ACCEPTED. Instrument in Female Self-obtained Vaginal Swabs and Male Urine Samples. Andrew Hardick 1. Justin Hardick 1.
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1 JCM Accepts, published online ahead of print on 30 August 2006 J. Clin. Microbiol. doi: /jcm Copyright 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Comparison between the Gen-Probe TMA Trichomonas vaginalis Research Assay and a Real-time PCR for Trichomonas vaginalis using the Roche Lightcycler Instrument in Female Self-obtained Vaginal Swabs and Male Urine Samples Andrew Hardick 1 Justin Hardick 1 Billie Jo Wood 1 Charlotte Gaydos 1 1 Division of Infectious Diseases, Johns Hopkins University, School of Medicine, MD Corresponding author: Charlotte A. Gaydos, Dr.P.H. Associate Professor 1159 Ross Building 720 Rutland Ave. Baltimore, MD Phone: FAX: cgaydos@jhmi.edu Results from this analysis were presented in part at the International Society for Sexually Transmitted Diseases Research, Amsterdam, The Netherlands, July 10-13, Funding was in part from Gen-Probe, Inc., San Diego, CA.
2 Abstract This study compared two assays for Trichomonas vaginalis detection, Gen- Probe s TMA-TV and BTUB FRET PCR in self-obtained clinical samples from 611 patients. Infection status was defined as 2 positives by 2 different tests. Initial TMA-TV sensitivity was 96.7%; specificity was 97.5%. The TMA-TV assay was comparable to BTUB FRET PCR. Manuscript Trichomonas vaginalis (TV) is the most common parasitic sexually-transmitted infection in the world and is estimated to cause 3 million infections in the US annually year (1, 21). TV can cause vaginitis, cervicitis, preterm labor, urethritis and prostatitis (3,32,33). TV is cytopathic to vaginal cells and is associated with other STDs infections, including transmission of HIV (2, 7, 26, 29). Conventional methods for diagnosing TV are microscopic examination of wet mount preparations and culture-based systems. Both methods rely of the collection of viable organisms and suffer from poor sensitivity (25). More sensitive research-based PCRs for the diagnosis of TV have been described (9,11-16, 22, 24). Development of a commercially available amplification assay for TV on selfcollected samples would be highly desirable from a patient, clinical, and public health perspective (6). Other commercially available FDA cleared STD tests such as those for chlamydia and gonorrhea could be performed on the self-obtained samples (4,5,27,28). We report a comparison of a research transcription-mediated amplification assay for TV (TMA-TV), now available as an analyte specific reagent (ASR) (Gen-Probe Inc., San Diego, CA) with BTUB FRET PCR (BTUB), a real-time research PCR (9). 2
3 We screened 615 people attending two STD clinics; 611 had male urine samples (N=290) or duplicate female self-obtained vaginal swabs (SOVS) (N= 321) collected. Institutional Review Board approval was obtained, and the study was funded in part by Gen-Probe. BTUB PCR. For females, two SOVS for BTUB and TMA-TV testing were collected in random order. SOVS for BTUB testing were transported in a dry state and were rehydrated in 1 ml of Tris-EDTA buffer, of which 200 ul was used for DNA robotic extraction. Similarly 200ul of male urine was subjected to this automated DNA extraction (MagNA Pure LC instrument, Roche Diagnostics, Indianapolis, IN). The BTUB PCR assay design was based on FRET probe chemistry (Roche Diagnostics). Use of positive and negative controls, thermocycling protocol, data analysis, and specific sequences of primers and probes were previously published using the Roche LightCycler Instrument (9). TMA-TV. The SOVS were placed in Specimen Transport Media (Gen-Probe) and male urine was pipetted into Urine Specimen Transport Tubes for testing. The male urine and female SOVS were tested using target capture TMA and detection by the hybridization protection assay (HPA) in a manner similar to the other Gen-Probe APTIMA family of assays (Gen-Probe APTIMA COMBO 2 Package insert, IN Rev A, Gen-Probe Incorporated, San Diego, CA). Primers, probes and target capture oligomers were designed for TV based on the 16S rrna gene target. The cut-off for positive reactions was set by the investigators at 60,000 relative light units. Samples that tested positive by both the BTUB and TMA-TV were considered true positives. Discordant samples were adjudicated by another research PCR with 3
4 another primer set, TVK3 and TVK4, utilizing gel electrophoresis for end-point analysis (14). Results for vaginal wet preparation for motile trichomonads obtained during the clinic visit for females were also recorded for samples; no culture for trichomonas was performed. Samples positive by two of the three amplified tests or by one amplified test and a positive wet preparation result were also considered to be true positives for final sensitivity and specificity analysis. Overall, 59 samples were positive by BTUB and TMA-TV; 535 were negative by both; 15 were TMA-TV positive and BTUB negative; and 2 were TMA-TV negative and BTUB positive. Initially, compared only to BTUB results, TMA-TV sensitivity, specificity, PPV and NPV were 96.7%, 97.3%, 79.7%, and 99.6%, respectively (Table 1). After further testing of the 15 TMA-TV positive/btub negative samples, 10 were resolved as true positives; of 2 TMA-TV negative/btub positive samples, 1 female resolved as a true positive. Final TMA-TV sensitivity was 98.6% (69/70) and specificity was 99.1% (536/541) (Table 1). For males, final sensitivity and specificity was 100%. All male TMA-TV positive/btub negative samples were TVK3/4 positive (Table 2). Final prevalence in males was 4.5%. For females, final sensitivity and specificity were 98.2% (56/57) and 98.1% (259/264), respectively (Table 3). Of the 12 female TMA-TV positive/btub negative samples, 7/12 were TVK3/4 positive, 2/12 were TVK3/4 negative, 2/12 were of quantity not sufficient (QNS) for TVK3/4 analysis. They were graded true negatives based on wet preparation negative results. The remaining discordant sample (1/12) was QNS for further resolution. It was considered negative based on wet preparation results 4
5 (Table 3). Overall prevalence in women was 17.8%. The prevalence by vaginal wet preparation in the clinic was 12.1%; sensitivity was 68.4% (39/57). Kappa statistic analysis for comparison between TMA-TV and BTUB indicated an overall kappa value of For men, kappa value was 0.826, for women it was Kappa analysis indicated almost perfect" agreement. There was a high prevalence of 11.5% of T. vaginalis in our study population. Females had a higher prevalence (17.8%) for TV in our population than men (4.5%). This data is similar to our STD clinic population and Miller at el. (18, 30,31). Male urine and SOVS have been shown to be acceptable for the detection of TV (9,10,16,20,23,31). In our study, these self-obtained samples demonstrated a very high initial sensitivity and specificity for TMA-TV of 96.7% and 97.3% respectively, and the TMA-TV assay had excellent resolved sensitivity and specificity at 98.6% and 99.1%. Kappa statistic analysis between the two assays indicated an almost perfect agreement. There were very few discordant samples, although the TMA-TV identified more positives, indicating that the TMA-TV may be more sensitive. Additional testing was not performed on all the samples due to economic constraints. We recognize this potentially introduced some bias into the results (8,17,19). Culture, the gold standard, is inherently limited because it relies on viable organism for detection. Wet preparation and culture are subject to interpretation by the microscopist, whereas nucleic acid amplification assays offer may offer more accurate and objective results (16, 30). The Gen-Probe TMA-TV assay is commercially available as an ASR (analyte specific reagent) and offers laboratories a highly sensitive and 5
6 specific assay for use clinically; hopefully it will become available as a fully FDA cleared test. Reference List 1. Centers for Disease Control and Prevention Sexually transmitted disease surveillance, Atlanta, GA: U.S. Department of Health and Human Services, September CDC. 2. Cohen, M. S Sexually transmitted diseases enhance HIV transmission: No longer a hypothesis. Lancet 351 (suppl III): Cotch M.F., J.G. Pastorek, R.P. Nugent, S.L. Hillier, R.S. Gibbs, D.H. Martin, D.A. Eschenbach, R. Edelman, J.C. Carey, J.A. Regan, M.A. Krohn, M.A. Klebanoff, A.V. Rao, and G.G. Rhoads Trichomonas vaginalis associated with low birth weight and preterm delivery. Sex Transm Dis. 24: Gaydos C.A Nucleic Acid Amplification Tests for Gonorrhea and Chlamydia: Practice and Applications, p In J. M. Zenilman and Jr. R. C. Moellering (eds.), Infect Dis Clin N Am. Elsevier Saunders, Philadelphia. 5. Gaydos, C. A., T. C. Quinn, D. Willis, A. Weissfeld, E. W. Hook, D. H. Martin, D. V. Ferrero, and J. Schachter Performance of the APTIMA Combo 2 6
7 assay for the multiplex detection of Chlamydia trachomatis and Neisseria gonorrheae in female urine and endocervical swab specimens. J Clin Microbiol 41: Gaydos, C. A. and Rompalo A.M The use of urine and self-obtained vaginal swabs for the diagnosis of sexually transmitted diseases. Current Infect.Dis.Reports 4: Gilbert, R., G. Elia, D. Beach, S. Klaessig, and B. Singh Cytopathic effect of Trichomonas vaginalis on human vaginal epithelial cells cultured in vitro. Infect Immun 68: Green, T. A., C. M. Black, and R. E. Johnson Evaluation of bias in diagnostic test sensitivity and specificity estimates computed by discrepant analysis. J Clin Microbiol 36: Hardick, J., S. Yang, L. Lin, D. Duncan, and C. A. Gaydos Use of the Roche LightCycler Instrument in a Real-time PCR for Trichomonas vaginalis in urine samples from females and males. J.Clin.Microbiol 41: Hsieh, Y.-H., M. R. Howell, J. C. Gaydos, J. K. T. McKee, T. C. Quinn, and C. A. Gaydos Preference among female army recruits for use of selfadministered vaginal swabs or urine to screen for Chlamydia trachomatis genital infections. Sex. Transmit. Dis 30: ,
8 11. Jordan, J. A., D. Lowery, and M. Trucco TaqMan-based detection of Trichomonas vaginalis DNA from female genital specimens. J Clin Microbiol 39: Kaydos-Daniels, S. C., W. C. Miller, I. Hoffman, T. Bnda, W. Dzinyemba, F. Martinson, M. S. Cohen, and M. M. Hobbs Validation of a urine-based PCR-enzyme-linked immunosorbent assay for use in clinical research settings to detect Trichomonas vaginalis in men. J.Clin.Microbiol. 41: Kaydos, S. C., H. Swygard, S. L. Wise, A. C. Sena, P. A. Leone, W. C. Miller, M. S. Cohen, and M. M. Hobbs Development and validation of a PCRbased enzyme-linked immunosorbant assay with urine for use in clinical research settings to detect Trichomonas vaginalis in women. J.Clin.Micrbiol. 40: Kegne, P., F. Veas, N. Vidal, J. L. Rey, and G. Cuny Trichomanas vaginalis repeated DNA target for highly sensitive and specific polymerase chain reaction diagnosis. Cell and Molec Bio 40: Lawing, L. F., J. R. Hedges, and J. R. Schwebke Detection of trichomonosis in vaginal and urine specimens from women by culture and PCR. J Clin Microbiol 38:
9 16. Madico, G., T. C. Quinn, A. Rompalo, Jr. K. T. McKee, and C. A. Gaydos Detection of Trichomonas vaginalis infection by polymerase chain reaction (PCR). J Clin Microbiol 36: McAdam, A. J Discrepant analysis: how can we test a test? J.Clin Microbiol 38 : Miller, W. C., H. Swygard, M. M. Hobbs, C. A. Ford, M. S. Handcock, M. Morris, J. L. Schmitz, M. S. Cohen, Harris K.H., and J. R. Udry The prevalence of trichomoniasis in young adults in the United States. Sex Tansmit Dis 32: Miller, W. C Bias in discrepant analysis: when two wrongs don't make a right. J Clin Epidemiol 51: Newman, S. B., M. B. Nelson, C. A. Gaydos, and H. B. Friedman Female prisoners' preference of collection methods for testing for Chlamydia trachomatis and Neisseria gonorrhoeae infection. Sex Trans Dis 30: Petrin, D., K. Delgaty, R. Bhatt, and L. P. Garber Clinical and microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev 11:
10 22. Riley, D., M. Roberts, T. Takayama, and J. N. Krieger Development of a polymerase chain reaction-based diagnosis of Trichomonas vaginalis. J Clin Microbiol 30: Rompalo, A. M., C. A. Gaydos, N. Shah, M. Tennant, K. A. Crotchfield, G. Madico, T. C. Quinn, R. Daniel, K. V. Shah, J. C. Gaydos, and J. K. T. McKee Evaluation of use of a single intravaginal swab to detect multiple sexually transmitted infections in active-duty military women. Clin Inf Dis 33: Schwebke, J.R., L.F. Lawing Improved detection by DNA amplification of Trichomonas vaginalis in males. J. Clin. Microbiol. 40: Soper, D Trichomoniasis: Under control or undercontrolled? Amer J.Obstet.Gynecol 190: Sorvillo, F., L. Smith, P. Kerndt, and L. Ash Trichomonas vaginalis, HIV, and African-Americans. Emerg Infect Dis 7: Van Der Pol, B., D. Ferrero, L. Buck-Barrington, E. Hook III, C. Lenderman, T. C. Quinn, Gaydos CA, J. Moncada, G. Hall, M. J. Tuohy, and B. R. Jones Multicenter evaluation of the BDProbeTec ET system for the detection of Chalmydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs. J Clin Microbiol 39:
11 28. Van Der Pol, B., Quinn T.C., Gaydos CA, K. Crotchfelt, J. Schachte, J. Moncada, D. Jungkind, D. H. Martin, B. Turner, C. Peyton, and R. B. Jones Evaluation of the AMPLICOR and Automated COBAS AMPLICOR CT/NG Tests for the Detection of Chlamydia trachomatis. J Clin Microbiol 38: Wang, C. C., R. S. McClelland, and M. Reilly The effect of treatment of vaginal infections on shedding of human immunodeficiency virus type 1. J Infect Dis 183: Wendel, K. A., E. J. Erbelding, C. A. Gaydos, and A. M. Rompalo Trichomonas vaginalis polymerase chain reaction compared with standard diagnostic and therapeutic protocols for detection and treatment of vaginal trichomoniasis. Clin Infect Dis 35: Wendel, K. E., E. J. Erbelding, C. A. Gaydos, and A. M. Rompalo Use of urine polymerase chain reaction to define the prevalence and clinical presentation of Trichomonas vaginalis in men attending an STD clinic. Sex Transmit Inf 79: Zhang, Z. F. and C. B. Begg Is Trichomanas vaginalis a cause of cervical neoplasia? Results from a combined analysis of 24 studies. Int J of Epidem 23:
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13 Table 1. Comparison of TMA-TV results to BTUB FRET PCR results for Trichomonas vaginalis from 611 patients; 321 women and 290 men. Data type PCR Sensitivity Specificity % PPV % NPV % Initial α TMA-TV 96.7% (59/61) 97.3% (535/550) 79.7% (59/74) 99.6% (535/537) Female Male 98.0% (49/50) 90.1% (10/11) 95.6% (259/271) 98.9% (276/279) 80.3% (49/61) 76.9% (10/13) 99.6% (259/260) 99.6% (276/277) Resolved ß TMA-TV 98.6% (69/70) 99.1% (536/541) 93.2% (69/74) 99.8% (536/537) Female Male 98.2% (56/57) 100% (13/13) 98.1% (259/264) 100% (277/277) 91.8% (56/61) 100% (13/13) 99.6% (259/260) 100% (277/277) α Sensitivity and specificity of TMA-TV compared to BTUB FRET PCR ß Sensitivity and specificity where infection status is determined by two positive amplified results from TMA-TV, BTUB FRET PCR or alternate PCR using primer set TVK3 and TVK4 or wet preparation result Table 2. Additional testing of Male discordant samples 3 Male discordant samples: all initially TMA-TV Pos / BTUB FRET PCR Neg Repeat TMA-TV Repeat BTUB Primers TVK 3/4 True Pos / Neg 1. + True pos 2. NA + True pos 3. NA + + True pos 1 Male Discordant sample: initially TMA-TV Neg / BTUB Pos True neg 13
14 Table 3. Additional testing of female discordant samples for 12 Female Discordant samples: all initially TMA-TV Pos / BTUB Neg (QNS = quantity not sufficient) Repeat TMA Rpt. BTUB TVK 3/4 Wet Prep. Results True Pos or Neg True pos True pos True pos True pos 5. Not done True pos 6. Not done - + Not done True pos 7. Not done True pos True neg True neg Not done - True neg Not done - True neg QNS QNS - True neg Additional testing of female discordant samples 1 Female Discordant sample: initially TMA-TV Neg / BTUB Pos True pos 14
NIH Public Access Author Manuscript Sex Transm Infect. Author manuscript; available in PMC 2013 June 04.
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