Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens.

We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.     

Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.

AIMS--To evaluate the use of a duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples. METHODS--Genital swab specimens were obtained from both China (203 swabs) and Hong Kong (202 swabs). N gonorrhoeae and C trachomatis were detected in each specimen with a number of tests including enzyme immunoassays (IDEIA) and PCR assays using both single and double primer pairs. The primer pair for N gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was 390 base pairs long. For C trachomatis, the PCR product was 473 base pairs long, resulting from amplification of a sequence in the common 7.4 kilobase plasmid present in all serovars. For N gonorrhoeae, PCR results were also compared with those obtained by culture and Gram's smear of the discharges. RESULTS--For the 203 specimens collected in China, similar numbers of positive results (177) were obtained by both Gonozyme and duplex PCR for the detection of N gonorrhoeae. No discrepant results were found among the cultured specimens when Gonozyme and duplex PCR were compared. C trachomatis was detected in 47 specimens by duplex PCR, but was detected in only 28 by IDEIA. Of the 202 Hong Kong specimens, 46 were positive for N gonorrhoeae, detected by both Gonozyme and duplex PCR; 34 were positive for C trachomatis, 25 of which were detected by IDEIA and the remainder by duplex PCR. CONCLUSIONS--The duplex PCR assay is a satisfactory diagnostic tool for the simultaneous detection of N gonorrhoeae and C trachomatis in clinical swab samples. Further evaluation is suggested.     

Characteristics of the m2000 automated sample preparation and multiplex real-time PCR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

We evaluated a new real-time PCR-based prototype assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae developed by Abbott Molecular Inc. This assay is designed to be performed on an Abbott m2000 real-time instrument system, which consists of an m2000sp instrument for sample preparation and an m2000rt instrument for real-time PCR amplification and detection. The limit of detection of this prototype assay was determined to be 20 copies of target DNA for both C. trachomatis and N. gonorrhoeae, using serially diluted linearized plasmids. No cross-reactivity could be detected when 55 nongonococcal Neisseria isolates and 3 non-C. trachomatis Chlamydia isolates were tested at 1 million genome equivalents per reaction. Concordance with the Roche Amplicor, BDProbeTec ET, and Gen-Probe APTIMA Combo 2 tests was assessed using unlinked/deidentified surplus clinical specimens previously analyzed with these tests. For C. trachomatis, concordance for positive results ranged from 93.7% to 100%, while concordance for negative results ranged from 98.2% to 100%. For N. gonorrhoeae, concordance for positive and negative results ranged from 91.4% to 100% and 99.3% to 100%, respectively. A workflow analysis of the prototype assay was conducted to obtain information on throughput under laboratory conditions. At 48 samples/run, the time to first result for both C. trachomatis and N. gonorrhoeae was 4.5 h. A total of 135 patient specimens could be analyzed in 8.9 h, with 75 min of hands-on time. This study demonstrated the technical and clinical feasibility of the new Abbott real-time PCR C. trachomatis/N. gonorrhoeae assay.     

Direct comparison of the BD ProbeTec ET system with in-house LightCycler PCR assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae from clinical specimens

The commercial BD ProbeTec ET (BDPT) system for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis from clinical specimens was compared with our in-house LightCycler real-time PCR (LC-PCR) assays. Specimens initially positive by the BDPT system were retested by our LC-PCR assays. Our results for C. trachomatis testing indicate a 91.2% agreement when the results of 114 clinical specimens, initially positive by BDPT over a wide range of method-other-than-acceleration (MOTA) scores, were retested by our LC-PCR assay. The agreement between the two systems improved to 96% when only MOTA scores of >30,000 were retested by the LC-PCR assay. The overall agreement between the two systems for Neisseria gonorrhoeae detection from 155 clinical specimens was only 77.4%, with agreement particularly low (24.1%) for MOTA scores ranging from 2,000 to 19,999. Repeat testing of specimens with the BDPT only closely correlated with that seen by others demonstrating that reproducibility of the BDPT system for specimens initially within the MOTA score range from 2,000 to 9,999 is problematic, especially for Neisseria gonorrhoeae testing. With our study, we proposed an algorithm for C. trachomatis and N. gonorrhoeae testing which involves screening with the BDPT system followed by selective use of our in-house LC-PCR assays.     

Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis,Neisseria gonorrhoeae and Mycoplasma genitalium in urine

We evaluated the performance of the Bio-Rad real-time Dx CT/NG/MG® assay for detection of C. trachomatis, N. gonorrhoeae and M. genitalium on a collection of 441 urine samples from sexually transmitted infections, or travellers consultations and from anonymous sperm donors that were previously analysed with the Abbott RealTime CT/NG assay. Samples positive for C. trachomatis or N. gonorrhoeae with the Abbott assay had all previously been confirmed with an in-house real-time PCR assay. Samples positive for M. genitalium with the Bio-Rad assay were subsequently analysed by an in-house real-time PCR. On a total of 441 urines, 104 samples were positive for C. trachomatis, 12 were positive for N. gonorrhoeae and seven were positive for M. genitalium. After retesting of discrepant results, the test results were completely concordant, resulting in a calculated sensitivity and specificity of the Bio-Rad assay of 98.1 % and 100 % for C. trachomatis and of 91.7 % and 100 % for N. gonorrhoeae. Results for M. genitalium with the Bio-Rad assay were also concordant with the results of an in house PCR. We also evaluated the performance of automated nucleic acid extractions of urine samples with the NucliSENS easyMAG (bioMérieux) compared to the manual DNA extraction prescribed by the insert of the kit. The easyMAG extraction gave lower Ct values, relieved inhibition and had a lower hands-on time.     

Evaluation of the Abbott RealTime CT/NG assay in comparison to the Roche Cobas Amplicor CT/NG assay

Several commercial methods exist for the molecular detection of Chlamydia trachomatis and Neisseria gonorrhoeae in clinical samples. Here we evaluated the performance characteristics of the newly FDA-cleared Abbott RealTime CT/NG assay (where "CT" stands for Chlamydia trachomatis and "NG" stands for Neisseria gonorrhoeae) that uses the automated m2000 molecular platform. Results were compared to those of the Roche Cobas Amplicor CT/NG assay. A total of 926 cervical swab, 45 female urine, 6 male urethral swab, and 407 male urine specimens from 1,384 patients were examined. After resolving all Roche N. gonorrhoeae-positive results with two additional real-time PCR assays, we found that the agreement between the assays was excellent. For urine samples, there was 99.6% positive agreement and 97.7% negative agreement for C. trachomatis, and for male urine samples, there was 100% positive agreement and 99.7% negative agreement for N. gonorrhoeae. For cervical swab samples, there was 98.8% positive agreement and 98.5% negative agreement for C. trachomatis, and there was 96.6% positive agreement and 99.8% negative agreement for N. gonorrhoeae. In limiting dilution analyses, we found that the Abbot assay was more sensitive than the Roche assay for both C. trachomatis and N. gonorrhoeae. In addition, there appeared to be an enhanced ability of the Abbott assay to detect dual infections, especially in the presence of large amounts of N. gonorrhoeae and small amounts of C. trachomatis organisms. In summary, we conclude that the Abbott RealTime CT/NG assay is an accurate and automated new addition to the available testing options for C. trachomatis and N. gonorrhoeae.     

Evaluation of three automated nucleic acid amplification systems for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in first-void urine specimens

A total of 500 first-void urine specimens were tested for the presence of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids using ProbeTec ET reagents on a Viper platform (BD Diagnostics, Mississauga, Ontario, Canada), Aptima Combo 2 reagents on a Tigris platform (Gen-Probe, Inc., San Diego, CA), and Abbott RealTime CT/NG reagents on an m2000 platform (Abbott Molecular Diagnostics, Des Plaines, IL). The performance of the three assays for detection of N. gonorrhoeae was comparable, but detection of C. trachomatis by the three assays showed more variation. All three platforms were suitable for the detection of C. trachomatis and N. gonorrhoeae, but additional factors, such as maximum daily specimen throughput, are important in evaluating automated systems for C. trachomatis and N. gonorrhoeae detection in high-volume laboratories.     

Neisseria species identification assay for the confirmation of Neisseria gonorrhoeae-positive results of the COBAS Amplicor PCR

Screening assays for Neisseria gonorrhoeae 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. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive 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. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay.     

Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.     

A comparison of three real-time PCR assays for the confirmation of Neisseria gonorrhoeae following detection of N. gonorrhoeae using Roche COBAS AMPLICOR.

Three different real-time PCR assays were evaluated as confirmatory tests for Neisseria gonorrhoeae after initial screening using the COBAS AMPLICOR Chlamydia trachomatis and N. gonorrhoeae duplex assay. The target genes used for the confirmation were the gyr, cppB and 16S rRNA genes. Analytical specificity was determined by testing 60 strains belonging to different bacterial species and/or serogroups. The primers chosen from the 16S rRNA gene for confirmation of N. gonorrhoeae were highly specific, showed no cross-reactivity with other bacteria included in the study, and had an analytical sensitivity of 1 CFU. Of 192 clinical specimens that were positive for N. gonorrhoeae according to the COBAS AMPLICOR assay, 42 were confirmed as positive using the 16S rRNA gene target, 26 were confirmed using the cppB target, and 30 were confirmed using the gyr target. It was concluded that the real-time PCR assay targeting the 16S rRNA gene is a useful confirmatory assay to complement the COBAS AMPLICOR screening test for N. gonorrhoeae.     

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests

The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for N. gonorrhoeae and by Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis. A positive N. gonorrhoeae result was defined as a positive result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of N. gonorrhoeae culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of C. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities of N. gonorrhoeae culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of C. trachomatis EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of both N. gonorrhoeae and C. trachomatis in endocervical specimens.     

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

Two assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae were compared: the LCx Probe system (the LCx system; Abbott Diagnostic Laboratories, North Chicago, Ill.) and the COBAS AMPLICOR C. trachomatis/N. gonorrhoeae system (the COBAS AMPLICOR system; Roche Diagnostic Systems, Branchburg, N.J.). Endocervical swab specimens, male urethral swab specimens, and female and male urine specimens were collected from 503 female and 498 male visitors attending a sexually transmitted diseases clinic in Amsterdam, The Netherlands. Prevalences for C. trachomatis were 12.5% (63 of 503) and 10.0% (50 of 498) in females and males, respectively. The prevalences for N. gonorrhoeae were 1.2% (6 of 503) and 4.2% (21 of 498) in females and males, respectively. Both assays showed high values for sensitivity and specificity with regard to the detection of C. trachomatis in endocervical swab specimens, male urethral swab specimens, and female and male urine specimens. The sensitivities for the LCx system were 92.1, 90.0, 88.9, and 94.0% for each type of specimen, respectively; and the sensitivies for the COBAS AMPLICOR system were 96.8, 98.0, 82.5, and 92.0% for each type of specimen, respectively. Specificities ranged between 98.4 and 100%. The sensitivity of the LCx system for the detection of N. gonorrhoeae was 100% for female cervical swab and urine specimens and male urethral swab specimens, while for male urine specimens the sensitivity was 95.2%; the specificity was 100% for all types of specimens. For the detection of N. gonorrhoeae by the COBAS AMPLICOR assay, the sensitivity for female cervical swab and male urethral swab specimens was 100%, that for female urine specimens was 66.7%, and that for male urine specimens was 95.2%. However, the predictive values of a positive test for female cervical swab specimens and urine specimens were 31.6 and 36.4%, respectively. Sequence analysis of the amplimers obtained by an in-house 16S rRNA PCR of the solely COBAS AMPLICOR system-positive swab specimens revealed neither N. gonorrhoeae nor other Neisseria spp. The COBAS AMPLICOR assay was considered not suitable for screening for infections with N. gonorrhoeae. If this assay is used for detection of N. gonorrhoeae, confirmation of positive results by a reliable test is mandatory.     

Evaluation of the new AmpliSens multiplex real‐time PCR assay for simultaneous detection of Neisseria gonorrhoeae,Chlamydia trachomatis,Mycoplasma genitalium,and Trichomonas vaginalis

In this study, we performed an evaluation of the new CE‐marked multiplex real‐time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis‐MULTIPRIME‐FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA‐approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first‐void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2–99.6%), 81.9% (95% CI: 70.7–89.7%), 100% (95% CI: 40.2–100%) and 100% (95% CI: 16.5–100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6–100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non‐gonococcal Neisseria species were negative. In conclusion, the multiplex real‐time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis‐MULTIPRIME‐FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection.     

Utility of pooled urine specimens for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in men attending public sexually transmitted infection clinics in Mumbai, India, by PCR

Pooling urogenital specimens for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by nucleic acid amplification tests is an attractive alternative to individual testing. As pooling can reduce the costs of testing as well as labor, it has been advocated for use in resource-poor settings. However, it has neither been widely adopted nor evaluated for use in developing countries. We evaluated the practical use of pooling first-catch urine (FCU) specimens for the detection of C. trachomatis and N. gonorrhoeae from 690 men in Mumbai, India, by PCR. FCU, urethral smears, and swabs were collected from men seen at two sexually transmitted infection (STI) clinics. All laboratory testing was done at the Lokmanya Tilak General Hospital. Gram stain smears and culture isolation for N. gonorrhoeae were performed. Each FCU was tested individually and in pools using the Roche Amplicor PCR for C. trachomatis and N. gonorrhoeae with an internal control for inhibition. Specimen pools consisted of aliquots from five consecutively processed FCUs combined into an amplification tube. An optical density reading of > or =0.20 indicated a pool for which subsequent testing of individual samples was required. Prevalence by PCR on single specimens was 2.2% (15/690) for C. trachomatis and 5.4% (37/690) for N. gonorrhoeae. Compared to individual FCU results, pooling for C. trachomatis and N. gonorrhoeae had an overall sensitivity of 96.1% (50/52). Specificity was 96.5% (83/86) in that three pools required single testing that failed to identify a positive specimen. Pooling missed two positive specimens, decreased the inhibition rate, and saved 50.3% of reagent costs. In this resource-limited setting, the use of pooling to detect C. trachomatis and N. gonorrhoeae by PCR proved to be a simple, accurate, and cost-effective procedure compared to individual testing.     

Evaluation of the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA by the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN)

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA, combined with luminescence detection by a probe hybridization. In the performance tests, this system was able to detect 10 to 100 copies of C. trachomatis/N. gonorrhoeae DNA for only 3.5 hours, and was highly specific to C. trachomatis/N. gonorrhoeae without any cross-reaction to C. pneumoniae, N. lactamica, N. sicca or N. meningitidis. When we tested 60 clinical samples of urine and cervical swabs, the interpretive results were completely consistent with those obtained by Roche PCR system. Of 13 positive samples by the ICAN and PCR systems for C. trachomatis, four were negative by EIA method(IDEIA Chlamydia). These results indicate that the ICAN system is an efficient and sensitive system to simultaneously detect C. trachomatis/N. gonorrhoeae DNA.     

Evaluation of Chlamydia trachomatis and Neisseria gonorrhoeae detection in urine, endocervical, and vaginal specimens by a multiplexed isothermal thermophilic helicase-dependent amplification (tHDA) assay

We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae, which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples

Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.