Design
This study describes the development and evaluation of a confirmatory test for COBAS Amplicor-positive rectal and pharyngeal specimens obtained from men using the omp1 gene as an alternative target for amplification by PCR.

Participants
Fifty-two specimens (47 anal swabs and 5 throat swabs) that were C. trachomatis positive by COBAS Amplicor PCR from 43 men who participated in a screening program in male-only saunas in Melbourne, Australia, were included in the study. The throat and anal swabs from 15 men testing C. trachomatis C. trachomatis negative were also included.

Description of Tests and Diagnostic Standard
DNA was isolated from the clinical samples using the automated MagNA Pure LC (Roche Diagnostics) instrument. PCR for amplification of β-globin sequence was conducted for detection of inhibition and sample adequacy. Three PCRs targeting the four omp1 variable domains were used. PCR VD1-4 used primers P1/OMP2 and amplified an approximately 1,136 bp region of the omp1 gene. PCR VD1-4 was carried out with 20 μl of each of the 52 swab samples. PCR VD1/2 and VD3/4 used primers P1/CT6 and CT6/OMP2, respectively, and amplified approximately 679 and 482 bp fragments, respectively. VD1/2 and VD3/4 were nested PCRs carried out using 2 μl of the VD1-4 product as target DNA. PCRs VD1/2 and VD3/4 were used on samples that were negative by PCR VD1-4. PCR products were visualized by gel electrophoresis and ethidium bromide staining. The presence of a band of the estimated product size determined a positive omp1 PCR result.

Main Outcome Measures
The cumulative results of all three omp1 PCRs were compared to the COBAS Amplicor results.

Main Results
The results of the COBAS Amplicor and omp1 PCRs for the detection of C. trachomatis in 62 anal and 20 throat swabs from 58 men are shown in the table. Three anal swabs were positive by Amplicor and negative by omp1 PCR. All three were positive for β-globin amplification. Forty-nine (94%) of 52 Amplicor positive samples were confirmed by the omp1 PCRs. One patient who had an omp1 negative anal sample also had C. trachomatis detected from a urine sample that was collected at the same time and was positive for C. trachomatis by both Amplicor and omp1 PCRs.

Results of C. trachomatis Amplicor and omp1 PCR assays performed on anal and throat specimens from 58 men.

Authors’ Conclusions
This study demonstrates the utility of COBAS Amplicor PCR for detection of C. trachomatis in anal and throat samples. The multicopy plasmid target of the Amplicor assay offers high sensitivity. A lower sensitivity with the omp1 PCR was expected because omp1 is a single-copy target. Due to the lower sensitivity of omp1 PCR, some Amplicor positive samples were not confirmed. However, there is a high likelihood of the positive Amplicor PCR samples being true positives.

Source of funding: Public Health Research Projects 2002-03 (Communicable Diseases), the Victorian Department of Human Services, Australia.

For correspondence: Sepehr. N. Tabrizi, Department of Molecular Microbiology, The Royal Women’s Hospital, 132 Grattan St., Carlton, Melbourne, Victoria 3053, Australia. E-mail address: sepehr.tabrizi@sch.org.au.