Infections caused by Legionella spp. produce a spectrum of symptoms, from mild upper respiratory tract infections to pneumonia. Legionella is responsible for 2’X,to 15% of community-acquired pneumonias.

Because not all hospitals routinely test for Legionella, the actual incidence is likely higher. These microorganisms are also associated with nosocomial infections.

Most human cases of legionellosis are caused by L. pneumophila. Other important species are L.

micdadei, L. longbeachae, L. dumoffi, and L. bozemanii.

Virulence Factors

Differing presentations of Legionella infection may be influenced by several factors, including the organism’s

ability to enter, survive, and multiply within the host’s cells, especially bronchoalveolar macrophages, and

the ability to produce proteolytic enzymes. Legionella spp. cause intracellular infections in humans but also survive in an extracellular environment.


Infections Caused by Pathogenic Legionella

Clinical manifestations of Legionella infections include febrile disease with pneumonia (Legionnaires’ disease), febrile disease without pulmonary involvement (pontiac fever), and asymptomatic infection. The mode of transmission and the number of infecting organisms in the inoculum playa role in the clinical features of the infection. In addition, host factors, such as a suppressed immune system, chronic lung disease, alcoholism, and heavy smoking, predispose

individuals to Legionnaires’ disease.


Legionnaires’ Disease

Legionnaires’ disease typically presents in three major patterns: (1) sporadic cases, which are most common and usually occur in the community; (2) epidemic outbreaks, characterized by short duration and low attack

rates; and (3) nosocomial clusters, occurring in compromised patient populations. Pneumonia is the predominant

manifestation of Legionella disease, and the organisms are among the top four causes of communityacquired

bacterial pneumonia. Streptococcus pneumoniae is the most common cause of bacterial pneumonia.

Mycoplasma pneumoniae, Chlamydophila (Chlamydia) pneumoniae, and Legionella produce symptoms different from S. pneumoniae and cause a disease sometimes referred to as atypical pneumonia. The mortality rate for Legionnaire’s disease is 15% to 30% and may approach 50% in patients with nosocomial pneumonia, if the correct diagnosis is not made early.

Serogroup 1 accounts for most cases (approximately 85%) of infection by L. pneumophila. Other L. pneumophila serogroups, commonly 4 and 6, and L. micdadei are more frequently implicated in clinical

infections than the other Legionella spp. The incubation period for Legionnaires’ disease is 2 to 10 days.

Patients typically present with a nonproductive cough, fever, headache, and myalgia. Later, as pulmonary infiltrates develop, sputum may be bloody or purulent.

Rales, dyspnea, and shaking chills are clinical manifestations of progressing disease.

Dissemination via the circulatory system may lead to extrapulmonary infections with or without pneumonia.

Infections of the kidneys, liver, heart, central nervous system, lymph nodes, spleen, and bone marrow as well as cutaneous abscesses have been described. Bacteremia, renal failure, liver function abnormalities, watery diarrhea, nausea, vomiting, headache, confusion, lethargy, and other central nervous system abnormalities have been associated with these infections.


Pontiac Fever

The non pneumonic form of Legionella infection, Pontiac fever, usually has an incubation period of

about 2 days. Patients are previously healthy individuals who complain of flulike symptoms of fever,

headache, and myalgia that last 2 to 5 days and then spontaneously subside. The incidence of Pontiac fever in the general population is unknown. L. pneumophila is responsible for most cases of this illness.



Most members of the family Legionellaceae are found worldwide, occurring naturally in aquatic sources, such as lakes, rivers, hot springs, and mud. Some species have been recovered only from environmental

sources. Because Legionella spp. can tolerate chlorine concentrations less than 2 to 3 mg/L, they resist water treatment and subsequently gain entry into and colonize human-made water supplies. Hot water systems,

cooling towers, and evaporative condensers are major reservoirs. Other sources include cold water

systems, ornamental fountains, whirlpools, humidifiers, and industrial process waters. The factors that

contribute to the ability of Legionella spp. to colonize these sources include the following:

The ability to multiply over the temperature range of 20° to 43° C and survive for varying periods at

۴۰° to 60° C The capacity to adhere to pipes, rubber, plastics, and sediment and persist in piped water systems

even when flushed The ability to survive and multiply within free-living protozoa and in the presence of commensal bacteria and algae Legionella spp. are transmitted to human hosts from these environmental sources primarily via aerosolized particles, like those produced by normal tap water pressure. Most outbreaks of disease originate from potable water distribution contamination.

Other means of transmission include aspiration of contaminated water or secretions, direct inoculation by respiratory therapy equipment, and immersion of wounds in contaminated water. Transmission between humans has not been demonstrated.


Several methods such as direct examination, culture, and antigen and antibody detection are available for the laboratory diagnosis of infections caused by Legionella spp. Most laboratories utilize more than one method to maximize their diagnostic capabilities.


Specimen Collection and Handling

Specimens for culture and direct examination commonly include sputum, bronchoalveolar lavage, and bronchial washings. Ingram and Plouffe recommend that sputum purulence screens not be used and that all sputum specimens be accepted for culture. Transtracheal aspiration, lung tissue, blood, wound and abscess material, and pleural, peritoneal, and pericardial fluids may also be submitted. Water from environmental sources may be cultured for epidemiologic investigation.

Urine is collected for antigen detection. Respiratory secretions and body fluids (except blood) are submitted in sterile, leak-proof containers.

Small pieces of tissue may be overlaid with sterile water. Saline or buffer should not be used in processing or transporting specimens because of the inhibitory effects of sodium on Legionella spp. Because overgrowth of contaminating flora may inhibit growth of Legionella species when transport of the specimens is prolonged, specimens should be refrigerated if more than 2 hours pass between collection and processing.

When samples are to be transported to a reference laboratory, the samples are placed on wet ice. Samples should be frozen at _70° C if processing will be delayed for several days.

For blood cultures, the Isolator (Wampole Laboratories, Cranbury, NJ) system, which uses the lysis centrifugation method, is preferred. Approximately 10 mL of blood is collected in the adult Isolator collecting

tube and transported to the laboratory for processing.

Legionella spp. can also be isolated from Bactec or BacTAlert blood culture bottles (BD, Sparks, Md).

Urine specimens for antigen testing are collected in sterile, leak-proof containers and assayed within 24 hours of collection. If testing is delayed, specimens should be stored at 2° to 8° C or frozen at _20° C.


Microscopic Examination

Legionella spp. are pleomorphic, weakly staining, gram-negative rods that are approximately 1 to 2 Ilm by 0.5 Ilm in size. Extending the safranin counterstaining time to at least 10 minutes can enhance the staining intensity of the organisms. L. micdadei is weakly acid fast in tissue and stains best with the modified Kinyoun procedure (see Chapter 7). Other stains, including Diff-Quik (Baxter Scientific, Kansas City, Mo) and Giemsa, may be used to facilitate observation of the organisms.

Nonspecific-staining methods are most useful for examination of specimens from normally sterile sites

The faint-staining, pleomorphic gram-negative rods may be found outside of and within macrophages and

segmented neutrophils (Figure 19-27). The modified Kinyoun procedure can be used for tissue if L. micdadei

is suspected. The direct fluorescent antibody (DFA) test is discussed in the Rapid Methods section.

The DFA test also provides a useful method of confirming that an isolate is a Legionella sp. and for identifying the more common species and serogroups of the genus.

Isolation and Identification

Isolation Methods

Acid treatment of specimens contaminated with bacteria before inoculation enhances isolation of Legionella

spp. (Figure 19-28). In this procedure, an aliquot of the specimen is first diluted 1:10 with 0.2 N KCI-HCI and

allowed to stand for 5 minutes (procedure 19-1). Then the medium is inoculated with a portion of the acid- treated specimen. Inoculated medium is incubated at 35° to 37° C in air for at least 7 days. Usually within 3 to

۵ days, Legionella spp. are visible.

The single most important test for Legionnaires’ disease is culture of the organism. Legionella spp. are

fastidious, aerobic bacteria that are unable to grow on sheep blood agar (SBA) and require the amino acid

L-cysteine for growth. The organisms may appear as tiny colonies on chocolate agar (CHOC) that contains L-cysteine. However, buffered charcoal yeast extract (BCYE) agar is the recommended medium for Legionella

isolation and is available commercially as nonselective and semiselective media. Semiselective BCYE contains

polymyxin B, anisomysin, and either vancomycin (pAY) or cefamandole (PAC) and improves recovery of Legionella spp. from highly contaminated specimens, but it can also inhibit growth of some Legionella spp.

Consequently BCYE with and without antimicrobial agents should be used for culture.

Colonial Morphology

On BCYEmedium, colonies appear as grayish-white or blue-green, convex, and glistening, measuring approximately 2 to 4 mm in diameter. When these colonies are viewed with a dissecting microscope illuminated from above, they reveal a characteristic appearance (Figure 19-29). The central portion of young colonies has a “ground-glass” appearance, light gray and granular, whereas the periphery of the colony has pink and/or light blue or bottle green bands with a furrowed appearance. Plates should be examined daily, because older colonies lose these characteristic features and may be mistaken for other bacteria. Plates with suspicious colonies can also be illuminated with a longwave ultraviolet light (366 nm) and examined for differences in colonial autofluorescence to detect possible mixed Legionella infections (Box 19-1)



۱٫ Buffered charcoal yeast extract (BCYE) nonselective and selective plates

۲٫ KCI/HCI buffer, pH 2.2

Add 5.3 mL of 0.2 N HCI and 25 mL of 0.2 N KCI to 100 mL of distilled water. Adjust pH to 2.2 with HCI

or KCI.

Dispense 4.5 mL into small screw-capped tubes, and autoclave for 10 minutes at 121° C.

Store under refrigeration. Expiration 6 months.


۱٫ Prior to medium inoculation, perform the following

procedures to obtain the proper inoculum:

· Centrifuge liquid specimens in excess of 2 mL at

۳۰۰۰ rpm for 10 minutes and 50-mL water

samples at 3000 rpm for 30 minutes. Use the

sediment as inoculum.

· Homogenize pieces of tissue in 1 mL of sterile distilled water using a sterile tissue grinder.



· Prepare concentrates from Isolator or other blood culture bottles.

۲٫ Inoculate one BCYE plate and one selective BCYE

plate with an aliquot of the sample and streak for

isolation. Note that for water samples this step

should be omitted. Proceed to step 3.

۳٫ Additionally, specimens likely to be contaminated

with bacterial flora are processed as follows:

· Add 0.5 mL of specimen to 4.5 mL of sterile HCl-

KCI buffer pH 2.2 in a sterile screw-capped tube.

Place three to four sterile glass beads in the tube

if the specimen is excessively mucoid. Recap the

tube, mix the specimen with the buffer, and

break up the mucus using a vortex mixer.

· Let the suspension stand for 5 minutes.

· Pipet 0.1 mL of the suspension to a nonselective

BCYE plate and 0.1 mL to a selective BCYE plate.

· Spread the inoculum over the surface of each


۴٫ Incubate the plates aerobically at 35° to 37° C for

۷ days.

۵٫ Examine cultures daily using a dissecting microscope illuminated from above .

Identification Methods

Conventional Methods

The physical and biochemical properties of Legionella spp. are listed in Box 19-2.However, biochemical testing has limited value in the presumptive identification of isolates to the species level. The confirmation of the suspected colonies and their presumptive identification as Legionella spp. can be achieved in the microbiology laboratory using growth requirement for L-cysteine testing, Gram stain, and DFAtests (Figure 19-30).The confirmation of the suspected colonies and their presumptive

identification as Legionella spp. can be achieved in the microbiology laboratory using the following protocol:

Prepare a smear from the suspicious colony growing on BCYE medium, and perform a Gram stain.

Legionella spp. are thin, gram-negative rods that may show size variation from 2 to 20 11min length.

· Subculture the isolate to BCYEwith L-cysteine and to either SBAor BCYEwithout L-cysteine. Legionella spp. grow only on BCYEmedium supplemented with L-cysteine.

· Prepare smears from colonies that require L-cysteine for growth and test with polyvalent and monovalent conjugates to determine specific species and serogroup.

Definitive identification is usually performed at reference, public health, or state laboratories.

Rapid Methods Urine Antigen Test The Food and Drug Administration (FDA) has approved several different assay methods such as radioimmunoassay, microplate enzyme immunoassay, and rapid immunochromographic

assay, for Legionella antigen detection in urine specimens. The Binax NOW (Binax, Inc., Portland, Me) is a 15-minute immunochromographic assay that detects soluble antigen from L. pneumophila serogroup 1,

which is responsible for up to 85% of the cases of legionellosis. The test has a reported sensitivity of

۹۷٫۱% and a specificity of about 100%. The antigen can be detected as early as day 3 of the infection and can

persist up to a year; consequently the test is of limited value in persons with a recent history of Legionella infection.

Enzyme immunoassays are also used by many clinical laboratories. Compared with culture, when using

concentrated urine the assays, sensitivities range from 90% to 94% and the specificity ranges from 97% to

۱۰۰%. With a sensitivity of 95% and a specificity of 95%, the positive predictive value drops to 90.5%, which means 1 of every 10 positive test results is a false positive. Furthermore, as the prevalence of Legionella infections decline, the false-positive rate increases, making it more important to confirm results with culture,

especially when determining the dynamics of this organism in a given patient population.

In addition to persistent antigenuria following clinical disease mentioned earlier, prolonged secretion has

also been associated with immunosuppression, renal failure, and chronic alcoholism. Conversely, early antimicrobial intervention with macrolides may decrease antigen excretion in some patients. Despite some limitations, these rapid tests represent an important step forward in timely diagnosis of Legionella infections.

Direct Fluorescent Antibody Test The DFAtest is a rapid laboratory procedure for detection of the more

common species of Legionella found in clinical samples from the lower respiratory tract. Available are



isothiocyanate (FlTC)-labeled conjugates that detect all known serogroups of L. pneumophila (Genetic

Systems, Seattle, Wash) and L. pneumophila groups 1 to 7, L. bozeman ii, L. dumoffii, L. gormanii, L. longbeachae groups 1 and 2, L. micdadei, and L. jordan is. The conjugate binds to antigens on the cell surface, and these antigen-antibody complexes are detected using a fluorescence microscope. The organisms thus appear

as bright yellow to green, short or coccobacillary rods with intense peripheral staining (Figure 19-31).

Because of specificity and sensitivity issues, it is recommended that the DFA test should only be performed

when specifically requested by the clinician, the Gram stain or special stains reveal suspicious organisms, or a large number of neutrophils are present but no organisms are seen. The microscopist must have experience with the morphologic and staining features of Legionella spp. so atypical organisms will not be misidentified. The specificity of the DFAtest has been reported to be 94% to 99%, but some species of Pseudomonas, Bacteroides, Corynebacterium, and other bacteria can cross-react with the polyvalent conjugates. Some laboratories prefer not to use polyvalent reagents for direct specimen examination because of these cross-reactions.

The sensitivity of DFA testing for direct specimen examination is approximately 25~, to 80~) when compared with that of culture, so a negative result does not rule out Legionella infection. To visualize organisms, approximately 10,000 to 100,000 organisms/mL of specimen must be present. Other factors such as excessively

thick smears, which tend to obscure microorganisms, and the technical skill of the observer may influence the

sensitivity of the test. Consequently, DFAshould not be the only test used.

DNA Detection The first commercially available DNA test kit (Gen-Probe, San Diego, CaliO used an 12sl-labeled, single-stranded DNA probe that would hybridize with the target organism’s ribosomal RNA, and detection was by a gamma scintillation counter. However, this assay has been removed from the market. The LightCyclerpolymerase chain reaction (LC-PCR; Roche, Basel, Switzerland) has demonstrated a high degree of sensitivity and specificity; however, products are not currently available for clinical use.

Serologic Testing

The indirect fluorescent antibody OFA)assay is the most common method employed for the serologic diagnosis

of Legionnaires’ disease, although other methods such as enzyme immunosorbent assay are available. For IFA,

heat- or formalin-killed bacteria are fixed to a microscope slide. Higher titers can be seen with heat treated

organisms; however, less cross-reactivity occurs with formalin preparations. Cross-reacting immunoglobulins

have been reported in patients with an infection caused by gram-negative bacilli, Mycoplasma, and Chlamydia.

The sensitivity of serologic tests is reported to be 75% to 80%, with a specificity of 90% to 100%. The specificity of the test is enhanced when paired sera from patients with symptoms of legionellosis are tested. A fourfold rise in lFA titer to at least 1:128 from the acute serum phase, obtained within 1 week of onset of symptoms, to the convalescent serum phase, 3 to 6 weeks later, is evidence of recent infection. However, patients with disease may not demonstrate a rise in titer for 8 weeks or longer after symptoms commence


Legionella are gram-negative rods that are ubiquitous in aquatic environments and infect humans through the respiratory route to cause febrile disease with or without pneumonia. The laboratory diagnosis of Legionella infection is accomplished by cultivation on specialized media and rapid assays such as urine antigen testing and DFA. Excellent therapeutic response can be expected with macrolides as long as antimicrobials are started early in the clinical course.



Dominguez JA et al: Comparison of Binax Legionella urinary antigen enzyme immunoassay (ElA) with the Biotest

Legionella urine antigen EIA for detection of Legionella antigen in both concentrated and unconcentrated urine

samples, J Clin Microbio134:1579, 199

Dominguez J et al: Assessment of a new test to detect Legionella urinary antigen for the diagnosis of Legionnaires’

disease, Diagn Microbiollnfect Dis 41:199,2001.

Fields BS et al: Legionella and Legionnaires’ disease: 25 years of investigation, Clin Microbiol Reviews 15:506, 2002.

File TM et al: The role of atypical pathogens: Mycoplasma pneumoniae. Chlamydia pneumoniae, and Legionella pneumophila in respiratory infection, Infect Dis Clin North Am 12:569, 1998.

Finkelstein R et al: Diagnostic efficacy of a DNA probe in pneumonia caused by Legionella species, J Med Microbiol

۳۸: ۱۸۳, ۱۹۹۳٫

Goetz A, Yu VL: Screening for nosocomial legionellosis by culture of the water supply and targeting of high-risk

patients for specialized laboratory testing, Am J Infect Control 19:63, 1991.

Gray JJ et al: Serological cross-reaction between Legionella pneumophila and Citrobacter freundii in indirect immunofluorescence and rapid microagglutination tests, J Clin Microbiol 29:200, 1991.

Hack~an BA et al: Comparison of Binax Legionella urinary antIgen EIA kit with Binax RIA urinary antigen kit for

detection of Legionella pneumophila serogroup 1 antigen, J Clin MicrobioI34:1579, 1996.

figure 19-29 figure 19-30 figure 19-31 box 19-1 box 19-2 figure 19-27 figure 19-28

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