Use of Gen-Probe APTIMA Tests to Detect Multiple Etiologies of Urethritis and Cervicitis In Sexually Transmitted Disease Clinics C-086 Board 133 Charlotte A. Gaydos, Andrew Hardick, Justin Hardick, Mathilda Barnes, Billie Jo Wood Johns Hopkins University, Baltimore, MD

Abstract Background: In addition to Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), being commonly recognized causes of urethritis and cervicitis, other agents, such as Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG), can cause urethritis and cervicitis. The Gen-Probe APTIMA TMA technology can be used to detect each of the 4 pathogens. Methods: Urines from 290 men and self-administered vaginal swabs from 325 women were collected from STD clinic patients. Demographics, behavioral risk factors, signs, and symptoms were recorded. CT and GC were detected by APTIMA Combo 2. MG and TV were detected by real time research PCR-MG and B-TUB FRET-TV and by prototype Gen-Probe TMA-based assays for MG and TV (TMA-MG and TMA-TV). A patient was considered to be infected with MG and/or TV if both research PCR assay and prototype TMA assay were positive. Results: Prevalence: males (N=153) with urethritis: CT 32.7%, GC 24.2%, TV 5.2%, MG 22.4%; males (N=137) without urethritis: CT 6.6%, GC 0%, TV 1.5%, MG 7.3%. Prevalence: females (N=133) with cervicitis: CT 15.4%, GC 6.0%, TV 18.9%, MG 28.6%; females (N=192) without cervicitis: CT 8.3%, GC 3.7%, TV 12.7%, MG 13.2%. Kappa statistic comparison of PCR and TMA-MG and TMA-TV were excellent (0.941 and 0.858, respectively). Of infected women and men, 23.6% and 20.3% had >1 pathogen, respectively. In regression analysis, women with symptoms were more likely to have cervicitis (p=0.04) and men who were contacts of persons with STDs were more likely to have urethritis (p=0.05). Only MG was associated with cervicitis and all organisms except TV were associated with urethritis in men. Conclusion: Prevalence of any organism was higher in urethritis and cervicitis. Co-infections were common. TMA-based assays for MG and TV performed very well compared to research PCRs.

Introduction Definition: Cervicitis: Inflammation of the cervix Urethritis: Inflammation of the urethra Diagnosis: can vary Cervicitis: Discharge, friability, gram stain 10-30 PMNs Mucopurulent endocervical discharge (yellow exudate) Appearance of the inflamed endocervix on physical exam (discharge, edema, erythema) Urethritis: Urethral GS > 5 PMNs/HPF Urine sediment GS: 15/400X; Leukocyte Esterase

Urethritis and Cervicitis Incidence: Urethritis (CDC) over 4 million cases per year Gonococcal- 650,000 cases/yr Non Gonooccal-over 2 million cases/ yr Up to 65% are of unknown origin Cervicitis- estimated 4 million cases per year Reported to CDC, CT/GC 832,506 cases Up to 40% are of unknown etiology

Signs, Symptoms, Risk Factors Males Urethral discharge Dysuria Itching at end of urethra Females Discharge Abnormal vaginal bleeding Abdominal pain Mucopus Young age Multiple/new partners Sex/social networks Hx STD Race Geography Age 1st sex

Objectives To determine the prevalence and multiple etiologies of male urethritis and female cervicitis To determine the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) in patients from STD clinics To compare the research Gen-Probe APTIMA TMA for MG and TV with research PCRs. To determine the association of CT, GC, MG, and TV with cervicitis and urethritis

Mycoplasma genitalium Thought to be sexually transmitted; also found in respiratory tract Associated with NCNGGU in men and cervicitis in women recently Possible sequelae endometritis?, PID? adverse birth outcomes?? Smallest prokaryote, with no cell wall, and very difficult to culture

Trichomonas vaginalis More prevalent than CT or GC: CDC estimates 5 million cases/yr in U.S.; Not a reportable disease Men: may be asymptomatic (>50%), NCNGGU, may cause prostatitis, epididymitis; assoc w/ significant decrease in sperm motility and viability, isolated from 10% infertile men Women: asymptomatic (<50%) or cause frothy discharge, risk for cervical neoplasia, tubal infertility, post-hysterectomy infection, atypical PID preterm birth, low birth weight Risk factor for HIV transmission

Methods Men (290) and women (325) attending STD clinics Urethritis in men was dx’d by >5 PMN on Gram Stain Cervicitis in women was dx’d by cervical mucopus, friability, or discharge Men provided 2 urethral swabs, urine; Women provided 3 cx swabs, 2 vaginal swabs, urine Routine tests included APTIMA Combo2 (GenProbe) tests for CT and GC from male urine, female cervical, vaginal and urine samples

Methods Research PCRs were performed for T. vaginalis and M. genitalium on male urine and female vaginal samples Research APTIMA TMA- based assays (GenProbe) were performed for M. genitalium (TMA-MG) and for T. vaginalis (TMA-TV) on male urine and female vaginal samples A patient was considered to be infected with MG and/or TV if both research PCR assay and research TMA assay were positive.

Methods Trichomonas Research PCR* based on detection of the B tubulin gene real-time PCR Roche Light Cycler technology Mycoplasma Research PCR** utilized 2 targets in a diplex assay MgPa adhesion gene 16S rRNA gene 7900 Sequence Detection ABI Prisim *Hardick et al. JCM 41:5619-5622, 2003; *Madico et el. JCM 36:3205-3210, 1998 ** Hardick et al. JCM 44:1236-1240, 2006

Microbial Etiology Study Results: N= 325 females, 290 males CT 11.1 GC 4.6 TV 15.3 MG 19.2 None 60.9 Males: % CT 20.3 GC 12.8 TV 3.4 MG 15.2 None 57.6 Median Age 24 yr. Black 92.6% Symptoms 69.8% Median Age 25 yr Black 96.9% Symptoms 66.9%

Results Females: Males Urethritis N=153 No urethritis N=137 Cervicitis N=133 No cervicitis N=192 CT 15.0% 8.3% GC 6.0% 3.6% TV 18.8% 12.5% MG 28.6% 12.0% Males Urethritis N=153 No urethritis N=137 CT 32.7% 6.6% GC 24.2% 0 % TV 5.2% 1.5% MG 22.4% 7.3%

Results Kappa statistics for comparison between research PCRs for MG and TV and TMA-MG and TMA-TV were excellent (0.941 and 0.858, respectively) Co-infections of > 2 organisms occurred in 10.6% of all males; if infected with at least one pathogen, 30-40 % had a coinfection Co-infections with > 2 organisms occurred in 11.4% of all females; if infected with at least one pathogen, 30% had a coinfection

Association with Cervicitis Univariate Multivar Model 1 Model 2 OR p value OR p OR p CT 1.95 0.058 1.60 0.250 1.58 0.078 NG 1.69 0.316 1.01 0.983 0.93 0.911 TV 1.61 0.126 1.59 0.146 1.56 0.174 MG 2.64 0.0006 2.42 0.0026 1.56 0.0028 Age <25 yr 1.42 0.140 1.21 0.467 Contact 0.62 0.323 0.47 0.858

Association with Urethritis Univariate Multivariate Model 1 OR p value OR p CT 6.9 <0.0001 6.92 <0.0001 NG* 88.5 <0.0001 ----* TV 3.72 0.1087 4.29 0.08 MG 3.66 0.000 3.67 0.000 Age <25 yr 1.1 0.69 Contact 1.97 0.05 *NG could not be fit into the multivariate model due to collinearity between NG and urethritis that caused the model not to fit

Urethritis Infection in men with urethritis Infection in men without urethritis 33.1%

Cervicitis Coinfection in women with cervicitis Coinfection in women without cervicitis 47.4% 72.3%

Conclusions Prevalence of all organisms was higher in men with urethritis and in women with cervicitis in this population Co-infections were common; MG and TV were associated with urethritis and cervicitis Research TMA-based GenProbe assays for MG and TV performed very well compared to research PCR assays Use of newer tests such as TMA or PCR will allow better etiologic diagnostic capability in STD patients Even with increased diagnostic capability, a significant proportion of urethritis and cervicitis cases have no etiologic agent detected