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Mycoplasma genitalium, Mycoplasma genitalium, STD and STD and


molecular diagnostics molecular diagnostics
Ccile Bbar
USC Mycoplasmal and chlamydial infections in humans
French National Reference Center for Chlamydia
INRA - Universit Bordeaux Segalen
CHU de Bordeaux
Overview
Introduction
Association between M. genitaliuminfections and
disease
Diagnosis
Antimicrobial susceptibility testing and treatment
studies
Mycoplasma genitalium
An introduction
1980: Mycoplasma genitaliumisolated from 2 of 13 men
with NGU (nongonoccocal uretritis)
Very slow growth (>50 days)
Very few isolates available
Sexually transmitted bacterium, lacks a cell wall
1990s: development of PCR assays, allowed study of
disease association
1995: smallest genome known (580 kbp, 480 genes)
One of the first fully sequenced (Himmelreich, 1995)
Minimal requirements of life, concept of minimal cell
Mycoplasma genitalium
An introduction
Similar to M. pneumoniae
Morphology (tip structure)
Genetics
How much ? Prevalence and incidence
What ? Association with diseases
Who ? Risk factors
M. genitalium: an epidemiologists view
M. genitalium: prevalence and incidence
Prevalence
- General population
1 - 4%
1 - 6%
- Clinicpopulations
4 - 26%
4 - 38%
Incidence
- Universitywomen: 0.9 per 100 WY
- Kenya Female sex workers: 23 per 100 WY
Anagrius STI 2005, Hamasuna STI 2004, Ross STI 2009, Tosh JAH 2007, Oakeshott
CID 2010, Cohen STD 2007, Pepin STI 2005, Hancock STI 2010
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M. genitalium: disease association
Men Women
Nongonococcal Urethritis (NGU) Urethritis
Epididymitis Cervicitis
Prostatitis Endometritis, Salpingitis (PID)
Proctitis (MSM) Ectopic pregrancy
Pretermbirth
Infertility
M. genitalium: disease association
Men Women
NGU Urethritis
Epididymitis Cervicitis
Prostatitis Endometritis, Salpingitis (PID)
Proctitis (MSM) Ectopic pregrancy
Pretermbirth
Infertility
Association between M. genitalium and male NGU
Odds Ratio (95% CI) Male Urethritis
Cazanave et al, Med Mal Infect 2012
Manhart et al, Clin Infect Dis 2011
Association between M. genitalium and female disease
Clinical presentation
Frequently asymptomatic
Similar to Chlamydia trachomatis with some exceptions
- Mucopurulent discharge
- Fewer PID symptoms
Long duration of infection
- Up to 21-33 months
Association between M. genitalium and female disease
Odds Ratio (95% CI)
Cervicitis
Endometritis/PID
Infertility
PretermBirth
Cazanave et al, Med Mal Infect 2012
Manhart et al, Clin Infect Dis 2011
M. genitaliumand upper genital tract disease
Endometritis
M. genitalium was found in 9 of 58 women (16%) with
histological endometritis and in 1 of 57 women (2%) w/o
endometritis (Cohen, 2002)
In the PEACH study, M. genitalium was found in 15%
(CT 14%; NG 15%), (Haggerty, 2008)
M. genitalium found in the endometrium of 60% of those
positive in the cervix
Pelvic pain scores, clinical symptoms, and signs were
similar in MG and CT-positive women (Short et al., CID,
2009)
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M. genitaliumand upper genital tract disease
Salpingitis
(Mller et al., 2006)
123 Kenian women with acute salpingitis, confirmed by
laparoscopy
M. genitalium detected in the cervix and endometrium of 9
women (7%), but only in 1 specimen from fallopian tubes
(Cohen et al., 2005).
The MATIST project
Prevalence and risk factors associated with Chlamydia trachomatis,
Neisseria gonorrhoeae and Mycoplasma genitalium infectionsin French
pregnant women
BordeauxUniversityhospital - Januaryto June 2011: 1006 pregnant women
Age
(yo)
No.
specimens
tested
Prevalence of infection with
C. trachomatis M. genitalium N. gonorrhoeae
18-44 1006 2.5 % 0.8 % 0 %
18-24 166 7.9 % 2.4 % 0 %
25-29 317 1.3 % 0.6 % 0 %
30 523 1.5 % 0.4 % 0 %
Risk factors for M. genitalium infection:
- Younger age (OD = 9, p = 0.01)
- History of abortion (OD = 8.6, p = 0.01)
- Having 1st sexual intercourse after 20 yo (OD = 7.1, p = 0.03)
Risk Factors
Nbr recent partners
Short duration of
stable relationship
Age at sex debut
Black / Indigenous
Chlamydia
Young age
Hormcontraception
Smoking
Marital status
HIV infection
Condom use
Income
Bacterial vaginosis (1)
Diagnosis of M. genitalium infections
Only a direct diagnosis
Culture extremely fastidious
By nucleic acid amplification tests:
- a lot of in house PCRs, real-time PCR ++
- a few monoplex (Roche, Diagenode) and
multiplex tests (Bio-Rad, Seegene)
commercialized
- some specimens better than others:
FVU> urethral swabs in men
vaginal swabs >cervix>FVU in women
No rapid POC, no serology commercialized
Currently commercially available mono and multiplex
real-time PCR-based NAATs for M. genitalium 1 real-time PCR reaction, double-stranded probes method
3 bacteria: C. trachomatis, N. gonorrhoeae, M. genitalium
1 internal control, 96 tests
On the Dx real-time system and work station
Dx collection system
- for endocervical, vaginal
and anorectal swabs
- for urethral swabs
Dx Dx CT/NG/MG assay CT/NG/MG assay
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Clinical performances of the Bio-Rad Dx CT/NG/MG assay
for M. genitaliumdetection Le Roy et al., J Microbiol Methods 2012



Reference
method results
Bio-Rad Dx CT/NG/MG performances


Pos Neg
Sens. (%)
[ [[ [95% CI ] ]] ]
Spec. (%)
[ [[ [95% CI ] ]] ]
NPV
(%)
PPV
(%)

Male urines (n=259)
Pos 5 0
100
[ [[ [56.5-100] ]] ]
100 100

Neg 0 254
100
[ [[ [98.5-100] ]] ]


Urethral swabs
(n=7)
Pos 0 0 NA NA 100

Neg 0 7
100
[ [[ [64.6-100] ]] ]


Female urines
(n=180)
Pos 1 0
100
[ [[ [20.6-100] ]] ]
100 100

Neg 0 179
100
[ [[ [97.6-100] ]] ]


Female swabs
(n=212)
Pos 4 1
100
[ [[ [51.0-100] ]] ]
100 80
B
i
o
-
R
a
d

D
x

C
T
/
N
G
/
M
G

A
s
s
a
y


Neg 0 207
99.5
[ [[ [97.3-
99.9] ]] ]

Mycoplasma genitalium treatment
M. genitalium: antibiotic susceptibility testing
Intrinsic resistance
-lactams and other
antibiotics targeting
the cell wall
No susceptibility
testing done in routine
Active antibiotics
Renaudin et al , Antimicrob Agents Chemother, 1992
M. genitalium treatment
European guidelines, 2009
Acute NGU cervicitis
Azithromycin 1 g single dose
Doxycycline 100 mg x 2, 7 d
Chronic NGU
1) Extended 1.5 g azithromycin (5 d)
2) Moxifloxacin 400 mg, 10 d
or
M. genitaliummale NGU treatment studies
Open Scandinavian multicenter trial (Bjrnelius 2008)
Doxycycline 200 mg + 100 x 8 d: 22% cure rate (n=103)
Azithromycin 1 g x 1 d: 86% cure rate (n=56)
Azithromycin 500 mg + 250 mg x 4 d: 97% cure rate (n=60)
Randomized US trial (Mena CID 2009)
Doxycycline 200 mg x 7 d: 45% cure rate (n=31)
Azithromycin 1 g x 1 d: 87% cure rate (n=23)
Clinical cure in DOX group at 2-3 weeks but subsequent
recurrence
Randomized US trial (Schwebke CID 2011)
Doxycycline 200 mg x 7 d: 49% cure rate (n=149)
M. genitalium clearance rate 30.8%
Azithromycin 1 g x 1 d: 43.6% cure rate (n=156)
M. genitalium clearance rate 66.7%
Moxifloxacin 400 mg for 7-10 d in treatment failure after
AZM: 100 % cure rate (Bradshaw 2006; Jernberg 2008)
M. genitalium treatment studies
Response to treatment in men
Insufficient treatment leads to persisting or recurring
symptoms
Persistence of symptoms
- Patients having Mg eradicated: 17%
- Patients with Mg treatment failure: 91% (p<0.0001) (Bradshaw et al.
pLoS One 2008)
Men with persistent NGU after doxycycline treatment:
- 41% (32/78) were M. genitalium-positive (Wikstrm & Jensen, STI 2006),
- 68% (61/90) were M. genitalium-positive ( Sena et al., J Infect Dis, 2012).
Patients failing azithromycin 1g single dose cannot be
treated successfully with extended 1.5 g AZM (Jernerg STI 2008)
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8 clinical strains AZM-R (Bradshaw EID 2006)
- MIC AZM >32 mg/l, ERY >64 mg/l
- mutations 2058, 2059 in 23S rRNA
19 patients: Mg positive specimens for a strain AZM-R
- mutations 2058, 2059 in domainV of 23S rRNA (Jensen CID 2008)
Azithromycin 1 g single dose -> 13 - 33 % therapeutic failures
(Bradshaw EID 2006, Jensen CID 2008, Ito STI 2011, Shimada EID 2011)
M. genitalium: emergence of macrolide resistance (1)
Mutations in domain V of 23S rRNA
- A2058G/C, A2059G (E. coli numbering)
- Azithromycin1 g single dose
Selection of resistant mutants duringAZM treatment
Therapeutic failureif patient infected witha mutatedstrain
23S rRNA
Domain V
M. genitalium: emergence of macrolide resistance (2)
Description of macrolide resistance since 2005 for
M. genitaliumin Australia, Scandinavia, New-Zealand,
Japan and France
According to the primary treatment used in countries
- Sweden : DOX = 1st line TT for NGU and cervicitis
181 M. genitalium (+) STD-clinic attendees
3 (1.6%) had 23S rRNA mutations
- Danemark : AZM = 1st line TT for NGU and cervicitis
415 M. genitalium (+) GP and STD-clinic attendees
162 (39%) had 23S rRNA mutations
- France : AZM and DOX = 1st line TT for NGU and cervicitis
115 M. genitalium (+) STD-clinic attendees
13 (11.3%) had 23S rRNA mutations
M. genitalium: emergence of macrolide resistance (3)
Detectionof mutations associated to macrolide resistance
in M. genitaliumsince 2006
Annual prevalence: 10-15.4% Chrisment et al, J Antimicrob Chemother 2012
Macrolide resistance in M. genitalium
in Bordeaux, France
0%
(0/1)
0%
(0/10)
0%
(0/6)
10%
(1/10)
13.3%
(2/15)
15.4%
(2/13) 14.3%
(3/21)
12.8%
(5/39)
0
2
4
6
8
10
12
14
16
18
20
2003 2004 2005 2006 2007 2008 2009 2010
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Years of specimen collection
Acquired resistance to fluoroquinolones
- Few reports, Japan ++
- Target mutations (gyrase and topo IV)
Description of multidrug resistance
- Resistance to macrolides and fluoroquinolones
- 1
st
strain described for a Chinese patient:
AZM and MXF MIC >16 mg/l
- Few other cases described in Australia and Norway
M. genitalium : acquired resistance
to other antimicrobials
M. genitalium, emerging STI pathogen, a new
chlamydia ?
An accepted cause of male NGU and female cervicitis
Probably associated with sequelae in women
- PID
- Infertility
- Pretermbirth?
Relatively low general population prevalence
- screening programs not appropriate
- Testing and treating in high risky STI populations
Conclusion (1) Conclusion (1)
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Commercially available nucleic acid amplification
tests, multiplexPCRs for detection of STI pathogens
Treatment of M. genitalium infections
- Tetracyclines not useful, AZM single dose better
Extended 1.5 g AZM 85 to 95% effective
- Emergence of resistance to macrolides
Huge local differences in resistance rates
- Moxifloxacin10 d in case of AZM failure
Manhart et al, Clin Infect Dis 2011
Taylor-Robinson and Jensen, Clin Microbiol Rev 2011
Conclusion (2) Conclusion (2) Acknowledgments Acknowledgments
Sabine Pereyre
Charles Cazanave
Delphine Chrisment
Alain Charron
Hlne Renaudin
Bertille de Barbeyrac
Olivia Peuchant
Chlo Le Roy
USC EA 3671
Gynecology clinics, CHU de Bordeaux
Dominique Dallay
Jacques Horowitz
USMR, CHU de Bordeaux
Genevive Chne
University of Washington, USA
Lisa L. Manhart
Statens Serum Institut, Denmark
Jorgen J. Jensen
Conflict of Interest Conflict of Interest
Bio-Rad
Roche Diagnostics
Diagenode