1. John Harrison, Michelle Garassi, Sara Wierzbicki, William LeBar Hospital Consolidated Laboratories-Providence Hospital, Southfield, MI, Abstract There are currently three FDA approved nucleic acid amplification tests (NAAT) for the detection of C. trachomatis (CT) and N. gonorrhoeae (GC). Each of these tests has its own assay specific collection and transport device. Laboratories may receive multiple NAAT transport devices that have not been validated for use with their particular assay. The APTIMA® Combo 2 (AC2), APTIMA CT (ACT), and APTIMA GC (AGC) are target amplification nucleic acid probe tests that utilize target capture, transcription mediated amplification and hybridization protection to detect rRNA. ACT and AGC utilize different target sequences than AC2 and thus may be used for confirmation of positive NAAT results, as well as stand-alone tests. The purposes of this study were to evaluate M4 medium for use with the AC2, ACT and AGC assays and evaluate ACT and AGC as confirmatory assays for positive CT and GC NAAT results. Endocervical specimens (n = 500) were collected in M4 transport medium and tested by AC2, ACT, AGC and the Roche AMPLICOR Microplate CT and GC. assays (PCR). Patient specimens were considered positive if two of the three tests for CT or GC were positive. Based on this standard, 62 patients were considered positive for CT and 19 positive for GC. 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). For gonococcal detection sensitivities were 100% (19/19) for AC2 and PCR and 94.7% (18/19) for AGC. Specificities for AC2 and AGC were 100% (480/480) and 99.4 (477/480) for PCR. One patient sample tested equivocal for GC in all three assays. A subsequent sample collected from this patient was negative in all three assays. These results demonstrate that M4 is a suitable transport medium for the APTIMA Combo 2, APTIMA CT and APTIMA GC assays. In addition, the APTIMA CT and APTIMA GC are sensitive and specific assays suitable for confirmatory testing of positive CT and GC NAAT results. Chlamydia trachomatis and Neisseria gonorrhoeae infections are the most common reportable sexually transmitted infections in the United States today. It is estimated that over 3 million C. trachomatis infections occur annually among adolescents and young adults while in 2003, N. gonorrhoeae was second in frequency with 318,411 cases reported. One of the keys to the prevention of these infections rests on the ability to make a diagnosis based on accurate laboratory testing. There are currently three FDA approved nucleic acid amplification tests (NAAT) for the detection of C. trachomatis (CT) and N. gonorrhoeae (GC). Each of these tests has its own assay specific collection and transport device. Laboratories may receive multiple NAAT transport devices that have not been validated for use with their particular assay. The APTIMA® Combo 2 (AC2), APTIMA CT (ACT), and APTIMA GC (AGC) are target amplification nucleic acid probe tests that utilize target capture, transcription mediated amplification and hybridization protection to detect rRNA. The purposes of this study were to evaluate M4 medium for use with the AC2, ACT and AGC assays and evaluate ACT and AGC as confirmatory assays for positive CT and GC NAAT results. Methods Endocervical 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 AC2, ACT, AGC and PCR. The Roche Amplicor CT/NG microplate assay was performed and interpreted according to manufacturers instructions. The target capture portions of the AC2, ACT and AGC 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. There are no established RLU values to differentiate positive from negative test results for the ACT assay. For interpretation of the ACT and AGC assay data, the RLU cutoffs for the AC2 assay were used to determine the final test results (see below) Patient specimens were considered positive if two of the three tests for CT were positive. Results A total of 500 specimens were tested by AC2, ACT, AGC 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 and 480 negative for GC. The RLU values for the negative ACT and AGC 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 and 19 for GC. 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 ACT, AC2 and the consensus results for the detection of CT from M4 medium. The RLU values from the 2 samples negative by AC2 and ACT and positive by PCR samples were all <2,000 RLU. RLU values for the 2 samples with positive AC2 ACT and negative PCR results are shown below Use of M4 Transport Medium for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the Aptima® Combo 2 TM, APTIMA CT and APTIMA GC assays There was 99.8% agreement between the AC2, AGC and the consensus results for the detection of GC from M4 medium. The RLU value for the discrepant AGC negative, consensus positive specimen was 2,125. Our results demonstrate that M4 is a suitable transport medium for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in the APTIMA Combo 2, APTIMA CT and APTIMA GC assays. The performance characteristics of the ACT and AGC assays also demonstrate that they are acceptable for use as a confirmatory assay for AC2 and PCR.