John Harrison, Michelle Garassi, Sara Wierzbicki, William LeBar Hospital Consolidated Laboratories-Providence Hospital, Southfield, MI,

Evaluation of the Aptima® CT Assay as a Direct and Confirmatory Method for the Detection of Chlamydia trachomatis

Abstract Background: There are currently three FDA approved nucleic acid target amplification tests (NAATs) for the detection of C. trachomatis (CT). The APTIMA CT (ACT) Assay is a target amplification nucleic acid probe test that utilizes target capture, transcription mediated amplification and a hybrid protection assay to detect C. trachomatis rRNA. This assay can be used both as a direct test for CT as well as a confirmatory test for the detection of CT by other NAATs. The purpose of this study was to compare the performance of the ACT to the APTIMA Combo 2 (AC2) and the Roche AMPLICOR Microplate CT assay (PCR) as a direct assay, and to evaluate its performance as a confirmatory assay for positive CT results. Methods: Endocervical specimens (n = 500) were collected in M4 transport medium and tested by ACT, AC2 and PCR. Patient specimens were considered positive if two of the three tests for CT were positive. Results: Based on this standard, 62 patients were considered positive for CT. Both AC2 and ACT yielded 100% (62/62), sensitivity for the detection of CT and PCR resulted in a CT sensitivity of 96.7% (60/62). Specificities for CT with AC2 and ACT were 100% (438/438) and for PCR 99.5% (436/438).Conclusions: The APTIMA CT is a highly sensitive and specific nucleic acid amplification assay for the direct detection of C. trachomatis and can also be used as a confirmatory test for positive results obtained with other CT nucleic acid amplification tests.

Approximately 3 million Chlamydia trachomatis infections occur annually in the United States and the majority of persons infected with CT are asymptomatic. A CT NAAT performed on a female endocervical swab specimen or an intraurethral swab or urine specimen from a male are the preferred tests for diagnosis. Although these assays are exquisitely specific, the specificity of each assay is less than 100%. Therefore, by definition, false positive tests will occur. Confirmatory testing has been suggested by CDC in those cases in which a false positive test would result in adverse medical, social or psychological impact for a patient.

Approaches to additional testing include testing a second specimen, testing the original specimen with a different test using a different target or repeat testing. The most practical of these approaches would be to utilize a different target while maintaining the original test platform. However, until recently no manufacturer of NAATs offered this option. The APTIMA CT (ACT) Assay is a target amplification nucleic acid probe test that utilizes target capture, transcription mediated amplification and a hybrid protection assay to detect C. trachomatis rRNA. ACT utilizes a different target sequences than AC2 (16S rRNA vs 23S rRNA) and thus may be used for confirmation of positive NAAT results, as well as a stand-alone test.

MethodsEndocervical samples (n = 500) were obtained from patients presenting at an Emergency Department with symptoms of genital tract disease or at an OB/GYN clinic presenting for routine gynecologic care were collected in M4 transport medium and tested by ACT, AC2 and PCR.

The Roche Amplicor CT microplate assay was performed and interpreted according to manufacturer’s instructions.

The target capture portions of the AC2 and ACT assays were performed with 400 ųl of sample from the M4 samples. The remainder of the target capture, amplification, selection and detection assays were performed according to instructions for the AC2 assay.

AC2 results are automatically interpreted by the assay software and presented as individual results. A result may be negative, equivocal, positive or invalid as determined by the assay type and total RLU detected. The parameters for the CT portion of the AC2 assay are below.

There are no established RLU values to differentiate positive from negative test results for the ACT assay. For interpretation of the ACT assay data, the RLU cutoffs described above were used to determine the final test results. Patient specimens were considered positive if two of the three tests for CT were positive

ResultsA total of 500 specimens were tested by AC2, ACT and PCR. Although there are no established RLU values to distinguish positive and negative test results for the ACT, the numeric results correlated with the interpretive criteria for the AC2. The breakdown of RLU values for the ACT are shown in Tables 1 and 2.

Based on consensus results, 438 patient specimens were considered negative for CT. The RLU values for the negative samples all were within the negative range as defined for the Aptima Combo 2 assay.

Based on consensus results, 62 patient specimens were considered positive for CT. The RLU values for the positive all were within or exceeded the positive range defined for Aptima Combo 2.

The performance characteristics of the assays are shown below.

There was 100% agreement between the Aptima CT and Aptima Combo 2 assays, and 99.2% agreement between ACT, AC2 and PCR. The ACT and AC2 RLU values for the 4 discrepant assays are shown in Tables 3 and 4.

These results demonstrate that the Aptima CT assay is comparable to PCR and Aptima Combo 2 as a stand alone assay for the direct detection of Chlamydia trachomatis from endocervical samples. The performance characteristics of the ACT also demonstrate that is is acceptable for use as a confirmatory assay for AC2 and PCR. This study also validates the use of M4 as a transport device for the Aptima Combo 2 and Aptima CT assays,

CraigH:

Did you mean to say exquisitely sensitive?

CraigH:

Did you mean to say exquisitely sensitive?