Neisseria gonorrhoeae False-Positive Result Obtained from a Pharyngeal Swab by Using the Roche cobas 4800 CT/NG Assay in New Zealand in 2012

Abstract

cThe Roche cobas 4800 CT/NG assay is a commonly used commercial system for screening for Neisseria gonorrhoeae infection, and previous studies have shown the method to be highly sensitive and specific for urogenital samples. We present the first confirmed clinical N. gonorrhoeae false-positive result using the cobas 4800 NG assay, obtained from testing a pharyngeal swab sample and caused by cross-reaction with a commensal Neisseria strain. N ucleic acid amplification tests (NAATs) are widely used for the detection of gonorrhea. However, the specificity of these methods can be undermined by ongoing genetic exchange between species within the Neisseria genus, leading to commensal Neisseria strains acquiring Neisseria gonorrhoeae (NG) NAAT target sequences. For these reasons, supplementary "confirmatory" testing for N. gonorrhoeae NAATs has been widely adopted (1, 2). The Roche cobas 4800 CT/NG assay is a later-generation NAAT method, and the NG component of the assay utilizes a dual-target approach, using two assays to detect sequences within the directrepeat (DR-9) region (3). Performance data to date show excellent sensitivity and specificity for urogenital specimens (3-7), and it has been suggested that the assay does not require a second test to confirm urogenital positive results (6). Also, to our knowledge, there have been no definitive reports of the assay cross-reacting with commensal Neisseria strains (3, 8); while initial testing in a study by Tabrizi et al. (8) showed that the cobas 4800 NG assay cross-reacted with two commensal Neisseria strains, both were negative upon retesting using fresh cultures (8). Herein, we report the first clinical demonstration of a Roche cobas 4800 NG falsepositive result obtained from a pharyngeal swab sample and caused by a reproducible cross-reaction with a commensal Neisseria strain.On 17 September 2012, as part of the heightened awareness program sponsored by the New Zealand AIDS Foundation, selfreferred throat and rectal swabs and a first-void urine were received from a 38-year-old male presenting to a sexual health clinic in Auckland, New Zealand. The samples were tested by PCR for N. gonorrhoeae and Chlamydia trachomatis (CT) on the Roche cobas 4800 CT/NG assay at Labtests, Auckland, New Zealand (Table 1). The three specimens were all negative for C. trachomatis DNA; the urine and rectal swabs were also negative for N. gonorrhoeae DNA. The throat swab was positive for N. gonorrhoeae DNA with a cycle threshold value of 35.7, which was confirmed upon retesting at a second laboratory (Aotea Pathology, Wellington; cobas 4800 NG assay positive with a cycle threshold value of 35.3), and the patient was treated with 500 mg intramuscular ceftriaxone. As part of an ongoing study investigating the specificity of the cobas 4800 NG assay in our population, the specimen was subsequently referred for supplementary testing by the Abbott m2000 real-time PCR (Canterbury Health Laboratories) and in-house PCR methods targeting the gonococcal porA and opa genes (Aotea Pathology, Welli...

Is confirmatory testing of Rochecobas4800 CT/NG testNeisseria gonorrhoeaepositive samples required? Comparison of the Rochecobas4800 CT/NG test with anopa/papduplex assay for the detection ofN gonorrhoeae

“…The low-level nature of the cobas 4800 CT/NG test results from the majority of unconfirmed oropharyngeal samples (table 3—CT>37 with clinical samples rarely if ever seen above CT>40), may reflect the typically scant organism load at this anatomical site26 or may be due to less than 100% primer and/or probe target sequence homology 12. The inability to obtain NG-specific 16S or DR-9 sequence data from these samples could also reflect either possibility.…”

“…Unfortunately, the false-positives described by Upton et al ,12 were only cultured after the patient had been treated for NG infection on two occasions, and originated from an isolate identified as several Neisseria species using different identification techniques. The true clinical and analytical nature of these unconfirmed positive results, therefore, may only be possible by conducting a prospective study on appropriate samples, although definitive characterisation may prove elusive.…”

“…Furthermore, the first account of a clinical NG false-positive result from a pharyngeal specimen using the cobas 4800 Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) test has recently been reported in the literature 12. Nevertheless, due to the significantly superior sensitivity of NAATs for the detection of NG especially in the rectum and oropharynx, they have been recommended as the method of choice for extragenital NG diagnosis in high-risk patients, such as men who have sex with men without the need for confirmation 4 13–15.…”

Analytical Evaluation of GeneXpert CT/NG, the First Genetic Point-of-Care Assay for Simultaneous Detection of Neisseria gonorrhoeae and Chlamydia trachomatis

“…Several NAATs, including commercial assays, have experienced problems with false positivity and false negativity due to cross-reaction (4-7), mutations (8,9), and genetic exchanges with related organisms (10)(11)(12). In particular, diagnosis of extragenital specimens, such as pharyngeal and rectal samples, can result in suboptimal specificity due to cross-reaction with commensal Neisseria species or Neisseria meningitidis (4,5,7,(13)(14)(15), which result in low positive predictive values, especially in low-prevalence populations.…”

Supplementary Testing Is Not Required in the cobas 4800 CT/NG Test for Neisseria gonorrhoeae Weak-Positive Urogenital Samples

“…Concern about the interpretation of these results arises from issues with false-positive results in low-prevalence populations, test reproducibility when there are low genome copy numbers in the specimen, and the possibility of cross-reaction with commensal Neisseria species at extragenital sites (9,10). In order to address these issues, we undertook an investigation to determine the analytical sensitivity of the cobas 4800 CT/NG test and correlate this with the reproducibility of the results of porA and opa supplementary assays.…”