Mycoplasma genitalium, STD and molecular diagnostics Overview

Overview
Mycoplasma genitalium, STD and
molecular diagnostics
Cécile Bébéar
USC Mycoplasmal and chlamydial infections in humans
French National Reference Center for Chlamydia
INRA - Université Bordeaux Segalen
CHU de Bordeaux
• Introduction
• Association between M. genitalium infections and
disease
• Diagnosis
• Antimicrobial susceptibility testing and treatment
studies
Mycoplasma genitalium
Mycoplasma genitalium
An introduction
An introduction
• 1980: Mycoplasma genitalium isolated from 2 of 13 men
with NGU (nongonoccocal uretritis)
– Very slow growth (>50 days)
– Very few isolates available
• Similar to M. pneumoniae
– Morphology (tip structure)
– Genetics
• Sexually transmitted bacterium, lacks a cell wall
• 1990’s: 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
M. genitalium: prevalence and incidence
M. genitalium: an epidemiologist’s view
• Prevalence
- General population
• How much ? Prevalence and incidence
• What ? Association with diseases
1 - 4%
1 - 6%
- Clinic populations
4 - 26%
4 - 38%
• Who ? Risk factors
• Incidence
-
University women: 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
1
M. genitalium: disease association
Men
M. genitalium: disease association
Women
Men
Women
Nongonococcal Urethritis (NGU)
Urethritis
NGU
Urethritis
Epididymitis
Cervicitis
Epididymitis
Cervicitis
Prostatitis
Endometritis, Salpingitis (PID)
Prostatitis
Endometritis, Salpingitis (PID)
Ectopic pregrancy
Proctitis (MSM)
Ectopic pregrancy
Proctitis (MSM)
Preterm birth
Preterm birth
Infertility
Infertility
Association between M. genitalium and male NGU
Male Urethritis
Odds Ratio (95% CI)
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
Cazanave et al, Med Mal Infect 2012
Manhart et al, Clin Infect Dis 2011
Association between M. genitalium and female disease
Cervicitis
Odds Ratio (95% CI)
M. genitalium and 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)
Endometritis/PID
– 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)
Infertility
Preterm Birth
Cazanave et al, Med Mal Infect 2012
Manhart et al, Clin Infect Dis 2011
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M. genitalium and upper genital tract disease
Salpingitis
The MATIST project
Prevalence and risk factors associated with Chlamydia trachomatis,
Neisseria gonorrhoeae and Mycoplasma genitalium infections in French
pregnant women
Bordeaux University hospital - January to June 2011: 1006 pregnant women
No.
specimens
tested
C. trachomatis
M. genitalium
N. gonorrhoeae
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%
Age
(yo)
18-44
(Møller 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
Prevalence of infection with
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)
(Cohen et al., 2005).
Risk Factors
Diagnosis of M. genitalium infections
• Only a direct diagnosis
Nbr recent partners
Smoking
Short duration of
Marital status
stable relationship
HIV infection
Age at sex debut
Condom use
Black / Indigenous
Income
Chlamydia
Bacterial vaginosis (1)
Young age
Horm contraception
• 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
Dx CT/NG/MG assay
• 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
3
• Clinical performances of the Bio-Rad Dx CT/NG/MG assay
for M. genitalium detection Le Roy et al., J Microbiol Methods 2012
Reference
method results
Pos
B io-R ad Dx C T/NG/MG Assay
Male urines (n=259)
Urethral swabs
(n=7)
Female urines
(n=180)
Female swabs
(n=212)
Neg
Sens. (%)
[95% CI]]
100
[56.5-100]]
Pos
5
0
Neg
0
254
Pos
0
0
Neg
0
7
Pos
1
0
Neg
0
179
Pos
4
1
Neg
0
Mycoplasma genitalium treatment
Bio-Rad D x CT/NG/MG performances
Spec. (%)
[95% CI]]
NPV
(%)
PPV
(%)
100
100
NA
100
100
100
100
80
100
[98.5-100]]
NA
100
[64.6-100]]
100
[20.6-100]]
100
[97.6-100]]
100
[51.0-100]]
207
99.5
[97.399.9]]
M. genitalium treatment
M. genitalium: antibiotic susceptibility testing
• Intrinsic resistance
European guidelines, 2009
• Active antibiotics
β-lactams and other
antibiotics targeting
the cell wall
• Acute NGU – cervicitis
or
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
• No susceptibility
testing done in routine
Renaudin et al , Antimicrob Agents Chemother, 1992
M. genitalium male NGU treatment studies
•
– 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)
•
Response to treatment in men
• Insufficient treatment leads to persisting or recurring
symptoms
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
•
M. genitalium treatment studies
Open Scandinavian multicenter trial (Björnelius 2008)
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)
• 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 (Wikström & Jensen, STI 2006),
- 68% (61/90) were M. genitalium-positive ( Sena et al., J Infect Dis, 2012).
• 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)
• Patients failing azithromycin 1g single dose cannot be
treated successfully with extended 1.5 g AZM (Jernerg STI 2008)
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M. genitalium: emergence of macrolide resistance (2)
M. genitalium: emergence of macrolide resistance (1)
• Mutations in domain V of 23S rRNA
• 8 clinical strains AZM-R (Bradshaw EID 2006)
- MIC AZM >32 mg/l, ERY >64 mg/l
- mutations 2058, 2059 in 23S rRNA
- A2058G/C, A2059G (E. coli numbering)
- Azithromycin 1 g single dose
Selection of resistant mutants during AZM treatment
Therapeutic failure if patient infected with a mutated strain
• 19 patients: Mg positive specimens for a strain AZM-R
- mutations 2058, 2059 in domain V of 23S rRNA (Jensen CID 2008)
• Azithromycin 1 g single dose -> 13 - 33 % therapeutic failures
23S rRNA
(Bradshaw EID 2006, Jensen CID 2008, Ito STI 2011, Shimada EID 2011)
Domain V
Macrolide resistance in M. genitalium
in Bordeaux, France
M. genitalium: emergence of macrolide resistance (3)
20
Proportion of patients infected with a
23S rRNA mutated M. genitalium a in France
• Description of macrolide resistance since 2005 for
M. genitalium in 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
18
16
13.3%
(2/15)
14
15.4%
(2/13)
14.3%
(3/21)
12.8%
(5/39)
10%
(1/10)
12
10
8
6
4
2
0
0%
(0/1)
0%
(0/10)
0%
(0/6)
2003
2004
2005
2006
2007
2008
2009
2010
Years of specimen collection
• Detection of mutations associated to macrolide resistance
in M. genitalium since 2006
• Annual prevalence: 10-15.4% Chrisment et al, J Antimicrob Chemother 2012
M. genitalium : acquired resistance
to other antimicrobials
• Acquired resistance to fluoroquinolones
-
Few reports, Japan ++
-
Target mutations (gyrase and topo IV)
• Description of multidrug resistance
- Resistance to macrolides and fluoroquinolones
-
1st strain described for a Chinese patient:
AZM and MXF MIC >16 mg/l
-
Few other cases described in Australia and Norway
Conclusion (1)
• M. genitalium,
chlamydia ?
emerging
STI
pathogen,
a
new
• An accepted cause of male NGU and female cervicitis
• Probably associated with sequelae in women
-
PID
Infertility
Preterm birth ?
• Relatively low general population prevalence
- screening programs not appropriate
- Testing and treating in high risky STI populations
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Conclusion (2)
Acknowledgments
USC EA 3671
• Commercially available nucleic acid amplification
tests, multiplex PCRs 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
-
Moxifloxacin 10 d in case of AZM failure
Manhart et al, Clin Infect Dis 2011
Taylor-Robinson and Jensen, Clin Microbiol Rev 2011
Sabine Pereyre
Charles Cazanave
Delphine Chrisment
Alain Charron
Hélène Renaudin
Bertille de Barbeyrac
Olivia Peuchant
Chloé Le Roy
Statens Serum Institut, Denmark
Jorgen J. Jensen
University of Washington, USA
Lisa L. Manhart
Gynecology clinics, CHU de Bordeaux
Dominique Dallay
Jacques Horowitz
USMR, CHU de Bordeaux
Geneviève Chêne
Conflict of Interest
Bio-Rad
Roche Diagnostics
Diagenode
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