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Legionella bacteria are ubiquitous in aquatic environments and are a rare cause of community-acquired pneumonia. Despite being recognized as human pathogens for over 4 decades, the diagnosis of Legionnaires’ disease is often missed due to lack of clinical awareness and the fastidious nature of the organism. Although some molecular diagnostic panels for respiratory tract infections now include L. pneumophila as a target, there remains a need for clinical laboratories to perform Legionella culture, especially given Legionnaires’ disease’s increased incidence in recent years.

Public Health Importance of Legionella

The World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) both recognize the public health significance of Legionella. Since 2003, rates of reported cases of Legionnaires’ disease have risen in the °®¶¹´«Ã½ States, with , although the actual disease burden is probably much higher. The causes of this steady increase are unclear, but have been proposed. These include the increase in susceptibility of the population (due to age and other risk factors), aging plumbing infrastructure and increased testing for Legionnaires’ disease.

Several high-profile outbreaks of Legionella have made headlines in the past few years. , where a total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Interestingly, the presence of L. pneumophila isolates highly related to the outbreak strain in multiple environmental samples and from past Legionnaires’ outbreaks suggested that there may be a potentially pathogenic “endemic” strain in the Bronx community. Another widely publicized Legionella outbreak associated with cooling towers was at ., where 2 cooling towers were associated with 22 cases of Legionnaires’ disease among 3 park employees and 19 individuals who visited the park in 2017. A subsequent investigation revealed that the park did not follow proper guidelines to disinfect its cooling towers, which may have resulted in high levels of Legionella within the structures. Legionnaires’ disease outbreak can also be associated with hotel stays and cruise ship travel. In 2015, in water sources supplying the ship, leading to an outbreak among disembarking passengers. Upon investigation, on-deck whirlpools seem to be a common source of contamination.

As evidenced by these outbreaks, Legionella is a common contaminant of human-made water systems. Colonization in such water sources by the organism can lead to an outbreak of Legionnaires’ disease, especially in facilities that house individuals with high risk of developing severe disease, such as hospitals and long-term skilled nursing facilities. During the COVID-19 pandemic, certain businesses and facilities have been closed for a long time. Water systems that are not in operation facilitate growth of Legionella (and other biofilm-forming organisms). Once these facilities reopen, there is potential to see an increased number of cases of Legionnaires’ disease. 

Characteristics of Legionella

Legionella spp. are fastidious, gram-negative, aerobic, monopolarly-flagellated rods.  , there are 62 validly published species encompassing at least 70 serogroups. Aquatic (such as streams, rivers and ponds) and soil environments are thought to be natural reservoirs for these bacteria, while humans are accidental hosts. , with high tolerance to a wide range of temperatures (0–68 °C) and pH (5.0–8.5). , which may allow them to resist chlorination and other decontamination procedures. These qualities allow them to colonize and proliferate in building water supply systems, cooling towers and hot water systems. They are often isolated from shower heads, taps, whirlpool spas and respiratory ventilators. Devices that aerosolize water from contaminated sources may play a role in dispersing the organism, resulting in human infections. Once colonization in a water source is established, they are virtually impossible to completely eradicate. Among members of the genus, .

Diseases caused by Legionella are collectively named legionellosis. Legionnaires’ disease is the pneumonic form, while Pontiac fever generally manifests as a self-limited influenza-like illness. L. pneumophila is the most extensively studied pathogenic species in the genus. Similar to some other members of the genus, L. pneumophila is considered an intracellular parasite of such as Acathamoeba, Naegleria and Vermamoeba. 

Laboratory Diagnosis of Legionnaires’ Disease

The clinical presentation of acute pneumonia caused by Legionella, which may range in severity from mild to fatal, is not distinguishable from that of acute, community-acquired pneumonia of other etiologies. In addition to the awareness of signs and symptoms of Legionnaires’ disease, thorough history taking and recognition of risk factors for developing disease are key to determining whether an individual should be tested. that clinicians can use to  decide whether to send specimens for laboratory examination. Legionnaires’ disease should be included in the differential diagnosis for community-acquired pneumonia, , such as age ≥50 years, history of smoking, chronic lung disease, immunocompromised status, systemic malignancy or other underlying medical conditions. It is important to note that Legionella can also cause healthcare-associated infections, which should be considered if there appear to be clusters of cases in a 12-month period or there is a change in the quality of a facility's water supply that may lead to growth of Legionella.

Urinary Antigen Detection

One of the most used laboratory methods to diagnose Legionnaires’ disease is detection of Legionella antigen in urine. These tests are . The use of antigen tests has become increasingly commonplace and so little is understood about the principle of the assay that occasionally providers will order a urine culture for Legionella. These tests were originally enzyme immunoassays (EIAs), but have since been developed as lateral flow immunochromatographic tests, which are even easier to perform and provide rapid results. Legionella antigen can be detected one day after symptom onset and may persist for weeks.

A major limitation of urinary antigen tests is their inability to reliably detect Legionella antigens from organisms other than those in serogroup 1. Although most L. pneumophila from human specimens in the U.S. are of serogroup 1 (which accounts for ), other serogroups of L. pneumophila can also cause Legionnaires’ disease and could be missed if the laboratory diagnosis relies solely on urine antigen tests. Moreover, the , from 40% in mild cases to 100% in severe cases. If possible, a lower respiratory tract specimen should also be collected for Legionella culture to ensure detection and recovery of non-serogroup 1 Legionella. 

The prevalence of disease caused by non-serogroup-1 organisms is probably underestimated due to limitations of the urine antigen tests that are commonly used to establish a laboratory diagnosis for Legionnaires’ disease. In certain regions of the world, such as, L. pneumophila serogroup 1 was found to be associated with only 50% of community-acquired Legionnaires’ disease, while L. longbeachae accounts for up to 30% of all cases.

Direct Microscopic Examination

It may be possible to visualize Legionella directly in clinical specimens via microscopy. However, microscopic examination of primary specimens could be very challenging. Conventional staining with Gram stain is of very limited utility, even with an alternative counterstain, such as basic fuchsin. Commercially available direct fluorescent antibody kits allow clinical and public health laboratories to perform microscopic examination for Legionella directly from clinical specimens, although .

Nucleic Acid Amplification Tests (NAATs)

Although there is currently no U.S. Food and Drug Administration (FDA)-cleared commercial NAATs dedicated solely to the detection of Legionella, laboratory-developed tests have been successfully implemented in research and clinical laboratories. , including the 16S ribosomal RNA gene (rDNA), the 23S-5S spacer region, 5S rDNA or the macrophage inhibitor potentiator (mip) gene have been evaluated as PCR targets. In the past few years, for the detection of viral and bacterial pathogens in lower respiratory tract specimens. 

Culturing Legionella

For all cases suspected of Legionnaires’ disease, lower respiratory tract specimens, which may include . For sputum, it is important not to use the usual microscopic sputum screening criteria to determine whether a specimen is adequate for culture since specimens from patients with Legionnaires’ disease may not contain many white blood cells. Pleural fluid usually has low yield for Legionella recovery and concentration of specimens by centrifugation should be performed prior to culture inoculation. KCl acid wash treatment prior to inoculation can be performed to reduce growth of contaminating flora. Media commonly used for isolation of Legionella include buffered charcoal-yeast extract agar (BCYE-α) with and without antimicrobials. 

Visible Legionella colonies usually do not appear before day 3 of incubation, so it is important to examine all agar plates daily to identify other organisms that may

appear before Legionella growth can be seen. All growth on each plate should be examined using a dissecting microscope. Legionella colonies are usually circular with an entire edge and may have a distinctive appearance described in most textbooks as “ground-glass.” L. pneumophila colonies may also exhibit a faint yellow fluorescence when examined under UV light, while other Legionella species may have different fluorescence characteristics. Gram stain using basic fuchsin as a counterstain should demonstrate gram-negative pleomorphic rods, with cellular morphology ranging from small coccobacilli to filamentous rods. A presumptive identification can be obtained by the absence of growth following subculturing onto media lacking L-cysteine, which does not support the growth of Legionella. 

Although Legionella are generally inert biochemically, certain basic biochemical tests can rule out other organisms. These include catalase (positive or weakly positive), oxidase (variable) and urease (negative). Definitive identification may be performed using latex agglutination to determine serogroups, DFA, DNA sequencing or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF; although of this method in identification of non-pneumophila Legionella species). 

Legionella Testing in Public Health Laboratories

Public health departments are almost always involved in epidemiological investigation after clusters of Legionnaires’ disease cases are reported, and public health laboratories play a crucial role in identifying potential sources in each outbreak. Most public health laboratories can perform diagnostic testing on clinical specimens and surveillance testing on environmental samples. The that state and public health laboratories may use to develop Legionnaires’ disease testing and outbreak response capacities. Additionally, the program from the CDC offers accreditation for laboratory proficiency in performing environmental testing for Legionella.

During a visit to the site at which a possible Legionella outbreak is reported, investigators typically collect 1-10 L of water from all potentially contaminated sources in sterile bottles containing sodium thiosulfate (to neutralize disinfectants). They also collect swabs of surfaces on which the bacteria are likely to form biofilms, such as faucets, aerators, shower heads and collection basins in cooling towers. At the public health laboratory, samples are directly inoculated into growth media or filtered to concentrate the organism prior to inoculation. For samples with high bacterial load, acid treatment reduces the number of non-Legionella bacteria. The media used to grow Legionella in public health labs are very similar to those used in the clinical labs. formulated to detect enzymatic activities specific to Legionella can be used in conjunction with culture trays containing wells of different sizes to allow for concurrent detection and enumeration of Legionella based on the . Once Legionella is isolated, public health laboratories can perform typing to determine relatedness between environmental and human isolates to support epidemiological investigations.

The Role of Clinical Laboratories in Legionella Outbreak Investigations

Clinical laboratories also play an important role in Legionnaires’ disease outbreak investigation, as they almost always are the first to identify clusters of cases. Unfortunately, some clinical laboratories no longer maintain the capability to perform Legionella culture and rely solely on culture-independent methods, such as urine antigen tests or molecular assays. By the time a potential outbreak is identified, if the clinical laboratory has not already isolated Legionella bacteria from potentially involved cases, it is often already too late for investigators to obtain original specimens so that public health laboratories can attempt to recover the organism. To establish a potential causative association between a contaminated water source and Legionnaires’ infected cases, public health laboratories perform sequence-based typing (by Sanger and/or whole genome sequencing) to determine relatedness between Legionella isolates from clinical specimens and environment samples. Another benefit of Legionella culture is its capability to identify Legionella isolates that may otherwise be undetectable by culture-independent methods. 

Although Legionnaires’ disease is not commonly encountered, a laboratory diagnosis often carries epidemiological significance that may lead to life-saving remediation of contaminated water sources. Identification of potential cases by clinical laboratories, coupled with their ability to perform Legionella culture and prompt reporting to public health authorities, are crucial to the effort to protect the public from preventable morbidity and mortality caused by Legionnaires’ disease.

The statements and opinions expressed in this article are those of the author and do not necessarily reflect those of Los Angeles County Department of Public Health and Public Health Laboratories, nor of the American Society for °®¶¹´«Ã½.

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