Figure 1. Physician instructions for specimen collection

1. Infection Specimen/Test CS L-PAP n CT AC2 AC2 AMP BD + + + + 25 + + + –6 + + ––1 + ––– 1 GC + + + + 5 + + ––1 COMPARISON OF APTIMA COMBO 2, AMPLICOR AND PROBETEC FOR THE DIAGNOSIS OF C. TRACHOMATIS (CT) AND N. GONORRHOEAE (GC) IN SUREPATH L-PAP SAMPLES 1M. Chernesky, 1D. Jang, 1M. Smieja, 2J. Kapala, 2C. Doucette, 2J. Sumner, 3R. Ewert, 3D. MacEachern, 3C. Pritchard, 4C. MacRitchie 1St Joseph’s Healthcare/McMaster University, Hamilton, ON, 2Gamma Dynacare Medical Laboratories, Brampton, ON, 3Evergreen Health Centre, Toronto, ON, 4Hamilton Community Health Centre, Hamilton, ON, CANADA Figure 2. L-Pap Sample Preparation REVISED ABSTRACT Objectives: SurePath L-Pap samples have not received FDA clearance for CT or GC testing in approved NAATs. Laboratories are testing SurePath samples using validation protocols. We compared APTIMA Combo 2® [AC2], ProbeTec™ [PT] and AMPLICOR® [AMP] assays to detect CT and GC from SurePath L-Pap samples. Methods: A total of 296 women consented to collection of a cervical swab [CS] and 2 L-Pap collection vials. In the laboratory the 2 L-Pap samples were pooled to ensure homogeneity before processing for Pap cytology. The remaining L-Pap fluid was tested for CT and GC using published protocols for the 3 assays: [a] transferring l ml into specimen transport media before testing 400 µl in AC2 [Gen-Probe Inc.]; [b] placing 500 µl into 2 ml of PT diluent then testing 150 µl in PT [Becton Dickinson]; [c] pipetting 500 µl into a centrifugation procedure leading to testing of 50 µl in AMP [Roche]. The CS was tested by AC2. To compare the presence of analytes in L-Pap remnant versus the gradient sample left after cytological processing, both samples were tested by AC2. Results: The prevalence of CT was 11.2% [33/296] and GC was 2.0% [6/296]. Two patients were infected with both organisms. Twenty-five [75.7%] of the 33 CS-positive women [CT-positives] were positive in the L-Pap sample in all 3 of the CT assays. The sensitivity of AC2 was 97.0% [32/33], 93.9% [31/33] for AMP and 75.8% [25/33] for PT. One CT-positive patient was negative by all assays on the L-Pap remnant but positive by AC2 on the gradient after cytology processing. For GC, AC2 identified all 6 of the infections [100%] while AMP and PT missed one [sensitivity 83.3%]. Conclusions: Using published protocols for CT and GC testing of SurePath L-Pap samples demonstrated very high sensitivity and specificity for the AC2 and AMP assays. The protocol for PT testing of L-Pap samples used in this study can not be recommended. The remnant sample was slightly better than the gradient for CT and GC testing by AC2. METHODS Patient recruitment: 296 women (ages 15-29) from April 2008-January 2009 attending health centre/ OB/GYN clinic for routine care signed an IRB approved informed consent to participate in the LPTSP (Liquid Pap Trial-SurePath) study. Study participants with previous antibiotics use in the past 3 weeks and women pregnant past the first trimester were excluded. Physician collection: Each collection package included information/informed consent forms and collection kits, labeled with an unique study identifier. The physician collected three samples (Fig. 1): SurePath L-Pap sample following an established procedure using the Cervex-Brush® and immersed into the specimen vial, an APTIMA unisex swab (Gen-Probe Inc.) followed by the second L-Pap SurePath sample (see diagram). Samples were shipped the same day to Gamma Dynacare Medical Laboratories (GDC), Brampton site. Laboratory specimen handling/testing: The cytology technologist removed only the two L-Pap vials from the study package and carefully mixed both vials to ensure homogeneity. Both samples were divided evenly. One vial remained for Pap cytology at GDC and the other placed into the study package with the rest of the study samples and shipped immediately to St. Joseph’s Healthcare Infections Research Group (SJH), where the L-Pap sample was prepared for AC2, PT and AMP within 48 hours (Fig. 2). APTIMA Combo 2 (Gen-Probe Inc.): As described previously, after vortexing the SurePath vial, 1mL of L-Pap was transferred into the APTIMA STM tube. Both the cervical swab STM and L-Pap processed STM were tested in the DTS 400/ Leader HC+ system. BD ProbeTec (Becton Dickinson): As described previously, 0.5mL of L-Pap was transferred into the BD specimen dilution tube, inverted 3-4x and tested according to the manufacturers’ instructions. AMPLICOR CT/NG (Roche Diagnostics): The protocol was initially published (Cytyc Corporation 2003 in conjunction with Roche Molecular Diagnostic System) for the PreserCyt ThinPrep medium. Briefly, 0.5mL of L-Pap was centrifuged at 12,500 g for 10 min, decanted and the pellet was treated with CT/NG lysis buffer (Roche) and further resuspended into specimen diluent. A 50µl volume was used in the CT/NG mastermix. The amplification and detection with micro-well plates (MWP) was performed according manufacturers’ instructions. Sensitivity and Specificity calculations: Calculations were made based on the CS being positive or negative. Confidence intervals were calculated using confidence intercal analysis software (version 2.1.2, 2004; T. Bryant, University of Southampton, UK). PT AMP CT/NG AC2 Cervical swab into APTIMA STM 0.5 ml L-Pap into Eppendorf tube 0.5ml L-Pap into BD Specimen Dilution tube 1ml L-Pap into APTIMA STM RESULTS Table 1. Testing profiles for 33 infected patients Table 2. Sensitivity and Specificity of AC2, AMP and PT on SurePath L-Pap samples INTRODUCTION Liquid based cytology for the detection of cytolological abnormalities leading to cervical cancer has demonstrated advantages over conventional smear preparation and has been adopted by many laboratories. Currently there are three liquid based cytology mediums cleared by the US Food and Drug Administration for liquid-based Pap (L-Pap) cytology: PreservCyt® ThinPrep (Hologic), SurePath (BD Diagnostics-TriPath) and Cytotek MonoPrep (Monogen). All use broom, brush or spatula sampling of the cervix placed into their proprietary transport media. AC2 and AMP have FDA clearance for the use of ThinPrep L-Pap for C. trachomatis (CT) and N. gonorrhoeae (GC) testing. Our laboratory as well as others have been interested in using the SurePath L-Pap samples for testing CT/GC by APTIMA Combo 2 (Gen-Probe Inc.), BD ProbeTec (Becton Dickinson) and AMPLICOR CT/NG (Roche Diagnostics) assay. CT GC Assay %Sensitivity %Specificity %Sensitivity %Specificity (95% C.I) (95% C.I) ( 95% C.I) (95% C.I) AC2 97.0 (32/33) 100 (263/263) 100 (6/6) 100 (290/290) (84.2-99.5) (98.6-100) (61.0-100) (98.7-100) AMP 93.9 (31/33) 99.6 (262/263) 83.3 (5/6) 98.9 (287/290) (80.4-98.3) (97.9-99.9) (43.6-97.0) (97.0-99.6) PT 75.8 (25/33) 100 (263/263) 83.3 (5/6) 100 (290/290) (59.0-87.2) (98.6-100) (43.6-97.0) (98.7-100) Figure 1. Physician instructions for specimen collection 1. 2. 3. CONCLUSIONS • The prevalence of infection in our study population was 11.2% (33/296) for CT and 2% (6/296) for GC based on CS. • SurePath L-Pap testing demonstrated excellent sensitivity and specificity with the APTIMA Combo 2 and AMPLICOR assays. • 24.2% of the CT positives were missed using the ProbeTec protocol for SurePath samples. • Presented at the 18th International Society for STD Research (ISSTDR/BASHH) June 28th–July 1st, 2009, London, UK OBJECTIVE Insert the central bristles into the endocervical canal deep enough to allow the shorter bristles to fully contact the ectocervix. Push gently, and rotate the broom in a clockwise direction five times. To compare the performance of APTIMA Combo 2 (AC2), BD ProbeTec (PT) and AMPLICOR CT/NG (AMP) on SurePath L-Pap samples for the diagnosis of CT and GC.