INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is associated with considerable morbidity and mortality. The World Health Organization estimates that COPD will be the third leading cause of death worldwide by 2030 [
1]. The overall prevalence of COPD in subjects aged ≥ 40 years in Korea is estimated to be 13.4% [
2]. Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) are the most important prognostic factor; such exacerbations are associated with short and long term reductions in quality of life and lung function, as well as an increased risk of death [
3,
4]. The etiology of AECOPD is multifactorial; the condition is caused by complex interactions between the host immune system, respiratory viruses, and airway bacteria, all of which lead to an increase in the inflammatory burden within the airway [
5]. Previous data suggest that the etiology is unclear in nearly 30% of AECOPD cases; however, respiratory tract infection (50% to 60% of cases) and air pollution (10% of cases) are major causes [
6].
Atypical respiratory pathogens usually include
Mycoplasma pneumoniae, Chlamydia pneumoniae, and
Legionella pneumophila, although the exact role of these pathogens in AECOPD is yet to be elucidated. Atypical pathogens are detected indirectly by serological assays, or directly by cell culture or polymerase chain reaction (PCR). Most studies used a single method [
7-
13], whereas a few combined methods [
14-
16]. Although serology may suggest that atypical pathogens play a significant role in AECOPD, interpreting serology results is tricky and yields variable results [
7,
8,
17-
25]. Also, culturing atypical pathogens is not easy. There are some data regarding the use of PCR for diagnosing atypical pathogens, and PCR appears superior to serology in this respect [
26]. However, serology and PCR can yield discrepant results [
14-
16]. Indeed, a previous study revealed that, according to real-time PCR results, atypical pathogens do not play a significant role in stable COPD or AECOPD [
9]. Thus, the role of atypical pathogens in AECOPD remains controversial. The present prospective study was designed to include patients with a moderate AECOPD (defined as an increase in symptoms that required treatment with antibiotics and/or corticosteroids, but not hospitalization); the aim was to use serology and multiplex PCR to determine the role of atypical pathogens in AECOPD.
DISCUSSION
This study examined sputum samples from patients with AECOPD and used serological and multiplex PCR assays to assess the role of atypical pathogens. The serology results revealed that atypical pathogens were relatively uncommon in AECOPD. PCR revealed no direct evidence of atypical pathogens in AECOPD. Finally, we found no clinically significant differences between patients with and without atypical respiratory pathogens.
We enrolled patients with moderate AECOPD who attended outpatient clinics; however, most previous studies enrolled hospitalized patients with severe AECOPD [
7,
14,
18,
21,
28,
29]. Moderate AECOPD is defined as an increase in symptoms that requires treatment with antibiotics and/or corticosteroids, whereas a severe exacerbation is one that requires hospitalization [
30]. Although our study was part of a multicenter trial, all laboratory tests were performed at a single central laboratory to avoid technical differences that may cause inconsistent results.
The role of atypical pathogens in AECOPD is controversial because
M. pneumoniae and
C. pneumoniae are common causes of respiratory tract infections, and both show high seroprevalence of IgG antibodies in the general healthy population (up to 60% and 70%, respectively) [
31,
32]. In the present study, the seroprevalence of IgG antibodies against
M. pneumoniae, C. pneumonia, and
L. pneumophila was 74.2%, 84.8%, and 2.6%, respectively. Also, there are different diagnostic criteria for detecting atypical pathogens by serology. A definitive diagnosis is based on a 4-fold increase in the IgG titer between the acute and convalescent phases; however, some studies report that the IgM response may be nonspecific or absent, particularly in adults [
31,
32]. One study defined elevated IgM levels on day 1 as a probable infection [
15]. Another study defined an acute
M. pneumoniae infection as a significant increase in the
M. pneumoniae IgG titer, seroconversion in paired sera, or the presence of IgM antibodies against
M. pneumoniae [
26]. We defined a definite infection by atypical pathogens as a 4-fold or greater increase in the IgG titers and a probable infection as a positive IgM result on day 1.
Most previous studies used serologic techniques to detect atypical pathogens in AECOPD samples [
7-
13]. More recently, however, molecular techniques have been used to detect etiologic agents of AECOPD; indeed, several studies used PCR to detect atypical pathogens in AECOPD [
9,
14-
16].
A previous study based on serologic assays reported a possible relationship between
M. pneumoniae infection and AECOPD in 9% of hospitalized AECOPD cases [
28]. Other reports estimate the prevalence of
M. pneumoniae in AECOPD at between 0% to 16%, depending on the serologic method and diagnostic criteria used [
7,
11,
15]. Recent microbiological studies of patients hospitalized for AECOPD identified
M. pneumoniae in only 1.5% to 2.2% of cases using serologic methods [
14,
29]. Two previous studies of outpatient AECOPD did not detect
M. pneumoniae infection by PCR [
9,
15]; however, serological evidence was obtained in 16% of cases [
15]. Another prospective study attempted to detect
M. pneumoniae in hospitalized AECOPD using both serologic and PCR-based methods. The serologic assay identified
M. pneumoniae in two patients (2.2%) who were also PCR-positive; however, another 3/92 patients were PCR-positive alone [
14]. A recent study prospectively examined 50 cases (43 cases of hospitalized AECOPD) using real-time PCR and conventional procedures, including sputum culture. PCR detected
M. pneumoniae in four cases (8%) without any serologic evidence of infection [
16]. Here, we found that none of the moderate AECOPD patients was PCR-positive, and that only 5.6% were positive for Mycoplasma infection according to serological tests.
According to serological tests, the incidence of
C. pneumoniae among AECOPD is 4% to 16% [
8,
23-
25]. Another recent study detected only one positive result for
C. pneumoniae after serologic testing of 132 patients hospitalized for AECOPD [
29]. Here, we identified only one patient with a
C. pneumoniae infection by serology; this was the only patient that was IgM-positive at the initial visit. The PCR assay was negative. Indeed, another study found no PCR-based evidence of
C. pneumoniae infection in outpatient AECOPD [
9]. A recent prospective study diagnosed acute or presumed acute infection with
C. pneumoniae in 4/92 hospitalized AECOPD patients (4.3%) by serologic testing; two of the four were also PCR-positive whereas one was positive by PCR only [
14]. Another prospective study detected C. pneumonia in one patient by PCR, without any serologic evidence of infection [
16].
Studies of the association between
Legionella and AECOPD are relatively rare. A prospective study revealed serological evidence of
Legionella spp. in 16.7% of hospitalized AECOPD patients [
18]; however, another study detected
Legionella non-pneumophila DNA in only one sputum sample out of 126 outpatient AECOPD samples by real-time PCR [
9]. By contrast, another prospective AECOPD study did not detect
Legionella spp. by PCR [
14]. In the present study, we identified seven
L. pneumophila-positive samples in serologic assays and one in the urinary antigen test; however, PCR was negative for all 341 patients.
Although PCR is used to detect atypical pathogens in clinical laboratories, no clear guidelines regarding the true performance, drawbacks, and limitations of the technique have been published [
14]. PCR has been used to test for
M. pneumoniae infections for about 20 years, but the method has several limitations. First, PCR inhibitors in samples can lead to false-negative results. Second, contamination can cause false-positive results. Third, the time of sampling influences the results. Indeed, in contrast to serologic assays, the diagnostic accuracy of PCR may decrease at or beyond 7 days after disease onset [
26,
33]. The above could explain why PCR shows low sensitivity for detecting atypical pathogens in AECOPD. Finally, samples suitable for PCR may be difficult to acquire.
A previous study showed that PCR was superior to serology for the diagnosis of acute
M. pneumoniae infection and identified a high rate of persistent infection [
9]. The study examined
M. pneumoniae infection in children aged 10 to 16 years who showed acute respiratory symptoms during a community outbreak of Mycoplasma infection. The results revealed that PCR testing of respiratory secretions may provide an early diagnosis and be more sensitive than serologic techniques.
Another prospective study was conducted to compare the diagnostic value of an indirect immunofluorescence assay with that of PCR for the diagnosis of
M. pneumoniae in adults. PCR showed lower sensitivity than serology; therefore, the authors recommended the use of serology and PCR in parallel to confirm
M. pneumoniae infections in adults with community acquired pneumonia [
34].
A meta-analysis showed that commercial PCR tests generate consistent results with high specificity, but they show low/variable sensitivity for
M. pneumoniae. These findings suggest that, although commercial PCR tests may be superior for diagnosing
M. pneumoniae infection, they cannot completely replace serologic assays. Thus, PCR plus serology could be a good screening method for the reliable and accurate diagnosis of
M. pneumoniae infection [
35].
Although a previous study showed that the seroprevalence of
M. pneumoniae infection in a study population was significantly higher than that in the control group, the role (if any) played by
M. pneumoniae in AECOPD was not substantiated by culture isolation or PCR [
15]. Thus, there is a need for more studies of well-defined patient populations with AECOPD to establish a correlation between serological evidence of
M. pneumoniae infection and that provided by culture and PCR.
Few studies have examined correlations between different microbiologic techniques with respect to the detection of atypical pathogens [
14-
16]. Indeed, we found no correlation between serologic assays and PCR in this respect.
Another finding that we found interesting was that 10 of 28 moderate AECOPD patients (36%) who were positive for atypical pathogens also yielded at least one typical bacterial pathogen upon sputum culture. A previous study detected at least one additional respiratory pathogen (viral or bacterial) in 71% of hospitalized AECOPD cases with
M. pneumoniae [
7]. Another prospective study of patients with severe exacerbation and respiratory failure showed that 29% were mixed infections by bacterial pathogens [
11]. The prevalence of mixed infections varied, depending on the detection method and pathogen examined. One pathogen may exacerbate infection by another pathogen, or two pathogens may act independently.
The aim of the current prospective study was to use a combination of techniques (serologic assays and PCR) to determine the role of atypical pathogens in 341 AECOPD patients. This is a large cohort when compared with those in previous studies. However, a limitation of the present study is that we did not perform direct culture of atypical pathogens.
In conclusion, the serological prevalence of atypical pathogens in AECOPD was relatively low. These results, when combined with the negative PCR results, suggest that atypical pathogens play no (or a very limited) role in AECOPD, and that detection of atypical pathogens is not necessary in South Korea.