Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species

Tabrizi, Sepehr N.; Unemo, Magnus; Limnios, Athena; Hogan, Tiffany R; Hjelmevoll, Stig Ove; Garland, SM; Tapsall, John W.

doi:10.1128/jcm.01217-11

Cited by 118 publications

(86 citation statements)

References 39 publications

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“…Several commercially available real-time PCR assays enable this double detection (3). Trichomonas vaginalis is the most prevalent nonviral sexually transmitted disease pathogen in the world, but it remains poorly diagnosed, as are Mollicutes (Ureaplasmas and Mycoplasmas), which are difficult to cultivate (4)(5)(6).…”

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confidence: 99%

Assessment of Coinfection of Sexually Transmitted Pathogen Microbes by Use of the Anyplex II STI-7 Molecular Kit

et al. 2015

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f Anyplex STI-7 is a new molecular kit that detects seven sexually transmitted pathogens. Among 202 subjects screened for genital infection, 143 (70.4%) were diagnosed with at least one pathogen, in concordance with reference methods. In addition, the Anyplex STI-7 demonstrated coinfections, such as that with Ureaplasma parvum and Chlamydia trachomatis, in young women. Since the consequences of genital infections through sexually transmitted diseases (STDs) can be as severe as sterility and disseminated infections, molecular assays detecting bacterial pathogens, such as Chlamydia trachomatis and Neisseria gonorrhoeae, were shown to be relevant not only in patent infections but also in paucisymptomatic sexually active women (1, 2). Several commercially available real-time PCR assays enable this double detection (3). Trichomonas vaginalis is the most prevalent nonviral sexually transmitted disease pathogen in the world, but it remains poorly diagnosed, as are Mollicutes (Ureaplasmas and Mycoplasmas), which are difficult to cultivate (4-6). The latter bacteria are involved in early pregnancy loss, stillbirth, preterm birth, and neonatal morbidity, as well as male infertility (6-9), although they are also commonly identified in the vaginal flora of 40% to 80% of healthy women, depending on the population (6, 7). Among them, Ureaplasma parvum is an overlooked pathogen.The Anyplex II STI-7 kit (STI-7, Seegene, Eurobio) is a multiplex real-time PCR assay relying on a newly developed tagging oligonucleotide cleavage and extension technology (TOCE). This assay is marketed to simultaneously detect seven microorganisms involved in sexually transmitted infections: C. trachomatis (CT), N. gonorrhoeae (NG), T. vaginalis (TV), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and U. parvum (UP). Our study aimed to evaluate this assay's performances in diagnosing and screening for STDs in our hospital cohort. The results were compared to those of validated methods. In addition, we looked for the coexistence of pathogens, which is usually undetected. Two or more pathogens were highly prevalent, especially in sexually active young women with or without symptoms of infection.A total of 213 specimens from 202 patients were tested from January to June 2012. The specimens came from 94 vaginal swabs, 46 cervical swabs, 61 first-void urine samples, and 12 pelvic aspirated fluid samples. Cervical and urethral swabs were discharged in 3 ml of M4-RT medium (Abbott), and other samples were put into sterile tubes with no additives and transported to the lab within 12 h. All specimens were submitted to the Abbott RealTime CT/NG assay (Abb CT/NG ) used routinely in our lab for detection of CT and NG. Samples were processed and interpreted as recommended by the manufacturer using the Abbott m2000 apparatus.Results were given as positive, negative, or equivocal, the latter being retested and reclassified positive or negative. For the STI-7 assay, DNA was extracted from 190 l of the sample (M4-RT medium, urine, or...

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confidence: 99%

Assessment of Coinfection of Sexually Transmitted Pathogen Microbes by Use of the Anyplex II STI-7 Molecular Kit

et al. 2015

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“…e must strenuously disagree with Tabrizi et al that "Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites" (emphasis ours) (6). This may reflect Australian guidelines, but the generalization is unfortunate.…”

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confidence: 59%

Routine Confirmation of Positive Nucleic Acid Amplification Test Results for Neisseria gonorrhoeae Is Not Necessary

Schachter¹,

Chernesky²

2012

J Clin Microbiol

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We must strenuously disagree with Tabrizi et al. that "Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites" (emphasis ours) (6). This may reflect Australian guidelines, but the generalization is unfortunate. Tabrizi cites the Centers for Disease Control and Prevention (CDC) 2002 recommendation for confirmatory testing but ignores the conclusion of a 2009 CDC expert committee that supplementary testing does not improve nucleic acid amplification test (NAAT) results (1). Our objections are based on an important incorrect assumption behind supplementary testing, as well as Tabrizi's specific results. We evaluated confirmatory testing for chlamydial and gonococcal infections and found it does not improve NAAT results (3, 4). The basic concept of confirmatory testing is that true positives are confirmed by the secondary test and false positives are exposed as such. That is simply not true; all true positives are not confirmed. Thus, the clinical utility of routine confirmatory testing is questionable.Certainly there are special circumstances, such as when using NAATs on specimens from extragenital sites, that must be considered. When we had many pharyngeal false positives for gonococci with COBAS Amplicor CT/NG (Roche Molecular Systems), we recommended the test not be used for such specimens (5). That finding was not unexpected based on the literature. Both the Amplicor and ProbeTec (BD) package inserts acknowledge the potential for positive results caused by commensals. Thus, Tabrizi's positive results with nongonococcal Neisseria spp. in these assays were predictable. But finding low-level false positives with all the other assays (Abbott m2000 RealTime [Abbott Molecular], Cobas 4800 [Roche], and APTIMA COMBO 2 and APTIMA GC [GenProbe]) was surprising. Our evaluations of APTIMA assays with samples from genital and nongenital sites found no specificity problems. Confirmatory testing yielded specificities of Ն99.7% (which in part led to the conclusion that such testing was unnecessary) (3, 5). In the Tabrizi study, retesting was done with m2000, Cobas 4800, and APTIMA COMBO 2 using a new culture, not the initially positive specimen (no retests with APTIMA GC). The initial "false" positives in the 4 assays, which have 4 different targets, had equivocal or low positive signals. When the new cultures were tested, they were negative, meaning the initial positives did not reflect reactions with similar or identical sequences present in the genomes of closely related bacterial species. Tabrizi suggests mispriming as a cause. As presented, these results are open to another explanation. Both APTIMA assays had low-level positives with the same Neisseria meningitidis isolates. Such clustering is a red flag suggesting that the original positives were contaminated with low levels of Neisseria gonorrhoeae. Retesting of the original samples should have been done to exclude this possibility, as neither passage nor other NAAT results ...

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“…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.…”

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confidence: 91%

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

Upton¹,

Bromhead²,

Whiley

2013

J Clin Microbiol

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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...

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Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species

Cited by 118 publications

References 39 publications

Assessment of Coinfection of Sexually Transmitted Pathogen Microbes by Use of the Anyplex II STI-7 Molecular Kit

Assessment of Coinfection of Sexually Transmitted Pathogen Microbes by Use of the Anyplex II STI-7 Molecular Kit

Routine Confirmation of Positive Nucleic Acid Amplification Test Results for Neisseria gonorrhoeae Is Not Necessary

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

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