Confirmation by 16S rRNA PCR of the COBAS AMPLICOR CT/NG Test for Diagnosis of Neisseria gonorrhoeae Infection in a Low-Prevalence Population

Abstract

The COBAS AMPLICOR CT/NG test is widely used for the diagnosis of Neisseria gonorrhoeae infection using genital swabs or urine samples. Although highly specific, cross-reactivity occurs with some nonpathogenic strains of Neisseria and Lactobacillus species. In low-prevalence populations, even highly specific assays may require confirmatory testing of positive results. We assessed the positive predictive value (PPV) of this test in a low-prevalence (0.5%) setting. Genital and urine specimens testing positive using the COBAS AMPLICOR NG test were retested using an investigational 16S rRNA PCR assay. Additionally, 737 specimens were tested in parallel by both culture and the above PCR protocol. Of 9,772 specimens tested in-house, 168 were positive by the AMPLICOR test; in addition, 62 AMPLICOR-positive specimens were referred to our laboratory for confirmatory testing, yielding 230 positive specimens. Of these, 72 were confirmed positive by 16S rRNA PCR, yielding a specificity of 98.7% and a PPV of 31.3%. Specificity was similar for all specimen types, whereas PPV varied with prevalence: specimens from males, females, urine specimens, and genital swabs had PPVs of 70.8, 13.3, 51.9, and 20.1%, respectively. The PPV was higher when the initial AMPLICOR optical density (OD) was >3.5 versus initial and repeat OD readings in an equivocal zone of >0.2 to <3.5 (65.1 versus 10.1%; P < 0.001). On repeat testing of specimens with ODs in the equivocal zone, 54 gave ODs of >0.2 and <2.0, 35 gave ODs of >2.0 and <3.5, and 12 gave ODs of >3.5, with 3.7, 20, and 33.3% confirmed positive, respectively (P ‫؍‬ 0.004). Comparing PCR to culture as the "gold standard," specificity increased from 96.8 to 99.9% when 16S rRNA PCR was performed on specimens positive by the COBAS AMPLICOR NG test. Confirmatory testing with a more specific method such as 16S rRNA PCR should be considered in low-prevalence populations, especially for specimens with an OD in the equivocal zone.Neisseria gonorrhoeae remains the second most prevalent sexually transmitted bacterial infection after Chlamydia trachomatis, with approximately 650,000 new infections occurring annually in the United States (2). A significant proportion of these infections, especially in women, are asymptomatic and, if they remain undiscovered, may result in such long-term consequences as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy, and infertility (9). Furthermore, silent and untreated infection facilitates the spread of this organism to unsuspecting sexual partners. Therefore, accurate diagnosis of both symptomatic and asymptomatic infection is critical.A number of techniques have been developed to detect genital infections caused by N. gonorrhoeae. The current "gold standard" for diagnosis of infection is by culturing endocervical or urethral swab specimens on selective media. However, even under optimal laboratory conditions, the sensitivity of gonococcal cultures ranges from 85 to 95% for acute infection (17) and falls to approximately 50% for females with c...

Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

“…Therefore, if the rate of test positivity in the population is 1.0%, then one-half of the observed positive results may be false-positive results, providing a positive predictive value of only 50%. When testing urogenital specimens in a low prevalence (0.5%) Canadian population, Diemert et al 72 found that the specificity of the Amplicor N. gonorrhoeae PCR assay was 98.7%, whereas the positive predictive value was only 31.3%. Thus, if positive results were issued for this Canadian population without supplementary testing, then close to 70% of positive results issued would in fact be false-positive results.…”

“…Notably, numerous studies conducted worldwide have shown positive predictive values for the Amplicor PCR assay on urogenital specimens below the 90% CDC threshold. 23,55,72,88,89 Increasing the positive result cutoff of the Amplicor assay will increase the positive predictive value of the test. This is because commensal Neisseria strains cross-reacting with the Amplicor assay may generally be at a low concentration or may contain minor base variations with the Amplicor PCR oligonucleotides.…”

“…Thus, supplementary testing is the only appropriate approach. 57,72 Supplementary tests for the Roche Amplicor assay have included a 16S PCR assay provided by Roche as well as various in-house PCR methods targeting the cppB gene. 51,64,90 Roche later withdrew the 16S assay from the marketplace, so cppB gene-based PCR assays were widely adopted for confirmation.…”

Molecular techniques for clinical diagnostic bacteriology

“…In addition, susceptibility test can be performed (25). For some authors, in areas where the N. gonorrhoeae prevalence is low, the culture is preferred over testing gene amplification (22). Other authors did not observe differences in sensitivity and specificity kits depending on the prevalence (23).…”

Asymptomatic men: should they be tested for urethritis?

“…It is well recognised that some men with N gonorrhoeae are asymptomatic 2 and would be missed if the microorganism was not tested for, and Horner and Taylor-Robinson 22 advocate testing for both, but acknowledge the increased risk of falsepositives not only because it is a low-prevalence population 23 but also because some NAATs can detect commensal Neisseria species. [24][25][26] (2) As for (1) but examine and only undertake a Gram-stained urethral smear for those with a discharge. (3) As for (2) but include a leucocyte esterase test on those without a discharge.…”