Mycoplasma ve Chlamydophila

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Mycoplasma ve Chlamydophila KOAH ve Astim
Dr MARK WOODHEAD Honorary Senior
Lecturer University of Manchester MANCHESTER UK
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Mycoplasma and Chlamydophila Role in COPD and
Asthma
Dr MARK WOODHEAD Honorary Senior
Lecturer University of Manchester MANCHESTER UK
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16 September 2009
Besiktas 0 - Manchester United 1
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16 September 2009
Besiktas 0 - Manchester United 1
25 November 2009
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Mycoplasma pneumoniae
Pneumonia associated with cold agglutinins Filtrab
le agent identified virus visualised in
1957 Recognised as bacterium M. pneumoniae has
one of the smallest known genomes
1943 1944 1957 1962
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Chlamydophila pneumoniae
1965 1978 1983 1987 2002
TW-183 in eye of trachoma patient -
Taiwan IOL-207 in eye of trachoma patient
London Finland 32 cases in CAP
epidemic Chlamydia strain TWAR Canada 6 of
adult CAP, Chlamydia pneumoniae Reclassified as
Chlamydophila pneumoniae
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C trachomatis C muridarum C suis
Chlamydia
Chlamydiaceae
Chlamydophila
C pecorum C pneumoniae C psittaci C abortus C
caviae C felis
Chlamydiales
Parachlamydiaceae
P acanthamoeba
Simkaniaceae
S negevensis
?
WSU 86-1044
Int J Syst Evol Microbiol 200251251-253
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Chlamydophila pneumoniae
Koalas, Horses, the Giant Barred Frog and the
Puff Adder !
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Mycoplasma and Chlamydophila

• Predominantly intra-cellular existence
• Difficult to culture in lab
• Diagnosis often only by indirect methods
• No cell wall - not sensitive to ß-lactam
  antibiotics

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Transmission electron micrograph of chlamydophila
particles Note the reticulate bodies (R),
intermediate bodies (I), and highly condensed
elementary bodies (E). Bar, 270 nm
http//www.cdc.gov/ncidod/eid/vol6no2/reed.htm
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Mycoplasma and Chlamydophila Role in COPD and
Asthma

• Acute exacerbation
• Chronic disease pathogenesis

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Mycoplasma and Chlamydophila Role in COPD and
Asthma

• Acute exacerbation
• Chronic disease pathogenesis
• Microbiological diagnosis
• Antibiotic treatment studies

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113 Hospital admissions for COPD Exacerbation
Larsen et al Scand J Infect Dis 20094126-32
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168 EXACERBATIONS OF COPD
VIRAL INFECTION PRESENT IN 66 (39)
Rhinovirus 39 RSV 19 Coronaviru
s 7 Influenza A 6 Influenza B
3 Parainfluenza 1 Adenovirus 1
(Seemungal et al AJRCCM 20011641618-1623)
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MICROBIOLOGY OF SEVERE COPD
N 50 42 prior antibiotics 6 mortality PSB,
BAL, TBAS, serology

S pneumoniae H influenzae M catarrhalis Pseudomona
s sp GNEB Chlamydophila pneumoniae Chlamydophila
psittaci Coxiella burnetii Viruses No pathogen
8 22 8 18 12 18 3 3 16 28
Soler et al Am J Respir Crit Care Med
19981571498-1505
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COPD EXACERBATION Samsun, Turkey
Erkan et al Int J Chron Obst Pulm Dis
20083463-467
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COPD EXACERBATION
43 of 141 stable patients were blood
mononuclear cell C pneumoniae PCR ve C
pneumoniae ve had more exacerbations (2.03 vs
1.43/yr) 100 of 34 patients were PCR ve at
exacerbation Blasi et al Thorax 200257672-676
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COPD EXACERBATION
43 of 141 stable patients were blood
mononuclear cell C pneumoniae PCR ve C
pneumoniae ve had more exacerbations (2.03 vs
1.43/yr) 100 of 34 patients were PCR ve at
exacerbation Blasi et al Thorax 200257672-676
26 of 110 stable patients were serum C
pneumoniae IgG ? 16 None were respiratory
secretion PCR ve C pneumoniae ve had No more
exacerbations 21 of 43 patients were PCR ve at
exacerbation Seemungal et al Thorax
2002571087-1089
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ATYPICAL PATHOGENS AND AECOPD A PCR BASED STUDY
Stable Phase 122 1 0 0
Exacerbation 126 1 0 0
Sputum samples (n) L pneumophila M
pneumoniae C pneumoniae
L pneumophila ves from different patients. Both
also grew S pneumoniae
Diederen et al Eur Resp J 200730240-244
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ASTHMA EXACERBATION
Frequency of atypical pathogens
Author Cosentini Allegra Lieberman
C pneumoniae 33 8 8
M pneumoniae 3 1 18
n 58 74 100
6 in control group 3 in control group plt0.0006
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ASTHMA EXACERBATION
C pneumoniae isolation 11 vs 4.9(controls) C
pneumoniae specific IgE 85 in culture ve vs
18-22 in culture negative Emre et al J Infect
Dis 1995172265-267
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ASTHMA EXACERBATION
C pneumoniae isolation 11 vs 4.9(controls) C
pneumoniae specific IgE 85 in culture ve vs
18-22 in culture negative Emre et al J Infect
Dis 1995172265-267 Refractory asthma C
pneumoniae culture ve 11 months Symptoms
resolved with prolonged erythromycin
Rx Hammerschlag et al Clin Infect Dis
199214178-182
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ASTHMA EXACERBATION
C pneumoniae isolation 11 vs 4.9(controls) C
pneumoniae specific IgE 85 in culture ve vs
18-22 in culture negative Emre et al J Infect
Dis 1995172265-267 Refractory asthma C
pneumoniae culture ve 11 months Symptoms
resolved with prolonged erythromycin
Rx Hammerschlag et al Clin Infect Dis
199214178-182 C pneumoniae acute serological
infection in 5.7 of acute asthma vs 5.7 of
controls Cook et al Thorax 199853254-259
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Mycoplasma and Chlamydophila in Asthma
Pathogenesis
Studies supporting role / total studies
Chronic stable asthma 16 / 20 Late onset
asthma 3 / 6 Acute asthma 5 / 6
Johnston Martin AJRCCM 20051721078-1089
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Terminal Attachment Organelle of Mycoplasma pneumo
niae
Waites et al Future Microbiol 20083635-648
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Role of Mycoplasma In Chronic Asthma
Waites et al Future Microbiol 20083635-648
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Mycoplasma / Chlamydophila
Asthma / COPD
Asthma / COPD
Mycoplasma / Chlamydophila
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Standardizing Chlamydia pneumoniae Assays
Recommendations from the Centers for Disease
Control and Prevention (USA) and the Laboratory
Centre for Disease Control (Canada)
Scott F. Dowell,1 Rosanna W. Peeling,5
Jens Boman,6 George M. Carlone,1
Barry S. Fields,1 Jeannette Guarner,1
Margaret R. Hammerschlag,4 Lisa A. Jackson,2
Cho-Chou Kuo,3 Matthias Maass,7
Trudy O. Messmer,1 Deborah F. Talkington,1
Maria Lucia Tondella,1 Sherif R. Zaki,1 and the
C. pneumoniae Workshop Participants There are
no wholly satisfactory serological methods for
diagnosis of C. pneumoniae infection Clin
Infect Dis 200133492-502
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PROBLEMS WITH SEROLOGY

• Need appropriately paired serum samples
• Acute infection
• IgM rise 2-3 weeks after onset
• IgG high titre only after 6-8weeks
• Reinfection no IgM and IgG rises in 1-2 weeks
• High background IgG levels in some communities
• Lack of standardised testing methods
• Shortage of high-quality reagents
• Subjective result interpretation
• False ve IgM results without prior adsorption
• IgA conjugate quality varies
• Culture ve, MIF ve occurs !

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PROBLEMS WITH CULTURE

• specificity depends on microscopists abilities
• uncertain role of centrifugation
• of serum free culture media
• of prolonged culture times
• pretreatment with polyethylene glycol and other
  agents
• centrifugation after inoculation
• number of culture passages not agreed
• few inclusions that fail to propagate ve ?

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CRITERIA FOR C PNEUMONIAE INFECTION
PCR
Four of 18 currently published assays met
proposed criteria for optimal validation
Each PCR run should include low positive
controls, and water controls every fifth
extraction
Clin Infect Dis 200133492-502
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PCR DIAGNOSIS OF C pneumoniae
Wellinghausen et al Int J Med Microbiol
2006296485-491
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ANTIBIOTIC ACTIVITY in vitro AGAINST C pneumoniae
Erythromycin
Doxycycline
Azithromycin
Clarithromycin
Ciprofloxacin
Levofloxacin
Moxifloxacin
.0001
.001
.01
.1
1.0
MIC ?g / ml
From Hammerschlag MR Eur Resp J 2000161001-1007
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Telithromycin in Acute Asthma
double-blind, parallel-group, randomized,
placebo-controlled, multicenter, study of 10
days of telithromycin (800mg/d) in 278 adults
PEFR
symptoms
Johnston et al NEJM 20063541589-1600
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Telithromycin in Acute Asthma
double-blind, parallel-group, randomized,
placebo-controlled, multicenter, study of 10
days of telithromycin (800mg/d) in 278 adults
PEFR
symptoms
end of Rx
p0.81
average
p0.28
Johnston et al NEJM 20063541589-1600
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Telithromycin in Acute Asthma
double-blind, parallel-group, randomized,
placebo-controlled, multicenter, study of 10
days of telithromycin (800mg/d) in 278 adults
PEFR
symptoms
end of Rx
p0.002
p0.81
average
p0.004
p0.28
Johnston et al NEJM 20063541589-1600
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Placebo-controlled study of Doxycycline in AECOPD
Day 10
Day 30
Daniels et al AJRCCM 2010181 150-157
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Placebo-controlled study of Doxycycline in AECOPD
Day 10
Day 30
Daniels et al AJRCCM 2010181 150-157
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Placebo-controlled study of Doxycycline in AECOPD
Day 10
Day 30
Daniels et al AJRCCM 2010181 150-157
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Placebo-controlled study of Doxycycline in AECOPD
Day 10
Day 30
Daniels et al AJRCCM 2010181 150-157
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Placebo-controlled study of Doxycycline in AECOPD
Daniels et al AJRCCM 2010181 150-157
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Long-term Antibiotics in COPD
randomized, double-blind, placebo-controlled study
of erythromycin administered at 250 mg twice
daily to patients with COPD over 12 months
Seemungal et al AJRCCM 20081781139-1147
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Long-term Antibiotics in COPD
randomized, double-blind, placebo-controlled study
of erythromycin administered at 250 mg twice
daily to patients with COPD over 12 months
Seemungal et al AJRCCM 20081781139-1147
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Antibiotics in Asthma and COPD

• anti-bacterial?
• anti-inflammatory?
• both?
• harmful effects?

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Mycoplasma and Chlamydophila Role in COPD and
Asthma Conclusions

• no gold standard for diagnosis
• many uncontrolled studies
• possible acute and chronic effects
• overall causative role likely to be small
• asthma and COPD may increase infection
  susceptibility
• antibiotic studies
• effects anti-atypical or anti-inflammatory?

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Persistent nasopharyngeal infection with C
pneumoniae for up to 18 years following acute
infection Hammerschlag et al Clin Infect Dis
199214178-182 Dean et al Scand J Infect Dis
199123387-388
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Presence of Chlamydia pneumoniae DNA In The
Cerebral Spinal Fluid Is A Common Phenomenon In
A Variety Of Neurological Diseases And Not
Restricted To Multiple Sclerosis Gieffers J et
alAnn Neurol 2001 May49(5)585-9
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PROBLEMS WITH PCR
Only four of 18 PCR assays used in published
reports met the proposed validation criteria
Clin Infect Dis 200133492-502
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CRITERIA FOR C PNEUMONIAE INFECTION
SEROLOGY ONLY MIF TEST RECOMMENDED
Acute infection, IgM of ?  1  16 or 4-fold
increase in IgG
Presumed past infection, IgG of ?  1  16
Possible acute infection, IgG of ?  1  512
CULTURE Respiratory secretions primary
isolation plus 2 additional passages
CULTURE Tissue secretions primary isolation
plus 4-6 additional passages
Only if strain can be subsequently propagated or
confirmed by additional test (eg PCR) should it
be classed as positive
Clin Infect Dis 200133492-502
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CRITERIA FOR C PNEUMONIAE INFECTION
IMMUNOHISTOCHEMISTRY
Each tissue block should be tested with 2
Chlamydia antibodies and 2 control antibodies
Each staining run should include 1 positive and
1 negative tissue control, each incubated with
the 4 antibodies used on the specimen of interest
Intracytoplasmic staining of macrophages,
endothelial cells, or smooth muscle cells in a
granular pattern may be considered positive
interpretation of a homogenous staining pattern
is controversial
Clin Infect Dis 200133492-502
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False-positive Chlamydia staining of smooth
muscle cell in an atheroma (arrow)
Clin Infect Dis 200133492-502