Analytical Sensitivity of the Urine-Based APTIMA® Trichomonas vaginalis molecular assay using the automated Tigris® Platform A.H. Freeman1, M.W. Pandori2, L.Rauch2, S.Liska2, S.S.Philip1, and J.D.Klausner1 1San Francisco Department of Public Health, STD Prevention and Control Section, San Francisco CA, USA 2San Francisco Department of Public Health Laboratory, San Francisco CA, USA

Background Trichomonas vaginalis infection is estimated to be the most common curable sexually transmitted infection (STI) in the United States and worldwide. Testing for Chlamydia trachomatis and Neisseria gonorrhoeae has largely moved to the use of nucleic acid amplification tests (NAATs), which exhibit high sensitivity & specificity compared to more traditional techniques, such as culture and microscopy. However, NAATs are not yet widely employed for detecting T. vaginalis.

Background The APTIMA® T. vaginalis (ATV) molecular assay (Gen-Probe, Inc., San Diego, CA) is available as an analyte specific reagent (ASR) test. ATV is typically performed manually, which would limit its usefulness to a busy laboratory.

Background The San Francisco Public Health Laboratory currently uses the Automated TIGRIS® platform to detect C. trachomatis and N. gonorrhoeae using the Aptima® Combo2 assay (Gen-Probe, Inc.)

Objectives To assess the analytical sensitivity (the detection limit of a particular assay) of ATV on the automated TIGRIS® system.

Methods Using a known concentration of T. vaginalis organisms (Biomed Diagnostics, White City, Oregon) in culture we prepared 2 sets of serial dilutions. Specimens were tested for T. vaginalis using ATV on the TIGRIS ® System. Analyte specific reagents were added to the general-purpose reagents prior to transcription-mediated amplification (Gen-Probe, Inc). Positive controls were T. vaginalis negative urine spiked with fluid from in-vitro cultured T. vaginalis.

Methods We determined the minimum detectable dilution of each series. Additionally one dilution series was also tested using a multiplex polymerase chain reaction (PCR) assay (Seegene Inc., Rockville, MD). We determined the minimum detectable dilution of each series.

ATV Serial Dilution Results Starting Concentration 3,570,000 trich/ml Dilution Trich/ml RLU(000) 1st Run 2nd Run 10-2 35700 4459 4792 10-4 357 4381 4782 10-5 35.7 4088 4332 10-6 3.57 3621 4176 10-7 0.357 77 113 10-8 0.0357 14 13 Neg Control 3 2 Limit of Detection RLU(000): relative light units / 1000

ATV Serial Dilution Results Starting Concentration 1,840,000 trich/ml Dilution Trich/ml RLU(000) 1st Run 2nd Run 10-2 18400 4247 5047 10-4 184 4052 4942 10-5 18.4 3643 4392 10-6 1.84 3404 4176 10-7 0.184 127 124 10-8 0.0184 43 27 Neg Control 6 1 Limit of Detection RLU(000):relative light units / 1000

ATV Serial Dilution Results With ATV, in both series, we detected organism at a dilution of 1 x 10-7. This corresponds to 0.357 and 0.184 T. vaginalis organisms per milliliter of sample, respectively. The estimated analytical sensitivity was 0.05 and 0.03 organisms per reaction.

PCR Results Using a starting concentration of 3,570,000 trich/ml, the PCR assay detected organism at a maximal dilution of 2 x 10-5. This corresponds to 71 organisms per milliliter of sample. Based on the single run, the estimated analytical sensitivity was 200 organisms per reaction.

Minimum number of organisms detectable per milliliter of sample by ATV and PCR Organism number

Limitations We tested only two series of dilutions by ATV and one by PCR. We conducted this study at a single site by one laboratorian. We do not know the correlation between the number of detectable organisms and clinical disease.

Conclusions Both ATV and PCR exhibited high analytical sensitivity and detected a low number of T. vaginalis organisms. ATV on the TIGRIS® was approximately 200-fold more sensitive than the multiplex PCR assay in this study.

Future Directions The use of ATV on the automated Tigris® System in clinical and public health laboratories might be feasible for routine diagnostic testing for T. vaginalis. Further evaluation is needed to confirm the potential utility of such systems as uniform testing platforms for C. trachomatis, N. gonorrhoeae, and T. vaginalis infection using urine, endocervical, or vaginal specimens.

Acknowledgements Staff at the San Francisco Department of Public Health Laboratory Research support was provided by Gen-Probe, Inc.