Rapid detection of pneumococcal antigens in sputa in patients with community-acquired pneumonia by coagglutination

This paper introduces the clinical use of coagglutination as a method for the rapid detection of pneumococcal antigens in patients with community-acquired pneumonia and compares it with the sputum Gram stain and culture methods. Among 105 patients, 50 (48%) were diagnosed as having pneumococcal pneumonia by at least one of the three methods. Of 95 sputa tested, 44 (46%) were found to be positive by the coaggulation test, 26 (27%) by Gram staining and only 16 (17%) by the bacterial culture method. The rate of detection of pneumococcal antigens was thus greater with coaggulation than with either of the other two methods. The differences were very significant (bothP<0.01). Our study indicates that the advantages of coagglutination over the traditional bacteriological methods are its speed, sensitivity, convenience and also its relative independence of antibiotic therapy. It thus provides a new dimension in the aetiological diagnosis of pneumococcal pneumonia.     

Antigen detection in oropharyngeal secretions for rapid diagnosis of pneumococcal pneumonia

To determine the value of detection of antigen in the oropharynx in the diagnosis of pneumococcal pneumonia, oropharyngeal secretions were cultured for the presence ofStreptococcus pneumoniae and tested for the presence of pneumococcal antigen. Sputum (if available) collected on the same day was also investigated for the presence of antigen. Detection of pneumococcal antigen was found to be directly related to the severity of pneumococcal carriership or infection (p<0.0001) and was not related to culture results. Patients with pneumococcal pneumonia had the highest antigen detection rate (38 %), followed by patients with pneumonia of unknown etiology (32 %) and patients with an acute lower respiratory tract infection due toStreptococcus pneumoniae (20 %). Pneumococcal carriers had a detection rate of only 9 %. Antigen could be detected in only one patient of the control groups. Although antigen detection in sputum was superior to that in oropharyngeal secretions, concordant results were obtained in 8 (40 %) and 6 (36 %) patients with pneumococcal pneumonia and pneumonia of unknown etiology respectively. The results strongly suggest that pneumococcal carriage seldom leads to a detectable level of antigen, and that antigen detection in the oropharynx appears to be of additive value in the diagnosis of pneumococcal pneumonia.     

Incidence of community-acquired pneumonia caused byChlamydia pneumoniae in Italian patients

The incidence ofChlamydia pneumoniae as a cause of community-acquired pneumonia was evaluated in a one-year prospective study in 108 patients with community-acquired pneumonia. The bacteriological diagnosis was based on culture of sputum or bronchial aspirate and examination of acute and convalescent phase sera forMycoplasma pneumoniae, Legionella pneumophila andChlamydia pneumoniae. A definitive microbiological diagnosis was obtained in 58 (54 %) patients.Chlamydia pneumoniae was the causative agent in 14 patients (13 %) on the basis of positive serological tests; in 10 of the 14 patientsChlamydia pneumoniae was also detected by means of an indirect immunofluorescence test using pharyngeal swab specimens. In conclusion,Chlamydia pneumoniae seems to be a common etiological agent of community-acquired pneumonia, as increasingly reported in the last six to seven years.     

Genomic Load from Sputum Samples and Nasopharyngeal Swabs for Diagnosis of Pneumococcal Pneumonia in HIV-Infected Adults

Quantitative lytA real-time PCR (rtPCR) results from nasopharyngeal (NP) swabs distinguish community-acquired pneumococcal pneumonia (CAP) from asymptomatic colonization. The use of an optimized cutoff value improved pneumococcal etiology determination compared to that of traditional diagnostic methods. Here, we compare the utility of lytA rtPCR from induced sputum and from NP swabs. Pneumococcus was considered the cause of CAP in HIV-infected South African adults if blood culture, induced-sputum culture or Gram stain, urine antigen test, or whole-blood lytA rtPCR revealed pneumococcus or if lytA rtPCR from NP swabs gave a result of >8,000 copies/ml. lytA rtPCR was also performed on induced sputum. Pneumococcus was detected by lytA rtPCR from sputum in 149 (67.1%) of 222 patients with available induced sputum, whereas the results of either Gram stain or culture of sputum were positive in 105 of 229 patients (45.9%; P < 0.001). The mean copy numbers from sputum were higher when the sputum cultures were positive than when the sputum cultures were negative (7.9 versus 5.6 log₁₀ copies/ml; P < 0.001). Against the composite diagnostic standard, a cutoff value of 10,000 copies/ml for good-quality sputum lytA rtPCR had a sensitivity of 78.1% and a specificity of 80.0%. This cutoff value performed similarly to the previously identified cutoff value of 8,000 copies/ml for NP swab lytA rtPCR (area under the curve receiver operating characteristic [AUC-ROC], 80.4% for sputum of any quality versus 79.6% for NP swabs). The AUC-ROC for good-quality sputum was 83.2%. Overall, lytA rtPCR performs similarly well on induced sputum as on NP swabs for most patients but performs slightly better if good-quality sputum can be obtained. Due to the ease of specimen collection, NP swabs may be preferable for the diagnosis of pneumococcal pneumonia.     

Evaluation of a latex test for rapid detection of pneumococcal antigens in sputum

A latex agglutination test (Directigen) for detection of pneumococcal capsular polysaccharide antigens in sputum was evaluated in comparison to culture and Gram stain using 121 sputum specimens. The sensitivity, specificity and rate of agreement of the latex test compared to culture were 90 %, 79 % and 81 %, respectively. The results suggest that the latex test may be useful for detecting pneumococcal antigen in sputum.     

Aetiology and Clinical Presentation of Mild Community-Acquired Bacterial Pneumonia

A prospective study was initiated to analyse the bacterial aetiology and clinical picture of mild community-acquired pneumonia in Slovenia using the previously described Pneumonia Severity Index. Radiographically confirmed cases of pneumonia in patients treated with oral antibiotics in seven study centres were included. An aetiological diagnosis was attempted using culture of blood and sputum, urinary antigen testing for Streptococcus pneumoniae and Legionella pneumophila, and antibody testing for Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila in paired serum samples. One hundred thirteen patients were evaluable for clinical presentation and 109 for aetiological diagnosis. At least one pathogen was detected in 62.4% patients. The most common causative agents were Mycoplasma pneumoniae in 24.8%, Chlamydia pneumoniae in 21.1%, and Streptococcus pneumoniae in 13.8% of patients. Dual infection was detected in 8.3% of patients. Most patients suffered from cough, fatigue, and fever. Patients with atypical aetiology of pneumonia differed from those with typical bacterial pneumonia or pneumonia of unknown aetiology in age, presence of dyspnea, and bronchial breathing on lung auscultation. Patients with pneumococcal, chlamydial, and mycoplasmal infections differed in age, risk class, presence of dyspnea, bronchial breathing, and proteinuria. There was an overlap of other clinical symptoms, underlying conditions, and laboratory and radiographic findings among the groups of patients classified by aetiology. Since patients with mild community-acquired pneumonia exhibit similar clinical characteristics and, moreover, since a substantial proportion of cases are attributable to atypical bacteria, broad-spectrum antibiotic treatment seems to be recommended.     

Predicting pneumococcal community-acquired pneumonia in the emergency department: Evaluation of clinical parameters

The aim of this study was to quantify the value of clinical predictors available in the emergency department (ED) in predicting Streptococcus pneumoniae as the cause of community-acquired pneumonia (CAP). A prospective, observational, cohort study of patients with CAP presenting in the ED was performed. Pneumococcal aetiology of CAP was based on either bacteraemia, or S. pneumoniae being cultured from sputum, or urinary immunochromatographic assay positivity, or positivity of a novel serotype-specific urinary antigen detection test. Multivariate logistic regression was used to identify independent predictors and various cut-off values of probability scores were used to evaluate the usefulness of the model. Three hundred and twenty-eight (31.0%) of 1057 patients with CAP had pneumococcal CAP. Nine independent predictors for pneumococcal pneumonia were identified, but the clinical utility of this prediction model was disappointing, because of low positive predictive values or a small yield. Clinical criteria have insufficient diagnostic capacity to predict pneumococcal CAP. Rapid antigen detection tests are needed to diagnose S. pneumoniae at the time of hospital admission.     

Diagnosis of pneumococcal pneumonia by antigen detection in sputum.

Pneumococcal polysaccharide was detected by counterimmunoelectrophoresis in the sputum of 20 of 26 (77%) adults with community-acquired pneumonia and a positive sputum culture for Streptococcus pneumoniae. The test was negative in 29 pneumonia patients with negative sputum culture for S. pneumoniae.Pneumococcal antigen was also detected in the sputum of six of nine adults with chronic bronchitis and a positive sputum culture, but was not detected in expectorated respiratory secretions of 22 pneumococcal carriers with colds. Pneumococcal antigen could also be detected in sputum by immunodiffusion; antigen titers varied from 1:2 to 1:256. These results strongly suggest that the detection of pneumococcal antigen in respiratory tract secretions indicates infection caused by S. pneumoniae.     

Utility of gram staining for evaluation of the quality of cystic fibrosis sputum samples

The microscopic examination of Gram-stained sputum specimens is very helpful in the evaluation of patients with community-acquired pneumonia and has also been recommended for use in cystic fibrosis (CF) patients. This study was undertaken to evaluate that recommendation. One hundred one sputum samples from CF patients were cultured for gram-negative bacilli and examined by Gram staining for both sputum adequacy (using the quality [Q] score) and bacterial morphology. Subjective evaluation of adequacy was also performed and categorized. Based on Q score evaluation, 41% of the samples would have been rejected despite a subjective appearance of purulence. Only three of these rejected samples were culture negative for gram-negative CF pathogens. Correlation between culture results and quantitative Gram stain examination was also poor. These data suggest that subjective evaluation combined with comprehensive bacteriology is superior to Gram staining in identifying pathogens in CF sputum.     

Comparison of three methods for detection of pneumococcal antigen in sputum of patients with community-acquired pneumonia

In a prospective study of 249 patients with community-acquired pneumonia, three tests for the detection of pneumococcal antigen in sputum were compared: a coagglutination test for detecting capsular antigens (Cap-CoA), a sandwich enzyme immunoassay (PnC-EIA) and a coagglutination test (PnC-CoA), both the latter detecting the pneumococcal C-polysaccharide common to all pneumococcal types. Sixty-three patients had culture-positive pneumococcal pneumonia, 45 pneumonia caused by other bacteria and 141 pneumonia of viral or unknown etiology. The sensitivity of Cap-CoA (63%) and PnC-CoA (65%) was somewhat higher than that of PnC-EIA (49%), but not significantly so. The specificity was 96–98% for all three methods. Using PnC-CoA 66 patients with possible pneumococcal infection were detected, the diagnosis being verified by culture in 41. Using Cap-CoA 59 such patients were detected, the diagnosis being verified in 40, and using the PnC-EIA 47 such patients were detected, the diagnosis being verified in 31. Antigen was found almost as often in non-purulent as in purulent samples, and as often in washed as in non-washed purulent samples. However, antibiotic treatment before the sputum sample was obtained resulted in significantly lower sensitivity of both PnC-CoA and Cap-CoA. This study confirms the high sensitivity and specificity of methods for pneumococcal antigen detection in sputum. Since CoA is easier and quicker to perform, and cheaper than the EIA, either PnC-CoA or Cap-CoA would seem to be the technique of choice for detection of pneumococcal antigen, whereby all sputum samples, including non-purulent samples, can be used.     

Usefulness of real-time PCR for lytA,ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.     

Quantitation of pneumococcal C polysaccharide in sputum samples from patients with presumptive pneumococcal pneumonia by enzyme immunoassay.

Although the Gram stain and culture of expectorated sputum are considered standard methods for the diagnosis of presumptive pneumococcal pneumonia, these methods remain relatively insensitive and nonspecific. We developed an enzyme immunoassay (EIA) for the quantitation of pneumococcal C polysaccharide (PnC) in the sputum of patients with presumptive pneumococcal pneumonia. Of 34 patient sputum samples collected within 24 h of the first radiographic report of pneumonia, 12 grew Streptococcus pneumoniae on culture. By using a cutoff point of 0.5 micrograms of PnC per ml of sputum, all 12 specimens were positive (sensitivity, 100%) by EIA. PnC levels ranged from 1.43 to 57.53 micrograms/ml. Blood samples from 18 of the 34 patients were cultured. S. pneumoniae grew in the culture of a blood sample from one patient, whose sputum also had the highest PnC level. Of 22 sputum samples from patients with pneumonia that did not grow S. pneumonia, two were positive by EIA (specificity, 90.1%). Sputa from both patients had low levels of PnC (2.7 and 4.5 micrograms/ml), and both patients had received antibiotics before sputum collection. The positive predictive value of the quantitative EIA was 85.7%. Quantitation of PnC has the potential for improving the accuracy of sputum examination for S. pneumoniae, monitoring disease severity and the effectiveness of antibiotic therapy, and differentiating between those patients with invasive pneumococcal disease and those who are carriers of S. pneumoniae.     

Microbial Aetiology of Community-Acquired Pneumonia in Hospitalised Patients

 Adult patients hospitalised with community-acquired pneumonia were studied prospectively to determine the microbial aetiology of pneumonia. Between April 1996 and March 1997, blood and sputum samples were collected for culture. Throat swabs were obtained for isolation of viruses and for detection of antigens of Chlamydia pneumoniae, influenza viruses A and B, respiratory syncytial virus and parainfluenza virus. Antibodies against Legionella spp., Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci, Coxiella burnetii, influenza viruses A and B, respiratory syncytial virus, adenovirus and parainfluenza virus were tested in serum samples. Two hundred eleven patients were included in the study; paired sera were available from 152 patients. Blood culture was positive in 23 (10.9%) patients, Streptococcus pneumoniae being the bacterium isolated most frequently. A fourfold or greater rise or fall in the Chlamydia pneumoniae IgG and/or IgM antibody titre was found in 20 (9.5%) patients and a high antibody titre (≥1 : 512) in the first and/or the second serum sample in 18 (18.5%) patients. Antibodies confirming acute Mycoplasma pneumoniae infection were found in 12 (5.7%) patients, Legionella spp. in six (2.8%), Chlamydia psittaci in two and Coxiella burnetii in one. Three patients had pulmonary tuberculosis. Only two patients had a virus present in the throat swab (adenovirus in one patient and echovirus in the other), and in nine patients, viral antigen was detected. Acute viral infection was confirmed in 51 (24.1%) patients. Bacterial pneumonia was diagnosed in 84 (39.8%) patients, 23 of whom had concurrent viral infection. Acute viral pneumonia without any other identified pathogen was diagnosed in 28 patients. Streptococcus pneumoniae and Chlamydia pneumoniae were the most frequently identified microorganisms.     

Detection ofChlamydia pneumoniae in clinical specimens by polymerase chain reaction using nested primers

A nested primers strategy was used to develop a two-step PCR test for the direct species-specific detection of the 16s rRNA gene ofChlamydia pneumoniae. This test was applied to 58 nasopharyngeal or oropharyngeal swab specimens collected from patients in studies of community-acquired pneumonia and in a local outbreak of respiratory disease. Twelve patients (21 %) showed evidence ofChlamydia pneumoniae infection in serological tests (7/56; 13 %), culture (8/58; 14 %) or PCR (10/58; 17 %). Nested PCR but not single-step PCR was found to be as sensitive as culture or serology for detection of infection with this organism. In summary, nested PCR can be useful in direct testing of clinical specimens forChlamydia pneumoniae, making additional DNA purification steps unnecessary.     

Evaluation of a real-time polymerase chain reaction assay for the diagnosis of pneumococcal and meningococcal meningitis in a tertiary care hospital

This study evaluated the performance of a real-time polymerase chain reaction (PCR) assay in comparison with Gram staining and culture of cerebrospinal fluid for the diagnosis of meningococcal and pneumococcal meningitis in patients with suspected bacterial meningitis. The sensitivity for detection of Neisseria meningitidis in cerebrospinal fluid was 87% (20/23) for the PCR assay, 27% (6/22) for Gram staining, and 17% (4/23) for culture. The sensitivity for detection of Streptococcus pneumoniae in cerebrospinal fluid was 100% (14/14) for the PCR assay, 62% (8/13) for Gram staining, and 36% (5/14) for culture. Therefore, we recommend that real-time PCR of cerebrospinal fluid for detection of N. meningitidis and S. pneumoniae become a part of the routine diagnostic procedure for patients with suspected bacterial meningitis.     

Comparison of immunological methods for diagnosis of pneumococcal pneumonia in biological fluids

Five immunological tests were evaluated for their ability to detectStreptococcus pneumonine antigen in serum and urine simultaneously as a means of rapid diagnosis in 40 patients with bacteremic or non-bacteremic pneumococcal pneumonia or pneumonia with other etiologies. Serum and urine were screened in parallel with counterimmunoelectrophoresis (CIE), two commercial latex agglutination kits — the Slidex pneumokit (LA-SPK) and the BactigenStreptococcus pneumoniae kit (LA-Bac) — the coagglutination Phadebact Pneumococcus test (CoA) and a newly developed enzyme-linked immunosorbent assay (ELISA) containing the immunoglobulin G fraction from rabbit pneumococcal antiserum. The detection rate for accumulated serum in bacteremic patients was 18 % for LA-Bac, 24 % for CIE, 47 % for LA-SPK and CoA and 76 % for ELISA, whereas antigenuria was present in only 29 % for LA-SPK, 24 % for CIE, 19 % for CoA, 14 % for LA-Bac and 5 % for ELISA. Detection by ELISA of pneumococcal antigen in severely ill patients can predict bacteremia and rapidly confirm the diagnosis of pneumococcal pneumonia if sputum and results of blood cultures are not available.     

Sputum gram stain assessment in community-acquired bacteremic pneumonia.

A prospective study was performed over a 4.5-year period to determine the ability of a sputum Gram stain to predict the cause of community-acquired bacterial pneumonia. A blood culture isolate, rather than a sputum culture, served as the reference standard to provide precise identification of the etiologic agent. The study population comprised 59 bacteremic adults who expectorated a valid sputum sample. Data are presented that indicate that a physician, aided by the morphology of the stained sputum, could theoretically select appropriate monotherapy approximately 94% of the time when selective, defined criteria for the microbiology of valid sputum are met. Three of the five patients with pneumonia caused by Haemophilus influenzae, however, had sputum stains that suggested alternative pathogens. This study reaffirms that the Gram-stained sputum is a reliable, but not infallible, guide to direct initial antibiotic therapy in adults with community-acquired bacterial pneumonia.     

Early recognition ofStreptococcus pneumoniae in patients with community-acquired pneumonia

The objective of this study was to assess the predictive value of signs, symptoms, and rapidly available laboratory parameters for pneumococci in community-acquired pneumonia (CAP). A prospective study on patients with CAP who were admitted to hospital was conducted. Clinical and laboratory data were collected according to a protocol. Two hundred sixty-eight patients aged 18 years or older, not living in a nursing home or not admitted to hospital within one week of this admission, with a new infiltrate on the chest radiograph consistent with pneumonia were included. According to microbiological and serological tests, patients were allocated to one of two aetiological groups,Streptococcus pneumoniae or other pathogens. Seventy-three variables were examined for a correlation with one of the aetiological categories by means of univariate and multivariate analysis. The resulting discriminant function was considered a clinical test for which posttest probabilities for pneumococcal pneumonia were calculated.Streptococcus pneumoniae was demonstrated in 79 patients and other pathogens in 83; no pathogens were detectable in 106 patients. The variables cardiovascular disease, acute onset, pleuritic pain, gram-positive bacteria in the sputum Gram stain, and leucocyte count correctly predicted the cause of CAP in 80% of all cases in both groups. Depending on the prevalence ofStreptococcus pneumoniae, posttest probabilities for pneumococcal pneumonia were up to 90%. It is concluded that data on history, together with the result of the Gram stain of sputum and the leucocyte count, can help to distinguishStreptococcus pneumoniae from other pathogens causing CAP.     

Comparison of Sputum and Nasopharyngeal Aspirate Samples and of the PCR Gene Targets lytA and Spn9802 for Quantitative PCR for Rapid Detection of Pneumococcal Pneumonia

We aimed to compare sputum and nasopharyngeal aspirate (NpA) samples and the PCR gene targets lytA and Spn9802 in quantitative PCR (qPCR) assays for rapid detection of pneumococcal etiology in community-acquired pneumonia (CAP). Seventy-eight adult patients hospitalized for radiologically confirmed CAP had both good-quality sputum and NpA specimens collected at admission. These samples were subjected to lytA qPCR and Spn9802 qPCR assays with analytical times of <3 h. Thirty-two patients had CAP with a pneumococcal etiology, according to conventional diagnostic criteria. The following qPCR positivity rates were noted in CAP cases with and without pneumococcal etiology: 96% and 15% (sputum lytA assay), 96% and 17% (sputum Spn9802 assay), 81% and 11% (NpA lytA assay), and 81% and 20% (NpA Spn9802 assay), respectively. The mean lytA and Spn9802 DNA levels were significantly higher in qPCR-positive sputum samples from cases with pneumococcal etiology than in qPCR-positive sputum samples from CAP cases without pneumococcal etiology or qPCR-positive NpA samples from cases with pneumococcal etiology (P < 0.02 for all comparisons). For detection of pneumococcal etiology, receiver operating characteristic curve analysis showed that sputum specimens were superior to NpA specimens as the sample type (P < 0.02 for both gene targets) and lytA tended to be superior to Spn9802 as the gene target. The best-performing test, the sputum lytA qPCR assay, showed high sensitivity (94%) and specificity (96%) with a cutoff value of 10⁵ DNA copies/ml. In CAP patients with good sputum production, this test has great potential to be used for the rapid detection of pneumococcal etiology and to target penicillin therapy.