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Sexually Transmitted Diseases:
doi: 10.1097/OLQ.0b013e31824e26ff
Original Study

Evaluation of the Roche cobas® CT/NG Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Male Urine

Taylor, Stephanie N. MD*; Liesenfeld, Oliver MD; Lillis, Rebecca A. MD*; Body, Barbara A. PhD; Nye, Melinda PhD; Williams, James BS§; Eisenhut, Carol MD; Hook, Edward W. III MD; Van Der Pol, Barbara PhD§,**

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Author Information

From the *Louisiana State University Health Sciences Center, New Orleans, LA; Roche Molecular Systems, Pleasanton, CA; Laboratory Corporation of America, Burlington, NC; §Indiana University School of Medicine, Indianapolis, IN; DCL Laboratories (Laboratory Corporation of America), Indianapolis, IN; University of Alabama at Birmingham and Jefferson County Health Department, Birmingham, AL; and **Indiana University School of Public Health, Bloomington, IN

The authors thank the staff of all testing (LabCorp, Burlington, NC; Infectious Diseases Lab, IN University School of Medicine, Indianapolis, IN; DCL Medical Laboratories, Indianapolis, IN; LSU Health Sciences Center, New Orleans, LA) and collection sites (University of Alabama at Birmingham, Birmingham, AL; Lyndhurst Clinical Research, Winston-Salem, NC; Women's Health Practice, Champaign, IL; Marion County Health Department, IN University School of Medicine, Indianapolis, IN; Quality of Life Medical & Research Center, LLC, Tucson, AZ; Axis Clinical Trials, Los Angeles, CA; Planned Parenthood Southeastern PA, Philadelphia, PA; Planned Parenthood, Mar Monte, Sacramento, CA; Healthcare Clinical Data, Inc., North Miami, FL; Planned Parenthood Houston and Southeast Texas, Inc. [Planned Parenthood Gulf Coast Inc.], Houston, TX; LSU Health Sciences Center, Section of Infectious Diseases, New Orleans, LA). They thank Rui Li for expert statistical support.

Supported by Roche Molecular Systems, Pleasanton, CA. S.T. has received research support or received honoraria from Gen-Probe, Cepheid, Roche Molecular Systems and Becton Dickinson; O.L. is an employee of Roche Molecular Systems; B.B. has served on advisory boards sponsored by Roche Molecular System, Roche Diagnostics, and Becton Dickinson; M.N. has served on an advisory board sponsored by Gen-Probe; E.H. has received research support or honoraria from Roche Molecular Systems, Becton-Dickinson, Gen-Probe, Abbott Molecular Diagnostics, Siemens Molecular, and Cepheid and has served as a consultant for Abbott Molecular Diagnostics; B.V. has received honoraria or research support from Abbott Molecular Diagnostics, Beckman Coulter, Becton Dickinson, Cepheid, Idaho Technologies, Inc., PinPoint Medical, Roche Diagnostics, and Roche Molecular Systems.

Correspondence: Stephanie N. Taylor, MD, 517 N Rampart St, New Orleans, LA 70112. E-mail: staylo2@lsuhsc.edu.

Received for publication September 21, 2011, and accepted February 1, 2012.

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Abstract

Background: The Roche cobas® CT/NG test (c4800), performed on the cobas 4800 system, is a new diagnostic assay using an automated workstation to isolate nucleic acids from clinical specimens and a real-time instrument for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). This study compared the performance characteristics of the c4800 with the Becton Dickinson ProbeTec™ CT/GC Qx assay (Qx) and Gen-Probe® Aptima Combo 2 (AC2) assay for the detection of CT and NG in male urine using patient-infected-status (PIS).

Methods: Urine and urethral swabs were obtained from men attending STD, family planning, or OB/GYN clinics from 11 geographically distinct locations. Aliquot order was randomized for urine specimens between AC2, c4800, and Qx. Urethral swabs were randomized between AC2 and Qx. Urethral swabs were only used to define PIS and were not tested on the c4800. A participant was considered infected if the 2 comparator assays with different molecular targets had positive results from either sample type.

Results: A total of 790 men were screened, with 768 evaluable for CT and NG. Symptoms were reported in 296 (38.5%) participants. For urine, the overall sensitivity and specificity of the c4800 assay for CT were 97.6% and 99.5%, respectively, when compared with PIS. Sensitivity and specificity for NG were 100% and 99.7%, respectively.

Conclusions: The c4800 has excellent sensitivity and specificity for male urine specimens when compared with PIS. Assay performance was similar in symptomatic and asymptomatic men and was equivalent to nucleic acid amplification tests that are currently on the market.

The importance of testing for gonorrhea and chlamydia to prevent complications and to reduce risk for HIV acquisition and transmission is well established.15 Because these infections are asymptomatic in up to 50% of men and 70% of women, screening is a crucial element of control efforts.3,6 In 2009, >1.5 million total cases of Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) were reported, with 464,140 of these infections being reported in men.7,8 These rates reflect high disease burden, increased NG and CT screening, improved reporting, and increased use of nucleic acid amplification tests (NAATs) for detection.9

NAATs were developed in response to the poor sensitivity and complications associated with culture of fastidious organisms, such as NG and CT. These tests target species-specific DNA for amplification and detection of these organisms. The performance of NAATs for the detection of NG and CT is outstanding. These commercially available tests demonstrate excellent sensitivity and specificity in a variety of urogenital sample types, including urine.1015 Urine samples are much less invasive when compared with urethral swabs and are the preferred specimens for screening in men, particularly asymptomatic men. Urine-based testing has the potential to increase screening, detection, and treatment of these infections. In addition, urine-based testing may have advantages in terms of its capacity to contribute to expansion of clinical screening services. Urine testing has been shown to increase patient throughput at busy STD clinics by permitting “express visits” for screening purposes.16

The cobas® CT/NG test (Roche Molecular Systems, Pleasanton, CA) (c4800) performed on the cobas 4800 system is a new fully automated CT/NG assay that is designed for use in routine clinical microbiology laboratories. The performance on urine and swab specimens has recently been reported, and the new assay addresses challenges that were inherent in the Roche COBAS AMPLICOR.17,18 First, there were problems with false-negative results due to sequence variation. Specifically, a variant of CT with a 377-bp deletion in the cryptic plasmid was discovered in Sweden in 2007 that was not detected with the assay. The new assay comprises 2 target sequences for detecting CT. Second, with regards to NG, there was cross-reaction with other strains of Neisseria species. The newly designed NG assay targets a direct repeat region called DR-9. This target is repeated 3 times on the NG genome and has 2 highly conserved sequence variations. The c4800 uses this dual approach for both organisms. In this multicenter study, the performance of the c4800 in male urine specimens was evaluated by comparison with a rotating patient-infected-status (PIS) using the Gen-Probe Aptima Combo 2 (AC2) and the Becton Dickinson ProbeTec CT/GC Qx (Qx) assays as references. The c4800 adds an excellent NAAT to those currently available for detection of CT and NG.

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METHODS

Study Population

Participants were enrolled from 11 geographically distinct specimen collection sites, including OB/GYN practices, family planning, and STD clinics (see Acknowledgments for complete listing), as part of the vaginal, endocervical, and urine screening VENUS trial for CT/NG.18 Men were included if they were 14 years of age or older and willing and able to provide written, informed consent (informed consent of parent or guardian for minors). Participants were excluded if they had been previously enrolled in the study or used antimicrobials active against CT or NG during the preceding 21 days. Symptoms indicative of CT or NG infection were elicited and included dysuria/pain during urination, urethral discharge, or testicular/scrotal pain or swelling. All other participants were classified as asymptomatic. Institutional Review Board approval for the study protocol was obtained from each participating site. The c4800 was not used in the diagnosis or treatment of participants.

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Specimen Collection, Transport, and Storage

Participants were assigned unique ID numbers, and all specimens were collected at one visit to the clinic or office; each sample was identified with a unique number that included the subject ID. From each male participant, 2 urethral swabs were collected in randomized order to minimize sample quality variation using the Qx or AC2 collection kits. Urethral swabs were only used to define PIS and were not tested on the cobas 4800. Collection of a first-catch urine sample followed collection of urethral swabs according to the manufacturer's package insert instructions. The urine specimen was divided into 3 aliquots, with the required volumes being placed into each assay's urine transport tube. All specimens were stored and tested according to their respective manufacturer's package inserts.9,12 No samples were frozen. Testing was performed at 4 testing sites in the United States (LabCorp, Burlington, NC; Infectious Diseases Lab, IN University School of Medicine, Indianapolis, IN; DCL Medical Laboratories, Indianapolis, IN; and Infectious Diseases Lab, LSU Health Sciences Center, New Orleans, LA).

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cobas CT/NG Test

The c4800 uses a dual-target approach: CT primers CP102 and CP103 are used to define a sequence of approximately 206 nucleotides within the cryptic plasmid DNA of CT (the cryptic plasmid is a multiple copy gene with an unknown function and a minimum of 10–20 copies per organism). In addition, CT primers CTMP101 and CTMP102 define a sequence of approximately 182 nucleotides within the gene encoding for the major outer membrane protein A of CT. The target region for detection of CT was designed to allow detection of the Swedish new variant.

NG primers NG514 and NG519 define a sequence of approximately 190 nucleotides from a highly conserved direct repeat region of NG called DR-9. In addition, another set of NG primers, NG552 and NG579, defines a second sequence of approximately 215 nucleotides identified as a conserved sequence variant from this region. There are 3 copies per genome depending on whether the strain is homogeneous DR-9, DR-9Var or heterogeneous of DR-9/DR-9Var; the function is unknown. Processed samples are added to the amplification mixture in a multiwell plate in which polymerase chain reaction (PCR) amplification occurs fully automated.

The CT/NG internal control (IC) is a combination of 2 noninfectious recombinant plasmid DNAs, each with primer binding regions identical to those of either the CT or the NG genomic target sequences. Both recombinant plasmid DNAs have an identical randomized internal target sequence, and a unique probe binding region that differentiates the CT/NG IC from target amplicon to ensure independent amplification of both the CT/NG IC and the CT and NG target DNAs. The CT/NG IC reagent is included in the c4800 and is introduced into each sample on the instrument during sample processing. Selective amplification of target nucleic acid from the specimen is achieved in the c4800 by use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate. The c4800 uses real-time PCR technology. The use of fluorescent probes provides for real-time detection of PCR product accumulation by monitoring the emission intensity of fluorescent dyes released during the amplification process. The amplification of CT targets, NG targets, and the CT/NG IC is measured independently and at different wavelengths. This process is repeated for a designated number of cycles, each cycle increasing the emission intensity of the individual reporter dyes.

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Determination of Patient-Infected-Status

Participants were designated as being infected with CT or NG if at least 2 NAATs with different target regions gave positive results in the urethral swab and/or the urine specimen (rotating PIS).

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Statistical Analysis

The statistical analyses were chosen based on recommendations in the statistical guidance from the Food and Drug Administration19 and in accordance with the guidelines published in CLSI EP12-A220 for evaluating qualitative test performance. Sensitivity and specificity of c4800 and the 2 comparator assays were calculated separately for detection of CT and NG by using PIS as the reference standard. The corresponding 2-sided 95% score confidence intervals were also calculated. Venn diagrams were plotted, separately by sample type, to compare the list of matched clinical specimens (i.e., same sample type for a given subject) tested positive from at least 1 NAAT. Fisher exact test was used to assess the statistically significant difference in performance estimates between symptomatic and asymptomatic groups. P values of <0.05 were considered statistically significant. All analyses were performed using SAS/STAT software, version 9.1 of the SAS System for Windows (SAS Institute Inc., Cary, NC).

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RESULTS

A total of 790 men were screened from 11 sites and 768 were enrolled. Table 1 outlines the demographics and baseline characteristics. Three hundred sixty-nine men (42.8%) were between 21 and 30 years of age, 497 (64.7%) men were black, and symptoms of urethritis were reported by 296 men (38.5%). A total of 187 men were enrolled from OB/GYN and Family planning upon presentation with their partners. Twenty-two men were not evaluable (3 withdrew consent after enrollment; 9 had errors in sample collection and/or storage; 10 had invalid cobas CT test results after the initial and repeated testing). Using the PIS, the prevalence of CT was 25% in symptomatic men and 11% in asymptomatic men. The prevalence of NG was 21.6% in symptomatic men and 1.4% among the asymptomatic men.

Table 1
Table 1
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With regards to the ICs, only 2 of 879 tests (includes repeated testing) had IC failures. The results were reported as invalid only if both CT and NG were tested negative. For the 2 cases with invalid IC results, CT and NG were tested and both were reported as positive.

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C. trachomatis

CT was identified in 126 men with 58.7% (74/126) of infections occurring in symptomatic men. Sensitivity and specificity of the c4800, AC2, and Qx assays for CT in men using the PIS are presented by sample type and symptom status in Table 2. Sensitivity of the c4800 was 97.6% in male urine compared with 96.8% for the AC2 and 98.4% for the Qx assays. Sensitivities for each of the 3 NAATs did not differ significantly by patient symptom status. Specificity of the c4800 was high (99.5%) and comparable with the specificities of the AC2 and Qx assays. Although efforts were made to enroll participants from low as well as high prevalence populations, there were no sites from which the prevalence of infection was less than 1%. The overall prevalence of CT was 16.4%, with site prevalence ranging from 1.4% to 26.1% with high positive and negative predictive values (Table 3). The high agreement among positive results for CT across the 3 assays in urine is shown in Figure 1A. There was also high agreement of positive results among the various tests performed on urine (c4800) and urethral swabs (AC2 and Qx assays) (Fig. 1B). Performance characteristics for the detection of CT did not show significant differences based on sampling order or clinic type (data not shown).

Table 2
Table 2
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Table 3
Table 3
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Figure 1
Figure 1
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N. gonorrhoeae

NG was identified in 71 men with 90.1% (64/71) of infections occurring in symptomatic men. Sensitivity and specificity of the c4800, AC2, and Qx assays for NG in men using the PIS are presented by sample type and symptom status in Table 4. Sensitivity of the c4800 was 100% in male urine and was identical to the comparator assays. Sensitivities for each of the 3 NAATs did not differ by patient symptom status. Specificity of the c4800 was high (99.7%) and comparable with the specificities of the AC2 and Qx assays. Enrollment and testing were performed in low as well as high prevalence populations. The overall prevalence of NG was 9.2%, with site prevalence ranging from 0% to 20% with high positive and negative predictive values (Table 5). The high agreement among positive results for NG across the 3 assays in urine is shown in Figure 2A. There was also a high agreement of positive results among urine and urethral swabs (Fig. 2B). Performance characteristics for the detection of NG did not show significant differences based on sampling order or clinic type (data not shown).

Table 4
Table 4
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Table 5
Table 5
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Figure 2
Figure 2
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DISCUSSION

The c4800 was evaluated in male urine specimens and compared with PIS, with the Food and Drug Administration-cleared second generation Qx and the AC2 assays used for reference. The c4800 demonstrated excellent sensitivities and specificities for both CT and NG when compared with PIS. In fact, there was no statistically significant difference between the c4800 and the AC2 and Qx. The 3 assays were clinically and statistically equivalent in performance.

Results obtained in the current study are similar to the results described by Rockett et al.17 When comparing the c4800 with the cobas AMPLICOR CT/NG and cobas TaqMan CT assays using 488 urine specimens submitted for CT and NG testing, the sensitivity, specificity, and negative and positive predictive values of the c4800 were 94.5%, 99.5%, 98.8%, and 97.7%, respectively, for CT, and 92.9%, 100%, 99.7%, and 100%, respectively, for NG. No cross-reactivity with nongonococcal Neisseria species, a weakness of some commercial assays for NG,21 was observed in the c4800.

In the current study, 61.5% of the men were asymptomatic and thus represented potential populations for whom screening might be recommended. There was no difference between the symptom status of the men and the sensitivity or specificity of the assay. A higher sensitivity was noted for urine compared with urethral swabs for CT in the reference assays, especially in asymptomatic men. This finding has been noted previously10 and demonstrates the value of urine specimens in the detection of CT and NG in screening situations. It also reflects the difficulty in obtaining urethral swabs from asymptomatic men and supports the CDC Laboratory Guidelines recommendation for urine NAATs as the preferred choice sample type in men.15, 2224 Urethral swabs were not tested on the c4800 in response to this recommendation. The c4800 can be used to screen men for CT and NG using noninvasive urine samples. This will result in the detection and treatment of more NG and CT infections and has the potential to contribute to better control of these infections.

Two strengths of this study were the high number of asymptomatic men enrolled and test performance in low prevalence areas and asymptomatic men. The lack of asymptomatic men was a weakness noted in a previous NAAT study.14 In addition to the symptomatic men with these infections, 11% of asymptomatic men had chlamydia and 1.5% of asymptomatic men had gonorrhea. The test performed equally in both high and in relatively low prevalence populations. Although efforts to identify low prevalence populations for gonorrhea test evaluation were successful, this was not the case for chlamydial infections. Thus, our data suggest that, perhaps due to the higher frequency of asymptomatic infections, chlamydial infections are widespread and further support the need to develop systematic screening recommendations and programs for at-risk men. Therefore, the c4800 can be used for both screening asymptomatic men who can be found to have high rates of infection and for the detection of CT and NG infections in symptomatic men.

In conclusion, in both symptomatic and asymptomatic men, the c4800 offers high sensitivity, specificity, positive and negative predictive values for the direct, qualitative detection of CT and NG in urine specimens. When a test has both high positive and negative predictive values, the value of that test is increased. The c4800 can be added to the list of available NAATs for screening and diagnostic purposes using easily obtained urine specimens in men.

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