1. Conclusions The analytical sensitivity of the APTIMA Trichomonas assay is less than 0.1 cell per reaction in all sample types tested. The APTIMA Trichomonas assay was 100% specific when tested against a panel of 43 strains of microorganisms that included closely related organisms and common urogenital flora. The APTIMA Trichomonas assay assay was 100% specific when tested against negative urine and vaginal swab specimens. The presence of 15% blood (v/v) in the sample did not affect the TV analyte signal. The IC prevented false-negative results in grossly bloody samples (25% v/v) by invalidating the result. For samples stored at ambient room temperature or above, TV rRNA can be detected by the APTIMA Trichomonas assay in raw urine for up to 5 days, in female and male urine-UTM for 36 days, in vaginal swab-STM specimens for 113 days, and in Cytyc PreservCyt LBC medium for 145 days. ANALYTICAL PERFORMANCE OF THE APTIMA ® TRICHOMONAS ASSAY ON THE AUTOMATED TIGRIS ® DTS ® SYSTEM D. Getman, B. Weinbaum, A. Aiyer, M. Catania, A. Worlock Gen-Probe Incorporated, San Diego CA USA Introduction Trichomoniasis is a sexually transmitted disease caused by the protozoan Trichomonas vaginalis (TV). In the United States, an estimated 5 to 7 million new infections occur each year. TV infections cause vaginitis in women and urethritis in men though infections are often asymptomatic. TV infections may lead to adverse health consequences, including pelvic inflammatory disease and an increased susceptibility to HIV infection. TV infection is most often confirmed by viewing a wet mount of a vaginal or urethral specimen and looking for motile cells, though this method has low sensitivity compared to culture and amplified nucleic acid testing [1-3] Study Objective This study evaluated the analytical sensitivity, clinical and analytical specificity, blood interference, and sample stability of the APTIMA Trichomonas (AT) assay, a Transcription Mediated Amplification (TMA) assay for TV rRNA detection that incorporates an internal control (IC). Materials and Methods Methods: Analytical sensitivity was evaluated by testing TV cell lysates diluted serially 1/10 with APTIMA Specimen Transport Medium (STM), negative pooled female and male urine samples, and Cytyc PreservCyt liquid cytology medium. Assay inhibition due to whole blood contamination, and assay cross reactivity with over 40 closely related microorganisms and common urogenital flora, was evaluated. Also assessed was the ability of the AT assay to detect 10 cell-equivalents per reaction of TV rRNA in pooled vaginal swab-STM matrix, pooled male urine-Urine Transport Medium (UTM) and female urine-UTM matrices, and PreservCyt-STM matrix, when stored at –70 C, -20 C, 4 C, 20 C, and 30 C. Amplicons for TV and IC were detected in the HPA reaction using Dual Kinetic Analysis (DKA; Figure 1 ). Calibrators are used to determine cutoff calculations for the IC and the analyte. For each reaction, the IC result was reported as either valid or invalid, whereas the TV result was reported as either reactive (i.e., positive) or non-reactive (i.e., negative). Reactions yielding a valid IC signal and a non-reactive TV result were considered to be true negatives. Table 1. Analytical Sensitivity of the APTIMA Trichomonas assay. Analytical sensitivity was determined by diluting a laboratory strain of TV into APTIMA Swab Transport Media (STM), urine-Urine Transport Medium (UTM) or Cytyc PreservCyt liquid cytology medium. OrganismIC Signal/Cutoff* APTIMA Trichomonas Assay Signal/Cutoff** TV Positive Control 2.1619.5 Derxia gummosa 2.000.16 Enterococcus faecalis 1.920.16 Kingella kingae 1.930.09 Moraxella osloensis 2.020.10 Neisseria gonorrhoeae 1.950.14 Neisseria cinerea 1.790.29 Neisseria elongata 1.850.08 Neisseria flava 1.920.10 Neisseria favescens 1.920.19 Neisseria lactamica 1.840.18 Neisseria meningitides A 1.920.10 Neisseria meningitides B 1.930.16 Neisseria meningitides C (1388) 1.580.31 Neisseria meningitides C (1389) 1.890.06 Neisseria meningitides C (1390) 1.960.12 Neisseria meningitides D 1.820.26 Neisseria meningitides W135 1.840.08 Neisseria meningitides Y 1.970.10 Neisseria mucosa 1.840.20 Neisseria polysaccharea 1.770.20 Neisseria sicca 1.900.11 Neisseria subflava 1.840.16 Chlamydia trachomatis 1.950.12 Chlamydia pneumoniae 1.560.35 Chlamydia psittaci (VR601) 1.910.11 Chlamydia psittaci (VR1369) 1.940.11 Trichomonas tenax 1.650.24 Pentatrichomonas hominis 1.690.16 Giardia intestinalis 1.790.07 Lactobacillis acidophilus 1.840.08 Lactobacillis brevis 1.810.20 Lactobacillis jensonii 1.760.22 Lactobacillis lactis 1.880.12 Ureaplasma urealyticum 1.890.16 Mycoplasma genitalium 1.690.32 Candida albicans 1.760.30 Candida glabrata 1.890.22 Candida parapsilosis 1.750.23 Candida tropicalis 1.830.09 E. coli 1.860.12 Gardnerella vaginalis 1.780.21 Staph. aureus 1.650.28 Staph. epidermidis 1.810.26 Table 2 Blood Interference in Vaginal Swab Specimens Two cells/reaction were tested in STM with 0, 10%, 15%, and 25% whole blood (v/v). Figure 1. Dual Kinetic Analysis showing the individual kinetic profiles of the IC and TV analyte probe signals. RLU = Relative Light Units. Table 5. Cross Reactivity Analysis of APTIMA Trichomonas Assay. A panel of 43 closely related microorganisms and common urogenital flora were tested to determine analytical specificity. Each organism of the 43-member cross-reactivity panel was tested at one million cells per reaction. Table 4. APTIMA Trichomonas assay detection of TV rRNA in various sample types at different storage conditions Table 4. APTIMA Trichomonas assay detection of TV rRNA in various sample types at different storage conditions. Each sample type was tested with 10 TV cells/reaction. N=5 for all conditions. * Non-reactive samples excluded. IC cutoff = 0.5 x mean Positive Control IC signal. Analyte cutoff = Average Negative Calibrator RLU + (0.04 x Average Positive Calibrator RLU) Table 3. Specificity of the APTIMA Trichomonas Assay in Negative Urine and Vaginal Swab Specimens. Table 3. Specificity of the APTIMA Trichomonas Assay in Negative Urine and Vaginal Swab Specimens. Sample reactivity status was confirmed by testing urine and vaginal swab clinical specimens from self-declared healthy asymptomatic donors using a highly sensitive alternate TMA nucleic acid test that targets a different region of TV rRNA (data not shown). References: 1. Krieger, J. N., et. al. (1988). JAMA. 259:1223-1227 2. Madico, G., Quinn T. C., Rompalo A., McKee K. T. and Gaydos, C. A. (1998). Journal of Clinical Microbiology. 36:3205-3210 3. Wendel, K. A., Erbelding, E. J., Gaydos, C. A. and Rompalo, A. M. (2002). Clinical Infectious Diseases. 35:576-580.Results 10210 Genetic Center Drive San Diego, CA 92121 (858) 410-8000 (858) 410-8870 (fax) ND, not done. APTIMA and TIGRIS DTS SYSTEM are trademarks of Gen-Probe Incorporated. PRESERVCYT and CYTYC are trademarks of Cytyc Corporation. *An IC S/CO 1.0 is valid. ** A TV (analyte) S/CO value 1.0 is reactive STM, Sample Transport Medium ND, Not Done P1.69