BacteriologyDesign and implementation of a protocol for the detection of Legionella in clinical and environmental samples
Introduction
Whereas the bacteria of the genus Legionella are responsible for an acute self-limiting nonpneumonic illness known as Pontiac fever, a far more significant public health issue is the potentially fatal pneumonic form of legionellosis, Legionnaires' disease. Legionella pneumophila is the most common pathogenic species within the genus Legionella, accounting for 90% of all reported cases of legionellosis in the United States (Fields et al., 2002). L. pneumophila serogroup 1 accounts for the majority of these (Rantakokko-Jalava and Jalava, 2001). Other species of Legionella that have been implicated in disease include Legionella dumoffii, Legionella micdadei, Legionella bozemanii, and Legionella longbeachae; however, they occur at much lower frequencies. As natural inhabitants of aquatic environments, legionellae are ubiquitous. However, their presence can be particularly problematic when found in the water distribution systems of healthcare facilities. Of particular concern is the risk of nosocomial and community-acquired infection of the immune compromised, the elderly, and individuals with underlying respiratory conditions via aerosolization of water from contaminated water supplies.
Currently, culture is considered to be the standard technique for the identification of Legionella. However, a single ideal test for Legionella does not exist. The fastidious nature of the organism and the prolonged incubation periods necessary for growth can make isolation and identification challenging (Ng et al., 1997) for the diagnostic laboratory. In its clinical manifestations, a pneumonia caused by Legionella is not always distinguishable from pneumonias of other bacterial etiologies (Mulazimoglu and Yu, 2001); this lack of specific symptoms can contribute to delay in isolation and identification of the causal agent. Rapid diagnosis is important because legionellosis can result in case fatality rates exceeding 15% to 20%, and patients with severe underlying disease are at particular risk (Edelstein, 1995). When environmental water samples are analyzed, legionellae can be difficult to isolate from samples that are grossly contaminated with other organisms. In such a situation, the source of the nosocomial or community-acquired infection could go unidentified as increasing numbers of individuals fall sick.
Rapid methodologies used for the identification of Legionella include urine antigen detection, direct fluorescent antibody (DFA), and polymerase chain reaction (PCR). The most widely used urine antigen detection test, which is considered to be specific for L. pneumophila serogroup 1, can miss as many as 40% of legionellosis cases. As when DFA testing is used, the sensitivity from respiratory samples for the diagnosis of Legionnaires' disease has ranged from 25% to 75% (Fields et al., 2002), and specificity can be an issue.
A rapid, sensitive, and specific method for the detection of Legionella is clearly important to identify infected individuals and to expedite cleanup of contaminated water systems so as to prevent additional cases of infection. Our laboratory performs Legionella testing for numerous hospitals and facilities in New York State each year in the context of outbreak and remediation investigations. To assist in this testing, we have designed a novel multiplex Taqman® real-time PCR assay to detect both the Legionella genus and, specifically, L. pneumophila serogroups 1 to 16. Our multiplex assay is the 1st to use the 23S rRNA gene as a target to detect 42 species and 19 serogroups of Legionella; this design enables rapid, sensitive, and specific detection of Legionella spp. and L. pneumophila in a single reaction from both environmental and clinical samples. In addition, we describe and assess a testing algorithm that combines this assay with culture and 16S rRNA gene sequence analysis. Use of this algorithm resulted in markedly improved sensitivity and specificity for the clinical and environmental Legionella samples received by our laboratory.
Section snippets
Bacterial strains
The bacterial strains were supplied by the American Type Tissue Culture Collection (ATCC), the Centers for Disease Control and Prevention (CDC), and the New York State Department of Health Wadsworth Center Bacteriology Laboratory Culture Collection. All Legionella strains tested (Table 3) were grown on buffered charcoal yeast extract (BCYE) agar and incubated at 37 °C in 5% CO2. A 1.0-McFarland suspension was made of each isolate, and each was heat treated at 95 °C for 20 min. The additional
Optimization of multiplex real-time PCR assay
Our multiplex assay was initially evaluated as 2 separate assays, 1 for the mip gene and 1 for the 23S rRNA gene. Primer, probe, and MgCl2 titrations were performed. Optimal concentrations for each component were determined by testing replicate samples of a 10−4 dilution of L. pneumophila serogroup 1 and plotting the concentration of each component against the Ct value. Final concentrations of 4 mmol/L MgCl2, 900 nmol/L of each primer, and 250 nmol/L of each probe were established via this
Discussion
Legionella bacteria are associated with potentially fatal pneumonias and can be found as natural inhabitants of aquatic environments such as water distribution systems, cooling towers, and fountains. Investigation and remediation of Legionella outbreaks are essential to ensure the rapid identification of a new or recurring source, as well as to assess whether the problem has been resolved, so that patients or the community are no longer at risk of acquiring a Legionella infection.
To improve the
Acknowledgments
The authors acknowledge the Wadsworth Center Molecular Genetics Core Facility for the synthesis of oligonucleotides and Dr William Wolfgang and Dr Adriana Verschoor for critical readings of this manuscript.
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