We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142 N. gonorrhoeae culturepositive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

Nucleic acid amplification assays such as the ligase chain reaction and PCR have encountered reproducibility problems. The initial extract and a newly extracted aliquot of urine specimens (n ‫؍‬ 120) which had signal-to-cutoff (S/CO) ratios above 0.80 by the LCx Chlamydia assay were retested. Nucleic acid was extracted from an additional urine sample for testing by the AMPLICOR PCR Chlamydia assay. Fifteen percent (18 of 120) of the urine specimens were negative by all repeat tests (initial mean S/CO ratio by the LCx Chlamydia assay, 0.93; S/CO ratio range, 0.80 to 3.30). Repeat testing of the 102 specimens with possible positive results by the LCx Chlamydia assay by use of the initially extracted aliquot confirmed the results for 95 (93.1%) of the specimens; repeat testing of a newly extracted aliquot confirmed the results for 87 (85.3%) of the specimens. Twenty specimens had discordant results by the two repeat LCx Chlamydia assays. A total of 78 of 102 (76.5%) of the specimens were positive by the AMPLICOR PCR, and the AMPLICOR PCR confirmed the results for 82.1% (78 of 95) and 89.6% (78 of 87) of the specimens positive by the two repeat LCx Chlamydia assays, respectively. Some of the discrepancies observed by multiple repeat tests may have been due to specimen mislabeling or contamination during performance of the procedure rather than to the LCx Chlamydia assay. Both assays suffered from a lack of reproducibility on repeat testing with a small proportion of specimens, probably due to the presence of low levels of DNA, the presence of variable amounts of amplification inhibitors, and the loss of DNA during extraction.For the past 8 years, clinical laboratories have become accustomed to using nucleic acid amplification (NAA) tests for the detection of Chlamydia trachomatis on swabs and in urine specimens from men and women (1-3, 5, 8, 10). These assays allow the effective management and treatment of C. trachomatis infections. The two NAA assays that have been in routine use the longest, the AMPLICOR PCR Chlamydia assay (Roche Diagnostics Systems, Branchburg, N.J.) and the LCx Chlamydia assay (Abbott Laboratories), have been reported to have reproducibility problems (4, 7).By February 2001, the Abbott Laboratories Diagnostics Division had received customer complaints concerning high rates of positivity for negative controls, resulting in invalid assay runs of the LCx Chlamydia assay, and positive patient specimens which did not test positive upon retesting. Abbott issued a Device Correction letter which stated the following: the specificity of the assay for some on-market lots of the test kit had dropped as low as 92%, but the test sensitivity remained in the normal range. The letter instructed LCx Chlamydia assay users to take the following actions: (i) interpret the results for samples with signal-to-cutoff (S/CO) ratios less than 0.80 as negative and report that C. trachomatis plasmid DNA was not detected and that the sample could be presumed to be negative for C. trachomatis by ligase chain reaction (LCR) ...

Specimen pooling to achieve efficiency when testing urine specimens for Chlamydia trachomatis nucleic acids has been suggested. We pooled endocervical swabs from 1,288 women and also tested individual swabs by ligase chain reaction (LCR). Out of 53 positive specimens, pools of 4 or 8 specimens missed two positives, providing 96.2% accuracy compared to individual test results. Dilution and positive-control spiking experiments showed that negative specimens with inhibitors of LCR in the pool reduced the signal. Conversely, two extra positives, detected only through pooling, were negative by individual testing but became positive after storage, suggesting that fresh positive specimens with labile inhibitors may be positive in a pool because of dilution of inhibitors. For this population of women with a 4% prevalence of C. trachomatis infection, substantial savings in cost of reagents (55 to 63%) and technologist time (50 to 63%) made pooling strategies a desirable alternative to individual testing.

Mocked samples of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) diluted in SurePath liquid-based Pap (L-Pap) fluid were detected by the APTIMA Combo 2 assay to end points 10-fold greater than dilutions in specimen transport media. Pooled L-Pap clinical specimens yielded CT-positive results after storage at room temperature for 10 days. Based on an infected patient standard for comparison, cervical swabs, urine, and SurePath L-Pap test samples collected with a SurePath cervical broom or ThinPrep cytobrush from 520 women then tested by APTIMA Combo 2 assay, detected 25 (4.8%) with CT, 5 (1.0%) with (GC), and 3 (0.6%) with both. Percent sensitivities (80-84), specificities (99.8-100), positive (99.5-100) and negative (99.2-99) predictive values of SurePath L-Pap for CT were validated as similar to those reported in a previously published multicenter trial. All values for GC were 100%. One collection device was not significantly better than the other.

Two endocervical swabs from each of 1,123 women were collected into manufacturer-supplied transport tubes and tested for Chlamydia trachomatis by a polymer conjugate-enhanced (PCE) enzyme immunoassay (EIA) (IDEIA PCE Chlamydia; DAKO) and a ligase chain reaction assay (LCx Chlamydia; Abbott). After confirmation by the EIA blocking test, the sensitivity of the IDEIA PCE remained at 91.8% and the specificity increased from 98.2 to 99.8% compared to LCx.Nucleic acid amplification (NAA) assays for Chlamydia trachomatis are more sensitive than culture (2, 15), enzyme immunoassay (EIA) (5, 17), and nucleic acid hybridization (3,8) and can be used successfully on noninvasive specimens such as urine (1, 11) and vaginal (4), vulvar (12), or introital (16) swabs. These qualities, together with high specificity, provide impetus for using NAA tests such as the ligase chain reaction (LCR) to screen asymptomatic populations at risk. The common limiting factors for NAA tests are increased costs for the assays and lower levels of throughput.The original 96-well-plate version of the EIA from DAKO, called IDEIA, was introduced in the 1980s. Following extensive evaluations compared with culture and NAA assays (6, 9), IDEIA was grouped with other EIAs, after confirmatory blocking and expansion of the "gold standard," as having a sensitivity of 60 to 70% (10). The manufacturer has since reconfigured the assay by incorporating dual amplification of the indicator system. This polymer conjugate enhancement (PCE) system consists of a dextran backbone to which anti-chlamydial lipopolysaccharide monoclonal antibodies and alkaline phosphatase are bound. For every immune complex interaction, multiple molecules of alkaline phosphatase are available to drive the signal generation in an enzyme-amplified color development system. The performance of the IDEIA PCE Chlamydia assay (DAKO) has been reported for detecting chlamydial antigens in female cervical and vaginal swabs (13,14) and in male first-void urine (14) compared to PCR (AMPLICOR). The purpose of our study was to compare the IDEIA PCE Chlamydia assay and the LCR test (LCx Chlamydia; Abbott) performed on endocervical swabs collected from women attending a birth control clinic.The study, which received ethics approval from the St. Joseph's Hospital investigational review committee, took place from April to November 1999. Two endocervical swabs were collected into manufacturer-supplied transport tubes from each of 1,123 consenting women of childbearing age attending a suburban clinic. The order of sampling was reversed after enrolling each 500 patients. All of the samples were held at 4°C, received in the laboratory within 24 h, and then processed within 48 h.The specimens were tested for C. trachomatis by LCR (LCx Chlamydia) and by EIA (IDEIA PCE Chlamydia) according to each manufacturer's instructions for cervical swab testing. All positive samples in the IDEIA PCE test were retested with and without IDEIA PCE Chlamydia blocking reagents. A reduction in the EIA signal of at least 40% con...