M genitalium

Mycoplasma genitalium
New Diagnostics for an Emerging Urogenital Pathogen
Damon Getman, Ph.D.
Director, Research and Development
Hologic, Inc
San Diego, USA
Mycoplasma genitalium
•First identified in 2 male NGU cases in 1981 (Tully et al)
•Small gram negative bacterium, no cell wall. Smallest genome
of any free-living organism (0.58 MB, ~480 genes)
•Very similar to other Mycoplasma spp. (M. pneumoniae, U. urealyticum)
•Very difficult to culture; immunofluorescent tests available,
but difficult to perform.
•NAAT is the only reliable way to diagnose.
Lind, Lancet 1982
Microorganisms recovered
from male urethral swabs
(%)
Jensen et.al., 1993
Why test for Mgen?
•High prevalence in STD clinic population (> Ct?)
•Causes inflammation of urogenital tract
•urethritis, cervicitis, prostatitis, salpingitis, PID
•association with tubal infertility
•Mgen found in 12% of PID, 40% of persistent PID cases
•any inflammation in UGT is a risk factor for HIV transmission
and HIV infection
• > 30 studies link Mgen with NGU in males
•Antibiotic Tx of UGT infections is organism-specific
•GC: cephalosporins and doxycycline
•CT: azithromycin or doxycycline or ofloxacin or levofloxacin
•TV: metronidazole, tinidazole
•Mgen: azithromycin, moxifloxacin (tet resistant)
Mgen Prevalence Worldwide
Study
Country
Mgen Prevalence
Remarks
Pepin, 2005
Ghana, Benin
Vandepitte, 2012
Uganda
Xiang, 2012
China
Salado-Rasmussen, 2014
Denmark
Oakeshott, 2010
United Kingdom
3.3%
Female Univ. students
Soni, 2010
United Kingdom
6.6%
MSM
Cazanava, 2013
France
3.8%
Females, HIV+
Gesink, 2012
Greenland
9.8%
Random sample, ages 15-65
Johnston, 2012
Honduras
18.3%
FSW
Tsunoe, 2000
Japan
12.6% FSW
1.1% ASx Preg.
Nakashima, 2014
Japan
7.7%
Males age 16-70, urine samples
Oliphant, 2013
New Zealand
8.4%
Females, STI clinic
Bjartling, 2012
Sweden
2.1%
Sx Females, Gyn clinic
Wetmore, 2011
United States
12.5%
Males with NGU
Gatski, 2011
United States
9.9%
Females, HIV+
Short, 2010
United States
5.6%
Young pregnant women
26.3%
FSW
14%
FSW
13.2%
FSW
3.8% females
10.3% males
Nationwide survey
Mgen Prevalence in Australia
Study
Region
Mgen
Prevalence
Bradshaw, 2006
Melbourne
5.4%
Males
9% in NGU
1% controls
McIver, 2009
Sydney
1.3%
Females, cervical swabs
Bradshaw, 2009
Melbourne
Couldwell, 2010
Sydney
4.5%
Males, symptomatic
Walker, 2011
Victoria, New
South Wales,
Australian Capital
Territory
2.4%
4% in sex. health clinics,
1.6% in general health
clinics
Couldwell, 2013
Sydney
38.6%
Mezzini, 2014
Adelaide
8.1%
Males, UTI
3.1% in MSM
9.1% in hetero/bisexual
Henning, 2014
Melbourne
13%
M/F, homeless ages 1225
2.1-3.6%
Remarks
MSM
Men with NGU
Mgen Prevalence in Men
NCNGU
Association between Mgen
and acute NCNGU in men
Taylor-Robinson, 2011
Sexual Transmission of Mgen
Evidence for concordant carriage of Mgen among
sexual partners
-Concordance rates = 35-65%
-Partners have same genetic strains (genotypes) of Mgen
-Mgen infection concordance rates similar to
those for C. trachomatis
(Keane et al; Anagrius et al; Falk et al.)
Mgen binds to human spermatozoa
Svenstrup et.al., Human Repro. 18, 2103, 2003
Mgen infection in Women
•Mgen can colonize primates after vaginal inoculation
•Mgen causes endosalpingitis and adhesions in
fallopian tubes of lower primates.
•Adheres strongly to human tubal epithelial cells
Human Fallopian Tube Morphology-Normal
Baczynska et. al., 2006
CT
GC
Baczynska et. al., 2006
Mgen
Baczynska et. al., 2006
M. hominis
Baczynska et. al., 2006
Mgen and upper genital tract disease
in Endometritis and PID
•Mgen found in 9/58 (16%) women with EM,
2% of women w/o EM (Cohen et al, 2002)
•5/56 (9%) women with PID Mgen positive,
0% in 80 controls (Uno et al., 1997)
•28/216 (13%) women with PID Mgen positive,
0% in 37 controls (Simms et al., 2003)
•29/132 (29%) of women with tubal infertility had Abs to Mgen,
compared to 6% of women with normal tubes (Clausen et al., 2001)
Mgen and Cervicitis Association
Odd Ratios Using All Diagnostic Criteria
Taylor-Robinson, 2011
Mgen and Cervicitis Association (PCR)
Odds Ratios by Diagnostic Criteria
Taylor-Robinson, 2011
Association of Mgen and HIV
N=2235
Mavedzenge et al, AIDS 2012
Aptima Mgen Transcription-Mediated Amplification Assay
Research Use Only
0.170 mL/vial (750 tests)
50 uL spike/GPR vial
APTIMA GPRs-250 test kit
TIGRIS
AMG Assay Bibliography
Justin Hardick, Julie Giles,1 Andrew Hardick, Yu-Hsiang Hsieh, Thomas Quinn, Charlotte Gaydos. Performance of the Gen-Probe Transmission-Mediated Amplification
Research Assay Compared to That of a Multitarget Real-Time PCR for Mycoplasma genitalium Detection. J Clin Microbiology 2006; 44(4): 1236–1240.
Jennifer K. H. Wroblewski,1 Lisa E. Manhart,2 Kathleen A. Dickey,3† Marie K. Hudspeth,3 and Patricia A. Totten. Comparison of Transcription-Mediated Amplification and
PCR Assay Results for Various Genital Specimen Types for Detection of Mycoplasma genitalium. J Clin Microbiology, 2006; 44(9): 3306–3312.
Suzanna C. Francis, Charlotte K. Kent, Jeffrey D. Klausner, Leah Rauch, Robert Kohn, Andrew Hardick, Charlotte A. Gaydos. Prevalence of Rectal Trichomonas vaginalis and
Mycoplasma genitalium in Male Patients at the San Francisco STD Clinic, 2005–2006. Sexually Transmitted Diseases, 2008, 35(9), 797–800.
Jill S. Huppert, Joel E. Mortensen, Jennifer L. Reed, Jessica A. Kahn, Kimberly D. Rich, Marcia M. Hobbs. Mycoplasma genitalium Detected by Transcription-Mediated
Amplification Is Associated With Chlamydia trachomatis in Adolescent Women. Sexually Transmitted Diseases, 2008; Vol. 35(3): 250–254.
Emily B. Hancock, MS, Lisa E. Manhart, PhD, Sara J. Nelson, MPH, Roxanne Kerani, PhD, Jennifer K. H. Wroblewski, MPH, and Patricia A. Totten, PhD Comprehensive
Assessment of Sociodemographic and Behavioral Risk Factors for Mycoplasma genitalium Infection in Women. Sexually Transmitted Diseases 2010; 37(12): 777-783.
Jane Hitti, Pedro Garcia, Patricia Totten, Kathleen Paul, Sabina Astete, King K. Holmes. Correlates of Cervical Mycoplasma genitalium and Risk of Preterm Birth Among
Peruvian Women. Sexually Transmitted Diseases 2010; 37(2): 81-85.
Stefanie L. Iverson-Cabral, Lisa E. Manhart, Patricia A. Totten. Detection of Mycoplasma genitalium-Reactive Cervicovaginal Antibodies among Infected Women. Clinical
and Vaccine Immunology, 2011; 18(10):1783–1786.
Lisa A. Cosentino, Tracy Campbell, Abi Jett, Ingrid Macio, Tracy Zamborsky, Ross D. Cranston and Sharon L. Hillier. Use of Nucleic Acid Amplification Testing for Diagnosis
of Anorectal of Sexually Transmitted Infections. J Clin Microbiology 2012; 50(6): 2005-2008.
Supriya D. Mehta, Charlotte Gaydos, Ian Maclean, Elijah Odoyo-June, Stephen Moses, Lawrence Agunda, Nicole Quinn, and Robert C. Bailey. The Effect of Medical Male
Circumcision on Urogenital Mycoplasma genitalium Among Men in Kisumu, Kenya. Sexually Transmitted Diseases, 2012; 39(4): 276-280.
Victoria L. Mobley, Marcia M. Hobbs, Karen Lau, Barbara S. Weinbaum Damon K. Getman, Arlene C. Sena. Mycoplasma genitalium Infection in Women Attending a
Sexually Transmitted Infection Clinic: Diagnostic Specimen Type, Coinfections, and Predictors. Sexually Transmitted Diseases, 2012; 39(9): 706-709.
Arlene C. Seña, Shelly Lensing, Anne Rompalo, Stephanie N. Taylor, David H. Martin, Laureen M. Lopez, Jeannette Y. Lee, Jane R. Schwebke. Chlamydia trachomatis,
Mycoplasma genitalium, and Trichomonas vaginalis Infections in Men With Nongonococcal Urethritis: Predictors and Persistence After Therapy. JID, 2012; 206: 357365.
Christine M Khosropour, Lisa E Manhart, Danny V Colombara, Catherine W Gillespie, M Sylvan Lowens, Patricia A Totten, Matthew R Golden, Jane Simoni. Suboptimal
adherence to doxycycline and treatment outcomes among men with non-gonococcal urethritis: a prospective cohort study. Sex Transmitted Infect 2013; 0:1–5.
doi:10.1136/sextrans-2013-051174
Tobian et al, Male circumcision and Mycoplasma genitalium infection in female partners: a randomised trial in Rakai, Uganda. Sex Transm Infect doi:10.1136/sextrans2013-051293
Sara Campbell1, Matt Psioda2, Jie Ting3, Nelly Mugo4, Jessie Kwatampora4, Michael Chitwa4, Hannah Gakure4, and Jennifer S. Smith5,6 Self-collection versus Physiciancollection for the Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas, and Mycoplasma genitalium Infections. Submitted to STD Jan 2014
AMG Assay
Analytical Sensitivity-Probit Analysis
Sample Matrix
Female Urine
Vaginal Swab
95% LOD
(CFU/Rxn)
50% LOD
(CFU/Rxn)
0.023
0.0005
(95% FL: 0.0114, 0.0712)
(95% FL: 0.0003, 0.0008)
0.023
0.0003
(95% FL: 0.0105, 0.0767)
(95% FL: 0.0001, 0.0005)
AMG Assay: Analytical Specificity (Cross Reactivity)
N=10 replicates
50k RLU cut-off
*Tested at a concentration equal to 1e6 CFU,
viral particles or copies of IVT per mL
Microbe*
Unspiked pool w/o microbes
Acinetobacter iwoffi
Actinomyces israelii
Alcaligenes faecalis
Atopobium vaginae
Bacteroides fragilis
Bifidobacterium adolescentis
Campylobacter jejuni
Candida albicans
Chlamydia trachomatis
Clostridium difficile
Corynebacterium genitalium
Enterobacter cloacae
Enterococcus faecalis
Escherichia coli
Fusobacterium nucleatum
Gardnerella vaginalis
Haemophilus ducreyi
Klebsiella pneumonia
Lactobacillus acidophilus
Lepyotrichia bucalis
Listeria monoctogenes
Mobiluncus curtisii
Mycoplasma hominis
Mycoplasma pneumoniae
Neisseria gonorrhoeae
Peptostreptococcs magnus
Prevotella bivia
Propionibacterium acnes
Psueudomonas aeruginosa
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus pyogenes
Trichomonas vaginalis
Ureaplasma Urealyticum
% Negative
Vaginal Swab Pool
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Urine Pool
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Mgen TMA Assay Results with STD Clinic
Patients at Johns Hopkins
M. genitalium study
Female vag. Swabs only
N=321
TMA +
TMA -
PCR +
65
2
PCR 4
250
Prevalence: 20.2%
Sensitivity:
97.00%
Specificity:
98.40%
Hardick et al, J Clin Micro 2006
Detection of STIs in Women with Cervicitis
Aptima Combo 2
Aptima TV
Aptima Mgen
Gaydos et al, Sex Trans Dis 2009
Mgen TMA Assay Results with
STD Clinic Patients at Johns Hopkins
M. genitalium study
Male urines only
N=286
TMA +
TMA -
PCR +
45
0
PCR 5
236
Prevalence: 15.7%
Sensitivity: 100.00%
Specificity:
97.90%
Hardick et al, J Clin Micro 2006
STI detection by Aptima TMA assays in 500 consecutive
rectal samples from MSM attending STD clinic
MG
TV
CT
GC
Sex Trans Dis, 2008
5.4%
0.6%
10%
11.4%
M. genitalium U.S. Prevalence Study
Prospective Multi-Center Collection
• Obtained 6,612 remnant clinical samples from Panther Aptima Combo 2 US
clinical study
– 7 geographically and ethnically diverse US clinical sites (OB/Gyn, FP, PH, STD Clinic)
– Subject self-reported symptomatic status
– N=580 males, 1332 females
• Current Mgen Study
– 6 different specimen types:
• Endocervical swab (female)
• Vaginal swab (female)
• First-catch urine (female)
• Liquid pap (female)
• First-catch urine (male)
• Urethral swab (male)
– 4,619 of the 6,612 samples were tested for M. genitalium by TMA
– Subset also tested with Aptima Trichomonas vaginalis assay
US Mgen Study
Subject demographic characteristics
Demographic
Characteristic
Age( total N=800)
14-17
18-20
21-30
31-40
41-50
51-60
61-70
71-78
Gender
Female
Male
Ethnicity
Hispanic
Non-Hispanic
Unknown
Race*
American Indian or
Alaska Native
Asian
Black or African
American
Native Hawaiian or
Pacific Islander
White
Unknown
Frequency (N)
Percent (%)
23
143
365
138
77
42
11
1
2.87
17.85
45.69
17.23
9.61
5.24
1.37
0.12
402
398
50.25
49.75
104
583
113
12.98
72.91
14.11
2
0.25
7
490
0.87
61.3
0
0
254
50
31.71
6.24
Prevalence of M. genitalium by subject age group
35
Male
% Mgen Positive
30
Female
25
20
15
10
5
0
14-17
18-20
21-30
31-40
Age (yrs)
41--50
51-60
61-70
71-78
M. genitalium TMA positivity rates by sample type
All Females
Female Urine
ThinPrep Liquid Pap
Endocervical Swab
Vaginal Swab
All Males
Male Urine
Male Urethral Swab
0
2
4
6
8
10
% Mgen Positive
12
14
16
18
Relative sensitivity of AMG assay for detection of M.
genitalium in Female Samples
Sample Type
Mgen Positive (n) Relative Sensitivity† (N=54)
Vaginal Swab
48
88.9%
Endocervical Swab
41
75.9%
ThinPrep Liquid Pap
37
68.5%
Female Urine
20
37.0%
VS+FU
49
90.7%
VS+ES
52
96.3%
VS+ES+FU
53
98.1%
VS+ES+FU+LP
54
100%
†Number
of specimens positive for M. genitalium for each specimen type or combination of
specimen types relative to the total number of women positive at any site by TMA
Relative sensitivity of AMG Assay for detection
of M. genitalium in Male Samples
Sample Type
Male Urine
Male Urethral Swab
MU+MUS
†Number
Mgen Positive
(n)
32
Relative Sensitivity†
(N=67)
47.8%
66
67
98.5%
100%
of specimens positive for M. genitalium for each specimen type or combination of
specimen types relative to the total number of women positive at any site by TMA
Symptomatic status by age group among
M. genitalium-positive subjects
100.0
90.0
80.0
Male
% Symptomatic
70.0
Female
60.0
50.0
40.0
30.0
20.0
10.0
0.0
14-17
18-20
21-30
31-40
41--50
Age (yrs)
51-60
61-70
71-78
Proportion of symptomatic subjects among
M. genitalium-positive female subjects
Vaginal Swab
Endocervical Swab
ThinPrep Liquid Pap
Female Urine
Male Urine
Male Urethral Swab
0.0
1.0
2.0
3.0
4.0
5.0
Symptomatic/Asymptomatic Ratio
6.0
M genitalium US Prevalence Study
Phase II
Evaluate Mgen Prevalence in relation
to CT, GC, TV Prevalence
Prevalence of Sexually Transmitted Organisms
in Female Urogenital Samples
Vaginal Endocervical Liquid
Swab
Swab
Pap
Organism N=318
N=301
N=785
Female
Urine
N=741
CT
9.4%
7.3%
8.4%
8.1%
GC
4.4%
3.0%
1.4%
1.5%
MG
21.7%
21.6% 11.2%
7.2%
TV
21.7%
23.3% 13.0%
11.5%
Co-Infection Rates of Sexually Transmitted Organisms Among Women Positive for a STI
Symptomatic Status Among Women with
Single Infections
Symptomatic Status Among Women with
Dual Infections
Prevalence (%) of Sexually Transmitted Organisms
in Male Urogenital Samples
PIS: Patient Infected Status = MU+ and/or US+
Co-Infection Rates of Sexually Transmitted
Organisms Among Men Positive for a STI
Male Urine
Samples
Male Urethral Swab
Samples*
Patient Infected
Status*
*TV Excluded from Analysis
Symptomatic Status Among Men with
Single Organism Infections
Conclusions
• APTIMA Mgen TMA assay is sensitive,
specific; substantial body of published
clinical data has established AMG as a
useful tool for M genitalium detection.
• M genitalium prevalence in US STI clinic
population rivals that of Chlamydia
• Strong evidence for Mgen as causative
agent in NCNGU in men and risk for HIV,
evidence growing for cervicitis, PID,
preterm birth
• Growing clinical consensus for M genitalium
diagnostic need - antibiotic Tx.
45