CHEST / 140 / 2 / AUGUST, 2011 401
life of affected patients but also imparts a signifi cant
economic burden on society. The term “refractory
asthma” (RA) is used for patients with persistent
asthma symptoms in whom comorbidities have been
treated, triggers addressed, compliance with treat-
ment evaluated, and alternative diagnoses excluded. 1
The link between bacterial processes and RA has
emerged as various phenotypes of chronic asthma
with persistent infl ammation have been recognized. 2-7
Many studies have implicated Mycoplasma pneumoniae
sthma is a prevalent and heterogeneous disease
that not only has a marked effect on the quality of
(Mp) in the initiation and persistence of asthma,
although the precise role it plays and its pathogenic
mechanisms remain elusive. 8 However, several limi-
tations exist in studies of Mp in asthma, including the
inability to consistently culture this organism, the
poor performance of Mp serology in defi ning active
infection, and the variable sensitivities of polymerase
chain reaction (PCR) assays in detecting Mp.
Recently, our group identifi ed a 68-kDa protein
unique to Mp called the community-acquired respi-
ratory distress syndrome toxin (CARDS Tx). CARDS
Tx is a highly immunogenic protein that possesses
Background: The role of Mycoplasma pneumoniae (Mp) in the initiation and persistence of asthma
remains elusive. Mp community-acquired respiratory distress syndrome toxin (CARDS Tx) is a
unique virulence factor that induces an intense lymphocytic response and exacerbates asthma in
animal models. We sought to determine the incidence of Mp infection and the presence of CARDS
Tx in subjects with refractory asthma (RA).
Methods: We conducted a prospective observational study in 64 subjects with RA. Respiratory
secretions (sputum, nasal lavage, and throat swab) and blood were analyzed for the presence of
CARDS Tx and P1 adhesin (P1) DNA by polymerase chain reaction (PCR), and CARDS Tx by anti-
gen capture. Serum IgM and IgG antibodies to CARDS Tx were determined by enzyme-linked
immunosorbent assay (ELISA).
Results: Thirty-three of 64 subjects (52%) tested positive for Mp: 29 of 33 by CARDS Tx vs 10 of
33 by P1 assays. Ten subjects followed longitudinally for up to 633 days tested persistently positive
for Mp. There were no signifi cant differences in Mp-specifi c IgG responses between Mp-positive
and Mp-negative groups. Eight of 10 subjects who tested persistently positive failed to mount a
substantial IgG response to CARDS Tx, and up to 8 weeks of clarithromycin failed to eradicate
Mp in fi ve subjects.
Conclusions: Subjects with RA may be chronically infected with Mp. PCR for CARDS Tx appears
to be the most sensitive method of identifying Mp infection. Despite the persistence of Mp in
subjects with RA, some subjects failed to mount an IgG response, and macrolide therapy was
insuffi cient to eradicate Mp. CHEST 2011; 140(2):401–407
Abbreviations: CARDS Tx 5 community-acquired respiratory distress syndrome toxin; ELISA 5 enzyme-linked
immunosorbent assay; Mp 5 Mycoplasma pneumoniae ; P1 5 P1 adhesin; PCR 5 polymerase chain reaction; RA 5 refractory
asthma; rCARDS Tx 5 recombinant community-acquired respiratory distress syndrome toxin; rP1 5 recombinant
P1 adhesin immunodominant carboxy domain
Persistence of Community-Acquired
Respiratory Distress Syndrome
Toxin-Producing Mycoplasma pneumoniae
in Refractory Asthma
Jay Peters , MD , FCCP; Harjinder Singh , MD ; Edward G. Brooks , MD ; Joseph Diaz , MD ;
Thirumalai R. Kannan , PhD ; Jacqueline J. Coalson , PhD ; Janet G. Baseman , PhD ;
Marianna Cagle , MA ; and Joel B. Baseman , PhD
MPN372) and P1 (annotated MPN 141) was performed as
CARDS Tx protein was detected and quantifi ed using antigen
capture enzyme-linked immunosorbent assay (ELISA) methods
as described previously. 10,12 The detection of antibodies against
recombinant CARDS Tx (rCARDS Tx) and recombinant P1
immunodominant carboxy domain (rP1) was performed using
Immunohistochemical staining was described previously. 10
Briefl y, cellular pellets from nasal lavage and sputum were col-
lected, and specimens were fi xed with 4% phosphate buffered
paraformaldehyde. After paraffi n embedding, 4- m m sections were
stained with rabbit anti-CARDS Tx polyclonal antibodies and
mouse anti-P1 monoclonal antibody (US Biologic; Swampscott,
Massachusetts) at 1:1,500 and 1:10 dilutions, respectively, fol-
lowed by polyclonal anti-rabbit or anti-mouse antibody-horse
radish peroxidase, and then developed with diaminobenzidine
(Vector Laboratories; Burlington, California) and counterstained
All results were expressed as mean ? SD. Data were tabulated
in SPSS, version 18.0 (SPSS Inc; Chicago, Illinois), and statistical
differences between group means were determined using para-
metric (two-tailed t test) or nonparametric (Mann-Whitney U test)
analyses (signifi cant differences , .05). Differences in categorical
data were determined using Pearson x 2 test.
Sixty-four adult subjects with RA were enrolled,
with a mean age of 44.8 years ( Table 1 ). Thirty-one
subjects were white, 25 Hispanic, and eight black.
Forty-three subjects (67%) were women. The pre-
dominance of women within the cohort is consistent
with other studies, which have shown a female pre-
dominance of adult subjects with severe asthma, 16
and the ethnic breakdown is proportional to our
regional population. Overall, the baseline postbron-
chodilator FEV 1 was 2.35 (73.6% predicted), the
mean IgE level was 599 mg/dL, the mean Asthma
Control Test score was 17.2, and the mean number of
asthma and allergy medications was 7.4. Forty-four
subjects (69%) were identifi ed as allergic asthmatics
by their referring physician. Comparisons between
subjects who were classifi ed as positive or negative for
Mp revealed signifi cant differences ( P , .05) in ethnic
distribution, with higher numbers of whites and fewer
Hispanics in the Mp-positive group ( Table 1 ).
Subjects were considered positive if Mp was detect-
able by PCR (CARDS Tx or P1) or by CARDS Tx
antigen capture on their initial study visit. Based on
this analysis, 31 subjects were negative, and 33 (52%)
were positive for Mp. Of the Mp-positive group, 29 of
33 subjects (88%) tested positive for CARDS Tx by
PCR, 10 of 33 (30%) tested positive for P1 by PCR,
and 52% tested positive by CARDS Tx antigen
adenosine diphosphate-ribosyltransferase activity simi-
lar to pertussis toxin. 9 We have subsequently devel-
oped assays to detect CARDS Tx by PCR and CARDS
Tx antigen-capture and to detect antibodies directed
against CARDS Tx. 10 CARDS Tx gene sequences are
more sensitive for the detection of Mp than other
sequences using PCR amplifi cation, including the P1
adhesin (P1) and the ATPase gene. 11-13
We studied 64 subjects with RA who had persistent
symptoms despite being under the care of an asthma
specialist and receiving optimal asthma therapy. The
purpose of this study was to identify the frequency of
Mp infection using both CARDS Tx PCR and con-
ventional P1 PCR, to evaluate antibody responses to
CARDS Tx and P1 proteins, and to detect CARDS
Tx protein concentrations within the airways of these
Materials and Methods
We conducted a prospective observational study in adult sub-
jects (aged 18-65 years) with RA defi ned by persistent symptoms
despite step 5 management of the National Asthma Education
and Prevention Program guidelines. An additional 91 subjects
undergoing diagnostic bronchoscopy for nonmalignant nonasth-
matic lung disease and 104 healthy control subjects were evalu-
ated. This study was approved by the institutional review board of
the University of Texas Health Science Center at San Antonio
(IRB No. 056-5012-271). Samples from nasal lavage and sputum
were collected with Copan fl ocked swabs (Copan Diagnostics;
Murrieta, California) and suspended in SP4 broth. 14 Serum and
respiratory samples were stored at 2 80°C until analysis.
Detection of Mp DNA, Protein, and Antibodies
Sputum and nasal lavage samples were homogenized prior
to extraction with dithiothreitol. DNA from airways and serum
samples was purifi ed using the QIAmp DNA Mini Kit (Qiagen;
Valencia, California). Real-time PCR for CARDS Tx (annotated
Manuscript received February 8, 2011; revision accepted April 13,
Affi liations: From the Department of Medicine (Drs Peters
and Singh), the Department of Pediatrics (Dr Brooks), the
Department of Microbiology and Immunology (Drs Kannan and
J. B. Baseman and Ms Cagle), and the Department of Pathology
(Dr Coalson), University of Texas Health Science Center at San
Antonio, TX; Asthma and Allergy Associates of South Texas
(Dr Diaz), San Antonio, TX; and the Department of Epidemiology
(Dr J. G. Baseman), University of Washington School of Public
Health, Seattle, WA.
Funding/Support: This study was supported by the National
Institutes of Health [Grant U19A1070412-01] and The Kleberg
Foundation to J. B. B.
Correspondence to: Jay Peters, MD, FCCP, 7704 Merton
Minter Blvd (111E), San Antonio, TX 78229; e-mail: peters@
© 2011 American College of Chest Physicians. Reproduction
of this article is prohibited without written permission from the
American College of Chest Physicians ( http://www.chestpubs.org/
CHEST / 140 / 2 / AUGUST, 2011 403
However, no signifi cant differences were detected in
IgG responses between the groups. Similarly, IgM
antibodies directed against rP1 were signifi cantly ele-
vated ( P , .05) in the persistent and positive groups
compared with the negative group at the initial visits,
but there were no differences in IgG responses. When
peak IgM and IgG responses were tabulated for the
persistent group, who were the only subjects with mul-
tiple samples taken, there was a signifi cant increase
in IgM levels to rCARDS Tx and rP1 ( P , .001) com-
pared with the negative group, but no signifi cant dif-
ferences in the IgG responses. Two subjects in the
persistent group were evaluated for immune defi ciency
and demonstrated normal total immunoglobulins and
normal antibody titers to tetanus and diphtheria.
The subjects’ physicians, who were notifi ed in the
event of persistent Mp infection, subsequently deter-
mined antibiotic therapy. Five subjects in the per-
sistent infection group received up to 8 weeks of
clarithromycin but had no discernible improvement
in their symptoms, rescue medication, Asthma Con-
trol Test scores, or spirometric values. All fi ve subjects
continued to have persistent detection of Mp in respi-
ratory secretions after completion of antibiotic ther-
apy. One of those subjects (shown in bold, dashed
lines in Fig 1 ) was followed for 633 days. On ini-
tial enrollment, the subject had high levels of anti-
rCARDS Tx IgM (1.2 OD405), high levels of Mp
detected by PCR to CARDS Tx (95,133 genomes/5 m L
nasal lavage) and P1 (39,887 genomes/5 m L), and high
levels of CARDS Tx antigen (2.3 pg/12.5 m L nasal
lavage or sputum), but no substantial increase in
IgG antibody to rCARDS Tx or rP1 ( Fig 1 ). After
completion of 8 weeks of clarithromycin, Mp was still
detectable by CARDS Tx PCR, and immunohis-
tochemical staining of sputum for CARDS Tx and P1
demonstrated surface-associated and intracytoplas-
mic Mp ( Fig 3 ). The immunostaining was similar to
that observed in airway epithelium from Mp-infected
The results of this study demonstrate that Mp is
often present in the airways of patients with RA and
can be detected by CARDS Tx assays described ear-
lier. In this cohort of patients with refractory asthma,
52% of subjects tested positive for Mp by PCR or
antigen capture, which was signifi cantly more frequent
than in subjects with nonasthmatic lung disease.
Only 30% of Mp-positive subjects were detected
by the conventionally used PCR assay to P1. If assays
using CARDS Tx had not been used in this study,
then 70% of subjects potentially infected with Mp may
have been misclassifi ed as Mp negative. This fi nding
capture. Only one subject was positive by P1 alone.
P1-negative specimens that were PCR positive to
CARDS Tx were submitted to an outside reference
laboratory (ARUP; Salt Lake City, Utah), which sub-
stantiated our P1 results. By comparison, 15 of 91 sub-
jects (16.5%) with nonasthmatic lung disease were
positive for CARDS Tx by PCR, and four of 91 (4.4%)
were positive by CARDS Tx antigen capture ( P , .05).
During the peak respiratory season, 104 healthy sub-
jects had airway specimens tested for CARDS Tx
by antigen capture, and only three of 104 (2.9%) were
The majority of positive subjects were analyzed
only during the initial visit. However, 10 of the 33
Mp-positive subjects were followed longitudinally and
analyzed at multiple visits. This subgroup exhibited
persistent detection of Mp DNA (CARDS Tx or P1)
and/or CARDS Tx by antigen capture for . 90 days
(average duration, 324 days; range, 91-633 days)
( Fig 1 ). Within this “persistent” group, all 10 were PCR
positive for CARDS Tx, one of 10 was PCR positive
for P1, six of 10 were positive by CARDS Tx antigen
capture on their initial visit, and all 10 were positive
for CARDS Tx antigen capture during the extended
period of observation.
We measured serologic responses to rCARDS Tx
and rP1 in most subjects ( Fig 2 , Table 2 ). The 10 sub-
jects (labeled “persistent” in Fig 2 and Table 2 ) who
persistently tested positive were analyzed separately
to determine them from the remaining subjects, who
were analyzed at only a single visit (designated as
“negative” and “positive”). IgM antibodies directed
against rCARDS Tx were signifi cantly elevated ( P , .01)
in both the Mp-positive and persistent groups com-
pared with Mp-negative subjects at the initial visit.
Table 1— Baseline Clinical Characteristics
of Study Population
Characteristic Mp PositiveMp NegativeTotal
FEV 1 b
FEV 1 %
44.1 ? 14.5
45.61 ? 12.3
44.8 ? 13.4
60.6 (20) a
2.45 ? 0.91
75.1 ? 24.8
700 ? 800
17.5 ? 5
6.7 ? 2.3 a
2.24 ? 0.71
71.9 ? 17.9
511 ? 521
17 ? 5.6
8.2 ? 2
2.35 ? 0.82
73.6 ? 21.6
599 ? 664
17.2 ? 5.2
7.4 ? 2.3
Data are presented as mean ? SD except for female sex and ethnicity,
which are expressed as % (No.). ACT 5 Asthma Control Test; B 5 black;
H 5 Hispanic; Mp 5 Mycoplasma pneumoniae ; W 5 white.
a P , .05 comparing Mp-positive with Mp-negative subjects.
b Postbronchodilator FEV 1 .
c Medications includes prescribed medications for asthma, allergic
rhinitis, and gastric refl ux disease.
has signifi cant implications for recently published
studies, in which macrolide or ketolide therapy was
based on less sensitive assays. 18,19 In the study of
telithromycin in acute exacerbations of asthma, 61% of
the 278 subjects met at least one criterion for atypical
infection; however, only three subjects were PCR
positive for Mp or Chlamydophila pneumoniae . 18
Sutherland et al 19 reported on the multicenter Myco-
plasma in Asthma study, which used a nested PCR to
detect P1 and Mp-specifi c 16S ribosomal RNA
sequences, and identifi ed 12/92 Mp-positive subjects
with RA. Although there have been no direct com-
parisons between this nested PCR assay and the
CARDS Tx PCR assay described here, a recent anal-
ysis by the US Centers for Disease Control and Pre-
vention evaluated three real-time PCR assays for the
detection of Mp in an outbreak of community-
acquired pneumonia. 20 Their data showed that real-
time PCR assays targeting the CARDS Tx gene were
the most sensitive in identifying positive specimens
and recommended that the assay be used as the ini-
tial screening marker in respiratory clinical specimens.
Of note, one subject in our study was P1 positive and
CARDS Tx negative on initial PCR screening. This
patient converted to negative on subsequent follow-up
visits and never demonstrated CARDS Tx by antigen
capture assay. This illustrates the diffi culty in dis-
tinguishing true-positive from false-positive results
when there is no clearly defi ned gold standard for the
diagnosis of Mp infection.
Serologic assays for Mp have demonstrated vari-
able clinical usefulness in the diagnosis of myco-
plasma infection. 21 Prior studies have shown that
CARDS Tx is highly immunogenic and results in dra-
matic seroconversion in pneumonia patients. 9 In the
current study, anti-rCARDS Tx antibody titers were
higher than anti-rP1 antibody titers. However, there
were no differences in IgG to either rCARDS Tx or
rP1 between those subjects who were Mp positive
and those who were Mp negative. This fi nding held
up even among subjects who were persistently posi-
tive for Mp as well as subjects among whom CARDS
Tx was identifi ed by antigen capture. IgM antibodies
directed against rCARDS Tx or rP1 were signifi cantly
elevated in both the Mp-positive and persistent
groups compared with Mp-negative subjects. How-
ever, the overall IgM responses still failed to rise to
the conventional standard of a three- to fourfold
increase in titer. Two subjects in the persistently posi-
tive group had robust IgM responses to rCARDS Tx
but did not exhibit substantial increases in IgG anti-
bodies. This lack of antibody response to Mp has
been reported by others. In a study by Petitjean et al 22
comparing four commercial Mp ELISAs in hospital-
ized subjects with upper or lower respiratory tract
infections, 22% to 48% of children and 17% to 25%
Figure 1. CARDS Tx PCR levels and serologic responses in 10 sub-
jects with persistent Mycoplasma pneumoniae infection. A, PCR.
B, CARDS Tx IgM. C, IgG titers. Each line represents an indi-
vidual case. The index case is shown in bold dashed lines. CARDS
Tx genome levels graphed at 10 2 1 genomes represent samples
that were below the detectable limits for the assay. CARDS
Tx 5 community-acquired respiratory distress syndrome toxin;
OD 5 optical density; PCR 5 polymerase chain reaction.
CHEST / 140 / 2 / AUGUST, 2011 405
responses were characterized by the rapid expression
of cytokines and chemokines with the concurrent
development of an intense lymphocytic infl ammatory
response. Additionally, signifi cant increases in both
airway obstruction and airway hyperreactivity corre-
lated with the degree of peribronchiolar and perivas-
cular airway infl ammation. 25
Although this study was not designed with a thera-
peutic arm, the primary treating physicians were
notifi ed when Mp was identifi ed within the airway of
their patient and of persistent infection. Five subjects
with persistent infection were treated with up to
8 weeks of clarithromycin therapy. We were unable
to detect clinical improvement or eradication of Mp
or CARDS Tx using this form of therapy. Whether
prolonged antimicrobial therapy would impact the
of adults had negative serology despite positive
PCR results to P1. In another study, Atkinson et al 23
identifi ed 7/9 (78%) pediatric subjects with asthma
who had negative IgG titers despite positive PCR
results to Mp 16S ribosomal RNA. In the setting of
Mp community-acquired pneumonia, up to 47% of
subjects who were culture positive, PCR positive, or
both, remained seronegative. 24 One might speculate
that the low levels of Mp infecting the airway in RA
could explain the poor immune responses noted in our
subjects; alternatively, these asthmatic subjects may
have been unable to mount an appropriate immune
response, resulting in a chronic airway infection.
We demonstrated previously that rCARDS Tx is a
potent inducer of pulmonary infl ammation in mice
and baboons. 25 rCARDS Tx-mediated infl ammatory
Figure 2. Serologic responses to CARDS Tx and P1 adhesin. Serum IgG and IgM directed against
CARDS Tx and P1 adhesin were analyzed by enzyme-linked immunosorbent assay at the initial visit for
all subjects. Data are expressed as OD 405. See Figure 1 legend for expansion of abbreviations.
remained persistently positive for Mp, and no imme-
diate differences were noted in their clinical or
immunologic parameters. Therefore, we cannot specu-
late as to whether there were unique characteristics,
immunologic or otherwise, in the subjects with per-
sistent infection. However, the fact that the infl am-
matory CARDS Tx protein was persistently identifi ed
in the airways of these subjects suggests that Mp and
the accompanying pathophysiology of infection and
intoxication may have played a pathogenic role in
the refractory nature of their disease.
The results of this study confi rm that Mp com-
monly exists within the airways of patients with RA
and can be detected optimally with assays targeting
CARDS Tx DNA and protein. The fact that 45%
(29 of 64) of the RA subjects in this study tested
positive by PCR to CARDS Tx, whereas only 16%
(10 of 64) tested positive by PCR to P1, further
demonstrates the superiority of CARDS Tx assays
when screening for Mp. Our failure to demonstrate
a distinguishing IgG response to Mp rCARDS Tx and
rP1in this group of severe asthmatics is relevant
and raises the possibility that some patients may be
unable to mount a robust immune response to Mp.
The role of Mp in both acute and chronic asthma
and other respiratory and extrapulmonary diseases
may need to be reassessed because many of the
prior studies in the literature have relied on sero-
logic diagnosis and less sensitive PCR assays in its
clinical course or eliminate the organism from the
airway remains speculative.
There were several limitations to this study. Ide-
ally, all subjects would have been followed longitudi-
nally and would have included a population of healthy
individuals and asthmatic subjects with various levels
of severity. Without this data, we are unable to deter-
mine if low genome values might be associated with
colonization. Another limitation is the diffi culty in
determining the precise timing of when the enrolled
subjects initially became infected with Mp. In fact,
none of the subjects reported either an acute respira-
tory illness or pneumonia during the 6-month period
prior to enrollment in the study. We chose our defi ni-
tion of persistent infection based on an epidemiologic
study of an acute outbreak of Mp infection, in which
the median duration of carriage was 7 weeks and 70%
of the subjects had no detectable Mp after 3 months. 20
Thus, according to this defi nition, 10 of our subjects
Table 2— Mp IgM and IgG Antibody Titers
Immunoglobulin AssayNegative Persistent Positive
CARDS Tx IgM
CARDS Tx IgM peak
CARDS Tx IgG
CARDS Tx IgG peak
P1 IgM peak
P1 IgG peak
0.103 ? 0.076 0.284 ? 0.335 0.207 ? 0.207
0.353 ? 0.311
0.284 ? 0.192 0.184 ? 0.161
0.294 ? 0.242
0.033 ? 0.020.068 ? 0.115 0.093 ? 0.087
0.089 ? 0.103
0.069 ? 0.062 0.046 ? 0.063 0.065 ? 0.081
0.069 ? 0.061
0.3 ? 0.227
Data are presented as mean ? SD . CARDS Tx 5 community-acquired
respiratory distress syndrome toxin; P1 5 P1 adhesin. See Table 1 for
expansion of the other abbreviation.
Figure 3. Sputum sample. A, Stained with rabbit anti-CARDS TX polyclonal antibodies (left) or control
rabbit antibody (right) (original magnifi cation 3 60). B, Stained with mouse anti-P1 monoclonal anti-
body (left) or control mouse antibody (right) (original magnifi cation 3 60 ). Both techniques demon-
strated mycoplasmas associated with mononuclear cells in the sputum of a subject (shown in bold dashed
lines in Figure 1 ) who had been treated with 8 weeks of macrolide therapy.
www.chestpubs.org Download full-text
CHEST / 140 / 2 / AUGUST, 2011 407
Author contributions : Dr Peters: contributed to study design,
data collection and analysis, and obtaining funding and was pri-
mary author of the manuscript.
Dr Singh: contributed to the oversight of acquisition and analysis
of data and preparation of the manuscript.
Dr Brooks: contributed to study design, immunologic assays, data
analysis, coauthorship, and review of the manuscript.
Dr Diaz: contributed to patient clinical data and review of the
Dr Kannan: contributed to design of study, data acquisition and
analysis, and preparation of the manuscript.
Dr Coalson: contributed to study design, core pathology, data
analysis, and review of the manuscript.
Dr J. G. Baseman: contributed to statistical analysis of the data
and preparation of the manuscript.
Ms Cagle: contributed to data acquisition and analysis and prepa-
ration of the manuscript.
Dr J. B. Baseman: contributed to study study, obtaining funding,
data collection and analysis, and preparation of the manuscript.
Financial/nonfi nancial disclosures: The authors have reported
to CHEST that no potential confl icts of interest exist with any
companies/organizations whose products or services may be dis-
cussed in this article.
Role of sponsors: No funding source had any role in the design
or conduct of the study or in collection, management, analysis,
interpretation of the data, or preparation of the manuscript.
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