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Journal of Clinical Microbiology, August 2007, p. 2460-2466, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02498-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA{triangledown}

Maria da Gloria S. Carvalho,1 Maria Lucia Tondella,1 Karen McCaustland,2 Luciana Weidlich,3 Lesley McGee,4 Leonard W. Mayer,1 Arnold Steigerwalt,1 Melissa Whaley,1 Richard R. Facklam,1 Barry Fields,1 George Carlone,1 Edwin W. Ades,1 Ron Dagan,5 and Jacquelyn S. Sampson1*

Division of Bacterial Diseases,1 Division of Scientific Resources, Centers for Disease Control and Prevention,2 Hubert Department of Global Health, Emory University, Atlanta, Georgia,4 Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, Brazil,3 Pediatric Infectious Diseases Unit, Soroka University Medical Center, Beer Sheva, Israel5

Received 13 December 2006/ Returned for modification 20 March 2007/ Accepted 22 May 2007

The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

* Corresponding author. Mailing address: Centers for Diseases Control and Prevention, 1600 Clifton Road, Mail Stop G05, Atlanta, GA 30333. Phone: (404) 639-3862. Fax: (404) 639-4043. E-mail: JSampson{at}CDC.gov

{triangledown} Published ahead of print on 30 May 2007.

Journal of Clinical Microbiology, August 2007, p. 2460-2466, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02498-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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