Improved performance of PACE 2 with modified collection system in combination with probe competition assay for detection of Chlamydia trachomatis in urethral specimens from males.

The Gen-Probe PACE 2 assay (GP) in combination with a modified collection system was compared with cell culture (CC) for the detection of Chlamydia trachomatis in urethral specimens from males. Analysis of discordant results was performed by PCR. The modifications, i.e., application of a more rigid swab type and a 50% reduction in the amount of transport medium, were made to improve the sensitivity of the assay. By using the modified GP on 302 urethral specimens from males, a sensitivity of 89.5% and a specificity of 100% were determined. In addition, performance of a probe competition assay on all GP samples with a result > 0.6 and < 1.0 times the cutoff factor (gray zone) detected three more true-positive samples. The sensitivity of GP in combination with the probe competition assay increased to 94.9%, with a specificity of 100%. This was identical to the performance of CC. The modified GP offers a very sensitive and specific alternative to CC

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay.

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR

Evaluation of Clearview and Magic Lite tests, polymerase chain reaction, and cell culture for detection of Chlamydia trachomatis in urogenital specimens.

The Clearview Chlamydia test (CV; Unipath Ltd., Bedford, United Kingdom), the Magic Lite Chlamydia test (ML; CIBA Corning, Medfield, Mass.), a polymerase chain reaction (PCR), and cell culture (CC) were evaluated for detection of Chlamydia trachomatis in urogenital specimens. Specimens were collected from 283 men and 724 women visiting the outpatient clinic for Sexually Transmitted Diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands. ML, PCR, and CC were all performed on the same sample to prevent swab-to-swab variability. CV was performed on a separate sample. Analysis of discordant results was performed by application of the following confirmatory assays: first, PCR on the CC, second, ML was repeated, and third, PCR was repeated by using a different DNA extraction protocol. If more than one test was positive, the sample was considered true positive. If only one test was positive, which was confirmed by the confirmatory assay, the sample was also considered true positive. By using these interpretations, the following results were obtained. The sensitivity and specificity of CV for samples from men were 60.4 and 86.3%, respectively. For samples from women, these values were 62.3 and 99.7%, respectively. The low specificity for samples from men was caused by unidentified substances in the swab that was used. The use of CV on samples from men is not recommended by the manufacturer. For samples from women, the specificity of CV was high, but the low sensitivity of CV limits its use for diagnostic purposes. The sensitivities of ML were low for samples from both men and women (68.8% and 50.9% respectively), while specificities were excellent for samples from both groups (100 and 99.9%, respectively). The low sensitivity of ML limits its diagnostic value. The PCR technique was highly specific for samples from both men (99.6%) and women (99.9%). The sensitivity of PCR, however, was unexpectedly low for samples from both groups (men, 87.5%; women, 79.2%), most likely because of the sample treatment method used. The sensitivity and specificity values of CC for samples from men were 95.8 and 100%, respectively. For samples from women, these values were 100 and 99.9%, respectively. In the present study, CC was the most reliable technique for the detection of C. trachomatis

Detection of Chlamydia trachomatis in male and female urine specimens by using the amplified Chlamydia trachomatis test.

The amplified Chlamydia trachomatis test (AMP-CT; Gen-Probe), a new diagnostic test for the detection of Chlamydia trachomatis, was evaluated with urine specimens from 1,000 patients visiting the outpatient department for sexually transmitted diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands, by comparing the results to those of cell culture. From February 1996 to July 1996, urine samples for the AMP-CT test and urethral swabs for cell culture were collected from 544 men, while cervical swabs from 456 women were also taken for cell culture. Positive test results were obtained for 130 (13%) of the patients. AMP-CT test and cell culture results were discordant for 70 (7%) specimens. Analysis of the samples with discordant results was performed by an in-house PCR. After resolution of the discordant results, the sensitivity, specificity, and positive and negative predictive values of the AMP-CT test were 84.3, 98.8, 89.6, and 98%, respectively, for samples from females and 100, 99.2, 93.1, and 100%, respectively, for samples from males, while for cell culture these values were 72.5, 99.2, 92.5, and 98%, respectively, for samples from females and 57.4, 99.0, 86.1, and 95.4%, respectively, for samples from males. We conclude that the AMP-CT test is a fast and reliable test for the detection of C. trachomatis in urine specimens from females and, in particular, males