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* From the Departments of Internal Medicine (Drs. Ruiz, Falguera, Galindo, and Porcel), Clinical Microbiology (Dr. Nogués), and Laboratory Medicine (Dr. Esquerda), Pleural Diseases Unit, Arnau de Vilanova University Hospital, Institut de Recerca Biomèdica de Lleida, Lleida, Spain; and Infectious Disease Service (Dr. Carratalá), Hospital Universitari de Bellvitge, Barcelona, Spain.
Correspondence to: José M. Porcel, MD, FCCP, Department of Internal Medicine, Arnau de Vilanova University Hospital, Avda Alcalde Rovira Roure 80, 25198 Lleida, Spain; e-mail: jporcelp{at}yahoo.es
Abstract
Study objectives: To determine whether the detection of pneumococcal antigen in pleural fluid augments conventional microbiological methods used for the etiologic diagnosis of pneumonia.
Methods: In this retrospective study, a rapid immunochromatographic test (ICT) [NOW Streptococcus pneumoniae assay; Binax; Scarborough, ME] was performed on pleural fluid samples from 34 patients with pneumonia due to S pneumoniae, 89 patients with effusions of nonpneumococcal origin, and 17 patients with pneumonia of unknown etiology. Data on blood cultures, pleural fluid cultures, and urinary antigen tests were recorded.
Results: The ICT test result was positive in 24 of 34 patients (70.6%) with pneumococcal pneumonia and negative in 83 of 89 patients (93.3%) without pneumococcal pneumonia. The sensitivity of the pleural ICT test was higher than that obtained for blood (37.5%) and pleural fluid cultures (32.3%), but lower than the detection of pneumococcal antigen in urine samples (82.1%). However, three patients with pneumococcal pneumonia and a negative ICT urine test result had a positive pleural fluid antigen detection result test. Previous antibiotic exposure did not influence pneumococcal antigen detection in either pleural fluid or urine specimens. Six additional patients with empyema due to anaerobes (three patients), Streptococcus viridans (two patients), and Enterococcus faecalis (one patient) had false-positive pleural ICT test results. Finally, the ICT assay finding was also positive in 5 of 17 patients (29.4%) with pneumonia without a definite microbiological cause.
Conclusions: The ICT test performed on pleural fluid samples augments the standard diagnostic methods of blood and pleural fluid cultures, even in the case of prior antibiotic therapy, and enhances the ICT urinary antigen assay.
Key Words: empyema • immunochromatographic test • pleural effusion • pneumococcal pneumonia
Community-acquired pneumonia (CAP) represents the most frequent cause of hospital admission and mortality of infectious origin in developed countries,1 and Streptoccoccus pneumoniae ranks first in the order of pathogens causing CAP.2
In clinical practice, there are a number of problems associated with demonstrating the microbial etiology of CAP by conventional methods. For instance, only one third of patients produce sputum suitable for culture, and results lack specificity due to nasopharyngeal carriage of pneumococci in healthy individuals.3 In addition, blood cultures are specific, but they have a low positivity rate (< 10%).45 About 10% of pneumonia cases are associated with pleural effusion,6 although a pathogen is recovered in less than half of those persons who undergo diagnostic thoracentesis.7 Finally, one third of patients with CAP have received antibiotics before the collection of biological specimens for microbial analysis,8 which may decrease the diagnostic yield by such conventional methods.9
The need for improved speed and accuracy during etiologic diagnosis of CAP has led to the development of a rapid urinary assay for detecting pneumococcal cell wall components common to all serotypes, namely, the NOW assay (Binax; Scarborough, ME).10 Until now, the test has been validated for urine and cerebrospinal fluid samples only. However, three recent studies have evaluated the NOW urinary antigen assay in pleural fluid samples from children1112 and adults13 with CAP. In all of them, the sensitivity of the pleural test was higher than that of pleural cultures, and authors suggested that the NOW test could be a valuable tool for the etiologic study of CAP, although a thorough comparison of the NOW operating characteristics between pleural and urine samples could be inferred from only one of these series.13
The aim of the present study was to ascertain whether the NOW test when performed on pleural fluid samples can add information to the findings of conventional methods (ie, blood cultures, pleural fluid cultures, and urinary antigen assay) for the identification of S pneumoniae as the cause of CAP.
Materials and Methods
Subjects and Study Design
The Ethics Committee of the Arnau de Vilanova University Hospital in Lleida, Spain, approved this retrospective study. We examined 140 pleural fluid samples that had been collected from patients who had undergone thoracentesis in our hospital between 2002 and 2006. Pleural fluids were selected randomly from our pleural fluid bank, where they were stored frozen at –80°C. The basis for randomization was a random number generator (http://www.randomization.com). Patients with pleural effusion were classified into the following three groups: group 1 included 34 patients with pneumococcal pneumonia; group 2 consisted of 89 patients with effusions of nonpneumococcal origin (nonpneumococcal pneumonia, 29 patients; tuberculosis, 20 patients; neoplasm, 23 patients; heart failure, 15 patients; cirrhosis, 1 patient; and post-coronary-artery bypass surgery, 1 patient); and group 3 comprised 17 patients with pneumonia of unknown etiology. Data on blood cultures, pleural fluid cultures, and urinary antigen tests were obtained from medical records.
Diagnostic Criteria
A parapneumonic effusion was defined as any pleural exudate associated with bacterial pneumonia. A diagnosis of CAP due to S pneumoniae was established when this microorganism was isolated from an uncontaminated sample (blood or pleural fluid), or when pneumococcal antigen was detected in unconcentrated urine samples by the NOW assay described in the next section. Sputum culture was not used as a "gold standard" test due to concerns regarding its specificity.14
A diagnosis of nonpneumococcal CAP was made on the basis of the identification of a microorganism other than S pneumoniae in blood or pleural fluid. The diagnosis of pneumonia of unknown etiology was based on negative results of all of the following: blood cultures; pleural fluid cultures; urinary antigen tests for S pneumoniae and Legionella pneumophila; and antibody testing for Mycoplasma pneumoniae, Chlamydia pneumoniae, L pneumophila, Chlamydia psittaci, Coxiella burnetti, and Influenza A virus in paired serum samples.
Tuberculous pleuritis was diagnosed if the results of Ziehl-Neelsen or Löwenstein cultures of pleural fluid, sputum, or pleural biopsy tissue samples were positive, or a pleural biopsy specimen showed granulomas in the parietal pleura. A pleural effusion was said to be malignant if malignant cells were demonstrated to be present on cytologic examination of pleural fluid or biopsy specimens. Finally, the diagnosis of heart failure was based on medical history, physical examination results, chest radiograph, ECG, or echocardiogram findings, and the response to diuretic therapy.
Pneumococcal Antigen Test
An immunochromatographic test (ICT) [NOW S pneumoniae antigen test; Binax] was used to detect S pneumoniae C-polysaccharides in urine and pleural fluid samples. Briefly, a swab was dipped into 200 µL of pleural fluid and inserted into the ICT device, according to the manufacturer’s instructions. Results were assessed by visual inspection after 15 min. A pink-to-purple color on both the sample and control lines indicates a positive antigen test result, whereas color on the control line alone indicates a negative test result. An absence of color on the control line indicates an invalid test. The reading of ICT results was performed and interpreted under blinded conditions by experienced personnel from the Department of Clinical Microbiology.
Statistical Analysis
Data are presented as proportions or means, as deemed appropriate. The sensitivity, specificity, and likelihood ratios of the pleural ICT test were calculated according to standard formulas using results obtained from groups 1 and 2. For between-group comparisons of proportions, the 
2 test or Fisher exact test were used when appropriate.
Results
The study population included 140 patients with pleural effusion, of whom 92 were men and 48 were women (age range, 18 to 93 years; mean [± SD] age, 56 ± 20 years). There were no differences in the patients’ mean age between study groups (group 1, 52 ± 20 years; group 2, 57 ± 20 years; and group 3, 58 ± 18 years; p = 0.22). However, men predominated in group 3 (16 patients; 94%) compared with groups 1 (19 patients; 56%), and 2 (57 patients; 65%; p = 0.02).
The ICT test result was positive in 24 of 34 pleural fluid samples from patients with pneumococcal pneumonia (group 1), providing a sensitivity of 70.6% (95% confidence interval [CI], 53.8 to 87.4%). Table 1 lists the readings of all microbiological results from this group. The addition of the pleural antigen test to bacterial cultures increased the yield of pleural fluid and blood samples by 38% and 33%, respectively. The ICT test result was positive in pleural fluid and negative in urine samples in three patients, whereas seven patients exhibited positive urine and negative pleural ICT test results. Patients exposed to prior antibiotic treatment had the same rate of pleural ICT positivity as nonexposed patients (16 of 22 patients [73%] vs 8 of 12 patients [67%], respectively; p = 0.71). Overall, thoracentesis was performed for a mean duration of 3.1 days (range, 0 to 15 days) after the administration of antibiotics. Similarly, previous antibiotic therapy did not significantly affect the frequency of urinary antigen detection (exposed patients, 16 of 20 patients [80%]; nonexposed patients, 7 of 8 patients [87.5%]; p = 1.00) [Table 1].
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The detection of pneumococcal antigen in the sputum,15 BAL fluid,16 cerebrospinal fluid,17 serum,18 and urine19 specimens as a method for the diagnosis of pneumococcal infection has been previously documented. However, it has seldom been performed in pleural fluid samples.111213 Early studies made use of a variety of techniques, such as counterimmunoelectrophoresis, agglutination tests, and enzyme immunoassays to identify S pneumoniae polysaccharide capsular antigen in pleural fluid samples. For example, in one series20 a counterimmunoelectrophoresis assay was positive in 6 of 16 patients (37%) with pneumococcal pneumonia, although no information was provided regarding test specificity. Boersma et al21 detected pneumococcal antigen by latex agglutination in 8 of 9 patients (89%) with pneumococcal pneumonia, but in only 1 of 12 patients (8%) with pneumonia caused by other etiologic agents. Finally, using a dot-enzyme-linked immunosorbent assay, Requejo et al22 obtained 91% sensitivity and 55% specificity for pneumococcal antigen detection among 840 pleural fluid samples from children with suspected CAP.
In contrast to the above-mentioned methods, which are laborious and time-consuming, the NOW method is a rapid and simple ICT test with a reported sensitivity of 50 to 80% and a high specificity for the detection of S pneumoniae antigen in urine specimens.1923 Moreover, it has the ability to detect antigen up to 1 month after pneumonia onset,24 and results are available in 15 min.
There have been three assessments published concerning the operating characteristics of the ICT test as used for S pneumoniae detection in pleural fluid samples. In one report,11 78 children with empyema, including 40 with pneumococcal infection, were evaluated. The sensitivity of the pleural ICT test was higher than that of latex antigen detection (97% vs 90%, respectively), whereas the specificity (95%) was similar for both techniques. Polymerase chain reaction and/or culturing techniques were used as the test standard. Also, in a pediatric population the NOW test result was positive on all 15 pleural fluid samples yielding S pneumoniae in culture, as well as on two samples that yielded Streptococcus oralis and Streptococcus salivarius in culture.12 In a third series,13 the ICT test result was positive in 15 of 19 parapneumonic pleural fluids of pneumococcal origin (79%), but only in 3 of 41 noninfectious pleural effusions (7.3%).
Our study, which represents the largest published series to date, validates the technical applicability of the ICT test on pleural fluid samples, and confirms its excellent sensitivity (70.6%) and specificity (93.3%) for the diagnosis of pneumococcal pneumonia. Importantly, we have demonstrated that antibiotic use before pleural sampling did not influence ICT test results, as previously reported for the urinary test.10 The addition of the pleural ICT test allowed us to detect 38% more pneumococcal pneumonia cases than pleural culture alone. In contrast with the study from Andreo et al,13 in which all 15 cases of positive ICT test results in pleural fluid exhibited positive pneumococcal antigen in urine, we found that information provided by pleural specimens may complement that of urine specimens and vice versa. Indeed, three patients with pneumococcal pneumonia and a negative NOW urinary antigen test yielded positive results for the detection of pneumococcal antigen in pleural samples.
We observed false-positive results in six patients with empyema due to microorganisms other than S pneumoniae, namely, anaerobes, S viridans and E faecalis. This finding can be explained by the cross-reactivity of cell wall antigenic components among the Streptococcus family, including noncategorizable streptococci (eg, viridans streptococci), anaerobic streptococci, and enterococci (morphologically indistinguishable from streptococci and immunologically similar to members of group D streptococci). The significance of five positive ICT test results in patients with pneumonia without an identified pathogen is uncertain. They may represent either a false-positive result (indicating problems with specificity) or a true-positive result of pneumococcal pneumonia undetected by standard microbiological methods. The second hypothesis is supported by the study of Ploton et al,12 in which 14 of 15 NOW-positive samples that were culture negative were shown to contain S pneumoniae DNA by polymerase chain reaction methods.
Our study has several limitations. Its retrospective design did not allow paired comparison for all samples (ie, two blood samples and six urine samples were unavailable from group 1 patients), which may somewhat influence ICT operational characteristics. In addition, we accepted urinary antigen tests as a "gold standard" for the diagnosis of pneumococcal pneumonia in adults. Nevertheless, although the specificity of the urinary ICT test is not absolute, it is reported to be > 90%, depending on the standard of comparison.19252627 Problems with specificity seem more pronounced in children who are nasopharyngeal carriers of pneumococci.2728 Although specificity appears to be higher for adults,27 colonization status as a potential source of false-positive results has not been systematically evaluated. Nonetheless, the Infectious Diseases Society of America ranks the recommendation for the use of the urinary ICT test as grade B-II.29
In conclusion, the detection of pneumococcal antigen in the pleural fluid of patients with parapneumonic effusions using the NOW assay may rapidly and accurately confirm etiology. Moreover, ICT testing of pleural fluid may provide additional diagnostic information beyond that obtained by the measurement of urine samples alone and vice versa. Therefore, this test should be considered whenever a pleural fluid sample is obtained in the setting of a parapneumonic effusion, particularly when the urine antigen test is not contributory.
Acknowledgements
We want to thank Rosa Biosca for her technical assistance in the performance of the pleural fluid antigen tests.
Footnotes
Abbreviations: CAP = community-acquired pneumonia; CI = confidence interval; ICT = immunochromatographic test
Authors have neither personal or financial support nor any conflict of interest
Received for publication July 30, 2006. Accepted for publication January 4, 2007.
References
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