Quantitative real-time Legionella PCR for environmental water samples: data interpretation

Abstract

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10(3) CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.

FIG. 1.

Linearity of quantitative Legionella mip PCR.

FIG. 2.

Comparison of Legionella culture results with quantitative Legionella 16S rRNA gene PCR and mip PCR results for water samples analyzed at Lyon (study 1) and Grenoble (study 2). Samples below the detection or quantification limit by culture or PCR were attributed the cutoff value. For culture versus mip PCR comparisons, samples yielding non-L. pneumophila legionellae by culture were excluded.

FIG. 3.

Use of receiver operating curves to determine quantitative Legionella 16S rRNA gene PCR (A) and mip PCR (B) cutoff values (GU/liter) for identification of samples containing ≥10³ CFU/liter legionellae among hot water system samples analyzed at Lyon (study 1) and Grenoble (study 2). For mip PCR (B), samples yielding non-L. pneumophila legionellae by culture (six samples in study 1 and one sample in study 2) were excluded. Se, sensitivity; Sp, specificity.