amplification assay and BD Affirm VPIII for detection of Trichomonas vaginalis in

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1 JCM Accepts, published online ahead of print on 19 January 2011 J. Clin. Microbiol. doi: /jcm Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved TITLE: Comparison of APTIMA Trichomonas vaginalis transcription-mediated amplification assay and BD Affirm VPIII for detection of Trichomonas vaginalis in symptomatic women: Performance parameters and epidemiologic implications RUNNING TITLE: TMA vs. Affirm for T. vaginalis detection AUTHORS: Sarah B. Andrea¹* and Kimberle C. Chapin 1,2,3 1 Department of Pathology; 2 Department of Medicine, Rhode Island Hospital, Providence, RI; 3 The Albert Brown Medical School, Providence, RI Preliminary findings were presented at the 110 th General Meeting of the American Society for Microbiology, San Diego, California, May 24, 2010 (Poster C-595). ATV reagents were provided by Gen-Probe Incorporated (San Diego, CA). *Corresponding Author. Mailing address: APC 11 Rm 1150, 593 Eddy Street, Providence, RI Phone: (401) Fax: (401) Sandrea@lifespan.org. WORD COUNT: Abstract: 228; Text:

2 ABSTRACT Trichomonas vaginalis (TV) is an underestimated sexually transmitted infection (STI) associated with numerous clinical sequelae. The true prevalence and clinical impact of trichomoniasis is unknown as current methods of detection exhibit poor sensitivity when compared to molecular amplification methods. Limited data exist comparing the BD Affirm VP III, hybridization assay, to the Gen-Probe APTIMA TV (ATV) transcriptionmediated amplification (TMA) assay for detection of TV. In this study, specimens from 766 patients were evaluated. Specimens were retrieved consecutively from patients with vaginal complaints and/or with history suggestive of STI. Study inclusion was dependent upon the request and collection of both a vaginal swab for Affirm and a specimen for APTIMA Combo 2 by the healthcare provider during the same office visit. Affirm was performed using the specific collection swab and transport provided for the test. The ATV assay was performed on remnant APTIMA Combo 2 specimens. A second ATV TMA assay, utilizing an alternate TV primer and probe set, was performed on all specimens positive by initial TMA and/or Affirm. Infected patient status was defined as a positive TV test result by at least 2 assays. Overall, 5.1% of subjects were positive for TV. TV was most prevalent in women years (11.9%), (7.7%), and (4.2%). The ATV assay was statistically more sensitive than the Affirm assay (100% vs. 63.4%, p<0.0001), identifying 36.6% more positive patients. KEY WORDS: Affirm VPIII, Bacterial vaginosis, Transcription-mediated amplification (TMA), Trichomonas vaginalis (TV), Trichomoniasis 2

3 INTRODUCTION Despite a worldwide prevalence rate likely to be double that of Neisseria gonorrhea (GC) and Chlamydia trachomatis (CT) combined, Trichomonas vaginalis (TV) is not currently a reportable disease in the US (1, 6). Recent literature suggests association between TV infection and the sequelae of other sexually transmitted infections (STIs) including: low birth weight, premature rupture of membranes and preterm labor, atypical pelvic inflammatory disease, infertility, prolonged carriage of human papilloma virus (HPV), and an increased risk for acquiring HIV (9, 10, 13, 18, 21, 31, 32). Current methods used for diagnosis of TV, including wet mount, rapid antigen test, and culture, have been shown to have poor sensitivity when compared to molecular amplification methods (5, 14, 17, 19, 24, 25, 27). In addition, variable performance occurs with antigen testing and wet mount depending on whether patients are symptomatic or asymptomatic (17, 26). The BD Affirm VPIII (Affirm) assay (Becton Dickinson, Sparks, MD) is the only Food and Drug Administration (FDA)-cleared direct specimen RNA probe-based diagnostic test designed to differentiate and identify pathogens associated with bacterial vaginosis, (Gardnerella vaginalis;bv) and vaginitis (TV and Candida species). The Affirm test is widely used; yet limited data exist comparing this test with other molecular methods (4). The purpose of this study was to evaluate the performance parameters of the BD Affirm with the Gen-Probe APTIMA Trichomonas (ATV) assay, a molecular diagnostic assay that uses transcription-mediated amplification (TMA) for the detection of TV. The ATV assay has recently completed clinical trials and has been submitted to the FDA for clearance. Testing was performed in a female population where Chlamydia trachomatis (CT), Neisseria gonorrhea (GC) and TV testing were being performed due to either 3

4 presentation of symptoms for BV, or in women with a history of STIs. TV prevalence by age in the population tested was compared to that of CT and GC. The population assessed is considered a low prevalence population for both CT and GC (28) MATERIALS & METHODS Subjects A total of 781 consecutive specimen pairs, consisting of one vaginal specimen and one cervical or urine specimen, were collected from 766 women from July 2009 to August 2010 during office visits with their healthcare provider. Only patients with specimen pairs, or test requests for both TV by Affirm and GC/CT by APTIMA were included in the study..the study design was approved by Lifespan Institutional Review Board (# ). Specimen collection and testing One vaginal specimen was collected from women during their office visit with the BD Affirm VPIII Ambient Temperature Transport System and was tested in the Affirm assay. A cervical specimen and/or a urine sample was collected during the same office visit with an APTIMA cervical swab or urine collection kit and tested in the APTIMA Combo GC/CT assay and subsequently in the ATV assay. Affirm Assay Vaginal specimens collected with the Affirm Ambient Temperature Transport System were used for testing by the Affirm VPIII. Affirm is an FDA-cleared hybridization assay that detects rrna for the three organisms associated with vaginosis/vaginitis: G. vaginalis for determination of vaginosis, C. albicans for yeast vaginitis, and TV for 4

5 trichomoniasis vaginitis. Specimens were tested and results were interpreted according to manufacturer instructions. APTIMA Combo 2 Assay This TMA-based assay is FDA-cleared to detect both CT and GC in one sample. The APTIMA Combo 2 assay was conducted as previously described (3, 20). Results of the APTIMA Combo GC/CT assay were used in epidemiological assessment of the population and prevalence of STIs. ATV Assay The ATV assay targets TV-specific 16S rrna. The ATV assay was performed on the remnant APTIMA Combo 2 specimen collected during the same office visit for GC/CT testing. The ATV assay was performed by using APTIMA General Purpose Reagents (GPR) and ATV analyte-specific reagent (ASR) oligonucleotides (Gen-Probe), which were reconstituted as described previously (24). Before March 1 st 2010, specimens were tested on the semi-automated direct tube sampling (DTS) platform (as described in the APTIMA Combo 2 package insert; Gen-probe Inc, San Diego); after that date, specimens were tested on the fully automated TIGRIS DTS. Positive specimens were re-tested to determine assay reproducibility. Samples with relative light unit (RLU) results >50,000 were considered positive. Samples with RLUs of 10,000 to 50,000 were considered indeterminate and retested in duplicate. If the specimen retested within this range the final result would have been called indeterminate. RLUs below 10,000 were considered negative and not retested. Confirmation of Initial Positive ATV Result and Discrepant Analysis 5

6 All initial positive ATV results were analyzed by an alternate GPR-based TMA assay that targets different TV rrna sequences (Alt-ATV assay). Specimens with an RLU >50,000 were considered positive. Performance parameters for the Alt-ATV assay at this institution were previously determined from 266 TV negative specimen pairs evaluated by both ATV and PCR(8). Alt-ATV sensitivity and specificity were 100 and 98.5%, respectively. Data Analysis A true positive result for TV infection was defined as a specimen with two positive test results for TV by two different molecular assays (Affirm & ATV, Affirm & Alt-ATV, and/or ATV & Alt-ATV). Age prevalence percentages were determined by dividing the number of unique infected patients by the total unique patients within an age group. Fisher s exact test was performed to calculate the statistical significance of general demographics and sensitivity/specificity using GraphPad Prism version 5.00 for Windows, (GraphPad Software; San Diego, CA). The null hypothesis was rejected if the p-value was <0.05. RESULTS Subject Population The study population consisted of 766 females with a mean age of 29.2 years (ranging from 13 to 88 years). 34.9% of women were negative for all organisms tested; G. vaginalis (BV) was the most prevalent organism found (41.3%), followed by Candida species (CA) (14.8%), TV (5.1%), CT (3.4%) and GC (0.7%) (Fig.1). While CT and GC infections were found only in women under 30 years, TV was the most prevalent in 6

7 women 36 to 45 (11.9%; 13/109) and women 51 to 60 (7.7%; 2/26) in addition to women under 30 (4.0%; 20/496) (Fig. 2); the median age of TV-positive women was 32 years. ATV and Affirm Assay Results Results and performance parameters for the Affirm and the ATV assays are shown in Table 1. Of the 781 samples analyzed with both assays, 41 were considered TV-positive and 740 were TV-negative (according to the infected status definition). The ATV assay detected all 41 TV-positive samples and produced no false positive or false negative results, yielding a sensitivity, specificity, PPV, and NPV of 100%. The Affirm assay, however, produced 15 false negative and 1 false positive samples, yielding a sensitivity of 63.4% (26/41), a specificity of 99.9% (739/740), a PPV of 96.3% (26/27) and a NPV of 98.0% (739/754). Specimens submitted for APTIMA were 74.5% (590/781) cervical and 24.5% (191/781) urine with 19 and 22 ATV positive results from each specimen type, respectively. There were no discrepant ATV results in patients with more than one APTIMA specimen type submitted during an office visit. All specimens testing positive on initial ATV repeated as positive in a second analysis of the same specimen with RLU clearly demarcated as >50,000. Additionally, no positive samples exhibited a RLU <100,000. Only 1 specimen exhibited an initial RLU in the indeterminate range (26,000); specimen was retested twice with subsequent RLUs of <10,000 for each and determined as negative. DISCUSSION We show herein that the ATV assay was statistically (p<0.0001) more sensitive than the Affirm assay (100% vs. 63.4%), detecting 36% more TV-infected women in the 7

8 population tested. Previous studies have compared ATV with non-molecular assays for TV detection such as wet mount and culture, and showed that ATV exhibited superior performance (15, 17, 22-24). Other studies have shown that ATV performance is comparable to that of non-commercial PCR assays (12, 24). This study is the first comparing ATV with another commercially available molecular assay (BD Affirm VP III assay) for TV detection side-by-side. One previously published abstract comparing Affirm and ATV performance in which TV was detected in both urine and vaginal genital specimens (30) confirms the results present in this study. This is important as both genital and urine specimens are useful specimens for TV detection (24). Not surprisingly and in accordance with published data (13), TV was more prevalent than CT or GC in the population tested. What was somewhat surprising was the high prevalence of TV at years of age in addition to the typical at-risk age group for STIs, years of age. Interestingly, women had no CT and GC infections but significantly more TV infection than women (p = 0.005, 0R = 3.1, 95% CI of 1.4 to 6.6). While this statistically significant double peak is consistent with symptomatic and asymptomatic general population data from our institution (7), other studies have also shown the unique prevalence of TV in an age group outside the recommended STI screening criteria (11, 16). While women also exhibited an increased TV prevalence this finding was not statistically significant when compared to any other age group. Lack of significance may be due to an inadequate number of women sampled within this age group. No wide-range prevalence studies have been conducted to date with amplification methods to determine the actual prevalence of TV by age. Determining TV prevalence in different age groups should help optimize TV testing 8

9 resources in high prevalence age groups. For example, in women performing TV testing in conjunction with CT/GC testing may be appropriate while in women 30 performing TV testing in conjunction with a cervical cancer screening with liquid cytology may be an optimal approach. A total of 316 (41.3%) patient specimens were found positive for BV by the Affirm assay. This seems quite high given the low prevalence of STIs in population studied; however all patients were symptomatic with vaginal complaints which may have selected for a higher infected patient group. In addition, the validity of these results is unknown since no comparative method was used for the diagnosis of BV. Among specimens identified as both BV positive and TV negative by the Affirm were 7 specimens that were in fact true TV positives. These specimens make up 46.7% (7/15) of Affirm false negative TV results. Thus, it is possible that some patients ended up being treated for the wrong etiology and/or with a less than optimal regimen. Issues related to recurrence of symptoms, continued transmission of TV, and the development of resistance are potential concerns if an incorrect diagnosis of BV is made in lieu of a correct diagnosis of TV. The lack of sensitivity in clinically diagnosing overlapping vaginosis/vaginitis symptoms supports the need to have a definitive test for TV vaginitis (2, 29). In this population with a low prevalence of STIs, the prevalence of TV infection (5.1%) was higher than that for GC and CT infections combined (4.1%), supporting the routine testing of TV. The ATV assay provided highly reliable data for TV detection from a sample already routinely collected for CT/GC testing. Therefore, the ATV assay could be considered as a useful companion assay to CT/GC testing in young women (<30 years 9

10 of age), and/or in conjunction with liquid cytology collected for cervical cancer screening in women 30 years of age Acknowledgements: TMA reagents and oligonucliotides as well as travel expenses and meeting fees for Sarah Andrea to attend the 110 th ASM GM were provided by Gen-probe (San Diego, CA). Downloaded from on November 14, 2018 by guest 10

11 211 REFERENCES American College of Obstetricians and Gynecologists (ACOG) Vaginitis. ACOG Practice Bulletin 72: Anderson, M. R., and A. Karasz How Do Clinicians Manage Vaginal Complaints? An Internet Survey. Medscape General Medicine Boyadzhyan, B., T. Yashina, J. H. Yatabe, M. Patnaik, and C. S. Hill Comparison of the APTIMA CT and GC Assays with the APTIMA Combo 2 Assay, the Abbott LCx Assay, and Direct Fluorescent-Antibody and Culture Assays for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Journal of Clinical Microbiology 42: Brown, H. L., D. D. Fuller, L. T. Jasper, T. E. Davis, and J. D. Wright Clinical evaluation of Affirm VPIII in the detection and identification of Trichomonas vaginalis, Gardnerella vaginalis, and Candida species in vaginitis/vaginosis. Infectious Diseases in Obstetrics and Gynecology 12: Caliendo, A. M., J. Jordan, A. Green, J. Ingersoll, R. J. Diclemente, and G. M. Wingwood Real-time PCR improves detection of Trichomonas vaginalis infection compared with culture using self-collected vaginal swabs. Infectious Diseases in Obstetrics and Gynecology 13: Centers for Disease Control and Prevention (CDC) February 3, , posting date. Reportable STDs in Young People Years of Age, by State. [Online.] 11

12 Chapin, K. C., S. B. Andrea, R. Dickenson, and E. Kojic Detection of Trichomonas vaginalis (TV) in a Varied Patient Population Using APTIMA Trichomonas Analyte Specific Reagents (ASRS), 18th ISSTDR, London. 8. Chapin, K. C., S. B. Andrea, J. Florczyk, and E. Kojic Comparison of Transcription Mediated Amplification (TMA) and Polymerase Chain Reaction (PCR) Methods for Detection of Trichomonas vaginalis from SUN Study Patients, p , 2010 CDC STD Prevention Conference, Atlanta, GA. 9. Cotch, M. F., J. G. I. Pastorek, R. P. Nugent, S. L. Hillier, R. S. Gibbs, D. A. Eschenback, R. Edelman, R. Carey, Joan A., K. M. A, and M. A. Klebanoff Trichomonas vaginalis Associated With Low Birth Weight and Preterm Delivery. Sexually Transmitted Diseases 24: Francis, S. C., C. K. Kent, J. D. Klausner, L. M. Rauch, R. P. Kohn, A. Hardick, and C. Gaydos Prevalence of Rectal Trichomonas vaginalis and Mycoplasma genitalium in Male Patients at the San Francisco STD Clinic, Sexually Transmitted Diseases 35: Freeman, A. H., K. A. Katz, M. W. Pandori, L. M. Rauch, R. P. Kohn, S. Liska, K. T. Bernstein, and J. D. Klausner Prevalence and Correlates of Trichomonas vaginalis Among Incarcerated Persons Assessed Using a Highly Sensitive Molecular Assay. Sexually Transmitted Diseases 37: Hardick, A., J. Hardick, B. J. Wood, and C. Gaydos Comparison between the Gen-Probe Transcription-Mediated Amplification Trichomonas vaginalis Research Assay and Real-Time PCR for Trichomonas vaginalis 12

13 Detection Using a Roche LightCycler Instrument with Female Self-Obtained Vaginal Swab Samples and Male Urine Samples. Journal of Clinical Microbiology 44: Heine, P., and J. McGregor Trichomonas Vaginalis: A Reemerging Pathogen. Clinical Obstetrics and Gynecology 36: Hobbs, M. M., D. M. Lapple, L. F. Lawing, J. R. Schwebke, M. S. Cohen, H. Swygard, J. Atashili, P. A. Leone, W. Miller, and A. C. Sena Methods for Detection of Trichomonas vaginalis in the Male Partners of Infected Women: Implications for Control of Trichomoniasis. Journal of Clinical Microbiology 44: Hollman, D., S. Coupey, A. Fox, and B. Herold Screening for Trichomonas vaginalis in high-risk adolescent females with a new transcription-mediated nucleic acid amplification test (NAAT): associations with ethnicity, symptoms, and prior and current STIs. Journal of Pediatric and Adolescent Gynecology 23: Huppert, J., B. E. Batteiger, P. Braslins, J. Feldman, M. Hobbs, H. Sankey, A. C. Sena, and K. Wendel Use of immunochromatographic assay for rapid detection of Trichomonas vaginalis in vaginal specimens. Journal of Clinical Microbiology 43: Huppert, J., J. Mortensen, J. Reed, J. Kahn, K. Rich, W. Miller, and M. Hobbs Rapid antigen testing compares favorably with transcriptionmediated amplification assay for the detection of Trichomonas vaginalis in young women. Clinical Infectious Disease 45:

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15 Setting of High Sexually Transmitted Disease Prevalence. Journal of Clinical Microbiology 46: Nye, M. B., J. R. Schwebke, and B. A. Body Comparison of APTIMA Trichomonas vaginalis transcription-mediated amplification to wet mount microscopy, culture, and polymerase chain reaction for diagnosis of trichomoniasis in men and women. American Journal of Obstetrics and Gynecology 200:188.e1-188.e Patel, S. R., W. Wiese, S. C. Patel, C. Ohl, J. C. Byrd, and C. A. Estrada Systematic Review of Diagnostic Tests fo Vaginal Trichomoniasis. Infectious Diseases in Obstetrics and Gynecology 8: Pattullo, L., S. Griffeth, L. Ding, J. Mortensen, J. Reed, J. Kahn, and J. Huppert Stepwise diagnosis of Trichomonas vaginalis infection in adolescent women. Journal of Clinical Microbiology 47: Piperaki, E.-T., M. Theodora, M. Mendris, L. Barbitsa, V. Pitiriga, A. Antsaklis, and A. Tsakris Prevalence of Trichomonas vaginalis infection in women attending a major gynecological hospital in Greece: a cross-sectional study. Journal of Clinical Pathology 63: Rhode Island Department of Health 2006, posting date. STD Epidemiology Summary: Rhode Island, [Online.] 29. Schwiertz, A., D. Taras, K. Rusch, and V. Rusch Throwing the dice for the diagnosis of vaginal complaints? Annals of Clinical Microbiology and Antimicrobials 5. 15

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17 TABLES Table 1. Comparison of molecular methods for detection of TV in symptomatic females a Diagnostic Method No. true positive No. false positive No. false negative No. true Sensitivity Specificity Predictive Value (%) negative % 95% CI % 95% CI Positive Negative Affirm VPIII ATV a: 781 specimen pairs were analyzed by Affirm VPIII and ATV Downloaded from on November 14, 2018 by guest

18 FIGURES Figure 1: Infections found in 766 symptomatic females using the Affirm a and APTIMA b assays 34.9% 0.7% 3.4% 5.1% a GV and CA b CT, GC, and TV 14.8% 41.3% BV CA TV CT GC Negative Downloaded from % 12.0% 10.0% 8.0% 6.0% 4.0% Figure 2: Age-dependent STI prevalence in 766 symptomatic females TV CT GC on November 14, 2018 by guest 2.0% 0.0% 15 (n=13) 16 to 20 (n=119) 21 to 25 (n=150) 26 to 30 (n=92) 31 to 35 (n=64) 36 to 40 (n=50) 41 to 45 (n=36) 46 to 50 (n=40) 51 to 55 (n=14) 56 to 60 (n=9) 61 (n=4) Age in Years