Utility of pneumococcal urinary antigen detection in diagnosing exacerbations in COPD patients

Respiratory Medicine (2010) 104, 397e403 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/rmed Utility of pneumococcal urinary antigen detection in diagnosing exacerbations in COPD patients Felipe Andreo a,b,c,d, Juan Ruiz-Manzano a,b,c,d, Cristina Prat e,f,g,h, Luis Lores c, Silvia Blanco e,f,g,h, Antoni Malet d, Xavier Gallardo d, José Domínguez e,f,g,h, * a Servei de Pneumologia, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, CIBER Enfermedades Respiratorias, Instituto de Salud Carlos III, Badalona, Spain b Servei de Microbiologia, Fundació Institut d Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, CIBER Enfermedades Respiratorias, Instituto de Salud Carlos III, Badalona, Spain c Servei de Pneumologia, Hospital de Sant Boi de Llobregat, Barcelona, Spain d Servei de Radiologia, Corporació Parc Taulí, Sabadell, Barcelona, Spain e Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Badalona, Spain f Servei de Microbiologia, Fundació Institut d Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain g Servei de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain h Servei de Microbiologia, CIBER Enfermedades Respiratorias, Instituto de Salud Carlos III, Badalona, Spain Received 20 December 2008; accepted 19 October 2009 Available online 5 November 2009 KEYWORDS Chronic obstructive pulmonary disease (COPD); Streptococcus pneumoniae; Urinary antigen; COPD exacerbation; Immunochromatographic test; Rapid diagnosis Summary The aim of this study was to evaluate the utility of pneumococcal urinary antigen detection (Binax Now Streptococcus pneumoniae Antigen Test) in diagnosing pneumococcal exacerbation of chronic obstructive pulmonary disease (COPD). Forty-six patients with S. pneumoniae isolation in sputum culture were studied (29 collected in stable period and 17 collected during exacerbation). In the 29 patients with samples collected in a stable period the antigen was detected in 3 cases (10.3%) using nonconcentrated urine (NCU), and in 12 cases (41.4%) using concentrated urine (CU). Regarding patients recruited during an exacerbation period, the antigen was detected in 3 cases (17.6%) using NCU, and in 13 cases (76.5%) when CU was used. For the evaluation of the specificity of the ICT test we also tested 72 cases in which pneumococcus was not isolated in the sputum sample. ICT was positive in 1 NCU and 9 CU of these patients. To have had at least one previous exacerbation (P Z 0.024), at least one exacerbation that required hospitalization (P Z 0.027), and a pneumonia episode in the year before (P Z 0.010) had statistically significant associated with the detection of specific antigen in CU. Using NCU, the only significant association was found when a previous * Corresponding author at: Servei de Microbiologia, Fundació Institut d Investigació en Ciències de la Salut Germans Trias i Pujol, Carretera del Canyet s/n, 08916 Badalona, Barcelona, Spain. Tel.: þ34 934978894; fax: þ34 934978895. E-mail address: jadomb@gmail.com (J. Domínguez). 0954-6111/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2009.10.013

398 F. Andreo et al. pneumonia in the year before had occurred (P Z 0.006). In summary, a positive result of pneumococcal urinary antigen from a COPD patient, in both bronchial exacerbation and pneumonia, should be evaluated with caution because the antigen detected could be related with previous infectious episode. ª 2009 Elsevier Ltd. All rights reserved. Introduction Chronic obstructive pulmonary disease (COPD) is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and/or emphysema. 1 Some patients with COPD are prone to frequent exacerbations which are a major cause of morbidity and mortality and an important determinant of health related quality of life. 2e4 Bacteria cause a substantial proportion of exacerbations of COPD which cause considerable morbidity and mortality. 5e7 Bacteria are isolated from sputum in 40e60% of acute exacerbations of COPD. The three predominant bacteria species isolated are non-type Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae, although Gram negative enteric bacilli, and Pseudomonas spp are also frequently isolated in patients with severe COPD. 8 Several new lines of evidence demonstrate that bacterial isolation from sputum during acute exacerbation in many instances reflects a cause-effect relationship. 4 Cultures of expectorated sputum from adults experiencing exacerbations of COPD reveal that S. pneumoniae is isolated from 7 to 26% of such samples. However, prevalence studies reveal that 20e40% of stable COPD patients are colonized at any time by this microorganism. 7,9,10 Therefore, the isolation of S. pneumoniae from sputum samples of chronic bronchitis patients only provides a probable etiological diagnosis of the exacerbation. In addition, pneumococcus is not usually isolated from the blood culture during exacerbation. At the moment, a reliable method to distinguish between colonization and clinical infection in COPD patients does not exist. In the last years, a new immunochromatographic (ICT) test (Binax Now S. pneumoniae Antigen Test, Portland, Maine, USA) was developed for detecting polysaccharide C (PnC) in urine samples 11e13 as well as serum 14 and pleural fluid samples. 15 The test has proven rapid, sensitive and specific in diagnosing pneumococcal pneumonia in adults. 11,12,16 Furthermore, concentrating the urine by selective ultrafiltration may elevate the utility of this test because it increases sensitivity. 17,18 TheintroductionofaS. pneumoniae urinary antigen assay in clinical practice has increased the rate of the etiological diagnosis of pneumococcal pneumonia. 18 The aim of this study was to assess the performance of the ICT method in diagnosing the pneumococcal bronchial exacerbation of COPD, detecting specific urinary antigen. Methods Group of patients We recruited prospectively COPD patients who attended to Hospital Universitari Germans Trias i Pujol (Barcelona, Spain) and Hospital de Sant Boi de Llobregat (Barcelona, Spain) for routine outpatients visits during a stable COPD period, or for exacerbation of the disease. All patients met standard criteria for the diagnosis of COPD. 1 Ethics approval for this study was provided by the Ethics Committee of the Hospital Universitari Germans Trias i Pujol, and of the Fundació Jordi Gol i Gurina (Barcelona. Spain). The severity of the disease was established by means of the forced expiratory volume in one second (FEV 1 ) following the standard recommendations for the diagnosis and treatment of patients with COPD. 19 The clinical diagnosis of COPD exacerbation was defined as increased breathlessness, and/or increased sputum volume, and/or new or increased sputum purulence. 20 Patients included in the study were analysed into groups according to the fact that they were recruited during an exacerbation or during a stable period of the disease, and on the basis of the result of the sputum culture. Urine samples were collected for S. pneumoniae antigen detection, being frozen at 20 C until testing. The characteristics recorded for the patients included in the study were the following: age, gender, smoking status, underlying diseases, cor pulmonale, years since evolution of COPD symptoms, basal dyspnoea, FEV 1, clinical symptoms, chest radiograph findings, microbiological results, home oxygen therapy, and prior antibiotic and/or steroid treatment. It was also recorded the number of exacerbations and the number of exacerbations that required hospitalization during the previous year, the occurrence of a previous pneumonia episode, and antibiotic treatment. The basal treatment for COPD and information about the evolution of the disease were also recorded. Patients that needed hospitalization to treat their exacerbation followed conventional treatment based on bronchodilators agents, systemic steroids, antibiotics and oxygen therapy. Inclusion criteria COPD patients with FEV 1 < 80% after bronchodilators agents, and/ airflow obstruction as evidenced by a ratio of FEV 1 to forced vital capacity (FVC) < 0.70 were included in the study. Exclusion criteria The exclusion criteria were the following: patients with clinical or radiological evidence of pneumonia at the moment of diagnosis, or with documented pneumonia in the previous two months, or with new chest radiographic infiltrates in the following 48 h. Patients with history of asthma, lung cancer causing airway obstruction, or other significant respiratory disease, including active tuberculosis; patients vaccinated against S. pneumoniae within the

Pneumococcal antigen detection in COPD 399 previous month. Patients with immunosuppression defined as HIV infection, leukopenia < 1000/mm 3 or neutropenia < 500/mm 3, chronic treatment with steroids ( 15 mg/day prednisone-equivalent during at least 3 months), immunosuppressor treatment in the 6 months before, hypogammaglobulinemia, hypoasplenia, or anatomic or functional asplenia, were also excluded. Bacteriological culture Gram s staining and a standard bacteriological culture with semi-quantification was done. For culturing, only good quality sputum samples containing >25 polymorphonuclear leukocytes and <10 squamous epithelial cells 100 field were according to MurrayeWashington criteria accepted. 21 Standard methods for culture and identification of microorganisms isolated from sputum culture were used. Pneumococcal antigen detection The presence of the PnC antigen of S. pneumoniae was determined using the Binax ICT method. Frozen urine samples were immediately thawed before the test. The detection of pneumococcal antigen was performed in nonconcentrated urine (NCU) and concentrated urine (CU) by selective ultrafiltration. 17 The samples were tested after being stored between 1 and 9 months. ICT was performed according to the instructions of the manufacturer. The results were read visually in 15 min. The test was interpreted according to the presence or absence of visually detectable pink-to-purple coloured lines. Bacterial etiological exacerbation of COPD The presumptive bacterial etiological diagnosis was determined when it complied with these criteria: 1) presence of a clinical compatible picture; 2) presence of a predominant type of microorganism after Gram staining and isolation of a predominant type of respiratory pathogen in the culture in sputum samples. Radiological studies In all cases of COPD exacerbation chest radiography was performed and, in patients who required hospitalization, serial chest radiography and thoracic computed tomography were done during the evolution of the exacerbation when it was necessary. The radiography from the previous year was revised in order to rule out any possible pneumonia not detected. Statistical analysis We used the McNemar test to compare the results obtained by the ICT test in NCU and CU. The differences in the ICT results according to the different defined parameters studied were calculated using Pearson Chi-squared test. Differences were considered significant when the p value was less than 0.05. All analyses were made with SPSS statistical software for Windows (SPSS version 15.0; SPSS Inc., Chicago; IL, USA). Results We studied 46 patients (29 collected in stable period and 17 collected during exacerbation) who had a S. pneumoniae isolated in the sputum culture. Table 1 shows the main characteristics of these patients. From these patients it was detected antigen in 6 cases using NCU, and in 25 cases in CU. The overall number of positive results using CU was significantly higher than using NCU (P < 0.0001). In the 17 patients recruited during an exacerbation period, the antigen was detected in 3 cases (17.6%) using NCU, and in 13 cases (76.5%) when CU was used (Table 2). Regarding the 29 patients in a stable period the antigen was detected in 3 cases (10.3%) using NCU, and in 12 cases (41.4%) using CU. The differences between the number of positives using CU in patients during a stable period and patients with exacerbation were also significant (P Z 0.023). In contrast, this difference using NCU was not significant (P Z 0.483). For the evaluation of the specificity of the ICT test we also tested 27 patients (13 in stable period and 14 during exacerbation) in which other predominant microorganism was isolated in the sputum sample (7 H. influenzae,4 M. catarrhalis, 5 Pseudomonas aeruginosa, 1 P. aeruginosa and Aspergillus fumigatus, 3Staphylococcus aureus, 2Escherichia coli, 1 Table 1 General characteristics of patients with S. pneumoniae isolated in the sputum culture (n Z 46). Parameter N (%) Age (years) Mean and standard deviation 68.54 (10.18) Gender Female 1 (2.2) Male 45 (97.8) Smoking status Active smoker 19 (41.3) Ex-smoker 27 (58.7) COPD status Stable 29 (63) Exacerbation 17 (37) Underlying diseases Yes 25 (54.3) No 21 (45.7) Heart failure 12 (48) Hepatopathy 5 (20) Diabetes 10 (40) Cancer 5 (20) Hematological disease 1 (4) Neurological disease 3 (15) Mild bronchiectasis 13 (52) FEV 1 Mean and standard deviation 1.004 (0.37) FEV 1 (%) Mean and standard deviation 35.15 (11.96) FEV 1 (%)/FVC Mean and standard deviation 50.57 (8.89)

400 F. Andreo et al. Table 2 Pneumococcal urinary antigen detection in patients with COPD attending to the result of the sputum culture. Result sputum culture Status COPD Positive pneumococcal urinary antigen detection S. pneumoniae NCU (%) CU (%) Stable 3/29 (10.3) 12/29 (41.4) Exacerbation 3/17 (17.6) 13/17 (76.5) Other microorganisms than S. pneumoniae Stable 0/13 (0) 2/13 (15.3) Exacerbation 0/14 (0) 5/14 (35.7) No predominant pathogen isolated Stable 0/26 (0) 1/26 (3.8) Exacerbation 1/19 (5.3) 1/19 (5.3) Citrobacter freundii, 1Stenotrophomonas maltophilia, 1 Neisseria meningitidis, 1 Klebsiella pneumoniae, and 1 Enterobacter aerogenes); and 45 patients (26 in stable period and 19 during exacerbation) in which a normal flora was isolated from the sputum sample. The ICT was positive in 7 CU of patients with predominant pathogen in the sputum culture (2 stable and 5 exacerbated patients), and in 1 NCU (exacerbation) and 2 CU (1 stable and 1 exacerbation) of patients without predominant pathogen (Table 2). Using the result of the sputum culture as gold standard, the sensitivity of the urinary antigen detection for diagnosing pneumococcal exacerbation in COPD patients using NCU and CU was 17.6% and 76.5%, respectively; and the specificity in exacerbated patients using NCU and CU was 96.9% (1/33) and 81.8% (6/33), respectively (Table 2). The number of positive results was significantly higher in patients with isolation of pneumococcus in the sputum samples than in the other groups of patients (other pathogens and normal flora) in both NCU (P Z 0.024) and, CU (P < 0.001) respectively. Among all patients with isolation of S. pneumoniae in the sputum sample, we found statistically significant association with the detection of specific antigen in CU, to be younger than 65 yr (P Z 0.005), to have had at least one previous exacerbation (P Z 0.024), at least one exacerbation that required hospitalization (P Z 0.027), and patients with a pneumonia episode within the previous year (P Z 0.010). Using NCU the only significant association was found with a previous pneumonia within the previous year (P Z 0.006). However, the association was nearly significant with previous exacerbation (P Z 0.057) and with exacerbation that required hospitalization (P Z 0.058). When analysing the results of the stable patients, statistically significant association was found with the fact of being younger than 65 yr in NCU (P Z 0.038) and CU (P Z 0.019), and with a previous case of pneumonia using NCU (P Z 0.022). Regarding exacerbated patients, association was found using CU in patients with previous inhalated steroids treatment (P Z 0.018) (Table 3). Discussion Bacterial infection in COPD patients contributes significantly in the pathogenesis and clinical evolution of the disease. 7,22 It has been described that patients who suffer exacerbations have significantly lower health status. 23,24 The development of reliable methods to define the etiology of the infectious exacerbations will be definite for treating adequately the patients. S. pneumoniae is one of the main bacteria involved in exacerbation of the COPD patients. 7 Detection of pneumococcal urinary antigen has been presented as an alternative method for the diagnosis of pneumococcal pneumonia in adults. 11,16,18,25 In contrast, its utility for diagnosing exacerbation of pneumococcal etiology has not been extensively studied. Our results showed that the ICT detects antigen, specially using CU, in patients diagnosed of pneumococcal exacerbation by means of sputum culture. Nevertheless, it was also possible to detect antigen in patients during a stable period of the disease, and also in patients that pneumococcus was not isolated, both during exacerbation and during stable period. In this sense, the detection of PnC antigen by ICT does not allow to distinguish between colonization and infection. Although some potential cross-reactivity has been described with few Streptococcus spp, 26 the high clinical specificity of the ICT test reported in previous studies, 11,12,25 and the fact that in our study the proportion of positive results in patients with a S. pneumoniae isolation in the sputum samples was statistically significant higher than in the other group of patients, it allows us to assume that the ICT test is detecting specific pneumococcal antigen. The result of the sputum culture during an exacerbation, only gives a presumptive etiological diagnosis. Therefore, we could not reject a real pneumococcal infection in patients without S. pneumoniae isolation but with a positive ICT result. In addition, we could not be totally sure of the pneumococcal etiology although the pneumococcus was isolated in the sputum culture. Positive results of pneumococcal urinary antigen detection in COPD patients without pneumonia have also been reported. Murdoch DR et al. 27 tested the ICT test in 97 NCU of COPD patients, being in 49 cases collected during an exacerbation, and in 48 cases during a stable period of the disease. S. pneumoniae was isolated from nasopharyngeal and/or sputum samples in 3 cases of exacerbated COPD patients. Antigen was only detected in 3 cases of exacerbated patients. In only one of them, S. pneumoniae was

Table 3 Association between main characteristics of the COPD patients with S. pneumoniae isolation in the sputum samples, and the urinary antigen detection by means of the immunochromatographic test. Variables Number of positive results by ICT test All patients Stable Exacerbation NCU P CU P NCU P CU P NCU P CU P Age < 65 yr 3/11(27.3) 0.108 10/11(90.9) 0.005 c 2/6 (33.3) 0.038 c 5/6 (83.3) 0.019 c 1/5 (20) 0.870 5/5 (100) 0.140 > 65 yr 3/35 (8.6) 15/35(42.9) 1/23 (4.3) 7/23(30.4) 2/12(16.7) 8/12(66.7) Smoking status Active smoker 3/18 (16.7) 0.432 11/18 (61.1) 0.346 2/11 (18.2) 0.241 6/11 (54.5) 0.210 1/7 (14.3) 0.761 5/7 (71.4) 0.682 Ex-smoker 4/28 (14.3) 15/28 (53.6) 1/18 (5.5) 6/18 (33.3) 2/10 (80) 8/10 (80) Underlying disease Yes 4/25 (16) 0.292 14/25 (56) 0.977 1/13 (7.7) 1 4/13 (30.8) 0.234 3/12 (25) 0.218 10/12 (83.3) 0.301 No 1/18 (5.6) 10/18(55.6) 1/13 (7.7) 7/13 (53.8) 0/5 (0) 3/5 (60) Bronchiectasis Yes 1/13 (7.7) 0.596 6/13 (46.2) 0.401 0/10 (0) 0.245 3/10 (30) 0.315 1/3 (33.3) 0.432 3/3 (100) 0.290 No 4/30(13.3) 18/30 (60) 2/16(12.5) 8/16 (50) 2/14(14.3) 10/14(71.4) Prior inhaled steroid Yes 4/34 (11.8) 0.376 20/34 (58.8) 0.247 1/21 (4.8) 0.699 8/21 (38.1) 0.873 3/13 (23.1) 0.356 12/13 (92.3) 0.018 c No 0/6 (0) 2/6 (33.3) 0/3 (0) 1/3 (33.3) 0/3 (0) 1/3 (33.3) FEV 1 < 35 5/25 (20) 0.224 16/25 (64) 0.375 2/14 (14.3) 0.636 7/14 (50) 0.671 3/11 (27.3) 0.243 9/11 (81.8) 0.770 > 35 1/16 (6.3) 8/8 (50) 1/12 (8.3) 5/12 (41.7) 0/4 (0) 3/4 (75) Previous exacerbation a Yes 5/19 (26.3) 0.057 8/21 (38.1) 0.024 c 2/7 (28.6) 0.099 5/7 (71.4) 0.117 3/12 (25) 0.425 9/12 (75) 0.469 No 1/21 (4.8) 14/19 (73.7) 1/19 (5.3) 7/19 (36.8) 0/2 (0) 1/1 (50) Hospitalization a,b Yes 4/16 (25) 0.058 12/16 (75) 0.027 c 1/4 (25) 0.171 3/ 4 (75) 0.173 3/12 (25) 0.425 9/12 (75) 0.469 No 1/23 (4.3) 9/23 (39.1) 1/21 (4.8) 8/21 (38.1) 0/2 (0) 1/1 (50) Previous pneumonia a Yes 3/7 (42.9) 0.006 c 7/7 (100) 0.010 c 1/ 2 (50) 0.022 c 2/2 (100) 0.096 2/3 (40) 0.142 5/5 (100) 0.119 No 2/34 (5.9) 16/34 (47.1) 1/23 (4.3) 9/23 (39.1) 1/10 (10) 7/11 (63.6) a In the year before. b Exacerbation that required hospitalization. c Differences were statistically significant. Pneumococcal antigen detection in COPD 401

402 F. Andreo et al. isolated from respiratory samples. Therefore, the antigen was detected in the 33.3% (1/3) of exacerbated patients with isolated S. pneumoniae from respiratory samples, and in the 4.3% (2/46) of exacerbated patients without S. pneumoniae isolation. Indeed, the authors considered interesting to repeat the study with a group of patients with COPD who had a high rate of documented S. pneumoniae colonization and/or infection. In our study, we studied 118 NCU of COPD patients, being in 50 cases collected during an exacerbation process. The antigen was positive in 17.6% (3/17) of patients with isolated S. pneumoniae from sputum samples, and in the 3% (1/33) of patients without S. pneumoniae isolation. Although the overall results are similar in exacerbated patients in comparison with the Murdoch et al. 27 study, they did not detected positive results in stable patients. However, in only one case of the 48 patients recruited in a stable period S. pneumoniae was isolated from respiratory samples. In contrast, in our study we have studied 29 cases, obtaining positive results in 3 of them. There are some theories and hypothesis for justifying the results obtained in patients during a stable period. On the one hand, the persistent colonization of the respiratory mucosa with S. pneumoniae could be responsible of the detection of pneumococcal antigen in urine. It has been proven that ICT test is not useful for diagnosing pneumonia in children. The colonization by S. pneumoniae in the nasopharynx in children, specially under 2 years old, induces positive pneumococcal urinary antigen detection. 28,29 In COPD patients S. pneumoniae is frequently recovered from the sputum, even in the absence of clinical infection. 7 Therefore, in the lower airways of adults with COPD colonized with S. pneumoniae, there are continuous division and shedding of cell wall fragments into the airways. 7 These antigens could be cleared from the airways by the immune system and released finally by the urine. On the other hand the presence of the S. pneumonie antigen in urine samples could represent a low grade infection. In fact, in some cases, in the routinely medical controls, patients underestimate the symptoms. In this case the early detection of antigen could be a benefit for patients. This potential explanation requires further investigation to be validated. On the other hand, it has also been described the long persistence of specific pneumococcal antigen in urine after a pneumococcal pneumonia. 18,25,27,30 It has been described in a previous study that in a pneumococcal pneumonia the lung may contain up to 2 g or even more capsular polysaccharide. 31 The release of large amounts of antigen from the lung to the bloodstream and their total clearance from the human body will require a long time. In our experience, it is possible to detect antigen more than 6 months after the diagnosis of pneumonia. 30 COPD is the most common underlying disease in patients with pneumonia, being COPD a frequent condition associated with the development of pneumonia. S. pneumoniae has been described as the leading cause of community-acquired pneumonia in COPD patients. 32 It is also possible that, after an acute pneumococcal exacerbation of the COPD, the time of excretion of the antigen could be also long. In fact, previous exacerbation episodes have been associated with the fact of obtaining a positive ICT test. The main limitation of the current evaluation is that we studied a small group of patients. This fact could influence the statistical analysis and avoid performing multivariate analysis. Despite this limitation, our study provides relevant data related to the clinical utility of the PnC antigen detection by ICT in urine samples in COPD patients. Even thought, further studies including more patients could be adequate for confirming the results. In summary, although the test was able to detect pneumococcal antigen during an exacerbation, it was also possible to detect antigen during stable period. Therefore, it may be difficult to consider the test as a reliable method for diagnosing pneumococcal exacerbation. This fact could be due to a previous exacerbation and or pneumonia episode and with the long time excretion of antigen by the urine. Consequently, its utilization for diagnosing pneumococcal exacerbation or pneumonia in COPD patients should be also used cautiously, because the antigen detected could be residual and not being related to the present episode. Longitudinal studies should be considered in the future for providing additional information in this issue. Acknowledgements We thank the nurse and nurses aides staff of the Hospital de Sant Boi and the Hospital Universitari Germans Trias i Pujol for technical assistance. Financial support This work was supported by a grant from the Societat Catalana de Pneumologia (SOCAP) and the Fundació Catalana de Pneumologia (FUCAP). Conflicts of interest None of the investigators have any financial interest in or a financial conflict with the subject matter or materials discussed in this manuscript. None of the Scientific Societies, neither Binax Inc. (Portland, Maine, USA) had a role in the study design, conduct, collection, management, analysis, or interpretation of the data, or preparation, review, or approval of the manuscript. References 1. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO global initiative for chronic obstructive lung disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163:1256e76. 2. Zalacain R, Sobradillo V, Amilibia J, et al. Predisposing factors to bacterial colonization in chronic obstructive pulmonary disease. Eur Respir J 1999;13:343e8. 3. Monso E, Rosell A, Bonet G, et al. Risk factors for lower airway bacterial colonization in chronic bronchitis. Eur Respir J 1999; 13:338e42. 4. Anzueto A, Sethi S, Martinez FJ. Exacerbations of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2007;4:554e64.

Pneumococcal antigen detection in COPD 403 5. Stockley RA. Lung infections. 1. Role of bacteria in the pathogenesis and progression of acute and chronic lung infection. Thorax 1998;53:58e62. 6. Patel IS, Seemungal TA, Wilks M, Lloyd-Owen SJ, Donaldson GC, Wedzicha JA. Relationship between bacterial colonisation and the frequency, character, and severity of COPD exacerbations. Thorax 2002;57:759e64. 7. Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. Clin Microbiol Rev 2001;14:336e63. 8. Monso E, Ruiz J, Rosell A, et al. Bacterial infection in chronic obstructive pulmonary disease. A study of stable and exacerbated outpatients using the protected specimen brush. Am J Respir Crit Care Med 1995;152:1316e20. 9. Eller J, Ede A, Schaberg T, Niederman MS, Mauch H, Lode H. Infective exacerbations of chronic bronchitis: relation between bacteriologic etiology and lung function. Chest 1998;113:1542e8. 10. Miravitlles M, Espinosa C, Fernandez-Laso E, Martos JA, Maldonado JA, Gallego M. Relationship between bacterial flora in sputum and functional impairment in patients with acute exacerbations of COPD. Study group of bacterial infection in COPD. Chest 1999;116:40e6. 11. Dominguez J, Gali N, Blanco S, et al. Detection of Streptococcus pneumoniae antigen by a rapid immunochromatographic assay in urine samples. Chest 2001;119:243e9. 12. Murdoch DR, Laing RT, Mills GD, et al. Evaluation of a rapid immunochromatographic test for detection of Streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia. J Clin Microbiol 2001;39:3495e8. 13. Roson B, Fernandez-Sabe N, Carratala J, et al. Contribution of a urinary antigen assay (Binax NOW) to the early diagnosis of pneumococcal pneumonia. Clin Infect Dis 2004;38:222e6. 14. Dominguez J, Andreo F, Blanco S, et al. Rapid detection of pneumococcal antigen in serum samples for diagnosing pneumococcal pneumonia. J Infect 2006;53:21e4. 15. Andreo F, Dominguez J, Ruiz-Manzano J, et al. Usefulness of pneumococcal antigen detection in pleural fluid samples by immunochromatographic assay for diagnosis of pneumococcal pneumonia. Clin Microbiol Infect 2006;12:682e4. 16. Gutierrez F, Masia M, Rodriguez JC, et al. Evaluation of the immunochromatographic Binax NOW assay for detection of Streptococcus pneumoniae urinary antigen in a prospective study of community-acquired pneumonia in Spain. Clin Infect Dis 2003;36:286e92. 17. Blanco S, Prat C, Pallares MA, Matas L, Dominguez J. Centrifugal ultrafiltration method for rapid concentration of Legionella pneumophila urinary antigen. J Clin Microbiol 2004;42:4410. 18. Andreo F, Dominguez J, Ruiz J, et al. Impact of rapid urine antigen tests to determine the etiology of community-acquired pneumonia in adults. Respir Med 2006;100:884e91. 19. Celli BR, MacNee W. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J 2004;23:932e46. 20. Anthonisen NR, Manfreda J, Warren CP, Hershfield ES, Harding GK, Nelson NA. Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Ann Intern Med 1987; 106:196e204. 21. Murray PR, Washington JA. Microscopic and baceriologic analysis of expectorated sputum. Mayo Clin Proc 1975;50:339e44. 22. Rosell A, Monso E, Soler N, et al. Microbiologic determinants of exacerbation in chronic obstructive pulmonary disease. Arch Intern Med 2005;165:891e7. 23. White AJ, Gompertz S, Stockley RA. Chronic obstructive pulmonary disease. 6: the aetiology of exacerbations of chronic obstructive pulmonary disease. Thorax 2003;58:73e80. 24. Veeramachaneni SB, Sethi S. Pathogenesis of bacterial exacerbations of COPD. COPD 2006;3:109e15. 25. Marcos MA, Jimenez de Anta MT, de la Bellacasa JP, et al. Rapid urinary antigen test for diagnosis of pneumococcal community-acquired pneumonia in adults. Eur Respir J 2003; 21:209e14. 26. Werno AM, Murdoch DR. Medical microbiology: laboratory diagnosis of invasive pneumococcal disease. Clin Infect Dis 2008;46:926e32. 27. Murdoch DR, Laing RT, Cook JM. The NOW S. pneumoniae urinary antigen test positivity rate 6 weeks after pneumonia onset and among patients with COPD. Clin Infect Dis 2003;37: 153e4. 28. Dominguez J, Blanco S, Rodrigo C, et al. Usefulness of urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal pneumonia in children. J Clin Microbiol 2003;41:2161e3. 29. Faden H, Heimerl M, Varma C, Goodman G, Winkelstein P. Urinary excretion of pneumococcal cell wall polysaccharide in children. Pediatr Infect Dis J 2002;21:791e3. 30. Andreo F, Prat C, Ruiz J, et al. Persistence of Streptococcus pneumoniae urinary antigen excretion after pneumococcal pneumonia. Eur J Clin Microbiol 2008;28:197e201. 31. Coonrod JD, Drennan DP. Pneumococcal pneumonia: capsular polysaccharide antigenemia and antibody responses. Ann Intern Med 1976;84:254e60. 32. Torres A, Dorca J, Zalacain R, et al. Community-acquired pneumonia in chronic obstructive pulmonary disease: a Spanish multicenter study. Am J Respir Crit Care Med 1996;154:1456e61.