Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires’ disease

Abstract

The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently

“…The distribution of STs among culture-proven and culture-negative but NP-SBT positive patients appears to be the same for ST1 (9.8%/9.1% for SBT and NP-SBT, respectively); ST23 (18.5%/26.3%), and ST62 (4.8%/ 8.1%) appear a little more represented in NP-SBT, and ST47 appears a little less represented in NP-SBT (8.5%/3.0%). Furthermore, L. pneumophila strains detected by culture (∼65% of LD cases) or PCR (∼20% of LD cases) in another study show a similar distribution of STs (Mentasti et al 2012).…”

Low genomic diversity of Legionella pneumophila within clinical specimens

“…Ten colony picks were obtained [5] from single sputum samples of ten epidemiologically unrelated patients with sporadic Legionnaires' disease in England (Table 1). Isolates were stored at –80°C.…”

Microbial Growth in Drinking-Water Supplies: Problems, Causes, Control and Research Needs

“…Still, the detection of mild cases of LD and/or cases caused by other serogroups and other species is complicated, also because of the limitations of the culture methods. A more complete and rapid diagnosis can be achieved by the inclusion of PCR-based methods (Diederen et al 2005;von Baum et al 2008;Mentasi et al 2012). Improvements in reporting and surveillance can contribute to a further increase of the notification rate as well.…”