Use of Gen-Probe APTIMA Tests to Detect

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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
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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 (N153) with urethritis CT
32.7, GC 24.2, TV 5.2, MG 22.4 males (N137)
without urethritis CT 6.6, GC 0, TV 1.5, MG
7.3. Prevalence females (N133) with
cervicitis CT 15.4, GC 6.0, TV 18.9, MG
28.6 females (N192) 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 gt1
pathogen, respectively. In regression analysis,
women with symptoms were more likely to have
cervicitis (p0.04) and men who were contacts of
persons with STDs were more likely to have
urethritis (p0.05). 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.
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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 gt 5 PMNs/HPF
• Urine sediment GS 15/400X Leukocyte Esterase

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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

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Signs, Symptoms, Risk Factors

• Males
• Urethral discharge
• Dysuria
• Itching at end of urethra
• Females
• Discharge
• Dysuria
• Abnormal vaginal bleeding
• Abdominal pain
• Mucopus

• Young age
• Multiple/new partners
• Sex/social networks
• Hx STD
• Race
• Geography
• Age 1st sex

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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

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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

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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 (gt50),
• NCNGGU, may cause prostatitis,
• epididymitis assoc w/ significant
• decrease in sperm motility and viability,
• isolated from 10 infertile men
• Women asymptomatic (lt50) 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

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Methods

• Men (290) and women (325) attending STD clinics
• Urethritis in men was dxd by gt5 PMN on Gram
  Stain
• Cervicitis in women was dxd 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

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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.

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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 415619-5622, 2003 Madico
et el. JCM 363205-3210, 1998 Hardick et al.
JCM 441236-1240, 2006
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Microbial Etiology Study Results N 325 females,
290 males
Females 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
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Results
Females Cervicitis N133 No
cervicitis N192 CT 15.0 8.3 GC
6.0 3.6 TV 18.8 12.5 MG
28.6 12.0
Males Urethritis N153 No urethritis
N137 CT 32.7 6.6 GC 24.2 0
TV 5.2 1.5 MG 22.4
7.3
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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 gt 2 organisms occurred in 10.6
  of all males if infected with at least one
  pathogen, 30-40 had a coinfection
• Co-infections with gt 2 organisms occurred in
  11.4 of all females if infected with at least
  one pathogen, 30 had a coinfection

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Association with Cervicitis
Univariate Multivar Model 1 Model 2
OR p value OR p OR p CT 1.95 0.058 1.60 0.25
0 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 lt25 yr 1.42 0.140 1.21
0.467 Contact 0.62 0.323 0.47 0.858

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Association with Urethritis
Univariate Multivariate Model 1
OR p value OR p CT 6.9 lt0.0001 6.92 lt0.0001
NG 88.5 lt0.0001 ---- TV 3.72 0.1087 4.29
0.08 MG 3.66 0.000 3.67 0.000 Age lt25
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
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Urethritis
Infection in men with urethritis
Infection in men without urethritis
33.1
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Cervicitis
Coinfection in women with cervicitis
Coinfection in women without cervicitis
47.4
72.3
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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