Controls for Chlamydia trachomatis and Neisseria gonorrhoea Mark Manak, Ph.D., SeraCare Life Sciences Gaithersburg, MD USA SoGAT Clinical Diagnostic Meeting NIBSC, South Mimms, UK June 25, 2008
C. trachomatis (US) Chlamydia Most common reported STD Most infections are asymptomatic 2-17% incidence rate >5% sexually-active women in UK Causes urogenital infections leading to PID and infertility N. gonorrhoeae (US) Gonorrhoeae Second most common reported STD Widely variable incidence among countries 40% co-infected with C. trachomatis Mucosal infection leads to cervicitis, prostatitis, PID, sterility Increase in antibiotic resistance noted U.S. Centers for Disease Control and Prevention; www.cdc.gov
C. trachomatis Infection Elementary Body (EB) is infectious form 0.3-0.4µM Environmentally resistant Metabolically inert Columnar epithelium Endocytosis Reticulate Body (metabolically active) Binary fission replication (100-500) Converted to EB Released by reverse endocytosis 15 Serovars A, B, Ba, C Trachoma D-K Genital infection LGV 1-3 Lymphogranuloma venerum
N. gonorrhoeae Infection Targets non-ciliated mucous epithelium Endocytosis Exocytotic release to sub-epithelial tissue Present in infectious exudate Human only known natural host Almost exclusively by sexual contact 35% transmission efficiency 50-60% efficiency 90% develop symptoms <50% develop symptoms Antibiotic Resistance Chromosomal and plasmid-derived
Nucleic Acid Amplification Testing (NAAT) More sensitive than culture for screening and diagnosis Noninvasively collected specimens Urine (men or women) First-Catch Urine samples (20-30 ml) Transport in Sample Transport Media Unprocessed Specimen container Swab Female Endocervical swabs, Male urethral swab Transport Dry or in Sample Transport Media
C. trachomatis N. gonorrheae assays Commercial C. trachomatis/ N. gonorrhoeae NAATs Roche Amplicor 1 BD ProbeTec SDA 2, 3 Gen-probe APTIMA 4 Technology PCR SDA TMA Gene target CT Cryptic plasmid Cryptic plasmid 23S ribosomal RNA gene Gene target NG Cytosine DNA methyltransferase gene Multicopy pilin gene-inverting protein homologue 16S ribosomal RNA gene Analytical Sensitivity of Commercial N. gonorrhoeae NAATs (Manufacturer data) Assay Vol/assay Analytical sensitivity (Limit of Detection) Gen-Probe Aptima Combo 1 400μl 50 cells/assay; 125 cells/ml Roche COBAS Amplicor 2 50μl 100 CFU/ml urine; 400 CFU/ml swab BD ProbeTec ET 3 200μl 10 cells/assay; 50 cells/ml 1 Gen-Probe APTIMA Combo2 Product Insert, IN0156 rev. B 2 Roche COBAS Amplicor CT/NG Test for N. gonorrhoeae Product Insert, 10/2004, Rev. 5.0 3 BD ProbeTec ET CTNG Amplified DNA assay Product Insert. Rev. 0606
Urine vs. Swab Processing: COBAS
ACCURUN Controls Control Design Resemble clinical specimen Control for all aspects of processing and detection Non-Infectious Validated for Swab and Urine protocols Intended Use Accuracy: Can the test produce the correct result? Reproducibility/precision: Can it do this consistently? Robustness: Can all techs run this method reliably? QC Monitoring: Can errors be detected when they occur?
Accurun 341 CT/NG Positive Controls C. trachomatis Purified Elementary Bodies Serovar L2, strain 424 rdna homology across serovars N. gonorrhoeae (Zopf 1885) Trevisan 1885 NCTC 8375/ATCC 19424 Multi-Analyte Control Compatible with all assay platforms Suspended in stabilized transport medium 2 o C to 8 o C storage >5 yr real-time stability (Roche COBAS Amplicor) CE Mark
Quantitation Standard Plasmid Design Neisseria gonorrhea (NG) pora pseudogene (pap) Chlamydia trachomatis (CT) MOMP gene sequences. Quantitated in SeraCare CT/NG TaqMan assay
Standards Amplification plots and standard Curves CT 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 NG 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1
Quantitation of ACCURUN 341 Controls Prepare individual dilutions of CT and NG stocks Run dilutions on each platform following assay instructions Bracket additional sub-dilutions for continuity Convert dilutions to theoretical particle counts CT: TEM NG: A 550, Quantitated against standardized plasmid CT Stock: 6.0 x 10 8 Copies/ml = 1.1 x 10 8 EB/ml (EM) NG Stock: 1.6 x 10 8 Copies/ml = 1.0 x 10 8 cells/ml (A550) Determine C 50 and C 95 levels in each assay
% Positive score % Positive score Gen-Probe APTIMA Combo2 Assay Gen-Probe Aptima Combo CT LOD EB/mL % Pos 100% 1 0% 3 67% 80% 60% 40% 20% 0% 0 5 10 15 20 25 Particle count CT 10 100% 25 100% 50 100% 125 100% 250 100% 625 100% 1250 100% 2500 100% Ferrer et al. 2002 IUSTI 100% Positive at 1 EB/mL Gen-Probe APTIMA Combo2 Package Insert <10 EB/mL Gen-Probe Aptima Combo NG LOD Cell/mL % Pos 1 0% 100% 80% NG 3 0% 10 0% 25 0% 60% 40% 20% 50 0% 125 25% 250 50% 625 100% Gen-Probe APTIMA Combo2 Package Insert 125 NG cells/ml 0% 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Particle count 1250 100% 2500 100%
% Positive score % Positive score Roche COBAS Amplicor CT/NG Assay Roche Amplicor CT EB LOD EB/mL % Pos EB/mL % Pos 100% 80% 60% 40% 20% 0% CT 0 10 20 30 40 50 60 70 80 90 100 Particle count 0.1 0% 1 0% 3 0% 5 0% 10 17% 25 33% 50 67% 75 100% 100 100% 333 93.1% 400 98.1% 500 88.9% 500 95.5% 677 98.1% 833 84.5% 1,000 100% 2,000 98.5% 5,000 100% Chalker et al., J Clin Microbiol 43, 2005 Roche Amplicor NG LOD Cell/mL % Pos 0.1 33% 100% 80% 60% 40% NG 1 67% 3 100% 5 100% 10 100% 25 100% Roche COBAS Amplicor CT/NG N. gonorrhoeae Package Insert 100 CFU/mL Urine 400 CFU/mL Swab 20% 50 100% 0% 0 1 2 3 4 5 75 100% 100 100% Particle count
% Positive score % Positive score BD ProbeTec ET CT and NG Amplified DNA assay BD ProbeTec CT LOD EB/mL % Pos EB/mL % Pos 3 0% 333 45.8% 100% 80% 60% 40% 20% CT 10 8% 50 17% 125 33% 625 50% 1000 67% 1250 89% 2500 100% 400 55.3% 500 63.2% 500 80% 677 78.4% 833 31.4% 1,000 93.8% 2,000 100% 0% 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 Particle count 5000 100% 5,000 100% Chalker et al., J Clin Microbiol 43, 2005 100% 80% 60% 40% 20% 0% BD ProbeTec GC LOD NG 0 25 50 75 100 125 Particle count EB/mL % Pos 1 0% 5 17% 10 50% 25 67% 50 100% 125 100% 625 100% BD ProbeTec ET Package Insert LOD 95 : 50 cells/ml
Low-Positive CT and NG Nucleic Acid Controls Controls designed to challenge each type of assay Different sensitivities prevents use of a single control for all assays Assay C. trachomatis N. gonorrhoeae C 50 C >95 C 50 C >95 Aptima 3 10 250 625 Amplicor 38 75 1 3 ProbeTec 625 2500 10 50 3 Series of A341 Multi-Analyte CT and NG NAT Controls Targeted at 5 x C 50 values for each major platform Series 100 for Gen-Probe APTIMA Combo2 Series 200 for Roche COBAS Amplicor CTNG Series 400 for BD ProbeTec ET CTNG Amplified DNA Assay
Acknowledgements SeraCare Life Sciences Product Development Phil Moen, Ph.D. Bharathi Anekella, Ph.D Testing Lab Cathrine Huang, Ph.D. Patricia Nye Donna Capozzoli Diane McCoy Manufacturing Jim Doherty Joan Thurston ARUP Laboratories, Salt Lake City, UT Jon Lowe mmanak@seracare.com