructions now provided with the released cobas 4800 method. Notably, swab material should be diluted directly in the cobas PCR media, and not be prediluted in PBS as performed in this study. Also, the sample volumes used in this study (1–1.5 ml) are below the minimum sample volume of 4.0 ml now used for cobas 4800 testing. In fact, the low sample volumes used in this study may have contributed to ‘failed’ or ‘invalid’ specimens. However, this was not further investigated.
Notable performance characteristics of the new cobas 4800 system N gonorrhoeae assay were a specificity and positive predictive value of 100% for the Path-Qld urine specimens and 99.4% and 90.0% for the IMVS-SA swab specimens. Only one vaginal specimen was considered to have produced a false-positive result in the cobas 4800 N gonorrhoeae assay (sample K; table 2) on the basis that it was negative in the supplementary porA pseudogene test. However, the fact that this specimen was positive by the both the COBAS AMPLICOR and cobas 4800 N gonorrhoeae assays is more consistent with this specimen being a true N gonorrhoeae-positive specimen and a false-negative result in the porA pseudogene method due to sampling at low load. The poor specificity of the predecessor Roche COBAS AMPLICOR N gonorrhoeae assay is well recognised, with reported positive predictive values (PPVs) as low as 31%.1
To provide further evidence of this problem, we re-examined our data to calculate the overall PPV of the COBAS AMPLICOR method in this study; in total, 41 specimens were positive by the AMPLICOR method, of which only 25 were positive by the supplementary assays, providing a PPV for the COBAS AMPLICOR N gonorrhoeae assay of 61%. In this area of platform and assay weakness, the cobas 4800 N gonorrhoeae assay represents a clinically important improvement over the COBAS AMPLICOR N gonorrhoeae method. It should be noted, however, that positive specimens were oversampled in this study to allow for a meaningful assessment of sensitivity using the cobas 4800 platform, and that PPVs may be raised and negative predictive values lowered compared with those that might be expected in routine testing in a lower prevalence setting.
In addition, further studies are needed to establish the performance of the cobas 4800 CT/NG in other patient populations as well as on extragenital specimens, including throat and rectal swabs. Notwithstanding the results of this study, Australian testing guidelines recommend the use of supplementary testing for gonococcal NAATs.4
In conclusion, our results show the new cobas 4800 CT/NG assay is sensitive and specific for the detection of C trachomatis and N gonorrhoeae in urogenital specimens. In addition, the two-target system of the C trachomatis assay decreases the potential for sequence-related, false-negative results as experienced with the Swedish C trachomatis variant.
The cobas 4800 CT/NG test is suitable for high through-put identification of C trachomatis and N gonorrhoeae infections.
The two-target system of the cobas 4800 C trachomatis assay decreases the potential for sequence-related, false-negative results as previously experienced with the Swedish C trachomatis variant.
The cobas 4800 N gonorrhoeae assay was used to test a Neisseria species panel comprising 147 non-gonococcal isolates and no false-positive results were seen.
This study was conducted as part of the reference work of the Australian National Neisseria Network.
Funding Financial support was provided by Roche Diagnostics, Australia, for specimen transport and cobas 4800 CT/NG testing costs.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.