Rapid identification of Streptococcus pneumoniae in blood cultures by using the ImmuLex, Slidex and Wellcogen latex agglutination tests and the BinaxNOW antigen test.

Rapid identification of Streptococcus pneumoniae in blood culture (BC) bottles is important for early directed antimicrobial therapy in pneumococcal bacteraemia. We evaluated a new latex agglutination (LA) test on BC bottles, the ImmuLex™ S. pneumoniae Omni (Statens Serum Institut, Denmark), and compared the performance with the Slidex® pneumo-Kit (bioMérieux, France) and the Wellcogen™ S. pneumoniae (Remel, UK) LA tests, as well as the BinaxNOW® S. pneumoniae (Alere, USA) antigen test. The four tests were directly applied on 358 positive BC bottles with Gram-positive cocci in pairs or chains and on 15 negative bottles. Valid test results were recorded in all cases for ImmuLex and BinaxNOW and in 88.5 % (330/373) and 94.1 % (351/373) of cases for Slidex and Wellcogen, respectively. Based on bottles positive for S. pneumoniae by conventional methods, the sensitivity of ImmuLex was 99.6 %, similar to the other tests (range, 99.6-100 %). Based on bottles positive for non-pneumococcal pathogens, the specificity of ImmuLex was 82.6 %, in comparison to 97.6 % for Slidex (p < 0.01) and 85.4 % for Wellcogen (p = ns). The BinaxNOW test had a lower specificity (64.1 %) than any LA test (p < 0.01). On BC bottles positive for α-haemolytic streptococci, ImmuLex was positive in 12/67 (17.9 %) cases, Slidex in 2/59 (3.4 %) cases, Wellcogen in 11/64 (17.2 %) cases and BinaxNOW in 25/67 (37.3 %) cases. In conclusion, the ImmuLex test provides a valid and sensitive technique for the rapid detection of S. pneumoniae in BC bottles, similar to the other compared methods. However, the specificity was sub-optimal, since the test may cross-react with other Gram-positive bacteria.

The Uni-Gold™ Streptococcus pneumoniae urinary antigen test: an interassay comparison with the BinaxNOW® Streptococcus pneumoniae test on consecutive urine samples and evaluation on patients with bacteremia.

The performance of the recently commercialized Uni-Gold™ Streptococcus pneumoniae test for the detection of pneumococcal antigen in urine was studied in a multicenter study. First, we studied the interassay agreement between Uni-Gold™ and the BinaxNOW® S. pneumoniae urinary antigen test on 337 consecutive urine samples sent to the laboratory for the detection of pneumococcal antigen. The two tests performed similarly (κ = 0.82): both tests positive in 27 cases, both tests negative in 299 cases, and with divergent test results in 11 cases. Secondly, the tests were run on urine samples from 203 patients with bacteremia, including 51 patients with pneumococcal bacteremia. The sensitivities and specificities were 67 and 86 % for Uni-Gold™, and 57 % and 94 % for BinaxNOW®, respectively. The false-positivity rate was significantly higher for Uni-Gold™ compared with BinaxNOW® in patients with Escherichia coli bacteremia (15 vs. 2.1 %, p = 0.04), and tended to be higher in patients with bacteremia with alpha-hemolytic streptococci (32 vs. 11 %, p = 0.13). When cases with E. coli and alpha-hemolytic streptococci were excluded from the analysis, the overall false-positivity rate was 9/85 (11 %) for Uni-Gold™ and 6/85 (7.1 %) for BinaxNOW®. In conclusion, the study showed that Uni-Gold™ was not inferior to BinaxNOW® for the detection of pneumococcal urinary antigen in patients with pneumococcal bacteremia. The specificity of Uni-Gold™ was suboptimal due to false-positive results in cases with E. coli and alpha-hemolytic streptococci bacteremia. However, in patient populations usually subjected to testing for pneumococcal urinary antigen, such as pneumonia and meningitis patients, bacteremia with these pathogens is uncommon. The diagnostic usefulness of the Uni-Gold™ test should be further evaluated.

Evaluation of a commercial multiplex PCR test (SeptiFast) in the etiological diagnosis of community-onset bloodstream infections.

The commercial polymerase chain reaction (PCR) test, SeptiFast, is designed to identify the DNA of individual bacterial and fungal pathogens in whole blood. We aimed to evaluate the usefulness of the test for the detection of community-onset bloodstream infections. We prospectively included adult patients who were subjected to blood culture (BC) at an infectious diseases department. For the evaluation, one BC/PCR set (two BC bottles and one PCR tube) per patient was used. When several sets were obtained and analyzed, the first set with any positive result was evaluated. Among 1,093 consecutively included patients, BC was positive in 138 and PCR was positive in 107. Fifty positive PCR results were supported by BC in the same BC/PCR set, ten were supported by other cultures, and, additionally, ten were supported by the clinical presentation. Compared with BC, PCR showed specificities and negative predictive values of >97% for all detectable pathogens. The following sensitivities and positive predictive values (PPVs) were noted: Staphylococcus aureus, 67% and 43%; Streptococcus pneumoniae, 12% and 67%; other Streptococcus species, 43% and 77%; Escherichia coli, 53% and 56%; and Klebsiella species, 43% and 23%. If support from other cultures and the clinical presentation were included in the reference standard, the PPVs for the detection of these bacteria were 57%, 100%, 92%, 75%, and 69%, respectively. Although the specificities were high, the low sensitivities and suboptimal PPVs noted in the present study discourage routine use of the test in its present form for the detection of community-onset bloodstream infections.

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.

Is quantitative PCR for the pneumolysin (ply) gene useful for detection of pneumococcal lower respiratory tract infection?

The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply-based PCR have been reported. The aim here was to study the performance of a quantitative ply-based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply-based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at >or=10(3) genome copies/mL in 61% and 71% of the subjects, at >or=10(5) genome copies/mL in 40% and 58% of the subjects, and at >or=10(7) genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply-based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 10(3) genome copies/mL, of 84% and 66% at a cut-off of 10(5) genome copies/mL, and of 53% and 90% at a cut-off of 10(7) genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply-based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply-based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated.

Usefulness of aetiological tests for guiding antibiotic therapy in community-acquired pneumonia.

The goal with antibiotic therapy in community-acquired pneumonia (CAP) is to cure the patient, ideally without causing side effects and without contributing to the further development of antibiotic resistance. Although patients with severe CAP should be treated with broad-spectrum antibiotics, patients with non-severe CAP should preferably receive pathogen-directed therapy. Rapid aetiological tests, such as sputum Gram stain and urinary antigen tests, are useful for targeting initial pathogen-directed therapy. Non-rapid tests, such as cultures, can subsequently support a switch from initial broad-spectrum therapy to narrow-spectrum therapy and direct therapy changes in case of treatment failure. As conventional diagnostic methods often fail to identify the aetiology of CAP, PCR (polymerase chain reaction) tests for respiratory pathogens have become useful and should be further developed. Based on the test specificities, aetiological tests may provide diagnoses with varying reliability, i.e. definite aetiologies (e.g., blood culture and Legionella urinary antigen test), probable aetiologies (e.g., sputum culture and PCR for Mycoplasma pneumoniae), or possible aetiologies (e.g., culture of nasopharyngeal secretions and PCR for Streptococcus pneumoniae). A definite or probable aetiology can often be used to target antibiotic therapy.

Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage.

The present study assessed the diagnostic usefulness of a multiplex PCR (mPCR) for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae applied to bronchoalveolar lavage (BAL). Fibreoptic bronchoscopy was performed on 156 hospitalised adult patients with lower respiratory tract infection (LRTI) and 36 controls. BAL fluid was analysed with bacterial culture and mPCR. By conventional diagnostic methods, S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae were aetiological agents in 14, 21, 3.2 and 0% of the LRTI patients, respectively. These pathogens were identified by BAL mPCR in 28, 47, 3.2 and 0.6% of cases, respectively, yielding sensitivities of 86% for S. pneumoniae, 88% for H. influenzae, 100% for M. pneumoniae and 0% for C. pneumoniae, and specificities of 81, 64, 100 and 99% for S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae, respectively. Of the 103 patients who had taken antibiotics prior to bronchoscopy, S. pneumoniae was identified by culture in 2.9% and by mPCR in 31%. Among the controls, mPCR identified S. pneumoniae in 11% and H. influenzae in 39%. In lower respiratory tract infection patients, bronchoalveolar lavage multiplex PCR can be useful for identification of Streptococcus pneumoniae, Mycoplasma pneumoniae and Chlamydophila pneumoniae. The method appears to be particularly useful in patients treated with antibiotics.

Detection of Panton-Valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects.

The prevalence of the Panton-Valentine leukocidin (PVL) gene in Staphylococcus aureus was investigated with a simple, reproducible and rapid real-time LightCycler SYBR Green I PCR assay. The PVL gene was detected in one isolate from 65 patients with S. aureus bacteraemia, in four isolates from 55 patients with respiratory tract infections, and in two isolates from 91 patients with cutaneous infections. In contrast, 15 of 25 cutaneous isolates of methicillin-resistant S. aureus (MRSA) were positive. All PVL-positive cutaneous MRSA isolates were community-acquired and comprised three different clones as determined by pulsed-field gel electrophoresis. The PVL gene was detected in isolates from patients with recurrent primary skin infections and S. aureus bacteraemia, but PVL did not seem to be an important virulence factor in the pathogenesis of staphylococcal bacteraemia.

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia.

OBJECTIVE: To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii. METHODS: The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR. RESULTS: Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample. CONCLUSIONS: IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii.