Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples

Abstract

The development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the Streptococcus pneumoniae autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (lytA), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the lytA primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100 % against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between S. pneumoniae and atypical Streptococcus mitis and Streptococcus oralis strains known to contain the lytA gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42 . 9 %, which was similar to a previously reported TaqMan pneumolysin PCR (43 . 8 %) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of S. pneumoniae in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.

Diagnosis of Streptococcus pneumoniae Infections in Adults with Bacteremia and Community-Acquired Pneumonia: Clinical Comparison of Pneumococcal PCR and Urinary Antigen Detection

“…The methods for the two pneumococcal PCRs, the pneumolysin-targeted TaqMan assay and autolysin-targeted LightCycler assay, have been described previously (14,15). All EDTA blood samples (200-l volume) were extracted using the MagNA Pure Total NA isolation system (Roche Diagnostics) by following the manufacturer's instructions.…”

“…In general, when performed on sputum samples they have good sensitivity but poor specificity (12,17). In order to overcome the latter problem, PCR assays have been evaluated on blood samples (5,8,14,15), giving excellent specificity but relatively poor sensitivity. Consequently, in an attempt to improve sensitivity but maintain good specificity, we have devised a dual-PCR testing protocol that tests for two pneumococcal targets (lytA and ply) in duplicate samples.…”

DNA bacterial load in children and adolescents with pneumococcal pneumonia and empyema

“…al. [17] have published an accurate meta-analysis and found that the studies were highly heterogeneously including different type of PCR methods [18,19], different target genes [20], most used stored frozen [21] and different samples (i.e. whole blood or plasma) [22], as well as different patients' characteristics and different clinical syndromes [23,24].…”

Diagnosis of Invasive Pneumococcal Infection by Serotype-Specific Urinary Antigen Detection

“…New sensitive diagnostic methods would be valuable not only for determining the etiology of individual infections but also for monitoring the epidemiology of pneumococcal disease within the general population and vaccine recipients in particular. Application of PCR assays for the diagnosis of invasive pneumococcal disease has proven to be of limited success because they are insufficiently sensitive when applied to blood or urine and are not infection specific when applied to respiratory samples (12,19). A number of publications have described antigen detection assays (7,9).…”