ACCEPTED. Northwestern University Feinberg School of Medicine, Chicago, IL 5

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1 JCM Accepts, published online ahead of print on 14 March 2007 J. Clin. Microbiol. doi: /jcm Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. A Neisseria Species Identification Assay for the Confirmation of N. gonorrhoeae-positive COBAS Amplicor PCR Kathy A. Mangold, 1,5 MaryAnn Regner, 3 Mohammed Tajuddin, 1 Aamair M. Tajuddin, 5 Lawrence Jennings, 4,5 Hongyan Du, 2 and Karen L. Kaul 1,5* Department of Pathology and Laboratory Medicine 1, Center on Outcomes, Research and Education 2, Evanston Northwestern Healthcare, Evanston, IL; Department of Pharmacology 3, University of Colorado Health Science Center, Denver, Colorado; Department of Pathology and Laboratory Medicine 4, Children s Memorial Hospital, Chicago, IL and Department of Pathology, *Corresponding author: Karen L. Kaul, MD, PhD Northwestern University Feinberg School of Medicine, Chicago, IL 5 Evanston Northwestern Healthcare Department of Pathology and Laboratory Medicine 2650 Ridge Avenue Evanston, Illinois Phone: (847) Facsimile: (847) k-kaul@northwestern.edu Downloaded from on July 26, 2018 by guest Presented at the annual meeting of the Association for Molecular Pathology, Scottsdale, AZ, November 10-13, Running title: NG Confirmatory Assay Keywords: Real-time polymerase chain reaction, gonorrhea Text pages = 14; Tables = 4; Figures = 2

2 1 ABSTRACT Introduction/Background: Screening assays for Neisseria gonorrhoeae (NG) exhibit low positive predictive values, particularly in low prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. Materials & Methods: NsppID, a 16S rrna real-time PCR/melt curve assay developed to distinguish NG from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for NG, the cppb gene and pora pseudogene. 408 clinical specimens (324 female endocervical swabs, and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor CT/NG assay (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. Results: The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified NG in 102 (56%). 69/181 (38%) were positive for NG by pora pseudogene and 115/181 (64%) for cppb. However, cppb was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or pora pseudogene (2%) assays. The pora pseudogene assay had the highest specificity for both genders, but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppb due to poor specificity Conclusions: The NsppID assay is an accurate confirmatory assay for NG detection. In addition, the NsppID assay can identify the non-ng species responsible for the majority of false positive results from the COBAS Amplicor CT/NG assay. 1

3 23 INTRODUCTION Neisseria gonorrhoeae (NG) is the second most prevalent sexually transmitted bacterial infection after Chlamydia trachomatis (CT). (1) Many NG infections are asymptomatic, especially in women, and if left untreated can develop long-term consequences, including chronic pelvic pain, pelvic inflammatory disease, and ascending infection of the fallopian tubes. Accurate diagnosis of symptomatic and asymptomatic infection is required to prevent these complications and to control the transmission of infection. Molecular assays such as the COBAS Amplicor CT/NG have high sensitivity and specificity in detecting both CT and NG infections, and screening high prevalence populations results in positive predictive values >80%. However, even with very specific assays, screening of low prevalence populations lowers the positive predictive value of any test. For NG analysis of low prevalence populations performed by the COBAS Amplicor (6, 12, 32) CT/NG assay, unacceptable positive predictive values have been reported, particularly when the optical density (OD) reading is in the gray-zone of 0.2 to <3.5. (6) Repeat testing of the gray-zone and positive specimens has been shown to improve the specificity of the COBAS Amplicor CT/NG assay to some extent. (24) Still, several crossreacting non-pathogenic organisms, primarily other Neisseria spp., with homologous sequences are known to contribute to false positive results. (6, 7, 13, 24, 25) As recommended by 41 the Centers for Disease Control and Prevention, (2) confirmation of NG infection using independent testing for a different NG-specific target greatly improves the positive predictive value. (14) Other real-time PCR assays have been developed targeting genes reportedly specific for NG, with confirmation frequencies of <60% of the presumptive NG positive specimens. (22, 2

4 46 29, 31) Subsequent studies indicated that several real-time confirmatory assays may lack the 47 (5, 23) specificity and/or sensitivity required for accurate diagnosis. Also, none of these molecular assays identify the cross-reacting species responsible for false positive results with the COBAS Amplicor CT/NG assay. Thus, this study describes a newly developed assay, NsppID, that not only detects and identifies NG but also identifies cross-reacting Neisseria spp. in an effort to ultimately reduce the number of false positives, and compares NsppID to two previously published confirmatory assays. MATERIALS AND METHODS (21, 29) Specimens: Genomic DNA from Neisseria spp. were obtained either directly from American Type Culture Collection (ATCC, Manassas, VA) or extracted in-house from strains obtained from ATCC. Genomic DNA from seven NG strains (ATCC 53420, , 53423, 53424, 53425, ), six N. meningitidis strains (ATCC 53414, 53415, 53416, 53417, 53418, BAA-335) and one strain each of N. cinerea (ATCC 14685), N. lactamica (ATCC 23970), N. perflava (ATCC 14799), and N. subflava (ATCC 49275) were used in assay development and as controls during analytical assay validation of NsppID, a 16S rrna PCR/melt curve assay we developed that distinguishes NG from other Neisseria spp. Sample remaining after COBAS Amplicor CT/NG testing from 408 de-identified clinical specimens (324 endocervical swabs, and 84 male urine or urogenital swab samples) were used according to the protocol approved by the ENH Institutional Review Board COBAS Amplicor Testing: Clinical specimens were screened using the COBAS Amplicor CT/NG assay (Roche Diagnostics, Indianapolis, IN) according to the manufacturer s instructions. Specimens were considered negative if the initial OD readings were <0.2 and the OD of the inhibition control was 0.2. Specimens with initial OD 3

5 readings in the gray-zone of 0.2 to <3.5 or positive readings 3.5 were repeated twice more, and at least two of the three OD readings needed to be 2.0 before the specimen was reported as COBAS NG-positive. (24) DNA Extraction: Genomic DNA from Neisseria spp. strains were extracted in-house using Puregene DNA Extraction (Gentra Systems, Minneapolis, MN). Genomic DNA was extracted from 200 µl of the original clinical specimen using GENERATION DNA Purification Capture Columns (Gentra Systems) and/or High Pure Viral Nucleic Acid (Roche Diagnostics) according to the manufacturers instructions. Some M4RT specimens received had a significant ph color change due to increased acidity; therefore, 10 µl of 0.5 M NaOH was added to all M4RT aliquots before extraction to ensure an alkaline sample and improve the recovery of the sample DNA. Real-Time PCR: Our laboratory includes geographically separate areas for reagent preparation, sample preparation, PCR amplification, and amplicon analysis to avoid carryover contamination. The real-time assays included negative, positive and no template controls in each run. The NsppID assay was developed and validated using genomic DNA extracted from ATCC controls, either individually or combinations of NG with either N. meningitidis or N. perflava. Clinical validation included testing of the 408 clinical specimens to determine any Neisseria spp. present using the NsppID assay. These results were compared to the results from two previously published real-time PCR confirmatory assays targeting genes reportedly specific for NG [the cppb gene (21) and pora pseudogene (pap) (29) ]. All three assays were performed on the LightCycler instrument (Roche). The two previously published procedures were performed as described in the individual publications, and the NsppID assay is described below. Real-time PCR inhibition/detection failure was 4

6 92 93 assessed via amplification of exon 8 of the human p53 gene (in-house assay) or universal bacterial 16S rrna gene amplification NsppID real-time PCR assay: Consensus primers (NG767F16: 5 -AAA gcg Tgg gta gca A-3 and NG964R16: 5 -TTC TTC gcg TTg CAT C -3 ; sequence numbering from GenBank Accession number X07714) were designed after comparing several partial 16S rrna gene sequences for each of ten Neisseria spp. The FRET probe pair consisted of a FITC-labeled probe (NG839GR3: 5 -CAA CCT gat TgC TTA gta gcg Tag CTA ACg- FITC 3 ) that was specific for N. subflava, N. flavescens and one strain of N. meningitidis, but reliably discriminated between the different Neisseria spp. by mean temperature of melting (T m ), and an LC640-labeled anchor probe (NG870RED: 5 LC640- gtg AAA TTg ACC gcc Tgg gga gta Cgg-phos 3 ) common to most Neisseria spp. (Figure 1). Each 20 µl-reaction mix contained 5 µl of sample DNA, 2 µl of 10 FastSTART DNA Hybridization Probe reagent (Roche), 3 mm MgCl 2 (final concentration), 0.2 mm of NG767F16 primer, 1.0 mm of NG964R16 primer, 0.2 mm of NG839GR3 probe, and 0.4 mm of NG2RED primer. Increased sensitivity was observed when the master mix was predigested with HpaII restriction enzyme (1 U per reaction; 37 C incubation for 10 minutes, followed by enzyme inactivation by heating to 65 C for 20 minutes) before addition of the specimen DNA. This restriction digest eliminated any genomic DNA in the master mix, including residual DNA found in commercial Taq polymerases, and has been shown to improve sensitivity in other bacterial 16S rrna PCR assays (data not shown). (18) PCR conditions consisted of an initial denaturation at 95 C for 10 minutes followed by amplification for 50 cycles of 10 seconds at 95 C, 15 seconds at 55 C and 7 seconds at 72 C with fluorescence acquisition at the end of each extension step. Amplification was 5

7 immediately followed by a melt program consisting of 15 seconds at 95 C, 15 seconds at 40 C, and a stepwise temperature increase of 0.2 C/second until 75 C with fluorescence acquisition at each temperature transition. The fluorescence data were analyzed using the F2/F1 setting. This assay for Neisseria spp. differentiation required approximately one hour to complete, in addition to the sample preparation time. Melt curve analysis was used to determine the species-specific T m based on values determined from the respective ATCC controls. Although we did not test a strain of N. flavescens, the sequence information obtained from GenBank would indicate that it would also be included with the group of N. subflava, N. perflava and N. meningitidis. Inter-assay Comparison: Since no accurate gold standard was available, a consensus NG result was defined as positive when at least two of the three confirmatory assays yielded positive NG results. This imperfect standard approach has been used in other studies (4, 21, 26) when the ideal gold standard does not exist, with under-estimation of the test s accuracy in most situations. (33) The Chi-square homogeneity test was used to determine if the distribution of NG positives was the same between the three confirmatory assays.(8) Sensitivity, specificity, the predictive values of both positive and negative results were calculated and compared using large sample binomial z test; confirmatory assay was also calculated. (28) (8, 20) the accuracy of each 133 RESULTS The NsppID assay was developed as a confirmatory test to determine the presence of NG or other cross-reacting Neisseria spp in clinical specimens that tested NG-positive using the COBAS Amplicor CT/NG assay. In the analytical validation of the NsppID assay, the 6

8 consensus primers were able to amplify 16S rrna gene sequences from all ATCC isolates of NG, N. lactamica, N. cinerea, N. subflava, N. perflava, and N. meningitidis tested, and FRET probes to a unique sequence between the primers allowed species differentiation using melt curve analysis (Figure 2 and Table 1). The analytical sensitivity of the assay was calculated to be 4 genome copies of NG (10 fg DNA) per reaction for this multicopy gene target (data not shown). Reactions containing DNA from both NG and either N. meningitidis or N. perflava indicated that NG could still be detected in the melt curve analysis even in the presence of 40-fold concentrations of the non-ng species (data not shown). The average T m in the melt curve analysis for each Neisseria spp. is as follows: NG ~58.0 C; N. lactamica ~51.5 C; N. cinerea ~55.0 C; N. meningitidis ~62.5 C or ~64.5 C; N. perflava ~62.5 C; and N. subflava ~62.5 C. Clinical validation was performed on a total of 408 ENH patient specimens screened using the COBAS Amplicor CT/NG assay, yielding 181 NG-positive specimens and 227 randomly selected NG-negative specimens for this study. All 408 specimens were positive for human DNA and/or universal bacterial gene amplification, indicating no substantial PCR inhibition (data not shown). The results of testing all 408 specimens using the NsppID assay are shown in Table 1. The NsppID assay was able to detect a Neisseria spp. in 150 of the 181 COBAS-positive specimens (83%), but identified NG in only 102 (56%). Thirty-one specimens did not amplify detectable amplicons for any Neisseria spp.; this could indicate a lack of sensitivity, problems with real-time PCR and fluorescent detection from M4 media, or it may also reflect cross-reacting organisms present in the COBAS-positive specimens other than Neisseria spp., such as Lactobacillus. (25) Dual infections were identified in six specimens, 7

9 with NG being one of the infectious agents in four of the six. Of the 111 clinical specimens that were positive for NG in the NsppID assay, 90 (81%) had initial OD readings 3.5, 20 (18%) had readings in the gray zone of 0.2 to <3.5, and only 1 specimen (1%) had an initial reading < 0.2 in the COBAS Amplicor assay. Comparisons between the NsppID, cppb and pap assays are shown in Table 2. The numbers of NG positive specimens amongst the 181 COBAS-positive specimens according to the other two confirmatory tests were 69 (38%) positive for pap and 115 (64%) for cppb. However, cppb was also positive in a significant number (15%) of COBAS-negative specimens as compared to either the NsppID (4.0%) or pap (2%). This may be due to the detection of non-ng spp. carrying the cryptic plasmid pjd1; (4, 17, 29) 13% of cppb positives yielded non-ng spp. in the NsspID assay. Since the NsppID assay detects and identifies other Neisseria spp. in addition to NG, while the other two confirmatory assays only detects NG, the percentage of COBAS-positive specimens with no resolution is only 17% using the NsppID assay as compared to 37% and 61% for cppb and pap assays, respectively. Using positive results in at least two of the three confirmatory assays as a consensus NG positive specimen, we can determine the sensitivity and specificity for the detection of NG in all four PCR assays. Comparisons between the COBAS Amplicor, NsppID, cppb and pap assays are shown in Table 3. Of the 181 COBAS-positive specimens, 95 were also consensus NG positive, and the percentage concordance differed along gender lines 45 of 61 male specimens (74%) were concordant whereas only 51 of 120 female specimens (43%) agreed. These results are in agreement with other published studies analyzing COBAS Amplicor detection of NG in low prevalence populations, (6, 14, 21, 32) and highlight the need of 8

10 a confirmatory test to ensure accurate reporting of infections. Six of the 227 COBASnegative specimens (3%) were consensus NG positive, all from female specimens. Five of the six were in the gray-zone of the COBAS Amplicor assay on the initial run but were categorized as negative after repeat testing. Although 3% is approximately the false negative rate reported in the COBAS Amplicor CT/NG product information, it is higher than the prevalence rate of 0.6% in our population. Statistical analysis indicates a significant difference between the NsppID and pap assays, with the NsppID assay being more sensitive and less specific than pap (p < 0.001; χ² Η = 33.8, df = 1); the cppb assay had significantly lower sensitivity and specificity than the other two assays (p < 0.01; χ² Η = 10.69, df = 1). Limiting the analysis to only those specimens that were NG positive by COBAS Amplicor testing, both NsppID and pap assays identified NG more accurately than cppb with higher sensitivity and specificity (p < 0.001; χ² Η = , df = 1), but the NsppID assay did not significantly exceed pap in accuracy (p > 0.1; χ² Η = 2.032, df = 1). Statistical assessment of clinical assays include common parameters of sensitivity, specificity, positive and negative predictive values, and accuracy, as shown in Table 4. The key statistic for confirmatory assays after an initial screen is predictive value of a positive result (PPV). The pap assay has the highest PPV for both genders (97% in female specimens and 100% in male specimens), followed closely by NsppID (90% in female specimens and 97.8% in male specimens). However, the sensitivity of pap is significantly lower than that of NsppID (p < 0.001), particularly in female specimens (59% vs. 92% in female specimens and 87% vs. 100% in male specimens, respectively). Therefore the accuracy of pap is lower (overall, 85% vs. 92%). An added feature not represented in these statistics is that the 9

11 NsppID assay is capable of identifying non-ng species responsible for false positive results of the COBAS Amplicor testing, reducing the number of specimens with unresolved COBAS positive results. Although the sensitivity of the cppb assay is higher than that of pap, the lack of specificity of the cppb assay makes it significantly lower in PPV, NPV and accuracy than the NsppID assay and lower in PPV than the pap assay (p < 0.001). DISCUSSION The COBAS Amplicor assay targets the NG cytosine methyltransferase gene. This assay has been shown to be a reliable target in a widely-used screening assay in asymptomatic patients, as NPVs greater than 99% occur with assays with sensitivities greater than 98%, regardless of the prevalence. (13, 14, 25) However, using this and similar assays in low prevalence populations significantly lowers the PPV of NG analysis below satisfactory levels, requiring the application of a second assay to confirm the existence of NG in patient specimens. (12) The COBAS Amplicor CT/NG assay used in the initial screen and the three confirmatory assays used in this study are all nucleic acid amplification tests (NAATs), but each of the four has a different genetic target reportedly specific for NG and can be considered independent assays, fulfilling the CDC requirement for a second independent test for confirmation. (2) Since this study was designed to find the best NG confirmatory assay after screening with COBAS Amplicor, the gold standard for designating NG positive specimens was positivity for NG in at least two of the three confirmatory tests. Generally, bacterial isolation using selective culture media would be used as the gold standard. However, successful culture of NG requires stringent collection, transport and storage of clinical specimens, and often produces false negative results. (3, 14, 16, 27) Under optimal laboratory conditions, the sensitivity of culture is 85-95% of acute NG infections (19) but 10

12 drops to 50% of chronically infected females. (3) In a high prevalence population (6.6% of women and 20.1% of men), the sensitivities of culture were 84.8% for endocervical specimens, 92.7% from symptomatic male urethral specimens, and 46.2% from asymptomatic male urethral specimens. (16) Additionally, the sensitivity of culture for NG has been reported as 88.9% for on site collection/testing compared with 77.8% for off-site testing. (11) Due to these and other limitations, culture results were not included in this study. We encountered unexpected difficulty with amplification of some samples, though whether this was due to PCR inhibition, interference with fluorescence detection, or other issues remains unclear. Significant effort went into determining the best DNA extraction method to use with M4RT transport media, the most common specimen received for clinical CT/NG testing in our laboratory. In our hands, the most successful extraction used was High Pure Viral Nucleic Acid extraction (Roche), followed closely by GENERATION Capture Column extraction (Gentra). Recently, the EZ1 Virus kit (Qiagen, Valencia, CA) has also been used successfully (data not shown). All 408 specimens analyzed in this study showed successful amplification of human and/or universal bacterial genes. However, endocervical samples, even with the most successful extraction method used, have a lower positivity rate for identification of NG or other species than urine or urethral samples We developed a real-time PCR assay (NsppID) that could identify NG as well as at least five other Neisseria spp. using genomic DNA from ATCC as controls, in order to confirm positive NG specimens and identify false COBAS Amplicor positives due to cross-reacting Neisseria spp. Repeated testing of seven strains of NG and four other Neisseria spp. resulted 11

13 in distinct melting curve T m peaks for NG and two other non-ng Neisseria spp. (N. cinerea and N. lactamica), and a single overlapping peak for three other non-ng Neisseria spp. (N. meningitidis, N. perflava, and N. subflava). Dual infections were also detected using the NsppID assay, with the capability of detecting NG even when the non-ng Neisseria spp. was 40x the concentration of NG. Therefore, the detection of non-ng Neisseria spp. in COBAS Amplicor NG-positive specimens without detecting an NG peak would indicate the positive result in COBAS was most likely due to a cross-reacting Neisseria spp. If no amplification occurred in the NsppID assay, either NG was present but below the sensitivity of the NsppID assay, or an organism besides Neisseria is cross-reacting in the COBAS Amplicor assay to produce a false NG-positive result. Some species of Lactobacillus have been reported as causing false NG-positive in the COBAS Amplicor CT/NG assay. (25) Based on sequence comparisons, it is highly unlikely that any Lactobacillus spp. would be detected in the NsppID assay presented in this report and is a possible cause for no amplification from specimens positive for NG after COBAS Amplicor CT/NG screening that are negative using the NsppID assay. Several other real-time NG confirmatory assays have been published. (22, 29, 31) The cppb gene has frequently been used as an NG-specific target in PCR assays, (10) but the specificity for NG of the cppb gene has recently been challenged. (4, 5, 15, 17, 23, 29) Although found on the cryptic plasmid pjd1 that is often at higher copy numbers than chromosomal genes, there are some NG strains that are cppb-negative by PCR due to lack of the plasmid or chromosomal integration. (4, 5, 15, 17, 23) Adding to its lack of specificity, the cppb gene has also been identified in Neisseria spp. other than NG as well. (4, 17, 29) The pap assay that detects the pora pseudogene has reportedly a greater specificity for NG but slightly lower 12

14 analytical sensitivity than the cppb assay. (29, 30) In this study, we compared the NsppID assay with these two previously published real-time PCR confirmatory assays; real-time confirmatory assays based on a third target that is also used commercially for NG detection (the opa gene) were recently published but not included in this comparison. (9, 22, 29, 30) The application of any of the three confirmatory assays studied to the COBAS Amplicor NG-positive results significantly improves the PPV beyond that of COBAS Amplicor alone (from 53% to 73-99% overall). The differences in improvement were most evident in the female specimens. The PPV for female specimens tested by COBAS Amplicor alone was only 43%. The COBAS plus pap results from female specimens had the highest PPV at 97%, higher than 90% for NsppID (p =.0410) and dramatically higher than 62% found using the cppb assay (p < 0.001). Although the PPV for pap is higher than either of the other confirmatory assays due to its specificity, the lower sensitivity of this assay resulted in negative results for over 40% of the consensus NG positive female specimens, compared to less than 8% that were not detected using the NsppID assay. With the added feature of being capable of identifying non-ng species responsible for false positive results of the COBAS Amplicor testing, the NsppID assay also reduced the number of specimens with unresolved COBAS positive results. Since the development and clinical validation of this test, our laboratory has utilized NsppID for confirmation of all repeatedly NG positive COBAS Amplicor specimens. The specimen is reported as positive for NG if an NG-specific melt peak is observed, and is reported as negative if a melt curve peak associated with a Neisseria spp. other than NG is detected without an accompanying NG peak. If no amplification is detected in using the NsppID assay, the specimen is reported as equivocal, and a repeat 13

15 specimen is recommended for NG testing by either molecular diagnostics analysis or microbial culture. Since implementing this strategy, we have tested over 15,000 specimens with over 200 specimens that were positive by the COBAS Amplicor assay. We are able to confirm 50% of the COBAS-positive NG results. Otherwise, findings are reported as negative in 17%, or reported as equivocal (33%) if the specimen does not amplify or clearly indicate in the NsppID assay the species present. The positivity rate has dropped from 0.9% to 0.3% of specimens submitted for NG testing from asymptomatic females, and from 3.9% to 3.1% of specimens from symptomatic males, as is predicted. Therefore, in our low-prevalence (0.6% overall) population, the COBAS Amplicor CT/NG screen plus the NsppID confirmatory assay have been used together to achieve high NPV and high PPV while greatly reducing the number of equivocal specimens that require retesting of the patient. ACKNOWLEDGEMENTS This study was funded in part by a grant from Roche Diagnostics Corp., Indianapolis, IN, and the Dept. of Pathology & Laboratory Medicine of ENH. We thank the technologists in the ENH Microbiology laboratory for their invaluable assistance in providing control strains of Neisseria spp., and the technologists in the ENH Molecular Diagnostics laboratory for clinical specimens and data. 14

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19 22. Tabrizi, S. N., S. Chen, J. Tapsall, and S. M. Garland Evaluation of opa-based real-time PCR for detection of Neisseria gonorrhoeae. Sex Transm Dis 32: Tapsall, J. W., E. A. Limnios, N. L. Nguyen, I. Carter, G. Lum, K. Freeman, S. N. Tabrizi, S. M. Garland, D. M. Whiley, T. P. Sloots, and I. W. Chambers Cryptic-plasmid-free gonococci may contribute to failure of cppb gene-based assays to confirm results of BD ProbeTEC PCR for identification of Neisseria gonorrhoeae. J Clin Microbiol 43: Van Der Pol, B., D. H. Martin, J. Schachter, T. C. Quinn, C. A. Gaydos, R. B. Jones, K. Crotchfelt, J. Moncada, D. Jungkind, B. Turner, C. Peyton, J. F. Kelly, J. B. Weiss, and M. Rosenstraus Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results. J Clin Microbiol 39: van Doornum, G. J., L. M. Schouls, A. Pijl, I. Cairo, M. Buimer, and S. Bruisten Comparison between the LCx Probe system and the COBAS AMPLICOR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections in patients attending a clinic for treatment of sexually transmitted diseases in Amsterdam, The Netherlands. J Clin Microbiol 39: Van Dyck, E., M. Ieven, S. Pattyn, L. Van Damme, and M. Laga Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests. J Clin Microbiol 39: Van Dyck, E., H. Smet, L. Van Damme, and M. Laga Evaluation of the Roche Neisseria gonorrhoeae 16S rrna PCR for confirmation of AMPLICOR PCR-positive 18

20 samples and comparison of its diagnostic performance according to storage conditions and preparation of endocervical specimens. J Clin Microbiol 39: Wassertheil-Smoller, S Biostatistics and epidemiology: a primer for health professionals. Springer-Verlag, New York. 29. Whiley, D. M., P. J. Buda, J. Bayliss, L. Cover, J. Bates, and T. P. Sloots A new confirmatory Neisseria gonorrhoeae real-time PCR assay targeting the pora pseudogene. Eur J Clin Microbiol Infect Dis 23: Whiley, D. M., P. P. Buda, K. Freeman, N. I. Pattle, J. Bates, and T. P. Sloots A real-time PCR assay for the detection of Neisseria gonorrhoeae in genital and extragenital specimens. Diagn Microbiol Infect Dis 52: Whiley, D. M., G. M. LeCornec, I. M. Mackay, D. J. Siebert, and T. P. Sloots A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler. Diagn Microbiol Infect Dis 42: Zenilman, J. M., W. C. Miller, C. Gaydos, S. M. Rogers, and C. F. Turner LCR testing for gonorrhoea and chlamydia in population surveys and other screenings of low prevalence populations: coping with decreased positive predictive value. Sex Transm Infect 79: Zhou, X. H., N. A. Obuchowski, and D. K. McClish Statistical Methods in Diagnostic Medicine. John Wiley & Sons, New York, NY. 19

21 Table 1. NsppID assay results from 408 patient specimens using DNA extracted either by Gentra GENERATION Capture Column or by Roche High Pure Viral methods. Note that dual infections are also identified using the NsppID assay. NsppID COBAS Amplicor Final Negative Positive No amplification 186 (82%) 31 (17%) N. lactamica 0 (0%) 0 (0%) N. subflava/n. perflava/n. meningitidis 27 (12%) 39 (22%) N. cinerea 2 (1%) 7 (4%) N. subflava/n. perflava/n. meningitidis & N. cinerea 3 (1%) 2 (1%) NG 8 (4%) 98 (54%) NG & N. subflava/n. perflava/n. meningitidis 1 (0%) 4 (2%) 1

22 Table 2. Results from 408 patient specimens screened using the COBAS Amplicor CT/NG assay and the three NG confirmatory assays. A lower Ct limit of 35 cycles was used for the cppb assay instead of the 40 cycles used for the other confirmatory assays, since a higher limit resulted in an exceedingly large percentage of consensus negative specimens to be positive by cppb (up to 56.3%). NsppID pap cppb Assay Negative COBAS Amplicor Final Positive No amplification 186 (82%) 31 (17%) Non-NG only 32 (14%) 48 (27%) NG 9 (4%) 102 (56%) Negative 222 (98%) 111 (61%) Positive 5 (2%) 70 (39%) Negative 194 (86%) 66 (36%) Positive 33 (15%) 115 (64%) 1

23 Table 3. Results from 408 patient specimens screened using the consensus result of at least two of the three confirmatory assays as the gold standard. A lower Ct limit of 35 cycles was used for the cppb assay instead of the 40 cycles used for the other confirmatory assays, since a higher limit resulted in an exceedingly large percentage of consensus negative specimens to be positive by cppb (up to 56.3%). Assay COBAS Amplicor NsppID pap Negative Consensus NG Result Positive Negative 221 (97%) 6 (3%) Positive 86 (48%) 95 (52%) No amplification 213 (69%) 4 (4%) Non-NG only 80 (26%) 0 (0%) NG 14 (5%) 97 (96%) Negative 306 (100%) 1 (0%) Positive 29 (29%) 72 (71%) cppb Negative 248 (81%) 12 (12%) Positive 59 (19%) 89 (88%) 1

24 Table 4. Sensitivity, specificity, predictive value of a positive result (PPV), predictive value of a negative result (NPV), and accuracy of the three confirmatory assays in testing COBAS Amplicor NG-positive specimens. NsppID pap cppb Female Male Female Male Female Male Sensitivity 92% 100.0% 59% 87% 86% 89% Specificity 87.0% 94% 99% 100% 61% 75% PPV 90% 98% 97% 100% 62% 91% NPV 94% 100% 76% 73% 86% 71% Accuracy 89% 98% 82% 90% 72% 85% 1

25 Legends for Figures Figure 1. Sequence comparisons between ten Neisseria spp. encompassed by the FRET probes used in the NsppID assay. The underlined sequences are exact matches to the probe sequences and therefore have the highest theoretical T m. The sequence numbering listed for the two probes is from GenBank Accession number X Figure 2. Melt curve analysis in the NsppID assay using the ATCC Controls, showing speciesspecific T m peaks. 1

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28 JOURNAL OF CLINICAL MICROBIOLOGY, July 2007, p Vol. 45, No /07/$ doi: /jcm ERRATUM Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae- Positive Results of the COBAS Amplicor PCR Kathy A. Mangold, MaryAnn Regner, Mohammed Tajuddin, Aamair M. Tajuddin, Lawrence Jennings, Hongyan Du, and Karen L. Kaul Department of Pathology and Laboratory Medicine, Center on Outcomes, Research and Education, Evanston Northwestern Healthcare, Evanston, Illinois; Department of Pharmacology, University of Colorado Health Science Center, Denver, Colorado, Department of Pathology and Laboratory Medicine, Children s Memorial Hospital, Chicago, Illinois; and Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois Volume 45, no. 5, p , Page 1404: Fig. 1 should appear as shown below, with the sequence for only one N. meningitidis strain being underlined for the FITC-labeled probe NG839Gr3. The legend of the figure is correct as published. 2353