ABSTRACT
A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.
Streptococcus pneumoniae infections are often difficult to diagnose accurately, as it is not uncommon for cultures to have a negative test result, particularly after antibiotic administration (10, 22). Several rapid-detection tests for S. pneumoniae have been described (13). The “gold standard” diagnostic method continues to be culture, but appropriate quality samples are not always obtainable. Diagnosis by means of noninvasive samples with new, sensitive methods would be beneficial for determining the etiology of pneumococcal infections. The rapid Binax Now S. pneumoniae urinary antigen test (Binax, Portland, ME) has been approved by the U.S. Food and Drug Administration for the diagnosis of pneumococcal pneumonia. It is highly specific and moderately sensitive for adults (2, 3, 7, 14, 16, 19, 27); however, the Binax Now S. pneumoniae test lacks specificity in children due to pneumococcal antigen cross-reactions in nasopharyngeal carriers (8, 9). This is of particular concern in developing countries, where nasopharyngeal colonization rates are high (1, 11, 17).
Pneumolysin (PLY) is a 53-kDa protein found in all pneumococcal strains; its encoding gene exhibits very limited sequence variability, thus making this molecule an ideal diagnostic target. Specific methods for PLY detection in sputum by Western blotting (31), in urine by agglutination (25), and in respiratory and nonrespiratory samples by nested PCR (20) have been reported but have not been incorporated into routine clinical practice due to insufficient sensitivity and lack of robustness. Successful diagnostic methods targeting PLY have been described but have important limitations because they depend on samples, such as sputum (32) or pleural fluid (12), that are not always available. This study evaluates the usefulness of a PLY-specific enzyme-linked immunosorbent assay (ELISA) (PLY-ELISA) for pneumococcal infection diagnosis in adults and children. We have also tested the persistence of this antigen in urine samples of 11 adult patients with pneumococcal pneumonia.
MATERIALS AND METHODS
Patient groups.Samples were obtained from 481 patients admitted to three hospitals in Asturias, Spain (Hospital Universitario Central de Asturias, Hospital Monte Naranco, and Hospital San Agustín), between January 2003 and December 2006 and recruited as part of a prospective, controlled clinical study investigating novel diagnostic tools for serious community-acquired bacterial infections. Of the 383 adults, 245 were male and 138 were female (mean age, 60.03 years; range, 18 to 99 years), and of the 98 children, 57 were male and 41 were female (mean age, 4.8 years; range, newborn to 16 years). Samples were collected upon admission or during the first day of hospitalization and frozen at −70°C until use. Samples from healthy adults were obtained from volunteer subjects at the Faculty of Medicine. Samples from healthy children were obtained during routine pediatric check-ups at a health care center in Lugones (Asturias, Spain). Ethical approval was obtained from the Hospital Universitario Central de Asturias Research Ethics Committee. Informed consent was obtained from adult patients and from the parents or tutors of the children. Fifteen adult urine samples were obtained by M. Falguera at the Hospital Universitari Arnau de Vilanova (Lleida, Spain). In children, a nasopharyngeal swab sample was taken using a calcium alginate-tipped swab on a flexible aluminum wire (TRANSWAB; Medical Wire and Equipment Co., Ltd., Corsham, Wilts, England). The specimens were brought back to the laboratory within 4 h for processing. None of the healthy controls had been hospitalized for any reason during the previous month, nor they had received any antibiotic treatment during the previous week; no respiratory symptoms were present.
Adult patients included in the study were classified into five main groups (see Table 2). The pneumococcal infection patient group included 76 urine samples from patients with pneumonia (n = 47), probable pneumococcal pneumonia (n = 12), septicemia (n = 8), meningitis (n = 5), arthritis (n = 1), brain abscess (n = 1), breast abscess (n = 1), and puerperal infection (n = 1) due to S. pneumoniae. One patient diagnosed with pneumococcal pneumonia was also positive for Mycobacterium tuberculosis. The nonpneumococcal infection patient group included 56 urine samples from 31 patients with nonpneumococcal pneumonia caused by Mycoplasma pneumoniae (n = 7), Legionella pneumophila (n = 6), Chlamydia pneumoniae (n = 6), Pseudomonas aeruginosa (n = 5), Haemophilus influenzae (n = 1), Haemophilus parainfluenzae (n = 1), Staphylococcus aureus (n = 2), Pneumocystis jiroveci (n = 1), and Moraxella catarrhalis (n = 2); 5 patients with lung infections due to M. tuberculosis; 14 patients with urinary tract infections caused by Candida albicans (n = 1), Enterobacter cloacae (n = 1), Escherichia coli (n = 4), Enterococcus faecalis (n = 1), P. aeruginosa (n = 3), Proteus mirabilis (n = 1), Providencia stuartii (n = 1), Streptococcus agalactiae (n = 1), and Staphylococcus epidermidis (n = 1); 2 patients with hepatic infections caused by hepatitis C virus (HCV); 1 patient with endocarditis due to Streptococcus bovis; 1 patient with herpetic meningoencephalitis; and 1 patient with lung abscess caused by Staphylococcus aureus. The noninfectious disease patient group contained 12 samples from patients suffering lung cancer (n = 8), asthma (n = 2), and cardiac failure (n = 2). The unknown etiological disease patient group comprised 204 urine samples from patients with pneumonia of unknown etiology (n = 178), chronic obstructive pulmonary disease (n = 3), pleurisy (n = 2), fever of unknown origin (n = 6), and respiratory failure (n = 15). The healthy person group included 35 subjects without clinical or radiological signs of pneumonia or infections.
The pediatric population included in the study was classified into five main groups (see Table 3). The pneumococcal infection patient group included 32 urine samples from patients with pneumonia (n = 11), probable pneumococcal pneumonia (n = 6), septicemia (n = 1), meningitis (n = 2), otitis (n = 8), sinusitis (n = 2), conjunctivitis (n = 1), and a finger infection (n = 1) due to S. pneumoniae. The nonpneumococcal infection patient group included two cases of pneumonia caused by M. pneumoniae (n = 1) and H. influenzae (n = 1), gastroenteritis caused by Salmonella species (n = 3), tonsillitis caused by Streptococcus pyogenes (n = 3), septicemia caused by Enterococcus faecalis (n = 1), common cold (n = 3), and urinary infection caused by Escherichia coli (n = 2). The noninfectious diseases patient group included samples from children with allergic rhinitis (n = 6) and jaundice (n = 1). The unknown etiological disease patient group was made up of 31 urine samples from patients with pneumonia of unknown etiology (n = 17), respiratory failure (n = 8), otitis (n = 3), sinusitis (n = 1), septicemia (n = 1), and fever of unknown origin (n = 1). The healthy person group included 12 subjects without clinical or radiological signs of pneumonia or infections. Pediatric subjects were divided into S. pneumoniae nasopharyngeal carriers and noncarriers, and the carrier status was determined to be positive when S. pneumoniae was isolated from the nasopharyngeal swab sample culture.
Microbiological analysis.Culture of blood (ESP system; Difco Laboratories, Detroit, MI), cerebrospinal fluid, peripheral venous catheter, tracheal aspirates, sputum, urine, ascites, pleural fluid, pus from paracentesis, and joint effusion samples was performed according to standard microbiological methods (21). Identification of microorganisms was based on the usual criteria. Human immunodeficiency virus identification was performed by enzyme immunoassay and Western blotting, and HCV identification was done by a recombinant immunoblot assay. Influenza A virus, influenza B virus, parainfluenza virus types 1, 2, and 3, adenovirus, and respiratory syncytial viruses were detected by culture and shell vial assay. Complement fixation tests were performed for influenza viruses A and B, parainfluenza virus types 1, 2, and 3, adenovirus, and respiratory syncytial virus. A microimmunofluorescence method was used to detect antibodies against C. pneumoniae, Coxiella burnetii, Chlamydia psittaci, and L. pneumophila. An enzyme immunoassay was carried out to detect anti-M. pneumoniae immunoglobulin M (IgM); the diagnosis of acute infection criteria was retained when a positive IgM or a fourfold or greater rise in IgM and IgG titers was obtained on sequential sera sampled at 3-week intervals. Identification of M. tuberculosis was performed by Ziehl-Nielsen staining, culture, and specific hybridization probes. Nasal swabs were inoculated onto 5% horse blood agar supplemented with gentamicin (5 μg/ml) and incubated in 5% CO2 for 16 to 24 h. All S. pneumoniae isolates were identified using colony morphology, Gram stain, optochin susceptibility, and bile solubility. All urine samples were cultured and tested with the Binax Now Legionella pneumophila test. Cases with incomplete microbiologic results or for which such results were unavailable were included in the groups of “unknown etiological diseases” and discarded from test performance calculations.
Diagnostic criteria.Patients were classified as having pneumonia if they presented with an acute illness with the presence of new or progressive infiltrate on chest radiograph plus at least two of the following symptoms: fever, cough, shortness of breath, and pleuritic chest pain. All patients were classified as having pneumococcal pneumonia when S. pneumoniae was isolated from blood samples, pleural fluid, or peripheral venous catheter. Pneumococcal pneumonia was considered to be probable when S. pneumoniae was isolated from tracheal aspirates (≥106 CFU/ml) or sputum (ratio of polymorphonuclear neutrophils/squamous epithelial cells of ≥2 or Q [quality] score of ≥+1 on Gram stain). The diagnosis of urinary tract infection was considered to be positive if >105 CFU/ml of a single bacterium was isolated in urine cultures. Noninfectious diseases such as asthma, lung cancer, and cardiac failure were diagnosed by standard criteria.
PLY-ELISA.Prior to analysis, samples were thawed at room temperature, an aliquot was spun at 15,000 × g for 1 min, and the supernatant was removed for assay. PLY quantification was performed by ELISA as previously described (5). Triton X-100 was added to urine samples to a concentration of 2% (wt/vol); the samples were then incubated at 37°C for 30 min and centrifuged at 3,000 × g for 15 min. Flat-bottomed polystyrene Combiplate White Breakable plates (Labsystems, Helsinki, Finland) were coated with 1 μg/well of PLY-7 (IgG1κ, anti-PLY mouse monoclonal antibody) in phosphate-buffered saline (PBS) at 37°C for 3 h. Plates were washed at each step with PBS plus 0.1% Tween 20 and blocked with blocking buffer prepared according to the instructions provided by the manufacturer of the ELISA-Light chemiluminescent detection system (Tropix; Applied Biosystems, Bedford, MA). Samples were then added to the wells and incubated at 37°C for 1 h with shaking. After washing the plates six times, they were incubated with rabbit IgG polyclonal anti-PLY antibody diluted in blocking buffer and incubated at 37°C for 30 min. Anti-rabbit IgG alkaline phosphatase conjugate secondary antibody (Sigma Chemicals Co.) was diluted to 1:5,000 in blocking buffer and incubated as described above. Plates were loaded onto a Luminoskan RS (Labsystems) luminometer, and the wells were automatically filled with substrate/enhancer solution (0.4 mM chloro-5-substituted adamantyl-1,2-dioxetane phosphate with 1× Sapphire-II) and incubated at 37°C for 10 min. Triplicate samples of urine from each subject were studied on a single plate. Each plate contained six twofold dilutions of a known concentration of PLY. Some control wells on each plate had all reagents added except diluted PLY and a pool of negative urine samples obtained from 20 healthy subjects. All ELISAs were carried out with two types of control wells: blank wells had all reagents added except diluted PLY, whereas negative wells were loaded with a pool of negative urine samples obtained from 20 known healthy subjects. No significant differences were observed between relative light units (RLU) obtained from said controls. A cutoff value was calculated from the mean concentration plus twice the standard deviation of the control groups (healthy donors, noninfectious diseases, and nonpneumococcal infections). The cutoff value was 11.13 pg PLY/well. The PLY-ELISA test was judged positive only if the final RLU exceeded twice that for the control wells or the cutoff point.
Bacterial strain lysates.Cell lysates of L. pneumophila serotype 1 (OLDA, Bellingham, and Philadelphia strains), serotype 3, serotype 6, and serotype 10 were kindly provided by J. H. Helbig, Technical University of Dresden, Dresden, Germany. M. tuberculosis H37Rv and CSU 93 cell lysates were obtained from Colorado State University (Mycobacteria Research Laboratories TB Vaccine Testing and Research Materials Program). C. pneumoniae CWL 029 and M. pneumoniae FH cell lysates were purchased from Microbix Biosystems Inc., Toronto, Canada. Clinical isolates of S. agalactiae, P. aeruginosa, P. mirabilis, E. faecalis, E. coli, P. stuartii, C. albicans, E. cloacae, and S. aureus were obtained from patients at the microbiology laboratory of the Hospital Universitario Central de Asturias. They were harvested from solid cultures with a cotton swab, and suspensions containing 107 CFU/ml were prepared in PBS, boiled for 5 min, and centrifuged at 3,000 × g for 15 min before being tested for antigen detection by PLY-ELISA.
Binax Now S. pneumoniae test.The Binax Now S. pneumoniae test was performed for nonconcentrated urine samples obtained from all enrolled patients according to the manufacturer's instructions. The results were read visually after 15 min. The test was interpreted by the presence or absence of visually detectable pink to purple lines.
Statistical analysis.The confidence intervals (CIs) were calculated by Wilson's method. Frequencies were compared using the χ2 test with Yates correction. Statistical differences in RLU were calculated by one-way analysis of variance followed by the Bonferroni test. Statistical analyses were performed using GraphPad PRISM software (v.4.00 for Windows; GraphPad Software, San Diego, CA). The limit of statistical significance was a P value of 0.05.
RESULTS
Antigen detection in lysates of nonpneumococcal bacteria.The possibility of positive results in the PLY-ELISA due to cross-reactions with antigens from other microorganisms that might be present in the samples was assessed. Bacterial lysates (Table 1) containing 107 CFU/ml from 19 nonpneumococcal strains did not produce significant immune reactions, with the exceptions of S. agalactiae and P. aeruginosa.
PLY-ELISA data from lysates of different microorganisms
Detection of pneumococcal antigens in urine samples.The sensitivity of the PLY-ELISA was calculated for patients with S. pneumoniae pneumococcal infections proven by culture. Test specificity was calculated in diverse subject groups used as controls, including patients with a definitive etiologic diagnosis of nonpneumococcal infections, patients with noninfectious diseases, and healthy subjects. There were no significant differences in ages and genders between patients diagnosed with pneumococcal infections and the groups of subjects used as controls.
Table 2 shows the results of PLY-ELISA and the Binax Now S. pneumoniae urinary antigen test in adults. In order to know if the presence of nasopharyngeal S. pneumoniae could determine the performance of PLY-ELISA in children, 35 pneumococcal nasopharyngeal carriers and 63 noncarriers were selected (Table 3). The concentrations of PLY in urine samples of different subject groups were obtained (Table 4). The descriptive statistics for PLY-ELISA and Binax Now S. pneumoniae urinary antigen test of the same urine samples are shown in Table 5.
Comparison of PLY-ELISA results with Binax Now S. pneumoniae urinary antigen test analysis of the same urine samples from adults
Comparison of PLY-ELISA results with Binax Now S. pneumoniae urinary antigen test analysis of the same urine samples from children
Concentrations of PLY in urine samples of different subject groups
Descriptive statistics for PLY-ELISA and Binax Now S. pneumoniae urinary antigen tests of the same urine samples
Table 6 presents the etiologic diagnoses of patients with potential false-positive results by the PLY-ELISA. Three patients were shown to have legionellosis, two of which were diagnosed by antigen detection in urine using the Binax Now Legionella pneumophila test; the samples were also positive for pneumococcal antigen using the Binax Now S. pneumoniae test. Two patients presented pneumonia due to M. pneumoniae, and one patient presented pneumonia caused by C. pneumoniae. Patient 7 was diagnosed with pneumonia by H. influenzae, and urine samples were positive for pneumococcal antigen according to the Binax Now S. pneumoniae test. Patient 8 was diagnosed with a urinary infection, although she developed respiratory symptoms while hospitalized. Two false-positive results were obtained from the group of noninfectious disease patients. The patient with cardiac failure was an elderly woman and died some hours later. Patient 10 was a premature infant diagnosed with jaundice. In all cases, the PLY-ELISA was positive, presenting RLU values close to the established cutoff (data not shown).
Etiologic diagnose of patients with potential false-positive results by PLY-ELISA
Persistence of PLY in urine.To examine the hypothesis that free PLY in urine was quickly eliminated, the persistence of detectable antigens was tested in 11 adults with pneumococcal pneumonia. Patients were diagnosed on the bases of a blood culture positive for S. pneumoniae (n = 3) as well as a positive sputum or tracheal aspirate culture (n = 8). Sequential urine samples were collected every day for the first week after hospital admission. The Binax Now S. pneumoniae test remained positive in samples taken over the first 6 days, while significantly decreased RLU values by PLY-ELISA were obtained after 4 to 6 days of hospital admission (P < 0.05) (Fig. 1).
Persistence of PLY in urine samples from 11 pneumococcal pneumonia patients. Sequential urine samples were collected every day for the first week after hospital admission. The presence of PLY was determined by PLY-ELISA and compared with a negative urine sample pool (NU). A significant decrease in RLU values was obtained after 4 to 6 days of hospital admission. *, P ≤ 0.05.
DISCUSSION
To the best of our knowledge, this is the first study to evaluate PLY-ELISA for the diagnosis of pneumococcal infections in children and adults by urinalysis. The results of the present study revealed that PLY-ELISA is highly specific for children with pneumococcal diseases. The method described above would fail to detect approximately 34% of young patients with pneumococcus-provoked illness, but if used in conjunction with a sensitive test for C polysaccharides, it would be successful in determining the etiology of most serious pneumococcal diseases in children. Different studies have demonstrated that the Binax Now S. pneumoniae test lacks specificity to detect pneumococcal urinary antigen in children, since detectable pneumococcal C polysaccharide is present in the urine of healthy infants with upper respiratory tract colonization (8, 9, 17). In order to prove the specificity of PLY-ELISA in children, we selected children with nasopharyngeal S. pneumoniae colonization and a similar number with nondetectable pneumococcus colonization. The high level of cross-match false-positive results achieved with the Binax Now S. pneumoniae test is probably due to S. pneumoniae present in the nasopharynx (4, 23) and, in noncarriers, might be due to undetected low-level pneumococcal colonization or colonization by Streptococcus mitis (15). At present, there is no evidence to suggest that PLY is present in the urine of colonized infants. Therefore, the PLY-ELISA is useful for pediatric urine samples where rates of pneumococcal colonization are high. Given that we examined a small sample size of children, further studies should be conducted in larger pediatric populations with controlled immunization status to determine how the presence of different serotypes in nasopharyngeal carriers might influence the PLY-ELISA results. The strategy of using the Binax Now S. pneumoniae assay as a primary screen and the PLY-ELISA only on Binax-positive samples provides diagnostic accuracy of 55% in pneumococcal infections. It was possible to conduct the PLY-ELISA in 2 h 30 min when ELISA plates had been previously prepared with antigen, blocked, and maintained at 4°C until use. Moreover, the PLY-ELISA method could be transformed into an immunochromatographic test and, in combination with the sensitive Binax Now S. pneumoniae test, would limit false-positive results enough to make its use in screening cost-effective, enabling pneumococcal infections to be quickly and accurately diagnosed in children.
We previously tested the high specificity of the PLY-ELISA in urine from 14 adult patients with pneumococcal pneumonia (6). In the present report, the PLY-ELISA was successfully used to diagnose 56.6%, 62.5%, and 68.7% of the pneumococcal infections in adults and children with and without nasopharyngeal pneumococcal colonization. Several factors could be involved in the low sensitivity observed. Lacking data regarding PLY stability, improved results would be expected in samples processed after brief storage. Furthermore, a considerable heterogeneity in PLY production among pneumococcal clinical isolates (6, 28) could also mark a significant variation in the amounts that are released into the blood. The time window for detectable pneumococcal antigenemia has been related to different serotypes (26), and preliminary results revealed that PLY persists in the urine of pneumonia patients for a shorter time than polysaccharide antigens (see below). Further research should focus on enhanced sensitivity and the influence of antimicrobial therapy on ELISA of urine samples.
PLY can be detected in concentrated urine samples by Western blot analysis (6). The PLY-ELISA is more sensitive than Western blot analysis, since as little as 300 μl of sample enabled a PLY-ELISA to be performed without having to concentrate the sample. PLY urinalysis by latex agglutination assay (25) and detection of the PLY gene by nested PCR (20) have been proven to yield low sensitivity rates. In our study, we achieved greater sensitivity and good specificity in both adult and pediatric patients, even with the inclusion of many control urine samples obtained from patients with different bacterial and viral infections, from patients with noninfectious diseases, and from healthy subjects. We identified 10 patients with potentially false-positive results using PLY-ELISA. The observed positive result was probably associated with mixed infections (24, 29, 30). Two findings that strongly support this hypothesis are that M. pneumoniae, C. pneumoniae, and L. pneumophila lysates produced negative results in PLY-ELISA, whereas the Binax Now S. pneumoniae test was positive in three out of seven of the pneumonia cases diagnosed as nonpneumococcal pneumonia.
There is a limitation to the application of the Binax Now S. pneumoniae test for diagnosing pneumococcal diseases. C polysaccharide remains in urine until 1 month following infection; therefore, positive results can indicate past pneumococcal infection without being related to the patient's present clinical status (18). We have tested the persistence of PLY in sequential urine samples from a small number of pneumococcal pneumonia patients and observed a significant decrease in detectable antigens after treatment is initiated. Larger patient populations and further investigations are needed before we can state that this behavior can be extrapolated to all pneumococcal infections.
In conclusion, although the Binax Now S. pneumoniae urine test is generally more sensitive than the PLY-ELISA technique in detecting pneumococcal antigens in urine samples and has proven its usefulness in adults, its cross-reactivity in children makes the PLY-ELISA a complementary method that can be useful for the diagnosis of pneumococcal infections due to a lack of better alternatives.
ACKNOWLEDGMENTS
This work was supported in part by MEC-06-BIO2006-1533-C04-02.
We thank Carlos Aranaz (Servicio de Medicina Interna y Geriatría, Hospital Monte Naranco), Javier Rodríguez-Álvarez (Unidad de Microbiología, Hospital San Agustín), José María García-García (Sección de Neumología, Hospital San Agustín), Ana Fleites (Servicio de Microbiología II, Hospital Central de Asturias), Jaime Martínez González-Río (Servicio de Neumología I, Hospital Central de Asturias-Silicosis), and Luis Molinos (Servicio de Neumología I, Hospital Central de Asturias-Silicosis) for collecting samples and clinical data from patients. We also thank Priscilla A. Chase for revising the text.
FOOTNOTES
- Received 18 May 2007.
- Returned for modification 26 July 2007.
- Accepted 20 August 2007.
- Copyright © 2007 American Society for Microbiology