Sexually Transmitted Diseases:
Analytical Specificity and Sensitivity of the APTIMA Combo 2 and APTIMA GC Assays for Detection of Commensal Neisseria Species and Neisseria gonorrhoeae on the Gen-Probe Panther Instrument
Golparian, Daniel MSc*†; Tabrizi, Sepehr N. PhD‡; Unemo, Magnus PhD*†
From the *WHO Collaborating Centre for Gonorrhoea and Other STIs, National Reference Laboratory for Pathogenic Neisseria, Örebro, Sweden; †Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Örebro, Sweden; and ‡Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville, Victoria, Australia
The authors are grateful to Gen-Probe for providing the diagnostic kits used in the study.
Supported by the Örebro County Council Research Committee, the Foundation for Medical Research at Örebro University Hospital, Sweden, and Scientific Affairs, Gen-Probe Incorporated, San Diego, CA.
All authors declare that they have no conflicts of interest.
Correspondence: Magnus Unemo, PhD, WHO Collaborating Centre for Gonorrhoea and Other STIs, Department of Laboratory Medicine, Microbiology, Örebro University Hospital, SE-701 85 Örebro, Sweden. E-mail: firstname.lastname@example.org.
Received for publication August 4, 2012, and accepted October 10, 2012.
Abstract: Genetic detection of Neisseria gonorrhoeae is replacing culture for increased diagnostic sensitivity. Specificity of several nucleic acid amplification tests is suboptimal. Herein, the Gen-Probe APTIMA Combo 2 and APTIMA GC assays had 100% specificity and 100% sensitivity after confirmatory testing, when testing 298 isolates of nongonococcal Neisseria and related species and 205 gonococcal isolates.
Neisseria gonorrhoeae is estimated to annually cause 106 million new gonorrhea cases among adults globally.1 A high proportion of infections are asymptomatic, particularly among women and extragenital infections, which may lead to serious sequelae such as pelvic inflammatory disease, ectopic pregnancy, and infertility. Therefore, highly sensitive and specific laboratory testing is crucial for appropriate diagnosis and subsequent adequate treatment of infected patients and their partners.
During the past decade, nucleic acid amplification tests (NAATs) have rapidly replaced culture for the detection of gonococci in many countries worldwide owing to advantages such as superior sensitivity (particularly for pharyngeal and rectal samples),2–7 and use of noninvasive specimens. In addition, NAATs allow for automation and high throughput, as well as simultaneous detection of several infectious agents.2,8–16 Disadvantages, as compared to culture, include the high cost, that no commercial NAAT is licensed by the US Food and Drug Administration (FDA) for extragenital specimens, inability to perform antimicrobial susceptibility testing, and suboptimal specificity of several NAATs that cross-react particularly with other Neisseria species, for example, the Cobas Amplicor CT/NG (Roche Molecular Systems, Branchburg, NJ), BD ProbeTec ET and GC Qx (Becton Dickinson, Sparks, MD).2,10,15–22 This suboptimal specificity, especially for extragenital samples, results in low positive predictive value (PPV), particularly in low-prevalence populations.
At present date, several commercial gonococcal NAATs have received FDA clearance, that is, APTIMA Combo 2 (AC2) and APTIMA GC test (AGC) (Gen-Probe, San Diego, CA), Cobas Amplicor and Cobas 4800 CT/NG (Roche Molecular Systems), BD ProbeTec ET and GC Qx (Becton Dickinson), and RealTime CT/NG (Abbott Laboratories, Des Plaines, IL). Disquietingly, a recent study22 reported false low-level sporadic positives in all of these assays when spiking samples with nongonococcal isolates. The Gen-Probe AC2 and the supplementary AGC, targeting different sequences of the 16S rRNA gene, have shown excellent sensitivity and specificity in numerous previous studies examining clinical samples.3–7,12,13,23–30 However, in the recent analytical study,22 the AC2 test initially showed false low-level sporadic positivity for Neisseria meningitidis isolates (4/75 tested) and Neisseria sicca (1/16 tested), as did AGC for N. meningitidis isolates (3/75 tested). Nevertheless, these false low-level positives could not be verified in repeated testing of new cultures.22
To examine this further, in the present study, the analytical specificity and sensitivity of the Gen-Probe AC2 and AGC assays were comprehensively evaluated, by testing of 298 nongonococcal (meningococci, commensal Neisseria, and related species) and 205 gonococcal isolates and using a strict algorithm for confirmatory testing of all initially equivocal and false-positive or false-negative results (Fig. 1).
The tested isolates included comprehensively species-verified gonococcal clinical isolates (n = 189) and World Health Organization reference strains (n = 16)31,32 from 1991 to 2011 (of which 40 isolates were examined also in Tabrizi et al.22), which represented geographically (global distribution) and genetically different strains; meningococcal clinical isolates (n = 194) and reference strains (n = 9) from 1974 to 2011 that represented geographically and genetically different strains of all major clones spreading worldwide; and isolates of 11 commensal Neisseria species (n = 85) and 3 related species (n = 10) (Table 1). Isolates were cultured and prepared for testing as previously described22 with minor modifications; that is, 10 pure colonies (higher specificity challenge) for nongonococcal isolates and 2 pure colonies (lower sensitivity challenge) for gonococcal isolates were suspended in 0.5 mL phosphate-buffered saline. Before any testing, a comprehensive algorithm for confirmatory testing to verify or reject all equivocal results and false-positive and false-negative results was designed (Fig. 1). Finally, all tests were run in duplicate on the fully automated Gen-Probe Panther instrument, and results were interpreted as recommended by the manufacturer.
The final results of the present study are summarized in Table 1. Briefly, 4 nongonococcal isolates initially showed a low-level false-positive reaction. That is, according to the interpretation of AC2 and AGC results described in the package inserts, Neisseria flavescens (n = 1; in AC2) and N. sicca (n = 1; in AGC) showed equivocal results, Neisseria lactamica (n = 1) and N. sicca (n = 1) showed low positive (in both the AC2 and AGC) results. Those were also showing the same false-positive results in repeated testing from the original dilutions. However, after reculture of the bacterial strains from frozen stocks and repeated comprehensive species verification, testing from new dilutions of all these 4 strains showed negative results. Furthermore, this procedure was repeated twice with negative results, and the original dilutions were also shown to be contaminated, that is, contained gonococcal nucleic acid according to all methods used in the genotypical verification (Fig. 1) as well as gonococcal molecular epidemiologic typing. Accordingly, both the AC2 and AGC had 100% specificity and 100% sensitivity (Table 1).
In the AC2 and AGC package inserts, equivocal but not low positive results are recommended to be retested using the same method. The Gen-Probe AC2 test is also the only FDA-cleared gonococcal NAAT for which a supplementary NAAT (AGC) is available. In the previous analytical study,22 showing low-level false-positive reactions in both AC2 and AGC, it was not possible to perform appropriate confirmatory testing, and the false-positive AGC results were never repeated because the testing was performed in an external laboratory. Accordingly, in the present study, we wanted to substantially challenge the analytical specificity of AC2 and AGC, using also a comprehensive algorithm for confirmatory testing, which is essential to use in this type of studies (Fig. 1). The final results showed that both the AC2 and the AGC had 100% specificity and 100% sensitivity. Nevertheless, 4 nongonococcal isolates initially showed equivocal (n = 2) or low positive (n = 2) results, which, however, in the confirmatory testing could be clearly verified as due to contamination with gonococcal nucleic acid. This highlights the possibility and ease of getting low-level false positives due to contamination, especially when using exceedingly sensitive NAATs, which is also stressed in the package inserts for AC2 and AGC. Accordingly, it is essential with adequate internal and external quality assurance including appropriate quality controls (positive and negative run controls as well as environmental controls), strict decontamination procedures, and complete separation of possible gonococcal culture from areas where gonococcal NAATs are prepared or run and to verify equivocal results as well as, ideally, also low positive results with repeated testing from primary sample. It would also be valuable to have a definition of not only equivocal results but also low positives in AC2, which is defined in AGC. To our best knowledge, only one nongonococcal isolate that shows false-positive cross-reaction in the APTIMA assays has ever been verified. This meningococcal isolate carried one copy of an N. gonorrhoeae 16S rRNA gene (M. Walcher et al. 21st ECCMID; Milan, Italy; May 7–10, 2011). Surveillance on a regular basis for identification of similar strains, in particular from extragenital sites, is important.
In general, using highly sensitive NAATs, rare low-level false positives due to amplicon or genomic contamination in the laboratory, or even laboratory errors (mislabeling and pipetting, etc) may be exceedingly hard to avoid. In addition, the specificities of available gonococcal NAATs substantially differ,2,10,15–22 and the choice of NAAT, particularly if also pharyngeal and rectal specimens are analyzed, should be made with great care to avoid false-positive results. The prevalence of gonorrhea in the population being tested must also be considered, and a PPV of greater than 90% is recommended when using NAATs to detect N. gonorrhoeae. If this PPV cannot be reached with a single NAAT, the results should be verified with a supplementary NAAT with different target; for example, the AC2 can be verified with the AGC.15,16,21,33 Nevertheless, it is also important to acknowledge that true low-level positives may result in negative results in the supplementary testing due to degraded nucleic acid (if the same specimen is tested), variation in sampling (if new specimen is sampled), or lower sensitivity of the supplementary NAAT applied,27,29 and accordingly, in some high-risk population, further diagnostics and/or surveillance of the patients may be necessary.
In conclusion, validated, quality assured, sensitive, and specific laboratory assays are crucial for accurate diagnosis of gonorrhea and subsequent appropriate management of patients. Nucleic acid amplification tests have dramatically improved the sensitivity for the detection of N. gonorrhoeae, especially in pharyngeal and rectal samples,2–7 as well as simplified the diagnostics. In this study, the analytical specificity and sensitivity of the Gen-Probe APTIMA Combo 2 and APTIMA GC tests were substantially challenged, and both assays displayed 100% specificity and 100% sensitivity.
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