HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Sheppard, C. L. Articles by George, R. C. Search for Related Content PubMed PubMed Citation Articles by Sheppard, C. L. Articles by George, R. C. Agricola Articles by Sheppard, C. L. Articles by George, R. C.

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

¹Respiratory and Systemic Infection Laboratory (RSIL), Health Protection Agency (HPA) Central Public Health Laboratory (CPHL), 61 Colindale Avenue, London NW9 5HT, UK ²Vaccine Evaluation Unit, Microbiology Department, Gloucestershire Royal Hospital, Gloucester GL1 3NN, UK

Correspondence Carmen L. Sheppard carmen.sheppard{at}hpa.org.uk

Received September 8, 2003
Accepted December 10, 2003

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.

Abbreviations: CSF, cerebrospinal fluid; IPC, internal process control.

The sequence of the IPC PCR product and a comparison of autolysin crossing points and pneumolysin crossing thresholds are available as supplementary data in JMM Online.

This article has been cited by other articles:

				K. Y. Fukushima, K. Yanagihara, Y. Hirakata, K. Sugahara, Y. Morinaga, S. Kohno, and S. Kamihira Rapid Identification of Penicillin and Macrolide Resistance Genes and Simultaneous Quantification of Streptococcus pneumoniae in Purulent Sputum Samples by Use of a Novel Real-Time Multiplex PCR Assay J. Clin. Microbiol., July 1, 2008; 46(7): 2384 - 2388. [Abstract] [Full Text] [PDF]

				M. d. G. S. Carvalho, M. L. Tondella, K. McCaustland, L. Weidlich, L. McGee, L. W. Mayer, A. Steigerwalt, M. Whaley, R. R. Facklam, B. Fields, et al. Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA J. Clin. Microbiol., August 1, 2007; 45(8): 2460 - 2466. [Abstract] [Full Text] [PDF]

				K. Y. Fukushima, Y. Hirakata, K. Sugahara, K. Yanagihara, A. Kondo, S. Kohno, and S. Kamihira Rapid Screening of Topoisomerase Gene Mutations by a Novel Melting Curve Analysis Method for Early Warning of Fluoroquinolone-Resistant Streptococcus pneumoniae Emergence J. Clin. Microbiol., December 1, 2006; 44(12): 4553 - 4558. [Abstract] [Full Text] [PDF]

				J. P. Leeming, K. Cartwright, R. Morris, S. A. Martin, M. D. Smith, and on behalf of the South-West Pneumococcus Study Gro Diagnosis of Invasive Pneumococcal Infection by Serotype-Specific Urinary Antigen Detection J. Clin. Microbiol., October 1, 2005; 43(10): 4972 - 4976. [Abstract] [Full Text] [PDF]

				S. Yang, S. Lin, A. Khalil, C. Gaydos, E. Nuemberger, G. Juan, J. Hardick, J. G. Bartlett, P. G. Auwaerter, and R. E. Rothman Quantitative PCR Assay Using Sputum Samples for Rapid Diagnosis of Pneumococcal Pneumonia in Adult Emergency Department Patients J. Clin. Microbiol., July 1, 2005; 43(7): 3221 - 3226. [Abstract] [Full Text] [PDF]