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Evaluation of Cepheid SmartCycler and EuroClone Duplicareal time Dual Easy CT assay for confirmation of Chlamydia trachomatis

Evaluation of Cepheid SmartCycler and EuroClone Duplicareal time Dual Easy CT assay for... BioscienceHorizons Volume 6 2013 10.1093/biohorizons/hzt005 Research article Evaluation of Cepheid SmartCycler and EuroClone real time Duplica Dual Easy CT assay for confirmation of Chlamydia trachomatis 1,2* Tolulope Oyebanji Faculty of Applied Science, University of Sunderland, Sciences Complex, City Campus, Chester Road, Sunderland, Tyne and Wear SR1 3SD, UK Gateshead Health NHS Foundation Trust Sheriff Hill, Queen Elizabeth Hospital, Gateshead, Tyne and Wear NE9 6SX, UK *Corresponding author: Microbiology Department, Scarborough General Hospital, Woodlands Drive, Scarborough, North Yorkshire YO12 6QL, UK. Tel: +44 07960391320. Email:tolu.oyebanji@live.co.uk 1 2 2 Supervisors: Lewis Bingle ; Nicole Gaukrodger and Natasha Waterhouse Chlamydia trachomatis, a member of the Chlamydiaceae, is the most common cause of bacterial sexually transmitted infec- tions worldwide. The application of real-time polymerase chain reaction (PCR) in laboratory diagnostic testing for C. tracho- matis is becoming increasingly popular. Modern approaches sometimes involve a combination of different test technologies for screening and confirmation; this indeed is a Clinical Pathology Accreditation (CPA) recommendation. Multiple real-time PCR platforms and assays are currently available, each of which comes with slight variation in their performances. Before incorporating any of these into routine clinical use, accuracy and reliability must be assessed. This study evaluated the per- real time formance characteristics of the Cepheid SmartCycler used in conjunction with the EuroClone Duplica Dual Easy CT assay for the confirmation of Roche COBAS TaqMan positive tests by comparing results obtained by each method. Consecutive clinical C. trachomatis positive (n = 67) and random negative (n = 17) urogenital samples received at a general district hospi- tal in the North East of England over a 6-month period were examined. Of the total 67 TaqMan-positive samples, 62 (92.5%) of them were also positive by the SmartCycler/EuroClone. There were 5 discrepancies in total (7.5%) and 17 samples were negative on both Roche TaqMan and SmartCycler/EuroClone. The sensitivity and specificity of the method were 92.5 and 100%, respectively. To further assess its specificity, the SmartCycler/EuroClone method was challenged with additional organisms and proved to possess the ability of avoiding false-positive results by correctly identifying these as C. trachomatis DNA negative. Finally, it was found that confirmation of positive C. trachomatis by the SmartCycler/EuroClone method increases the turn around time of chlamydia investigation by 2-folds. In conclusion, the SmartCycler/EuroClone method gave good concordance with the established Roche TaqMan method and proved to be highly suitable for confirmation testing of C. trachomatis positive samples. The remarkable specificity and acceptable sensitivity also confirms its suitability and reliability for this purpose. Keywords: Chlamydia trachomatis, SmartCycler, Cepheid, EuroClone, evaluation, real-time PCR Received 14 January 2013; revised 18 April 2013; accepted 19 April 2013 Introduction common cause of bacterial sexually transmitted infections (Unemo et al., 2010), with an annual record of 50–70 million Chlamydia trachomatis, a member of the Chlamydiaceae cases worldwide (Paavonen and Lehtinen, 1996). Although (Demkin and Zimin, 2005; Unemo et al., 2010), is the most difficult to stain, they are Gram-negative, obligate intracellular © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 6 2013 bacteria (Demkin and Zimin, 2005; Pickett et al., 2005) that exhibit a complex and unique developmental cycle (Paavonen and Lehtinen, 1996; Pickett et al., 2005). Chlamydia trachomatis often remains undetected and untreated for an extended period of time due to the high per- centage of asymptomatic cases (Jaton, Bille and Greub, 2006). This potentially leads to complications such as pelvic inflammatory disease; hence the key approach is the early identification of infected individuals before disease progres - sion (Paavonen and Lehtinen, 1996; Jaton, Bille and Greub, 2006). Consequently, a significant emphasis is placed on screening. The National Chlamydia Screening Programme reported that 70 700 people were screened in the North East of England between April and December 2010. A project by Low et al. (2007) for the Chlamydia Screening Studies group shows that proactive screening for chlamydia in young adults using home-collected specimens was feasible and acceptable (Low et al., 2007). In this regard, rapid testing methods are essential in order to respond to this public health mandate. Figure 1. T ypical amplication curve of RT-PCR. Cycle threshold Modern approaches to laboratory diagnostic testing for C. (ct value) is the point at which the amplification curve of a reaction trachomatis sometimes involve the combinations of different crosses the threshold and hence, it is the point at which fluorescence is detected in the sample. Here, sample A with a ct value of 20 contains a test technologies for screening and confirmation ( Black, higher number of starting template than sample B whose ct value is 30. 1997). The use of an alternative method to confirm positive C. trachomatis is a Clinical Pathology Accreditation (CPA) recommendation. While in vitro tissue culture has in the past software and increased multiplexing (Logan and Edwards, been the gold standard, the application of real-time poly- 2009), which allow simultaneous detection of multiple tar- merase chain reaction (RT-PCR) in investigating chlamydia is gets (Low et al., 2007). However, there are variations relating fast growing, replacing conventional PCR and other nucleic to the performance of each of these platforms and assays. acid amplification tests (NAATs), due to the many advan - tages that it presents over the previously used methods Most of today’s commercially available NAATs target (Quinn et al., 1996; Rockett et al., 2010). both a sequence on the chlamydia cryptic plasmid (harboured by most strains) and the genome sequence (Pickett et al., Though the key principle of the reaction remains 2005; Unemo et al., 2010) due to previously reported prob- unchanged from the traditional PCR (developed in 1983 by lems with false-negative results caused by a 377 bp deletion Karry Mullis) (Shipley, 2006), RT-PCR is based on the detec- in the cryptic plasmid of the new Swedish variant of C. tra- tion of fluorescent signal emitted from fluorescently labelled chomatis, nvCT (serovar E). Employing the most widely used probes or primers. The increase in fluorescence is directly detection chemistry, TaqMan probes, the EuroClone proportional to the increase in the amplicon (Shipley, 2006) real time Duplica Dual Easy CT assay targets the cryptic plasmid and this basis provides a relative means of DNA quantifica - as well as the 16S ribosomal RNA (Demkin and Zimin, tion by reference to a generated standard curve (Theaker, 2005). On the other hand, the Roche COBAS® TaqMan® 2011) (Fig. 1). CT Test, v2.0 assay which also employs the TaqMan detec- RT-PCR occurs in closed system reactions, eliminating the tion chemistry targets the MOMP gene genomic DNA in requirement for post-PCR manipulations such as the use of addition to the cryptic plasmid (Roche TaqMan kit insert). gels for detection (Shipley, 2006); hence is less prone to con- The TaqMan detection probe is based on the use of the 5′ tamination (Heid et al., 1996; Jaton, Bille and Greub., 2006). nuclease activity of Taq polymerase to cleave a non-extend- It does not depend on the final amount of amplicon since it ible hybridization probe during the extension phase of PCR. allows continuous collection of fluorescent signal over a Dual-labelled fluorogenic hybridization probes are employed range of PCR cycles (Shipley, 2006). in this approach, where one fluorescent dye, usually FAM Today, multiple platforms and assays co-exists in the mar- (i.e. 6-carboxyfluorescein, usually excited at 488 nm), serves ket place, commonly used ones include Roche COBAS® as a reporter which emits fluorescent signal that is monitored TaqMan® 48 and the Cepheid SmartCycler (Shipley, 2006; in an assay. The other, usually TAMRA (i.e. 6-carboxy-tetra- Low et al., 2007). Since the invention of the 7700 instrument, methylrhodamine), serves as a quencher which is responsible the first PCR instrument by ABI ( Shipley, 2006), advances in for the initial quenching of the reporter signal (Shipley, 2006; RT-PCR instrumentation and assay include faster thermocy- Low et al., 2007) (mechanism illustrated in Fig. 2). A cling times, higher throughput, flexibility, more user-friendly less-expensive option is the use of DNA intercalating dyes 2 Bioscience Horizons • Volume 6 2013 Research article advanced nucleic acid analyser (HANAA; 30 cm × 30 cm in size) with a combination of 16 independent thermocyclers, which conduct rapid heating and cooling of plastic reaction tubes, sufficient for four simultaneous reactions ( Higgins et al., 2003; Low et al., 2007). This analyser was originally invented by the US army specifically to detect bioterrorism-related outbreaks of anthrax; however, it is now used for the detection of other pathogens (Low et al., 2007) including C. trachomatis and MRSA. Although it has been in use for approximately 7 years, the application of the SmartCycler for detecting C. trachomatis is recent. The real time EuroClone Duplica Dual Easy CT assay is CE marked and IVD approved for use on the Cephied SmartCycler. However, there are presently no published evaluations of the SmartCycler/EuroClone method. The EuroClone Duplica assay uses the TaqMan chemistry as previously described. The kit contains ready-to-use amplification reagents as well as internal control (IC) of amplification, which allows the validation of the reaction. While FAM serves as a reporter for the target sequence, another fluorophore contained in this assay, HEX (hexa-chloro-fluorsceine) serves as reporter for the other probe which detects the IC. Figure 2. A TaqMan probe assay. The reporter dye fluorescence when free in solution is suppressed by the quencher. During primer This study aimed to assess the performance characteristics annealing, the TaqMan probe binds to the complementary sequence of the SmartCycler/EuroClone method in comparison with at a temperature higher than the primer on the same strand. The 5′ that of the Roche TaqMan method which has been well nuclease activity of the Taq polymerase displaces and degrades the established in this clinical laboratory for the screening of probes, so that the reporter dye is released from the probe molecule. female cervical swabs, male urethral swabs and male/female The reporter and the quencher are no longer in close proximity; therefore, the full signal of the reporter is released. urine samples. The first objective was to compare the qualita - tive results obtained from both methods. Previous evaluation of the Roche TaqMan method yielded a 95% sensitivity and specificity, but equivalent data have not been provided for the such as SYBR Green; however, this is less specific ( Higgins EuroClone Duplica kit; hence, this study aimed to assess the et al., 2003). assay sensitivity. The specificity of the assay was also exam - As with most other clinical conditions, accurate diagnosis ined by adapting the method described by Rockett et al. of chlamydia is vital to avoid risk of falsely labelling a patient (2010). In summary, it was anticipated that the reliability of with a sexually transmitted infection (STI). Accordingly, this method in confirming the Roche TaqMan positive results prior to incorporation of a diagnostic method into routine would be established. clinical use, assessment of its performance must be ensured. Parameters for evaluating a test are detailed by the Analytical Materials and methods validity, Clinical validity, Clinical utility, Ethical, legal or social implications model. Analytical validity measures the Clinical specimens accuracy and precision of the test. Clinical validity is the accuracy with which a test identifies a patient’s clinical status A total of 86 clinical specimens from both symptomatic and (Van Der Schouw, Verbeek and Ruijs, 1992). asymptomatic patients submitted to the Microbiology depart- ment of a General District Hospital in the North East of In the clinical laboratory where this study was conducted, England for C. trachomatis testing were used in this study. screening and confirmation was done using the same RT-PCR The specimens that were collected over a 6-month period method: the Roche COBAS® TaqMan CT Test, v2.0, used in comprised 27 (31.4%) urine samples (4 female and 23 male), conjunction with the Roche COBAS® TaqMan analyser. 57 (66.3%) female cervical swabs and 2 (2.3%) male ure- This method is henceforth referred to as the Roche TaqMan thral swabs. Patient groups included those attending a sexual method. However, in order to conform fully to the CPA rec- health clinic, various GP patients and hospital inpatients. ommendation, an alternative method to use for the confirma - tion of Roche TaqMan positive tests was sought. This study Specimen collection and transport aimed to evaluate the alternative assay and platform prior to incorporation into routine use. The Cepheid SmartCycler Samples were collected from the patients by clinicians; urethra real instrument used in conjunction with the EuroClone Duplica and cervical swabs were suspended and transported in 3 ml time Dual Easy CT assay is a commercially available handheld M4RT® MicroTest culture transport system media (a general 3 Research article Bioscience Horizons • Volume 6 2013 Table 1. Selected thermal profile on the smart cycler multipurpose transport medium, contained in Remel tubes, which is also used for culture of viruses and chlamydia). Urine samples were transported in sterile non-boric acid containers. Fluorescent Time Temperature (°C) No. of cycles acquisition Extraction of nucleic acid 5 min 95 1 DNA was extracted from all samples using the NorDiag 15 s 95 Bullet instrument (with the NorDiag BUGS’n BEADS STI- ×10 Off fast kit), a platform that automates the procedure for the 60 s 68 extraction and purification of nucleic acids from the bacteria 15 s 95 if present in the sample. Samples and reagents were brought ×50 On to room temperature prior to commencing the procedure as 60 s 54 outlined by the manufacturer. Amplification and detection on the Cepheid Reference method Smart Cycler Qualitative results obtained from the SmartCycler/EuroClone The reagents for the amplification reaction on the Cepheid method were compared with those from the Roche TaqMan real time SmartCycler were provided in the EuroClone Duplica method (Table 2). The sensitivity and specificity produced by Dual Easy CT assay. each method was also determined. Preparation of the master mix Test of specificity The master mix for each reaction was prepared according to The specificity of the SmartCycler/EuroClone method in com - manufacturer’s instructions by mixing together 10 µ l amplifi - parison with that of the Roche TaqMan was assessed by per- cation mix (containing hot start Taq DNA polymerase, forming the above assay on three non-chlamydial bacteria. nucleotides and MgCl ), 10 µ l oligo mix (containing primers These were Escherichia coli, Neisseria gonorrhoeae and and probes) and 1 µ l control 2 (the internal amplification Moraxella catarrhalis isolated from clinical specimens. The control). The amplification mix, oligo mix and control were organisms were selected on the basis that they can also be found provided in the EuroClone Duplica kit. in the human genital tract, as either commensals or pathogens. The method used for this investigation was an adaptation of Preparation of the reaction mix that used by Rockett et al. (2010). In this study, five standard For each sample, 5 µ l of DNA extract was added to the 21 µ l colonies taken from a 24 h single colony subculture of each master mix contained in the Cephied PCR reaction tube. isolate were suspended in 1 ml of distilled water. Twenty micro- litres of this suspension was then transferred into a Remel tube Quality control containing 980 µ l of viral transport media M4RT to provide similar conditions to the test samples. These were then extracted An IC (control 2) was used to validate the amplification pro - by an automated DNA extraction platform (Nordiag Bullet) cess within the instrument. In addition, the EuroClone kit and amplified on the SmartCycler as described previously. also contains Control 1 (a positive control of amplification) and a reaction blank. Five microlitres of the positive control was added to 21 µ l of master mix. Five microlitres of reaction Results blank was also added to another 21 µ l master mix. These acted as positive and negative controls of the procedure, A total of 86 samples that were initially tested by the Roche respectively. TaqMan method were re-tested by the SmartCycler/ EuroClone method. This comprises 69 (80.2%) consecutive Cepheid tubes were then capped after the addition of the Roche TaqMan positive specimens which were stored at specimen, control 1 or reaction blank before brief centrifuga- −20°C prior to re-extraction and retesting by the SmartCycler/ tion and then placed into the Cepheid SmartCycler. EuroClone method. Of these, 34 samples were re-extracted Thermal cycling and retested by the Roche TaqMan for confirmation before retesting on the SmartCycler. This was to validate changing The equipment was switched on and appropriate thermal protocols. The remaining 35 positive samples were only profile was selected prior to preparation of the reaction mix. tested once on Roche TaqMan. Out of the 34 specimens that The thermal profile selected is as shown in Table 1. were tested twice by the Roche TaqMan method, 2 specimens were excluded from further calculations because they had Once the reaction mix and controls were prepared and yielded an indeterminate (equivocal) result (positive first run placed into the heating block, the real-time PCR process was but negative second run) on the Roche TaqMan. Essentially, started. The PCR run lasted a total time of 1.5 h on the this implies that the decision is not reached as to whether the instrument. 4 Bioscience Horizons • Volume 6 2013 Research article patient is positive or negative by this method; therefore, clini- specificity were 92.5 and 100%, respectively, as determined by cians are advised to resample from the patient and submit for equations (1) and (2). The positive predictive value (PPV) that retest. From the 67 remaining positive specimens, 2 samples was determined by equation (3) was 100%; negative predictive were unresolved on the SmartCycler/EuroClone method. A value (NPV) that was determined by equation (4) was 77.3%. repeat test was performed and both yielded a positive result, TN hence were included among the ‘positives’. Seventeen ran- %Specificity = × 100 (1) () FP + TN domly selected Roche TaqMan negative samples (19.8%) were re-extracted and retested by the SmartCycler/EuroClone TP method to quality assure the method. %Sensitivity = ×100 (2) () TP + FN A summary of the comparison analysis of qualitative results of the SmartCycler/EuroClone method and the Roche TP TaqMan method is provided in Table 2. PPV% = ×100 (3) () TP + FP Out of the total 86 samples tested, 27 were urine specimens and the remaining 59 were swabs. Of the urine samples, 3 TN NPV% = ×100 (4) (13.6%) of those that were positive on the RocheTaqMan were () FN + TN negative when tested by the SmartCycler/EuroClone method. Of the Roche TaqMan positive swabs, two (4.4%) resulted where TN is the true negative; FN the false negative; TP the negative by the SmartCycler/EuroClone method. Table 3 shows true positive; FP the false positive (Source: Coulthard, a summary of the performance characteristic of the SmartCycler/ 2007). EuroClone; out of the total 67 Roche TaqMan positive sam- ples, 62 (92.5%) of them were also positive by the SmartCycler/ Clinical specificity EuroClone method. There were 5 discrepancies in total (7.5%) The additional organisms (E. coli, N. gonorrhoeae and M. and 17 samples were negative on both Roche TaqMan and catarrhalis) used in testing the clinical specificity of the SmartCycler/EuroClone. The analytical sensitivity and Table 2. Comparison of qualitative results of the SmartCycler/EuroClone and Roche TaqMan methods Reference results (Roche Additional TaqMan) results (Roche Totals Taqman Positive (n) Negative (n) equivocal) Smart cycler/EuroClone Positive 19 0 1 19 Urine (n = 27) Negative 3 4 7 Positive 43 0 43 Swab (cervical and urethra) (n = 59) Negative 2 13 1 15 Totals 67 17 2 84 Total excluding the sample(s) with the equivocal Roche TaqMan results. Two of these samples were initially unresolved on the SmartCycler/EuroClone method, but positive on the repeat test. Table 3. Performance characteristics of the SmartCycler/EuroClone method for urine and swab specimens Reference results (TaqMan) PPV% NPV% Sens% Spec% Positive Negative Total SmartCycler Positive 62 0 62 Negative 5 17 22 100 77.3 92.5 100 Total 67 17 84 PPV, positive predictive value; NPV, negative predictive value; Sens, sensitivity; Spec, specificity. 5 Research article Bioscience Horizons • Volume 6 2013 Table 4. Assay results for additional organisms tested by the A number of discrepancies (n = 5) were observed in this SmartCycler/EuroClone method study as explained in the Results section. On the note that Ct values are inversely proportional to DNA load (Rockett et al., Bacterium Assay result 2010); three of the discrepant results had Ct values >38 on the Roche TaqMan and are hence considered weak positive. E. coli Negative Further, one of these three samples had a Ct value of 41.8 on the Roche TaqMan and 46.4 on the SmartCycler/EuroClone. N. gonorrhoeae Negative This suggests two possibilities: either that the sensitivity of M. catarrhalis Negative SmartCycler/EuroClone is not as high as that of the Roche TaqMan when detecting minute starting quantities of DNA, or that these are indeed Roche TaqMan false positives. SmartCycler/EuroClone method all gave a negative result as The clinical specificity was well supported by the analyti - presented in Table 4. cal specificity of 100% and a correlation with the calculated PPV. The SmartCycler/EuroClone method proved able to Discussion avoid false positives by correctly identifying M. catarrhalis, E. coli and N. gonorrhoeae as C. trachomatis DNA negative. The results of the SmartCyler/Euroclone method gave good These additional organisms are commonly part of the genital agreement with the established Roche TaqMan method and flora that may also be present in clinical samples submitted proved to be suitable for the detection of C. trachomatis. for chlamydia testing; hence, we have demonstrated that the Most of the Roche TaqMan positive samples were also found SmartCycler/EuroClone can specifically discriminate between to be positive by the SmartCycler/EuroClone method. All other urogenital organisms and C. trachomatis. Studies such Roche TaqMan negative samples were also found to be nega- as Dicker et al. (2000) noted that co-infection of chlamydia tive on the SmartCycler/EuroClone (Table 4). with N. gonorrhoeae is common by including N. gonor- Sensitivity and specificity are important measures for diag - rhoeae as one of the control organisms; we have shown that nostic purposes because they allow the possibility of calculating the SmartCycler/EuroClone method is also able to discrimi- the probability of a disease being present in a particular patient nate between these two STIs. It should also be noted that (Van Der Schouw, 1992). Coulthard (2006) has provided a only three additional organisms were tested, using more uro- detailed definition of each of these: sensitivity was defined as genital organisms such as lactobacilli and Candida albicans, the percentage of true cases that resulted positive and specificity would have been advantageous. as percentage of unaffected individuals found to be test nega- Further explanation of the discrepancies seen in this study tive. He noted that although robust and often used to report the include DNA degradation; samples were archived at −20°C power of diagnostic tests, sensitivity and specificity parameters for up to 3 days before testing by the SmartCycler/EuroClone have limited clinical usefulness if used alone. Hence, they are and therefore some DNA degradation might have occurred; commonly converted to other terms such as PPV and NPV hence if these weak Roche TaqMan positive samples were which address the likelihood of a particular diagnosis from the truly positive, all the present C. trachomatis DNA might have perspective of a known test result. PPV is the fraction of true degraded while in archive, causing a negative result on the positive cases among all the patients with positive test results. SmartCycler/EuroClone assay. Notwithstanding, no attempts NPV, on the other hand, is the fraction of correctly diagnosed were made to retest these samples with a discrepant result true negative cases (Einstein, Boidian and Gill, 1997; Coulthard, due to limitations of fund. It would have been interesting to 2006). Other validation test parameters have also been sug- see how the result differs upon repeat testing; however, this gested (Van Der Schouw, 1992). The sensitivity of the Roche may not be worth it because oversampling of the specimen TaqMan in comparison with the BD ProbeTec ET test on urine may also lead to false-negative result. Furthermore, possibly specimens and endocervical swabs was reported to be 95.7 and TaqMan probes begin to degrade after repeated thermal 96.0%, respectively (Roche TaqMan kit insert); whereas, in cycles as the fluorophore begins to cleave due to repeated this study, the analytical sensitivity of the SmartCycler/ series of heating and cooling that they have been subjected to. EuroClone in comparison with the Roche TaqMan was 92.5%, resulting in a low NPV of 77.3. This is of considerable concern Although it can be argued that using Roche TaqMan as a because it suggests that in a population of 100 chlamydia posi- reference method of this study is reliable enough since this tive patients, ~20 of them will be missed, leading to disease method has been previously evaluated, an improved study progression and potential complications such as PID in women methodology would be to confirm all Roche TaqMan results, and sterility in men. A more reliable approach for determining particularly the positives, by sending them to the reference the sensitivity of an assay is the method used by Jaton, Bille and laboratory before testing by the SmartCycler/EuroClone Greub (2006), who determined the analytical sensitivity of their method. However, a problem with this is that it would fur- PCR assay by performing a 10-fold serial dilution of the posi- ther increase the gap of testing periods between the Roche tive control template. If resources allowed, this study would TaqMan method and the SmartCycler/EuroClone method, have also adopted this approach. subjecting sample DNA to further degradation. 6 Bioscience Horizons • Volume 6 2013 Research article Performance of other commercial methods have also been Acknowledgements impressive for the detection of C. trachomatis; a recent study which evaluated the Abbott RealTime CT/NG assay in com- First and foremost, I am indebted to all staff at the microbiol- parison with the Roche Cobas Amplicor CT/NG assay found ogy department of Queen Elizabeth hospital, Gateshead 99.6 and 97.7% positive and negative agreement respectively NHS Foundation Trust, for the opportunity granted me to for urine samples (Cheng, Qian and Kirby, 2011). Another complete this research project at their institute. Not only did recent study by Rocket et al. found that the sensitivity, speci- they let me use their facilities, but also provided tremendous ficity and NPVs and PPVs of the new Cobas 4800 CT/NG for support. My sincere appreciation to Miss Nicole Gaukrodger urine samples were 94.5, 99.5, 98.8 and 97.7%, respectively, and Mrs Natasha Waterhouse who both supervised my work for C. trachomatis, while for swabs, these were 92.0, 100, at the hospital and persevered my inquisitiveness. I am 99.5 and 100%, respectively. The performance characteristic equally grateful to Dr Lewis Bingle, who as my University of the SmartCycler/EuroClone assay is comparable to these project supervisor showed tremendous support and also per- widely known methods. Interestingly, this method has very severed my inquisitiveness. My gratitude also to Amy similar sensitivity and specificity with the Cobas 4800 CT/ Openshaw, Launch Diagnostic UK, for providing me with NG assay. additional information relevant to my study. A 2-fold increase in TAT (turn around time) was observed Author biography when positive chlamydia investigations were confirmed by the SmartCycler/EuroClone. TAT is defined as the total time Tolu graduated in July 2012 with a first class BSc in Applied taken between the submission of a sample for investigation Biomedical Science from the University of Sunderland. After and the issue of report. This observation is in fact as a result her second year, she completed a placement year in the of limiting testing by the SmartCycler/EuroClone method to Microbiology department at Queen Elizabeth Hospital, just twice per week, whereas testing was done daily on the Gateshead, where this study was subsequently undertaken. Roche TaqMan. Therefore, this does not reflect the speed of Towards the end of her final year, Tolu was awarded SfAM’s the method as reaction time on the SmartCycler analyser is (Society for Applied Microbiology) ‘Students into Work’ typically 1.5 h. Perhaps if this method was used for this pur- grant which gave her an opportunity to carry out an 8-week pose in a region with higher prevalence of the infection, this research project, which involved screening a library of syn- effect may not be seen. thetic chemical compounds for possible anti-pathogenicity To conclude, this study has demonstrated that the activities. She also took the opportunity to learn about and SmartCycler/EuroClone method can sufficiently confirm get involved in other areas of antimicrobial research. During Roche TaqMan positive in a clinical microbiology labora- this time, Tolu was also appointed as a Biomedical Scientist tory. The specificity of the method is remarkable, whereas the at Scarborough General Hospital. Her major interest areas sensitivity gives a little concern, but is still acceptable. The are in medical microbiology, particularly in diagnostic meth- discrepant results highlight the importance of confirming ods, antimicrobial and control of infection. positive results on an alternative method, supporting the rec- ommendation by the CPA. Using this method to confirm References positive chlamydia increases TAT, but this is peculiar to our laboratory and may differ in other laboratories, depending Black, C. M. (1997) Current methods of laboratory diagnosis of on their workload. Chlamydia trachomatis infections, Clinical Microbiology Reviews, 10, 160–184. Further work Cepheid (2011) SmartCycler system, accessed at: http://www.cepheid. c om/sy st ems-and-sof t w ar e/smar t c y cler -sy st em/ (11 D ec ember Cheng et al. pointed out that clinicians should be aware of 2011). the potential for inadequate detection of dual infections with some of the currently used NAAT methods; hence, Cheng, A., Qian, Q. and Kirby, J. E. (2011) Evaluation of the Abbott they suggested that clinical laboratories should thoroughly RealTime CT/NG assay in comparison to the Roche Cobas Amplicor evaluate multiplex PCR for adequacy in detecting dual CT/NG Assay, Journal of Clinical Microbiology, 49 (4), 1294–1300. infections (Cheng, Qian and Kirby, 2011). Therefore, Coulthard, M. J. (2006) Quantifying how tests reduce diagnostic uncer- assessment of the ability of the SmartCycler/EuroClone tainty, Archives of Disease in Childhood, 92, 404–408. method in detecting dual infections of C. trachomatis with N. gonorrhoeae is a point of interest. The SmartCycler Demkin, V. V. and Zimin, A. L. (2005) A new amplification target for PCR– should also be evaluated for use with other STI assays, for RFLP detection, Archives of Microbiology, 183, 160–165. investigations such as syphilis. Another point of interest was to see the cost difference between the two methods but Einstein, A. J., Boidian, C. A. and Gill, J. (1997) The relationships among my study was limited in this respect due to a lack of ade- performance measures in the selection of diagnostic tests, Archives quate costing information. of Pathology and Laboratory Medicine, 121 (1), 110–117. 7 Research article Bioscience Horizons • Volume 6 2013 Heid, C. A., Stevens, J., Livak, K. J. et al. (1996) Real time quantitative PCR, Pickett, M. A., Everson, J. S., Pead, P. J. et  al. (2005) The plasmids of Genome Research, 6, 986–994. Chlamydia trachomatis and Chlamydia pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plas - Higgins, J. 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NHS vital signs 2010/11: new variant of Chlamydia trachomatis: genome sequence, morphol- Primary Care Trust (PCT) and Strategic Health Authority (SHA) spe- ogy, cell tropism and phenotypic characterization, Microbiology, 156 cific tables 1st April to 31st December 2010 (available online 3rd (1), 1394–1401. February 2011), accessed at: (15 December 2011). Van der Schouw, Y. T., Verbeek, A. L. and Ruijs, J. H. (1992) ROC curves for Paavonen, J. and Lehtinen, M. (1996) Chlamydial pelvic inflammatory the initial assessment of new diagnostic tests, Family Practice, 99 (4), disease, Human Reproduction Update, 2 (6), 519–529. 506–511. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Evaluation of Cepheid SmartCycler and EuroClone Duplicareal time Dual Easy CT assay for confirmation of Chlamydia trachomatis

Bioscience Horizons , Volume 6 – Jun 4, 2013

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BioscienceHorizons Volume 6 2013 10.1093/biohorizons/hzt005 Research article Evaluation of Cepheid SmartCycler and EuroClone real time Duplica Dual Easy CT assay for confirmation of Chlamydia trachomatis 1,2* Tolulope Oyebanji Faculty of Applied Science, University of Sunderland, Sciences Complex, City Campus, Chester Road, Sunderland, Tyne and Wear SR1 3SD, UK Gateshead Health NHS Foundation Trust Sheriff Hill, Queen Elizabeth Hospital, Gateshead, Tyne and Wear NE9 6SX, UK *Corresponding author: Microbiology Department, Scarborough General Hospital, Woodlands Drive, Scarborough, North Yorkshire YO12 6QL, UK. Tel: +44 07960391320. Email:tolu.oyebanji@live.co.uk 1 2 2 Supervisors: Lewis Bingle ; Nicole Gaukrodger and Natasha Waterhouse Chlamydia trachomatis, a member of the Chlamydiaceae, is the most common cause of bacterial sexually transmitted infec- tions worldwide. The application of real-time polymerase chain reaction (PCR) in laboratory diagnostic testing for C. tracho- matis is becoming increasingly popular. Modern approaches sometimes involve a combination of different test technologies for screening and confirmation; this indeed is a Clinical Pathology Accreditation (CPA) recommendation. Multiple real-time PCR platforms and assays are currently available, each of which comes with slight variation in their performances. Before incorporating any of these into routine clinical use, accuracy and reliability must be assessed. This study evaluated the per- real time formance characteristics of the Cepheid SmartCycler used in conjunction with the EuroClone Duplica Dual Easy CT assay for the confirmation of Roche COBAS TaqMan positive tests by comparing results obtained by each method. Consecutive clinical C. trachomatis positive (n = 67) and random negative (n = 17) urogenital samples received at a general district hospi- tal in the North East of England over a 6-month period were examined. Of the total 67 TaqMan-positive samples, 62 (92.5%) of them were also positive by the SmartCycler/EuroClone. There were 5 discrepancies in total (7.5%) and 17 samples were negative on both Roche TaqMan and SmartCycler/EuroClone. The sensitivity and specificity of the method were 92.5 and 100%, respectively. To further assess its specificity, the SmartCycler/EuroClone method was challenged with additional organisms and proved to possess the ability of avoiding false-positive results by correctly identifying these as C. trachomatis DNA negative. Finally, it was found that confirmation of positive C. trachomatis by the SmartCycler/EuroClone method increases the turn around time of chlamydia investigation by 2-folds. In conclusion, the SmartCycler/EuroClone method gave good concordance with the established Roche TaqMan method and proved to be highly suitable for confirmation testing of C. trachomatis positive samples. The remarkable specificity and acceptable sensitivity also confirms its suitability and reliability for this purpose. Keywords: Chlamydia trachomatis, SmartCycler, Cepheid, EuroClone, evaluation, real-time PCR Received 14 January 2013; revised 18 April 2013; accepted 19 April 2013 Introduction common cause of bacterial sexually transmitted infections (Unemo et al., 2010), with an annual record of 50–70 million Chlamydia trachomatis, a member of the Chlamydiaceae cases worldwide (Paavonen and Lehtinen, 1996). Although (Demkin and Zimin, 2005; Unemo et al., 2010), is the most difficult to stain, they are Gram-negative, obligate intracellular © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 6 2013 bacteria (Demkin and Zimin, 2005; Pickett et al., 2005) that exhibit a complex and unique developmental cycle (Paavonen and Lehtinen, 1996; Pickett et al., 2005). Chlamydia trachomatis often remains undetected and untreated for an extended period of time due to the high per- centage of asymptomatic cases (Jaton, Bille and Greub, 2006). This potentially leads to complications such as pelvic inflammatory disease; hence the key approach is the early identification of infected individuals before disease progres - sion (Paavonen and Lehtinen, 1996; Jaton, Bille and Greub, 2006). Consequently, a significant emphasis is placed on screening. The National Chlamydia Screening Programme reported that 70 700 people were screened in the North East of England between April and December 2010. A project by Low et al. (2007) for the Chlamydia Screening Studies group shows that proactive screening for chlamydia in young adults using home-collected specimens was feasible and acceptable (Low et al., 2007). In this regard, rapid testing methods are essential in order to respond to this public health mandate. Figure 1. T ypical amplication curve of RT-PCR. Cycle threshold Modern approaches to laboratory diagnostic testing for C. (ct value) is the point at which the amplification curve of a reaction trachomatis sometimes involve the combinations of different crosses the threshold and hence, it is the point at which fluorescence is detected in the sample. Here, sample A with a ct value of 20 contains a test technologies for screening and confirmation ( Black, higher number of starting template than sample B whose ct value is 30. 1997). The use of an alternative method to confirm positive C. trachomatis is a Clinical Pathology Accreditation (CPA) recommendation. While in vitro tissue culture has in the past software and increased multiplexing (Logan and Edwards, been the gold standard, the application of real-time poly- 2009), which allow simultaneous detection of multiple tar- merase chain reaction (RT-PCR) in investigating chlamydia is gets (Low et al., 2007). However, there are variations relating fast growing, replacing conventional PCR and other nucleic to the performance of each of these platforms and assays. acid amplification tests (NAATs), due to the many advan - tages that it presents over the previously used methods Most of today’s commercially available NAATs target (Quinn et al., 1996; Rockett et al., 2010). both a sequence on the chlamydia cryptic plasmid (harboured by most strains) and the genome sequence (Pickett et al., Though the key principle of the reaction remains 2005; Unemo et al., 2010) due to previously reported prob- unchanged from the traditional PCR (developed in 1983 by lems with false-negative results caused by a 377 bp deletion Karry Mullis) (Shipley, 2006), RT-PCR is based on the detec- in the cryptic plasmid of the new Swedish variant of C. tra- tion of fluorescent signal emitted from fluorescently labelled chomatis, nvCT (serovar E). Employing the most widely used probes or primers. The increase in fluorescence is directly detection chemistry, TaqMan probes, the EuroClone proportional to the increase in the amplicon (Shipley, 2006) real time Duplica Dual Easy CT assay targets the cryptic plasmid and this basis provides a relative means of DNA quantifica - as well as the 16S ribosomal RNA (Demkin and Zimin, tion by reference to a generated standard curve (Theaker, 2005). On the other hand, the Roche COBAS® TaqMan® 2011) (Fig. 1). CT Test, v2.0 assay which also employs the TaqMan detec- RT-PCR occurs in closed system reactions, eliminating the tion chemistry targets the MOMP gene genomic DNA in requirement for post-PCR manipulations such as the use of addition to the cryptic plasmid (Roche TaqMan kit insert). gels for detection (Shipley, 2006); hence is less prone to con- The TaqMan detection probe is based on the use of the 5′ tamination (Heid et al., 1996; Jaton, Bille and Greub., 2006). nuclease activity of Taq polymerase to cleave a non-extend- It does not depend on the final amount of amplicon since it ible hybridization probe during the extension phase of PCR. allows continuous collection of fluorescent signal over a Dual-labelled fluorogenic hybridization probes are employed range of PCR cycles (Shipley, 2006). in this approach, where one fluorescent dye, usually FAM Today, multiple platforms and assays co-exists in the mar- (i.e. 6-carboxyfluorescein, usually excited at 488 nm), serves ket place, commonly used ones include Roche COBAS® as a reporter which emits fluorescent signal that is monitored TaqMan® 48 and the Cepheid SmartCycler (Shipley, 2006; in an assay. The other, usually TAMRA (i.e. 6-carboxy-tetra- Low et al., 2007). Since the invention of the 7700 instrument, methylrhodamine), serves as a quencher which is responsible the first PCR instrument by ABI ( Shipley, 2006), advances in for the initial quenching of the reporter signal (Shipley, 2006; RT-PCR instrumentation and assay include faster thermocy- Low et al., 2007) (mechanism illustrated in Fig. 2). A cling times, higher throughput, flexibility, more user-friendly less-expensive option is the use of DNA intercalating dyes 2 Bioscience Horizons • Volume 6 2013 Research article advanced nucleic acid analyser (HANAA; 30 cm × 30 cm in size) with a combination of 16 independent thermocyclers, which conduct rapid heating and cooling of plastic reaction tubes, sufficient for four simultaneous reactions ( Higgins et al., 2003; Low et al., 2007). This analyser was originally invented by the US army specifically to detect bioterrorism-related outbreaks of anthrax; however, it is now used for the detection of other pathogens (Low et al., 2007) including C. trachomatis and MRSA. Although it has been in use for approximately 7 years, the application of the SmartCycler for detecting C. trachomatis is recent. The real time EuroClone Duplica Dual Easy CT assay is CE marked and IVD approved for use on the Cephied SmartCycler. However, there are presently no published evaluations of the SmartCycler/EuroClone method. The EuroClone Duplica assay uses the TaqMan chemistry as previously described. The kit contains ready-to-use amplification reagents as well as internal control (IC) of amplification, which allows the validation of the reaction. While FAM serves as a reporter for the target sequence, another fluorophore contained in this assay, HEX (hexa-chloro-fluorsceine) serves as reporter for the other probe which detects the IC. Figure 2. A TaqMan probe assay. The reporter dye fluorescence when free in solution is suppressed by the quencher. During primer This study aimed to assess the performance characteristics annealing, the TaqMan probe binds to the complementary sequence of the SmartCycler/EuroClone method in comparison with at a temperature higher than the primer on the same strand. The 5′ that of the Roche TaqMan method which has been well nuclease activity of the Taq polymerase displaces and degrades the established in this clinical laboratory for the screening of probes, so that the reporter dye is released from the probe molecule. female cervical swabs, male urethral swabs and male/female The reporter and the quencher are no longer in close proximity; therefore, the full signal of the reporter is released. urine samples. The first objective was to compare the qualita - tive results obtained from both methods. Previous evaluation of the Roche TaqMan method yielded a 95% sensitivity and specificity, but equivalent data have not been provided for the such as SYBR Green; however, this is less specific ( Higgins EuroClone Duplica kit; hence, this study aimed to assess the et al., 2003). assay sensitivity. The specificity of the assay was also exam - As with most other clinical conditions, accurate diagnosis ined by adapting the method described by Rockett et al. of chlamydia is vital to avoid risk of falsely labelling a patient (2010). In summary, it was anticipated that the reliability of with a sexually transmitted infection (STI). Accordingly, this method in confirming the Roche TaqMan positive results prior to incorporation of a diagnostic method into routine would be established. clinical use, assessment of its performance must be ensured. Parameters for evaluating a test are detailed by the Analytical Materials and methods validity, Clinical validity, Clinical utility, Ethical, legal or social implications model. Analytical validity measures the Clinical specimens accuracy and precision of the test. Clinical validity is the accuracy with which a test identifies a patient’s clinical status A total of 86 clinical specimens from both symptomatic and (Van Der Schouw, Verbeek and Ruijs, 1992). asymptomatic patients submitted to the Microbiology depart- ment of a General District Hospital in the North East of In the clinical laboratory where this study was conducted, England for C. trachomatis testing were used in this study. screening and confirmation was done using the same RT-PCR The specimens that were collected over a 6-month period method: the Roche COBAS® TaqMan CT Test, v2.0, used in comprised 27 (31.4%) urine samples (4 female and 23 male), conjunction with the Roche COBAS® TaqMan analyser. 57 (66.3%) female cervical swabs and 2 (2.3%) male ure- This method is henceforth referred to as the Roche TaqMan thral swabs. Patient groups included those attending a sexual method. However, in order to conform fully to the CPA rec- health clinic, various GP patients and hospital inpatients. ommendation, an alternative method to use for the confirma - tion of Roche TaqMan positive tests was sought. This study Specimen collection and transport aimed to evaluate the alternative assay and platform prior to incorporation into routine use. The Cepheid SmartCycler Samples were collected from the patients by clinicians; urethra real instrument used in conjunction with the EuroClone Duplica and cervical swabs were suspended and transported in 3 ml time Dual Easy CT assay is a commercially available handheld M4RT® MicroTest culture transport system media (a general 3 Research article Bioscience Horizons • Volume 6 2013 Table 1. Selected thermal profile on the smart cycler multipurpose transport medium, contained in Remel tubes, which is also used for culture of viruses and chlamydia). Urine samples were transported in sterile non-boric acid containers. Fluorescent Time Temperature (°C) No. of cycles acquisition Extraction of nucleic acid 5 min 95 1 DNA was extracted from all samples using the NorDiag 15 s 95 Bullet instrument (with the NorDiag BUGS’n BEADS STI- ×10 Off fast kit), a platform that automates the procedure for the 60 s 68 extraction and purification of nucleic acids from the bacteria 15 s 95 if present in the sample. Samples and reagents were brought ×50 On to room temperature prior to commencing the procedure as 60 s 54 outlined by the manufacturer. Amplification and detection on the Cepheid Reference method Smart Cycler Qualitative results obtained from the SmartCycler/EuroClone The reagents for the amplification reaction on the Cepheid method were compared with those from the Roche TaqMan real time SmartCycler were provided in the EuroClone Duplica method (Table 2). The sensitivity and specificity produced by Dual Easy CT assay. each method was also determined. Preparation of the master mix Test of specificity The master mix for each reaction was prepared according to The specificity of the SmartCycler/EuroClone method in com - manufacturer’s instructions by mixing together 10 µ l amplifi - parison with that of the Roche TaqMan was assessed by per- cation mix (containing hot start Taq DNA polymerase, forming the above assay on three non-chlamydial bacteria. nucleotides and MgCl ), 10 µ l oligo mix (containing primers These were Escherichia coli, Neisseria gonorrhoeae and and probes) and 1 µ l control 2 (the internal amplification Moraxella catarrhalis isolated from clinical specimens. The control). The amplification mix, oligo mix and control were organisms were selected on the basis that they can also be found provided in the EuroClone Duplica kit. in the human genital tract, as either commensals or pathogens. The method used for this investigation was an adaptation of Preparation of the reaction mix that used by Rockett et al. (2010). In this study, five standard For each sample, 5 µ l of DNA extract was added to the 21 µ l colonies taken from a 24 h single colony subculture of each master mix contained in the Cephied PCR reaction tube. isolate were suspended in 1 ml of distilled water. Twenty micro- litres of this suspension was then transferred into a Remel tube Quality control containing 980 µ l of viral transport media M4RT to provide similar conditions to the test samples. These were then extracted An IC (control 2) was used to validate the amplification pro - by an automated DNA extraction platform (Nordiag Bullet) cess within the instrument. In addition, the EuroClone kit and amplified on the SmartCycler as described previously. also contains Control 1 (a positive control of amplification) and a reaction blank. Five microlitres of the positive control was added to 21 µ l of master mix. Five microlitres of reaction Results blank was also added to another 21 µ l master mix. These acted as positive and negative controls of the procedure, A total of 86 samples that were initially tested by the Roche respectively. TaqMan method were re-tested by the SmartCycler/ EuroClone method. This comprises 69 (80.2%) consecutive Cepheid tubes were then capped after the addition of the Roche TaqMan positive specimens which were stored at specimen, control 1 or reaction blank before brief centrifuga- −20°C prior to re-extraction and retesting by the SmartCycler/ tion and then placed into the Cepheid SmartCycler. EuroClone method. Of these, 34 samples were re-extracted Thermal cycling and retested by the Roche TaqMan for confirmation before retesting on the SmartCycler. This was to validate changing The equipment was switched on and appropriate thermal protocols. The remaining 35 positive samples were only profile was selected prior to preparation of the reaction mix. tested once on Roche TaqMan. Out of the 34 specimens that The thermal profile selected is as shown in Table 1. were tested twice by the Roche TaqMan method, 2 specimens were excluded from further calculations because they had Once the reaction mix and controls were prepared and yielded an indeterminate (equivocal) result (positive first run placed into the heating block, the real-time PCR process was but negative second run) on the Roche TaqMan. Essentially, started. The PCR run lasted a total time of 1.5 h on the this implies that the decision is not reached as to whether the instrument. 4 Bioscience Horizons • Volume 6 2013 Research article patient is positive or negative by this method; therefore, clini- specificity were 92.5 and 100%, respectively, as determined by cians are advised to resample from the patient and submit for equations (1) and (2). The positive predictive value (PPV) that retest. From the 67 remaining positive specimens, 2 samples was determined by equation (3) was 100%; negative predictive were unresolved on the SmartCycler/EuroClone method. A value (NPV) that was determined by equation (4) was 77.3%. repeat test was performed and both yielded a positive result, TN hence were included among the ‘positives’. Seventeen ran- %Specificity = × 100 (1) () FP + TN domly selected Roche TaqMan negative samples (19.8%) were re-extracted and retested by the SmartCycler/EuroClone TP method to quality assure the method. %Sensitivity = ×100 (2) () TP + FN A summary of the comparison analysis of qualitative results of the SmartCycler/EuroClone method and the Roche TP TaqMan method is provided in Table 2. PPV% = ×100 (3) () TP + FP Out of the total 86 samples tested, 27 were urine specimens and the remaining 59 were swabs. Of the urine samples, 3 TN NPV% = ×100 (4) (13.6%) of those that were positive on the RocheTaqMan were () FN + TN negative when tested by the SmartCycler/EuroClone method. Of the Roche TaqMan positive swabs, two (4.4%) resulted where TN is the true negative; FN the false negative; TP the negative by the SmartCycler/EuroClone method. Table 3 shows true positive; FP the false positive (Source: Coulthard, a summary of the performance characteristic of the SmartCycler/ 2007). EuroClone; out of the total 67 Roche TaqMan positive sam- ples, 62 (92.5%) of them were also positive by the SmartCycler/ Clinical specificity EuroClone method. There were 5 discrepancies in total (7.5%) The additional organisms (E. coli, N. gonorrhoeae and M. and 17 samples were negative on both Roche TaqMan and catarrhalis) used in testing the clinical specificity of the SmartCycler/EuroClone. The analytical sensitivity and Table 2. Comparison of qualitative results of the SmartCycler/EuroClone and Roche TaqMan methods Reference results (Roche Additional TaqMan) results (Roche Totals Taqman Positive (n) Negative (n) equivocal) Smart cycler/EuroClone Positive 19 0 1 19 Urine (n = 27) Negative 3 4 7 Positive 43 0 43 Swab (cervical and urethra) (n = 59) Negative 2 13 1 15 Totals 67 17 2 84 Total excluding the sample(s) with the equivocal Roche TaqMan results. Two of these samples were initially unresolved on the SmartCycler/EuroClone method, but positive on the repeat test. Table 3. Performance characteristics of the SmartCycler/EuroClone method for urine and swab specimens Reference results (TaqMan) PPV% NPV% Sens% Spec% Positive Negative Total SmartCycler Positive 62 0 62 Negative 5 17 22 100 77.3 92.5 100 Total 67 17 84 PPV, positive predictive value; NPV, negative predictive value; Sens, sensitivity; Spec, specificity. 5 Research article Bioscience Horizons • Volume 6 2013 Table 4. Assay results for additional organisms tested by the A number of discrepancies (n = 5) were observed in this SmartCycler/EuroClone method study as explained in the Results section. On the note that Ct values are inversely proportional to DNA load (Rockett et al., Bacterium Assay result 2010); three of the discrepant results had Ct values >38 on the Roche TaqMan and are hence considered weak positive. E. coli Negative Further, one of these three samples had a Ct value of 41.8 on the Roche TaqMan and 46.4 on the SmartCycler/EuroClone. N. gonorrhoeae Negative This suggests two possibilities: either that the sensitivity of M. catarrhalis Negative SmartCycler/EuroClone is not as high as that of the Roche TaqMan when detecting minute starting quantities of DNA, or that these are indeed Roche TaqMan false positives. SmartCycler/EuroClone method all gave a negative result as The clinical specificity was well supported by the analyti - presented in Table 4. cal specificity of 100% and a correlation with the calculated PPV. The SmartCycler/EuroClone method proved able to Discussion avoid false positives by correctly identifying M. catarrhalis, E. coli and N. gonorrhoeae as C. trachomatis DNA negative. The results of the SmartCyler/Euroclone method gave good These additional organisms are commonly part of the genital agreement with the established Roche TaqMan method and flora that may also be present in clinical samples submitted proved to be suitable for the detection of C. trachomatis. for chlamydia testing; hence, we have demonstrated that the Most of the Roche TaqMan positive samples were also found SmartCycler/EuroClone can specifically discriminate between to be positive by the SmartCycler/EuroClone method. All other urogenital organisms and C. trachomatis. Studies such Roche TaqMan negative samples were also found to be nega- as Dicker et al. (2000) noted that co-infection of chlamydia tive on the SmartCycler/EuroClone (Table 4). with N. gonorrhoeae is common by including N. gonor- Sensitivity and specificity are important measures for diag - rhoeae as one of the control organisms; we have shown that nostic purposes because they allow the possibility of calculating the SmartCycler/EuroClone method is also able to discrimi- the probability of a disease being present in a particular patient nate between these two STIs. It should also be noted that (Van Der Schouw, 1992). Coulthard (2006) has provided a only three additional organisms were tested, using more uro- detailed definition of each of these: sensitivity was defined as genital organisms such as lactobacilli and Candida albicans, the percentage of true cases that resulted positive and specificity would have been advantageous. as percentage of unaffected individuals found to be test nega- Further explanation of the discrepancies seen in this study tive. He noted that although robust and often used to report the include DNA degradation; samples were archived at −20°C power of diagnostic tests, sensitivity and specificity parameters for up to 3 days before testing by the SmartCycler/EuroClone have limited clinical usefulness if used alone. Hence, they are and therefore some DNA degradation might have occurred; commonly converted to other terms such as PPV and NPV hence if these weak Roche TaqMan positive samples were which address the likelihood of a particular diagnosis from the truly positive, all the present C. trachomatis DNA might have perspective of a known test result. PPV is the fraction of true degraded while in archive, causing a negative result on the positive cases among all the patients with positive test results. SmartCycler/EuroClone assay. Notwithstanding, no attempts NPV, on the other hand, is the fraction of correctly diagnosed were made to retest these samples with a discrepant result true negative cases (Einstein, Boidian and Gill, 1997; Coulthard, due to limitations of fund. It would have been interesting to 2006). Other validation test parameters have also been sug- see how the result differs upon repeat testing; however, this gested (Van Der Schouw, 1992). The sensitivity of the Roche may not be worth it because oversampling of the specimen TaqMan in comparison with the BD ProbeTec ET test on urine may also lead to false-negative result. Furthermore, possibly specimens and endocervical swabs was reported to be 95.7 and TaqMan probes begin to degrade after repeated thermal 96.0%, respectively (Roche TaqMan kit insert); whereas, in cycles as the fluorophore begins to cleave due to repeated this study, the analytical sensitivity of the SmartCycler/ series of heating and cooling that they have been subjected to. EuroClone in comparison with the Roche TaqMan was 92.5%, resulting in a low NPV of 77.3. This is of considerable concern Although it can be argued that using Roche TaqMan as a because it suggests that in a population of 100 chlamydia posi- reference method of this study is reliable enough since this tive patients, ~20 of them will be missed, leading to disease method has been previously evaluated, an improved study progression and potential complications such as PID in women methodology would be to confirm all Roche TaqMan results, and sterility in men. A more reliable approach for determining particularly the positives, by sending them to the reference the sensitivity of an assay is the method used by Jaton, Bille and laboratory before testing by the SmartCycler/EuroClone Greub (2006), who determined the analytical sensitivity of their method. However, a problem with this is that it would fur- PCR assay by performing a 10-fold serial dilution of the posi- ther increase the gap of testing periods between the Roche tive control template. If resources allowed, this study would TaqMan method and the SmartCycler/EuroClone method, have also adopted this approach. subjecting sample DNA to further degradation. 6 Bioscience Horizons • Volume 6 2013 Research article Performance of other commercial methods have also been Acknowledgements impressive for the detection of C. trachomatis; a recent study which evaluated the Abbott RealTime CT/NG assay in com- First and foremost, I am indebted to all staff at the microbiol- parison with the Roche Cobas Amplicor CT/NG assay found ogy department of Queen Elizabeth hospital, Gateshead 99.6 and 97.7% positive and negative agreement respectively NHS Foundation Trust, for the opportunity granted me to for urine samples (Cheng, Qian and Kirby, 2011). Another complete this research project at their institute. Not only did recent study by Rocket et al. found that the sensitivity, speci- they let me use their facilities, but also provided tremendous ficity and NPVs and PPVs of the new Cobas 4800 CT/NG for support. My sincere appreciation to Miss Nicole Gaukrodger urine samples were 94.5, 99.5, 98.8 and 97.7%, respectively, and Mrs Natasha Waterhouse who both supervised my work for C. trachomatis, while for swabs, these were 92.0, 100, at the hospital and persevered my inquisitiveness. I am 99.5 and 100%, respectively. The performance characteristic equally grateful to Dr Lewis Bingle, who as my University of the SmartCycler/EuroClone assay is comparable to these project supervisor showed tremendous support and also per- widely known methods. Interestingly, this method has very severed my inquisitiveness. My gratitude also to Amy similar sensitivity and specificity with the Cobas 4800 CT/ Openshaw, Launch Diagnostic UK, for providing me with NG assay. additional information relevant to my study. A 2-fold increase in TAT (turn around time) was observed Author biography when positive chlamydia investigations were confirmed by the SmartCycler/EuroClone. TAT is defined as the total time Tolu graduated in July 2012 with a first class BSc in Applied taken between the submission of a sample for investigation Biomedical Science from the University of Sunderland. After and the issue of report. This observation is in fact as a result her second year, she completed a placement year in the of limiting testing by the SmartCycler/EuroClone method to Microbiology department at Queen Elizabeth Hospital, just twice per week, whereas testing was done daily on the Gateshead, where this study was subsequently undertaken. Roche TaqMan. Therefore, this does not reflect the speed of Towards the end of her final year, Tolu was awarded SfAM’s the method as reaction time on the SmartCycler analyser is (Society for Applied Microbiology) ‘Students into Work’ typically 1.5 h. Perhaps if this method was used for this pur- grant which gave her an opportunity to carry out an 8-week pose in a region with higher prevalence of the infection, this research project, which involved screening a library of syn- effect may not be seen. thetic chemical compounds for possible anti-pathogenicity To conclude, this study has demonstrated that the activities. She also took the opportunity to learn about and SmartCycler/EuroClone method can sufficiently confirm get involved in other areas of antimicrobial research. During Roche TaqMan positive in a clinical microbiology labora- this time, Tolu was also appointed as a Biomedical Scientist tory. The specificity of the method is remarkable, whereas the at Scarborough General Hospital. Her major interest areas sensitivity gives a little concern, but is still acceptable. The are in medical microbiology, particularly in diagnostic meth- discrepant results highlight the importance of confirming ods, antimicrobial and control of infection. positive results on an alternative method, supporting the rec- ommendation by the CPA. Using this method to confirm References positive chlamydia increases TAT, but this is peculiar to our laboratory and may differ in other laboratories, depending Black, C. M. (1997) Current methods of laboratory diagnosis of on their workload. Chlamydia trachomatis infections, Clinical Microbiology Reviews, 10, 160–184. Further work Cepheid (2011) SmartCycler system, accessed at: http://www.cepheid. c om/sy st ems-and-sof t w ar e/smar t c y cler -sy st em/ (11 D ec ember Cheng et al. pointed out that clinicians should be aware of 2011). the potential for inadequate detection of dual infections with some of the currently used NAAT methods; hence, Cheng, A., Qian, Q. and Kirby, J. E. (2011) Evaluation of the Abbott they suggested that clinical laboratories should thoroughly RealTime CT/NG assay in comparison to the Roche Cobas Amplicor evaluate multiplex PCR for adequacy in detecting dual CT/NG Assay, Journal of Clinical Microbiology, 49 (4), 1294–1300. infections (Cheng, Qian and Kirby, 2011). Therefore, Coulthard, M. J. (2006) Quantifying how tests reduce diagnostic uncer- assessment of the ability of the SmartCycler/EuroClone tainty, Archives of Disease in Childhood, 92, 404–408. method in detecting dual infections of C. trachomatis with N. gonorrhoeae is a point of interest. The SmartCycler Demkin, V. V. and Zimin, A. L. (2005) A new amplification target for PCR– should also be evaluated for use with other STI assays, for RFLP detection, Archives of Microbiology, 183, 160–165. investigations such as syphilis. Another point of interest was to see the cost difference between the two methods but Einstein, A. J., Boidian, C. A. and Gill, J. (1997) The relationships among my study was limited in this respect due to a lack of ade- performance measures in the selection of diagnostic tests, Archives quate costing information. of Pathology and Laboratory Medicine, 121 (1), 110–117. 7 Research article Bioscience Horizons • Volume 6 2013 Heid, C. A., Stevens, J., Livak, K. J. et al. (1996) Real time quantitative PCR, Pickett, M. A., Everson, J. S., Pead, P. J. et  al. (2005) The plasmids of Genome Research, 6, 986–994. Chlamydia trachomatis and Chlamydia pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plas - Higgins, J. A., Nasarabadi, S., Karns, J. S. et al. (2003) A handheld real time mid-curing agents, Microbiology, 151 (1), 893–903. thermal cycler for bacterial pathogen detection, Biosensors and Quinn, T. C., Welsh, L., Lentz, A. et al. (1996) Diagnosis by AMPLICOR PCR Bioelectronics, 18 (9), 1115–1123. of Chlamydia trachomatis infection in urine samples from women Jaton, K., Bille, J. and Greub, G. (2006) A novel real-time PCR to detect and men attending sexually transmitted disease clinics, Journal of Chlamydia trachomatis in first void urine or genital swabs, Journal of Clinical Microbiology, 34 (6), 1401–1406. Medical Microbiology, 55 (1), 1667–1674. Rockett, R., Goire, N., Limnios, A. et al. (2010) Evaluation of the Cobas Logan, J. and Edward, K. (2009) Chapter 2: An overview of PCR plat- 4800 CT/NG test for detecting Chlamydia trachomatis and forms. 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NHS vital signs 2010/11: new variant of Chlamydia trachomatis: genome sequence, morphol- Primary Care Trust (PCT) and Strategic Health Authority (SHA) spe- ogy, cell tropism and phenotypic characterization, Microbiology, 156 cific tables 1st April to 31st December 2010 (available online 3rd (1), 1394–1401. February 2011), accessed at: (15 December 2011). Van der Schouw, Y. T., Verbeek, A. L. and Ruijs, J. H. (1992) ROC curves for Paavonen, J. and Lehtinen, M. (1996) Chlamydial pelvic inflammatory the initial assessment of new diagnostic tests, Family Practice, 99 (4), disease, Human Reproduction Update, 2 (6), 519–529. 506–511.

Journal

Bioscience HorizonsOxford University Press

Published: Jun 4, 2013

Keywords: Chlamydia trachomatis SmartCycler Cepheid EuroClone evaluation real-time PCR

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