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Journal of Clinical Microbiology, May 2001, p. 1796-1801, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1796-1801.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Replicate PCR Testing and Probit Analysis for Detection and Quantitation of Chlamydia pneumoniae in Clinical Specimens

M. Smieja,1,* J. B. Mahony,1,2 C. H. Goldsmith,3 S. Chong,1 A. Petrich,1,2 and M. Chernesky1,2

Hamilton Regional Laboratory Medicine Programme1 and Departments of Pathology and Molecular Medicine2 and Clinical Epidemiology and Biostatistics,3 McMaster University, Hamilton, Ontario, Canada

Received 28 July 2000/Returned for modification 27 November 2000/Accepted 5 March 2001

Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae ATCC VR-1310. The proportion positive versus the C. pneumoniae concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested, P < 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median C. pneumoniae concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target C. pneumoniae DNA present.

* Corresponding author. Mailing address: Laboratory Medicine L424, St. Joseph's Hospital, 50 Charlton Ave. East, Hamilton ON L8N 4A6, Canada. Phone: 905-522-1155 (5140). Fax: 905-521-6083. E-mail: smiejam{at}mcmaster.ca.

Journal of Clinical Microbiology, May 2001, p. 1796-1801, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1796-1801.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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