Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens

Nucleic acid amplification of clinical specimens with low target concentrat ion has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydi a pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assa ys were compared using 10 replicates of 16 serial dilutions of C. pneumonia e ATCC VR-1310, The proportion positive versus the C. pneumoniae concentrat ion was modeled by probit regression analysis. To validate the model, 10 re plicates of 26 previously positive patient specimens of peripheral blood mo nonuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied,vith the concentrati on of C. pneumoniae in the sample. At concentrations above 5 infection-form ing 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 interva l, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 1 5 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 specimen s, 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 pe rforming 5 or 10 replicates considerably increased the sensitivity and repr oducibility of C. pneumoniae PCR and enabled quantitation for clinical spec imens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens w ith small amounts of target C. pneumoniae DNA present.