Evaluation of a rapid immunochromatographic test for detection of Streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia but is undoubtedly underdiagnosed. Isolation of S. pneumoniae from blood is specific but lacks sensitivity, while isolation of S. pneumoniae from sputum may represent colonization. We evaluated a new immunochromatographic test (NOW S. pneumoniae urinary antigen test; Binax, Portland, Maine) that is simple to perform and that can detect S. pneumoniae antigen in urine within 15 min. Urine samples from 420 adults with community-acquired pneumonia and 169 control patients who did not have pneumonia were tested. Urine from 315 (75%) of the pneumonia patients and all controls was tested both before and after 25-fold concentration, while the remaining 105 samples were only tested without concentration. S. pneumoniae urinary antigen tests were positive for 120 (29%) patients with pneumonia and for none of the controls. Of the urine samples tested with and without concentration, 96 were positive, of which 6 were positive only after concentration. S. pneumoniae antigen was detected in the urine from 16 of the 20 (80%) patients with blood cultures positive for S. pneumoniae and from 28 of the 54 (52%) patients with sputum cultures positive for S. pneumoniae. The absence of S. pneumoniae antigen in the urine from controls suggests that the specificity is high. Concentration of urine prior to testing resulted in a small increase in yield. The NOW S. pneumoniae urinary antigen test should be a useful adjunct to culture for determining the etiology of community-acquired pneumonia in adults.     

Real-time polymerase chain reaction assay for detection of Streptococcus pneumoniae in sputum samples from patients with community-acquired pneumonia.

BACKGROUND AND PURPOSE: A quantitative real-time polymerase chain reaction (PCR) method targeting the pneumolysin gene of Streptococcus pneumoniae in sputum specimens from patients with community-acquired pneumonia (CAP) was evaluated for the identification of pneumococci. METHODS: The applicability of the assay to clinical samples was evaluated by studying 140 sputum specimens from patients with CAP. Of the specimens, 96 (68.6%) were found to be positive by real-time PCR. The results were compared to culture findings. RESULTS: The sensitivity and specificity of the real-time PCR assay developed in this study as compared to those of the culture method were 97.2% and 60.9%, respectively. CONCLUSION: Real-time PCR assay was found to be a rapid and sensitive method for the detection of pneumococci.     

Evaluation of a commercial probe assay for detection of rifampin resistance inMycobacterium tuberculosis directly from respiratory and nonrespiratory clinical samples

A commercial assay (Inno-Line Probe Assay; Innogenetics, Belgium) was evaluated to determine its ability to detect rifampin resistance inMycobacterium tuberculosis directly from clinical specimens. Fifty-nine selected specimens (42 respiratory and 17 nonrespiratory) culture positive forMycobacterium tuberculosis were tested along with their corresponding isolates in culture. The results were compared with those obtained by in vitro susceptibility testing. The results of the line probe assay to detect rifampin resistance inMycobacterium tuberculosis present in clinical specimens and in cultured isolates were concordant for 58 of 59 (98.3%) isolates (95% confidence limits = 90.9–99.9%). The line probe assay failed only once, when a fecal specimen was tested; no amplification was observed due to the presence of inhibitory compounds. The most frequently observed mutation was His₅₂₆Asp (58.7%), followed by the His₅₂₆Tyr (23.9%); together, they represented 82.6% of rifampin-resistant samples. In conclusion, the Inno-Line Probe Assay is a rapid, useful method for detecting the presence ofMycobacterium tuberculosis complex and its resistance to rifampin directly from clinical specimens and culture. Moreover, since rifampin resistance is a potential marker for multidrug resistance inMycobacterium tuberculosis, this assay may constitute an important tool for the control of tuberculosis.     

Molecular detection of Mycoplasma pneumoniae in adults with community-acquired pneumonia requiring hospitalization

Mycoplasma pneumoniae infection was diagnosed in 18 (12.5%) of 144 adults hospitalized with community-acquired pneumonia. The infection was demonstrated by PCR in 15 patients and by serology, using two methods, in 10 patients. The mean age of the 8 patients with positive M. pneumoniae PCR and negative serology was significantly higher than that of the 10 patients with positive serology.     

Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility

We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of ≤0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.     

The role of Streptococcus pneumoniae in community-acquired pneumonia among adults in Europe: a meta-analysis

The primary objective of this meta-analysis was to estimate the prevalence of adult community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae in Europe, adjusted for possible independent covariates. Two reviewers conducted a systematic literature search using PubMed on English-language articles that involved human subjects with CAP during the period from January 1990 to November 2011 across European countries. A mixed-effects meta-regression model was developed and populated with 24,410 patients obtained from 77 articles that met the inclusion criteria. The model showed that the observed prevalence of S. pneumoniae in CAP significantly varies between European regions, even after adjusting for explanatory covariates, including patient characteristics, diagnostic tests, antibiotic resistance, and health-care setting. The probability of detecting S. pneumoniae was substantially higher in studies that performed more frequently a diagnostic polymerase chain reaction assay compared to all the other diagnostic tests included. Furthermore, S. pneumoniae was more likely to be confirmed as the cause of a CAP in studies with intensive care unit patients as compared to those with hospital- or community-treated patients. This study provides estimates of the average observed prevalence of S. pneumoniae, which could be used for projecting the health and economic benefits of pneumococcal immunization.     

Molecular detection of respiratory pathogens in community-acquired pneumonia involving adults

BackgroundCommunity-acquired pneumonia (CAP) causes substantial morbidity and mortality in adults worldwide. The etiology of CAP often remains uncertain, and therapy is empirical. Thus, there is still room for improvement in the diagnosis of pneumonia.MethodsAdults aged >20 years who presented at the outpatient or emergency departments of Linkou and Keelung Chang Gung Memorial Hospital with CAP were prospectively included between November 2016 and December 2018. We collected respiratory specimens for culture and molecular testing and calculated the incidence rates of CAP according to pathogens.ResultsOf 212 hospitalized adult patients with CAP, 69.3% were male, and the median age of the patients was 67.8 years. Bacterial pathogens were detected in 106 (50%) patients, viruses in 77 (36.3%), and fungal pathogens in 1 patient (0.5%). The overall detection rate (culture and molecular testing method) was 70.7% (n = 150). Traditional microbial culture yielded positive results in 36.7% (n = 78), molecular testing in 61.3% (n = 130). The most common pathogens were influenza (16.1%), followed by Klebsiella pneumoniae (14.1%), Pseudomonas aeruginosa (13.6%), human rhinovirus (11.8%), and Streptococcus pneumoniae (9.9%). Multiple pathogen co-infections accounted for 28.7% (n = 61), of which co-infection with K. pneumoniae and human rhinovirus comprised the largest proportion.ConclusionsMolecular diagnostic testing could detect 23.6% more pathogens than traditional culture techniques. However, despite the current diagnostic tests, there is still the possibility that no pathogen was detected.     

Diagnostic utility and clinical significance of naso- and oropharyngeal samples used in a PCR assay to diagnose Mycoplasma pneumoniae infection in children with community-acquired pneumonia

PCR assays of naso- and oropharyngeal samples among hospitalized children appear equally effective for the diagnosis of serologically confirmed community-acquired mycoplasmal pneumonia. However, the combination of results from both sites yields optimal sensitivity (57%), specificity (98%), and positive (92%) and negative (82%) predictive values when compared with Mycoplasma pneumoniae enzyme-linked immunosorbent assay.     

Low prevalence of Chlamydia pneumoniae in adults with community-acquired pneumonia

The incidence of community-acquired pneumonia (CAP) due to Chlamydia pneumoniae was determined in a prospective study of 546 adult patients with CAP included in the German CAP Competence Network (CAPNETZ) project. Three different PCR protocols for detection of C. pneumoniae in respiratory specimens were compared by a multicenter, inter-laboratory comparison involving three laboratories. A case was defined as a patient with a respiratory sample positive by PCR in at least two laboratories. CAP was caused by C. pneumoniae in 5/546 cases (0.9%). Antibody testing by microimmunofluorescence was done in 376 of 546 patients. All patients were negative for IgM antibodies. In the five PCR-positive patients, neither specific IgG nor IgA antibodies were found. Patients with CAP caused by C. pneumoniae had a lower median age (36 years) than the general study population (62 years). C. pneumoniae is currently a rare cause of CAP in adult patients in Germany. Analysis of a single serum sample is not useful for diagnosis of acute C. pneumoniae infection in CAP.     

Evaluation of several biochemical and molecular techniques for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and their detection in respiratory samples

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO(2) atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O(2) atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.     

Evaluation of semiautomated multiplex PCR assay for determination of Streptococcus pneumoniae serotypes and serogroups

A semiautomated method for the determination of five serotypes and three serogroups in Streptococcus pneumoniae was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.     

Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes

In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.     

Contribution of PCR for detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens

AIM OF THE STUDY: To evaluate the sensitivity of PCR versus culture of complex tuberculosis mycobacteria and to determine the delay between PCR results and identification of mycobacteria in culture. MATERIALS AND METHODS: Ninety-nine pulmonary and 66 extrapulmonary specimens were analyzed. Samples were inoculated on liquid (MGIT, Bactec) and solid media (Coletsos) and respectively incubated 6 and 12 weeks. Identification was performed by reverse hybridization of PCR products to their complementary probes immobilized on membrane strips (Genotype MTBC, HAIN). Specimens DNA detection was realized by PCR (Cobas Amplicor Mycobacterium tuberculosis test, Roche). RESULTS: Sensitivity of PCR for acid fast bacilli smear positive pulmonary (50/50) and extrapulmonary (7/7) specimens was 100%. Delay between PCR result and identification was 11 days for pulmonary specimens and 8 days for extrapulmonary specimens. Sensitivity of PCR for smear negative samples was, respectively, of 78.7% (37/47) and 51.8% (29/56) for pulmonary and extrapulmonary specimens. In case of PCR positive result of a smear negative sample, a gap of respectively 13 and 12 days was obtained for pulmonary and extrapulmonary specimens compared to identification. CONCLUSION: Positive PCR result for respiratory specimens allows a gap of 11 to 13 days in diagnosis in comparison with identification of mycobacteria in culture.     

recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci

Proper identification of Streptococcus pneumoniae by conventional methods remains problematic. The discriminatory power of the 16S rRNA gene, which can be considered the "gold standard" for molecular identification, is too low to differentiate S. pneumoniae from closely related species such as Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis in the routine clinical laboratory. A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing <95.8% interspecies homology. In addition, 6 signature nucleotides specific for S. pneumoniae were identified within the 313-bp recA fragment. We show that recA analysis is a useful tool for proper identification to species level within the S. mitis group, in particular, for pneumococci.     

Evaluation of a real-time PCR assay for the detection of the Klebsiella pneumoniae carbapenemase genes in microbiological samples in comparison with the modified Hodge test

Transfer of the bla(KPC) genes encoding the Klebsiella pneumoniae carbapenemase (KPC) are increasingly responsible for emerging carbapenem resistance. The modified Hodge test (MHT) is recommended for the detection of KPC. We compared MHT with a real-time polymerase chain reaction (PCR) assay targeting common subtypes of bla(KPC), using previously described forward and reverse primer sequences. The PCR product was detected using SYBR Green (Applied Biosystems, Foster City, CA) and confirmed by melt curve analysis. PCR was positive in 96% (52/54) of isolates that were MHT+, 90% (28/31) of MHT- isolates were PCR-, and the results were strongly correlated (P = .0001; Fisher exact test). The PCR assay is a sensitive, specific, and rapid test for detecting bla(KPC) genes. It could help optimize patient care by reducing the time taken to institute appropriate antimicrobial therapy and so help improve patient outcomes.     

Detection of Streptococcus pneumoniae in sputum samples by PCR.

A method for the detection of Streptococcus pneumoniae in sputum samples by PCR has been developed. The assay employs oligonucleotide primers specific for a portion of the autolysin gene lytA of S. pneumoniae. Other closely related streptococci, Haemophilus influenzae, and Moraxella catarrhalis do not give a positive result in the assay. The assay was capable of detecting between 10 and 100 CFU of S. pneumoniae in distilled water and 1.4 x 10(4) CFU/ml in simulated sputum samples. Sputum samples from 33 patients with acute pneumonia were collected and subjected to culture, PCR, and C-polysaccharide antigen detection by enzyme-linked immunosorbent assay (ELISA). A significant isolate of S. pneumoniae was isolated from 14 patients, of which 13 were positive by PCR and C-polysaccharide antigen ELISA. No positive results were obtained for the 19 patients in whom other pathogens or upper respiratory tract floras only were isolated. The sensitivity of the autolysin PCR is 92.8%, the specificity is 100%, the predictive value of a positive result is 100%, and the predictive value of a negative result is 95%. This suggests that autolysin PCR is suitable for the detection of S. pneumoniae in clinical samples.     

Detection and serotyping of Streptococcus pneumoniae from nasopharyngeal samples by PCR-based multiplex assay

We developed a multiplex PCR-based methodology for nasopharyngeal samples maintained in egg thioglycolate antibiotic and skim milk-tryptone-glucose-glycerol media to identify and serotype the most important serotypes of Streptococcus pneumoniae that cause invasive disease in children. This technique can be used to study the epidemiology of pneumococcal colonization and the effect of conjugate vaccines.     

Serotype distribution and antimicrobial profile of Streptococcus pneumoniae isolated from adult patients with community-acquired pneumonia in Jakarta,Indonesia

Streptococcus pneumoniae is one of the primary causes of community-acquired pneumonia. The vaccine serotypes were dominant and could be isolated in 14% of adult patients, with serotype 3 being the most predominant (25%), followed by 6A, 6B, and 7F. Approximately, 44% of the isolates showed resistance to tetracycline.