Real‐time PCR to supplement gold‐standard culture‐based detection of Legionella in environmental samples

• Published in: Journal of applied microbiology 2015-10, Vol.119 (4), p.1158-1169
• Main Authors: Collins, S., Jorgensen, F., Willis, C., Walker, J.
• Format: Article
• Language: English
• Subjects: Bacteriology; Bioassays; Colony Count, Microbial; culture; Environment; environmental; Environmental Microbiology; Humans; Legionella; Legionella - classification; Legionella - genetics; Legionella - growth & development; Legionella - isolation & purification; Legionella pneumophila; Legionnaires' Disease - microbiology; Microbiology; mip; Polymerase chain reaction; qPCR; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity and Specificity; ssrA; water; wzm
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.12911

Aims Culture remains the gold‐standard for the enumeration of environmental Legionella. However, it has several drawbacks including long incubation and poor sensitivity, causing delays in response times to outbreaks of Legionnaires' disease. This study aimed to validate real‐time PCR assays to quantify Legionella species (ssrA gene), Legionella pneumophila (mip gene) and Leg. pneumophila serogroup‐1 (wzm gene) to support culture‐based detection in a frontline public health laboratory. Methods and Results Each qPCR assay had 100% specificity, excellent sensitivity (5 GU/reaction) and reproducibility. Comparison of the assays to culture‐based enumeration of Legionella from 200 environmental samples showed that they had a negative predictive value of 100%. Thirty eight samples were positive for Legionella species by culture and qPCR. One hundred samples were negative by both methods, whereas 62 samples were negative by culture but positive by qPCR. The average log10 increase between culture and qPCR for Legionella spp. and Leg. pneumophila was 0·72 (P = 0·0002) and 0·51 (P = 0·006), respectively. Conclusions The qPCR assays can be conducted on the same 1 l water sample as culture thus can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples. Significance and Impact of the Study The assay could prove informative in public health investigations to identify or rule out sources of Legionella as well as to specifically identify Leg. pneumophila serogroup 1 in a timely manner not possible with culture.