Evaluation of the Digene Hybrid Capture II CT-ID Test for Detection of Chlamydia trachomatis in Endocervical Specimens

Patient population.A total of 587 females between the ages of 16 and 45 attending two Baltimore City sexually transmitted disease clinics were enrolled after informed consent was obtained. The study protocol was approved by the Johns Hopkins University and the Baltimore City Health Department ethical review boards. Two endocervical swabs and one Digene endocervical brush or swab were obtained from each woman enrolled in the study: one cervical swab for C. trachomatis culture, a cervical swab for PCR, and a cervical brush or swab for the Digene HC II CT-ID test. All enrolled women at the clinics were asked to consent to having the Digene cervical brush specimen obtained; if they did not consent to the brush, then a cervical swab specimen was obtained. For pregnant women, only cervical swabs were obtained. For nonpregnant women consenting to a brush specimen, the order of specimen collection was determined by patient identification numbers, i.e., for odd patient identification numbers, the Digene brush specimen was collected first, followed by the culture swab specimen, and visa versa for even patient identification numbers. The order of swab collection for pregnant women and women who did not consent to a brush specimen was alternated by odd and even patient identification numbers in the fashion stated above. A third cervical swab for PCR was collected after the specimens for culture and HC II CT-ID were obtained. The endocervical culture swab was placed in chlamydia transport vials containing sucrose-phosphate buffer, 10% fetal bovine serum, and antibiotics. The endocervical HC II CT-ID brush or swab was placed in 1.0 ml of specimen transport medium. The PCR swab was placed in AMPLICOR swab specimen transport medium (Roche Molecular Systems, Branchburg, N.J.). The chlamydia culture transport vials were transported at −70°C and stored at −70°C for 12 to 24 h until they were processed for culture. The HC II CT-ID swabs were transported at 2 to 4°C and stored in a −20°C freezer. If the Digene HC II CT-ID tests were to be done within 3 weeks of collection, the specimens could be held at 2 to 30°C for up to 2 weeks and at 4°C for one additional week. The PCR swabs were transported on ice and stored at 2 to 8°C until processed.

Cell culture.Chlamydia culture was performed in 96-well microtiter plates with cycloheximide-treated McCoy cell monolayers as described previously (15). After 48 h both wells were evaluated for chlamydia inclusions by immunofluorescence staining. One well was stained with monoclonal antibody to C. trachomatis major outer membrane protein (Microtrak Chlamydia Culture Reagent; Syva, San Jose, Calif.), and the other well was stained with antilipopolysaccharide monoclonal antibody (Sanofi Diagnostics Pasteur, Chaska, Minn.) (5).

Digene HC II CT-ID.HC II CT-ID was performed on endocervical specimens by using a new chemiluminescent, signal amplification-based solution hybridization assay. Specimens were prepared by the addition of denaturation reagent and a 45-min incubation at 65°C. Denatured specimens (DNA) were hybridized with a C. trachomatis-specific RNA probe cocktail for 1 h at 65°C. The RNA probes are homologous to the entire chlamydia cryptic plasmid sequence (7,500 bp) and approximately 39,000 bp of the C. trachomatis genome (4%). There are approximately 10 copies of the cryptic plasmid sequence per organism and a single copy of the remaining genomic sequences per organism.

The hybridization mixture was transferred to a microplate coated with antibodies specific for RNA-DNA hybrids and shaken for 60 min at room temperature to capture the hybrids. The microplate was decanted and reacted with alkaline phosphatase-conjugated antibodies specific for RNA-DNA hybrids for 30 min at room temperature. The capture microplate was decanted and washed six times to remove any unbound antibody conjugate. The signal is generated by the cleavage of a chemiluminescent substrate. Substantial signal amplification is achieved because several alkaline phosphatase molecules are conjugated to each antibody and multiple antibodies bind to each captured RNA-DNA hybrid. As the substrate is cleaved by the bound alkaline phosphatase, light is emitted and measured as relative light units with a luminometer. The cutoff value for the test is the mean number of relative light units RLU of three replicates of the positive control which is tested in each assay. The positive control is 1 pg of cloned cryptic plasmid DNA per ml in Digene specimen transport medium. The limit of detection for the HC II CT-ID test ranges from 50 to 2,500 elementary bodies/assay for the 15 C. trachomatis serovars.

PCR.Roche AMPLICOR CT PCR was performed in 96-microwell plates according to the manufacturer’s instructions and as described previously (9).

Discrepant analysis.Specimens that were culture negative but positive by HC II CT-ID and/or PCR were also analyzed by direct fluorescent-antibody (DFA) staining of sedimented culture transport media for elementary bodies (2). A patient was considered true positive for C. trachomatis if the culture was positive or if at least two of the following tests were positive: HC II CT-ID, PCR, or DFA staining.

Of the 587 females, 65 (11.1%) were considered true positives forC. trachomatis (Table 1). Among these 65 positive females, 62 (10.6% of the total) were HC II CT-ID positive and 3 were HC II CT-ID negative but culture positive, yielding a sensitivity for HC II CT-ID of 95.4% (Table2). An additional five specimens were HC II CT-ID positive, culture and PCR negative, and confirmed negative by DFA staining (HC II CT-ID specificity of 99.0%) (Table 2). Fifty-nine specimens were positive for PCR (Table 1). Of the remaining six true-positive specimens, two were culture positive only and four were also HC II CT-ID positive, thus yielding a sensitivity of 90.8% for AMPLICOR CT PCR (Table 2). Two specimens were PCR positive but culture and HC II CT-ID negative; these were also negative by DFA staining (AMPLICOR specificity of 99.6%) (Table 2). Fifty-three of the sixty-five true-positive females were positive by culture. Of the 12 HC II CT-ID- and PCR-positive, culture-negative specimens, only 2 were positive by DFA staining. The sensitivity of C. trachomatis culture was therefore 81.5% (Table 2), which was significantly lower than the sensitivities of HC II CT-ID and PCR (P < 0.01, McNemar’s chi-square statistic). The specificity of culture was 100%, by definition.

One hundred twenty-two specimens from the study were rerun on two separate occasions by using the HC II CT-ID assay to test reproducibility. The agreement between the two runs was 98.4%. The two runs gave highly reproducible results, with a kappa value of 0.948 and an r ² value of 0.96.

This paper describes the evaluation of the Digene HC II CT-ID signal amplification-based test for the detection of C. trachomatis from endocervical specimens. HC II CT-ID was highly sensitive and specific for the detection of C. trachomatis infection compared to culture and was comparable to a nucleic acid amplification-based method, PCR, known to be a highly sensitive technique for detecting C. trachomatis DNA in endocervical swabs (14).

The difference in the sensitivity of HC II CT-ID and AMPLICOR CT PCR with endocervical specimens was not statistically significant and could be a result of several factors. Different clinicians use different collection techniques. Reliable results are dependent on consistent specimen collection techniques; however, it is impossible to guarantee that there was no variability in the specified collection techniques for this or any other assay. In addition, the sensitivity of PCR could have been affected since it was always performed on the third cervical swab. Furthermore, obtaining a positive culture and a negative HC II CT-ID or AMPLICOR CT PCR result may also reflect variations in specimen collection or the presence of inhibitors which may cause false-negative results. For clinical purposes, retesting of all HC II CT-ID specimens to assess the presence of such possible inhibitors is not practical and therefore was not performed. However, such an evaluation would be an important step in determining the true reliability of an amplification assay.

The HC II CT-ID assay is well adapted for high-throughput screening, since 90 clinical specimens per microplate can be processed in 5 h. When only one specimen is used, however, the Digene HC II CT/GC test algorithm is an excellent test for screening people for the most common sexually transmitted diseases. For example, if the initial test forC. trachomatis and gonorrhea is negative, no further testing is required, and a cost savings results for low-prevalence populations. This more accurate screening and identification method has an immediate application in asymptomatic populations, where it may lead to a reduction in the spread of infection and in the risk for complications and sequelae.

Our results demonstrate that HC II CT-ID is more sensitive than culture in identifying chlamydial organisms in endocervical specimens. The assay is comparable to other currently available nucleic acid target amplification-based tests, such as PCR. In addition, the HC II CT-ID test provides a rapid and reproducible assay that is easy to use and efficient for the detection of C. trachomatis.