Chapter 34 Legionella and Legionella-like Organisms
Richard W. Gilpin, PhD
Associate Professor of Microbiology and Immunology
The Medical College of Pennsylvania
In, Levison, Matthew E (ed): The Pneumonias: Clinical Approaches to Infectious Diseases of the Lower Respiratory Tract
John Wright | PSG Inc. Boston, 1984
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Legionella pneumophila structure in thin section
Taxonomic Classification of Legionella and LLO
Legionella pneumophila Serogroup Strains
Physiological Classification of Legionellae
Distribution in Nature
DESCRIPTION OF CLINICAL DISEASE
Direct Fluorescent Antibody (DFA) Test
Indirect Immunofluorescence (IFA) Test
Other Diagnostic Tests
Legionella and Legionella-like organisms (LLO) are gram-negative bacilli which have a uniform width (0.3 to 0.9 µm) and length (2.5 µm). A few bacteria in some isolates are much longer. The bacteria usually are found as individual cells but may form chains or long filaments. Legionella and LLO have an outer and inner membrane structure typical of gram-negative bacteria (Figure 34-1). Electron lucent cytoplasmic vacuoles, which probably contain poly, 6-hydroxybutyrate, are often observed (Figure 34-1). These vacuoles appear as large bumps within Legionella in scanning electron micrographs. These bacteria do not form endospores and are not encapsulated. Legionella have a branched-chain fatty acid composition that is unusual for gram-negative bacteria and affords identification based on analysis of cellular fatty acids by gas-liquid chromatography.
A single polar flagellum has been found on isolates that have been grown on media that permits rapid growth. Flagellated Legionella have also been observed in human lung tissue in areas that do not have a dense population of Legionella or do not have a heavy infiltrate of inflammatory cell debris. Pili (fimbriae), one half the diameter of the flagellum, have also been observed by electron microscopic studies of Legionella grown in-vitro.
There are no unique morphological characteristics that can easily distinguish Legionella from other gram-negative rods. The cytoplasmic vacuoles, flagellum, and pili are not always observable and may be missing in some species.
Legionella and LLO from in-vitro cultures all stain gram negative. The pink color may be faint, however, unless the safranin or carbol fuchsin (preferred) counterstain is left on smears for at least one minute. Legionella in tissue or other clinical specimens will usually not take the gram stain. Therefore, the gram stain procedure has limited usefulness for identification of these bacteria in clinical material. Legionella and LLO are not considered to be acid-fast.
Legionella pneumophila structure in thin section
-available at https://www.researchgate.net/publication/293653644_Legionella_and_legionella-like_organisms
The Legionella were fixed with glutaraldehyde, followed by osmium tetroxide, dehydrated in ethanol, embedded in Epon and sectioned. Sections were stained with uranyl acetate and lead citrate. The wrinkled cell wall outer membrane and the inner cytoplasmic membrane are quite apparent. Two electron-lucent cytoplasmic inclusions can be seen. Magnification: 140,000 X.
There are two staining procedures that may identify bacteria such as Legionella in lung or other tissues. Various modifications of the Giemsa stain have produced variable results because of differentiating Legionella from a background of inflammatory cell debris. The Dieterle silver impregnation stain originally developed for staining spirochetes in tissue is useful, because the bacteria appear as dark red rods against a yellow to pink background of cellular debris. The Warthin-Starry silver impregnation method also produces satisfactory results. Other standard histological stains, including hematoxylin and eosin, are rarely useful.
Flagella may be stained by the Leifson flagellar stain, but this has limited application for clinical diagnosis. Beta-hydroxybutyrate inclusions of Legionella may stain blue-black with the Sudan Black B fat stain, but this also has limited diagnostic usefulness.
Legionella and LLO are unusual because they do not grow on the routine bacteriological media available in clinical microbiology laboratories. This includes sheep blood agar, chocolate agar, nutrient agar, Mueller-Hinton agar, trypticase soy agar, and all the selective/differential agar or broth media used for gram-negative enteric bacteria. There is some evidence to suggest that peroxides in the above culture media may inhibit the growth of Legionella. For this reason, the types of media for Legionella culture will be reviewed. The first in-vitro culture medium to be developed utilized a Mueller-Hinton agar base, modified by the addition of 1%o IsoVitalex and 1% hemoglobin. However, Legionella growth on this medium is slow. It was soon discovered that these bacteria grow better on a medium containing casein hydrolysate plus beef extractives or yeast extract when two filter-sterilized supplements, L-cysteine and ferric pyrophosphate-soluble were added. These media are known as Feeley-Gorman (FG) agar and charcoal (0.2% activated charcoal) yeast extract (CYE) agar, respectively. Some Legionella do not grow well on FG agar. Therefore, the current medium of choice is CYE agar modified by the inclusion of a buffer. The most commonly used filter sterilized broth medium is the same as CYE agar with the omission of activated charcoal and agar. Autoclaved broth medium may not support growth.
Further modifications of CYE agar have been made to differentiate between the various species of Legionella and inhibit growth of other bacteria. Buffered CYE agar medium [BCYE] containing glycine, alpha-ketoglutarate, cefamandole, polymyxin B, anisomycin, vancomycin, bromthymol blue, and bromcresol purple may permit isolation of Legionella from samples that contain high numbers of other bacteria. However, buffered CYE agar remains the first choice for primary isolation of Legionella and LLO. Stock cultures can be stored in the dark at room temperature on CYE agar slants for at least two months.
Legionella grow best on CYE medium in humid air supplemented with 2.5% CO2 at 35°C. Primary cultures from clinical sources may require five to seven days before colonies can be seen. Unfortunately, other bacteria present in a specimen may overgrow and/or inhibit the growth of Legionella during this prolonged incubation period. Primary clinical or environmental samples may need to be passed through guinea pigs and/or embryonated hen eggs before Legionella or LLO can be isolated on CYE agar. Legionella and LLO are aerobic bacteria that utilize amino acids as a source of carbon and energy. However, Legionella may use some carbohydrates as a carbon source.
Speciation of Legionella and LLO has moved rapidly during the last few years. There are currently nine proposed species; L. pneumophila, L. micdadei, L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. wadsworthii, L. jordanis, and L. oakridgensis. There are additional LLO isolates that have not been classified at this time. There seems to be a precedent for retaining the genus name, Legionella, for the isolates that have been characterized. The current literature also uses other taxonomic names, original isolate numbers or letter designations, and even different genus names. Therefore, the following tables have been compiled to show the taxonomic (Table 34-1) and physiological (Table 34-3) differences among the Legionella that have been investigated.
There are currently eight serogroups of L. pneumophila (Table 34-2). Most of the sporadic clinical cases of Legionnaires' disease studied through 1979 were caused by L. pneumophila, serogroup 1, followed by serogroups 2, 3 and 4. Data is incomplete on the frequency of serogroups 5 and 6 among clinical cases. There appears to be a Legionella common antigen, termed F-1, which is located on the surface of the bacterium.
The genome size of Legionella is in the range of 2.5 x 109 daltons and the guanine plus cytosine (GC) content is between 35% and 43%, with an average content of 39%. No genetic relationship to previously known bacteria has been established by DNA hybridization studies. Plasmids have been detected in some Legionella isolates, but what they code for is not known.
Taxonomic Classification of Legionella and LLO
Proposed Name Original Isolate Designation* Other Proposed Name
Legionella pneumophi!a Legionnaires' disease bacillus, Philadelphia, OLDA None
L. micdadei TATLOCK, HEBA, Pittsburgh pneumonia agent (PPA) Tatlockia micdadei L. pittsburghensis
L. bozemanii WJGA, MI-15 Fluoribacter bozemanae
L. dumoffii NY-23, Tex-K1 Fluoribacter dumoffii
L. gormanii LS-13 Fluoribacter gormanii
L. longbeachae Long Beach 4 None
L. jordanis BL-540 None
L. oakridgensis Oak Ridge 10 None
L. wadsworthii Wadsworth 81-716A None
*Italicized original isolate designation is the type strain for that species.
Legionella pneumophila Serogroup Strains
Bacterial Isolate Serogroup
Knoxville 1 1
Togus 1 2
Bloomington 2 3
Los Angeles 1 4
Dallas IE 5
Chicago 2 6
Chicago 8 7
Concord 3 8
Physiological Classification of Legionellae*
- available at https://www.researchgate.net/publication/293653644_Legionella_and_legionella-like_organisms
Distribution in Nature
Legionella and LLO are distributed ubiquitously in natural aquatic environments. Legionella are also found in man-made aquatic environments such as shower heads, faucets, hot water tanks, heat rejection devices like cooling towers and evaporative condensers used for air conditioning, and, most recently, recreational whirlpools. There does not seem to be a human or animal reservoir. Cases of legionellosis have been found in most areas of the United States and in many countries around the world.
Epidemiological investigations of legionellosis have been based on retrospective evaluations of patient sera and postmortem tissue. In studies of sera from adult populations, approximately 4% to 5% of the people have antibody titers to serogroup 1 L. pneumophila, which suggests possible past infection but not necessarily disease.
A retrospective study by England et al. (1981) of the first 1005 cases of documented sporadic legionellosis caused by L. pneumophila serogroups 1-4 produced many interesting results. Cases were reported in all states, except Alaska and South Dakota. The attack rate in 1978 was 2.4 cases per million population. Ages ranged from 16 months to 89 years with a median age of 55 years for males and 56 years for females.
The relative risk for males was 2.6-fold higher than for females. Currently, 250,000 cases of legionellosis are estimated to occur each year in the United States. Sporadic cases seem to be more common than outbreaks. The epidemiological relationships to age, underlying disease, immunosuppression, smoking, travel, and exposure to recent excavations are similar for both sporadic cases and outbreaks.
Sporadic cases and outbreaks of legionellosis in the United States and in other countries occur more frequently during the summer months. Isolation of L. pneumophila from natural lakes or ponds in the United States is highest during the months of May, June and July. Of 1005 sporadic cases during the 1977-1979 period, 73% occurred between the months of June and October, with most cases (43%) during the months of August and September.
Outbreaks of legionellosis have often been related to airborne transmission. The current evidence implicates the moist air exhaust of cooling towers, evaporative condensers, and other air-handling equipment as possible sources. Although L. pneumophila are often identified in concentrated water samples from cooling towers and evaporative condensers, the bacterium has not been isolated directly from the aerosol exhaust of air-conditioning equipment. Laboratory tests of the viability of L. pneumophila in aerosols indicate that the relatively short half-life of its viability is greater in humid air.
If cooling towers are a source of infective Legionella, there should be an increased risk for people who service or maintain the equipment. However, a serologic survey of mechanics, mechanic's helpers, and water treatment servicemen who maintain 3000 cooling towers in the New York metropolitan area failed to confirm this.
Legionella have been isolated from shower heads in hospitals and hotels. However, acquisition of legionellosis from these sources has not been proven conclusively.
The factor or factors responsible for the ability of Legionella to establish the disease state have yet to be identified: An early hypothesis involved endotoxin activity because most gram-negative rods have the lipid A-associated endotoxin in the lipopolysaccharide of their cell wall. Legionella do produce a strongly positive Limulus lysate test, a nonspecific in-vitro test for endotoxin. However, Legionella do not produce significantly positive results in the more specific in-vivo rabbit pyrogenicity and mouse lethality tests.
Exotoxins and/or exoenzymes have been suggested as possible virulence factors and include proteolytic, hemolytic and cytotoxic activities. Numerous other enzymes, including an enzyme that inhibits oxygen-dependent killing by phagocytic cells have been described in the literature.
Legionella do not seem to be highly infective once they have been passed several times in-vitro. Passage of in-vitro grown Legionella through hen embryonated yolk sac or guinea pigs restores a moderate level of virulence.
Investigation of the infectivity of Legionella aerosols for guinea pigs found that the geometric mean LD50 dose was approximately 3 x 104 bacteria per lung (Davis et al. 1982). The lethal dose by the intranasal or intraperitoneal routes is reportedly much greater. It is apparent from other reports that guinea pigs may be infected by a relatively low aerosol dose (100 to 200 bacteria) without developing lethal disease. Also, no cross-infection to a sentinel guinea pig placed in a cage with an infected guinea pig has been found. The infective dose for the healthy human host has not been determined. However, patients who have a compromised immune response are possibly at higher risk. Only one case of possible patient-to-patient transmission has been reported. However, the CDC recommends that known cases be placed in respiratory isolation.
Guinea pig peritoneal macrophages isolated from normal guinea pigs phagocytize yolk sac-grown Philadelphia 1 strain of L. pneumophila at a lower rate than do macrophages isolated from previously infected, convalescing guinea pigs. Adding immune sera increases the rate of phagocytosis by macrophages from both normal and immune guinea pigs. However, once Legionella are phagocytized, they begin to multiply in vacuoles and eventually kill the macrophages from both normal and convalescing guinea pigs. This is not influenced by the presence of immune sera. Legionella grown in-vitro are also phagocytized, but they are subsequently killed by the macrophages. Legionella within histiocytes or macrophages in tissue from both humans and guinea pigs are often observed to be intact with no evidence of bacterial lysis. There are undoubtedly some virulence-associated factors involved in the survival and multiplication of Legionella within macrophages, but they remain to be elucidated.
DESCRIPTION OF CLINICAL DISEASE
Legionellosis or Legionnaires' disease, whether occurring sporadically or in an outbreak, is basically an acute bacterial bronchopneumonia. A mild, non-pneumatic disease produced by Legionella has also been described (Pontiac fever).
The clinical aspects of severe legionellosis have been described by many authors. The incubation period ranges from two to 10 days. Pneumonia is accompanied by a high fever (39°C-41°C); nonproductive cough which may later become productive and possibly contain blood; headache; a change in mental status; nausea; diarrhea; and vomiting. Chest roentgenograms may initially show an infiltrate, usually confined to a lower lobe, which may progress to bilateral involvement. Pulmonary cavitation has also been reported. Renal and hepatic involvement have been frequently described. Legionella antigen has also been found in urine. Elevated serum lactic acid dehydrogenase, SOOT, and alkaline phosphatase are frequently reported. Initial leukocyte counts among sporadic cases vary from 4700 to 28,000 with an average of 9900/mm3. The red cell sedimentation rate may become elevated (> 55 mm/hr) in some cases.
Pathologic findings from fatal cases show a high frequency of bilateral pulmonary consolidation. There is often a dense fibropurulent pneumonia with bacteria at the edge of consolidated areas. Focal areas of hemorrhage are common. There is an acute inflammatory reaction, diffuse alveolar damage with intact Legionella present within alveolar macrophages. Immunocompromised patients may have a fibroserous pneumonia without much inflammation.
It is apparent that Legionella may spread by endobronchial, interstitial, endolymphatic, and hematogenous routes. Bacteria may spread with migrating phagocytic cells. Legionella have been found in hilar lymph nodes, spleen, bone marrow, kidneys, and pulmonary venules.
Direct Fluorescent Antibody (DFA) Test
This test is used for the early diagnosis of legionellosis. Specific antisera are produced by immunizing rabbits with a formalinized or heat killed vaccine without Freunds' adjuvant. The gamma-globulin fraction of the serum is conjugated with fluorescein isothiocyanate and used to identify Legionella in specimens by means of fluorescent microscopy. Rhodamine-conjugated normal rabbit serum is used as a diluent to counterstain autofluorescent debris. DFA reagents for L. pneumophila serogroups 1-6, L. bozemanii, L. dumoffi, L. gormanii, L. micdadei, and L. longbeachiae are now available from commercial sources. The DFA test is more specific and sensitive than the usual histologic staining procedures. It can be done on sputum, if available, or on other respiratory secretions, such as tracheal aspirates, or pleural fluid. Liquefaction and concentration of sputum may be helpful. This test can also be performed on microscope slide imprints of fresh or formalinized lung tissue or deparaffinized lung tissue sections. False-negative DFA tests may occur because of inadequate numbers of Legionella in the specimens.
The omission of Freunds' complete adjuvant from the vaccines and the inclusion of the rhodamine counterstain have eliminated much of the nonspecific staining associated with some of the early DFA tests. A recent report suggested, however, that patients with Bacteroides fragilis infection may produce a positive, cross-reactive DFA test to serogroup 1 L. pneumophila but not to serogroups 2-4. Also, Pseudomonas fluorescens, P. alcaligenes, and the Flavobacterium-Xanthomonas group were reported to stain positively with serogroup 1 antisera.
Preparation of specimens, examination of slides, and interpretation of DPA results are presented in the CDC Manual. Since there are very few organisms in lung exudates, observation of 5 fluorescing bacteria is considered positive. For clinical specimens, except lung exudates, 25 strongly fluorescing bacteria per smear is considered positive. If there are < 25 strongly fluorescing bacteria per smear, the actual numbers are reported. No strongly fluorescing bacteria per smear is reported as negative. Lung exudates are difficult to evaluate because of the presence of autofluorescing debris in the specimens (personal observation). Therefore, familiarity with the morphology of Legionella and experience with fluorescent microscopy are needed for proper interpretation. Appropriate controls must be run in parallel with test samples.
It is recommended that non-formalinized specimens examined by the DFA test also be cultured on buffered CYE [BCYE] agar with and without additional supplements. The combination of culture plus DFA test produces results that are more diagnostically useful than when each is used alone. A survey of sporadic cases found that nearly half of DFA-positive specimens were culture positive. Blood should also be cultured since Legionella have been isolated from the blood of a few patients with legionellosis. A diphasic (broth/agar) CYE blood culture medium is recommended but standard aerobic blood culture medium also has been used successfully.
There are two reasons that may explain false-negative cultures in patients with legionellosis. First, viable Legionella may not be present in respiratory secretions unless retrograde spread of the organism has occurred in the airway. Second, human anterior pharyngeal flora contaminating specimens of respiratory secretions may inhibit the growth of Legionella. For example, we have found that some isolates of Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and some viridians streptococci inhibit the growth of L. pneumophila Philadelphia 1 on buffered CYE agar. Culture of clinical specimens is recommended, however, because there have been reports of occasional positive cultures from specimens that were DFA-negative.
Various modifications of the culture medium are being evaluated at this time. Some of these modifications are designed to permit differentiation between some species of Legionella, while others contain antibiotics and other supplements to inhibit the growth of other bacteria and fungi while permitting the growth of Legionella. At this time, buffered CYE agar medium is recommended for primary isolation and is commercially available.
Laboratories with suitable biological containment facilities have successfully recovered Legionella by intraperitoneal injection of clinical specimens into guinea pigs. Culture of guinea pig spleen homogenates or peritoneal washings after three to five days directly on CYE agar or on CYE agar after passage through the yolk sac of embryonated hen eggs has often resulted in isolation of Legionella when direct plating results were negative. The procedures for in-vivo passage are presented in the CDC Manual.
Indirect Immunofluorescence (IFA) Test
This is a serological test for antibody to Legionella in patient’s serum. The IFA test can be used to confirm legionellosis when paired acute (within seven days of onset) and convalescent (20 to 40 days after onset) patient sera show a fourfold or greater rise in titer to 128. When only one serum sample is available, a single or standing titer of 256 is presumptive evidence of previous or current infection.
Patients with legionellosis seroconvert to a diagnostically positive IFA titer, unless they succumb to the disease before an antibody response is elicited or they have a defective immune response to the organism. However, seroconversion may take up to three weeks. Therefore, the IFA test has limited usefulness in the early diagnosis of legionellosis. The DFA test and culture produce positive diagnostic results earlier, but they may often be negative. In this instance, early diagnosis must be based on clinical signs and symptoms and later confirmed by the IFA test.
The IF A test procedure and interpretation of results is presented in the CDC Manual. State public health laboratories are equipped to perform this test. The antigens for the IFA test are Legionella that are grown on CYE agar and heat-killed. The bacteria are suspended in normal chicken yolk sac fluid and fixed to a microscope slide. Various dilutions of patient's serum are incubated with the antigen. The slide is then washed and incubated with fluorescein-conjugated anti-human serum to determine whether antibodies in the patient's serum attached to the known antigen. Fluorescence intensities are graded from 0 to 4 +. The highest dilution of the patient's serum that produces at least 1+, barely visible staining, is considered the end point. It is very important to include appropriate controls with all tests. As with the DFA test, some experience is required to evaluate staining intensities and background staining of yolk sac material, filamentous bacteria and other artifacts. The current commercially available IFA test kits using individual serogroup bacterial preparations seem to have a sensitivity of 85% and a specificity of 92%.
There are a variety of cross-reactions that may occur, however. Patient sera with antibody to other gram-negative bacteria may cross-react with Legionella. This cross-reactivity may be effectively blocked with immunosorbent from Escherichia coli strain 013:K92:H4. However, this procedure is not routinely used. No cross-reactions against L. pneumophila antigens have been found in sera from cases of Q fever, tularemia, or psittacosis, or in sera of patients with rheumatoid factor.
Other Diagnostic Tests
Several other tests for Legionella antigen and antibody have been developed and compared to the more established DFA and IFA tests. A brief description of these tests will suffice because most are not widely used. A microagglutination test has been developed for detection of IgM class antibody. A microenzyme-linked immunosorbent assay (ELISA) has been developed to detect antibodies to serogroup 1 L. pneumophilia and Legionella antigen in urine. Various agglutination tests, indirect hemagglutination, ELISA, radioimmunoassays (RIA), and counter-immunoelectrophoresis tests to detect Legionella antigen have been described. A guinea pig skin test reportedly can detect the presence of Legionella antigens when they are injected intradermally into immunized guinea pigs. Biochemical tests for the presence of Legionella enzyme activities are also under development.
In summary, laboratory tests are available for early diagnosis and retrospective confirmation of legionellosis. The most useful tests currently are the DFA and IFA tests in combination with culture on buffered CYE agar directly or after passage through guinea pigs and/or chicken embryo yolk sacs. The two fluorescent antibody tests require trained laboratory personnel if test results are to be correctly interpreted. Development of more selective culture media and diagnostic tests will probably continue for some time. Therefore, it is necessary to keep abreast of the literature and information available at state or local public health laboratories, the Centers for Disease Control and current reviews.
There have not been controlled clinical trials, but the evidence reported in the literature indicates that erythromycin and rifampin are active against Legionella with in-vitro assays, with in-vivo models of legionellosis, and in patients. Erythromycin gluceptate alone is given intravenously in amounts ranging from 0.5 to 1.0 g every six hours. As a patient's condition stabilizes, the dosage may be reduced, and erythromycin may be given orally. Antibiotic therapy is often continued for up to three weeks. The combination of erythromycin and rifampin is recommended if a patient is not responding to erythromycin alone. A considerable amount of supportive therapy must be given to patients with severe legionellosis.
In-vitro antimicrobial sensitivity studies with Legionella often produce results that are at variance with results of tests with animal models and clinical experience. The aminoglycosides, penicillins, cephalosporins, tetracycline, and chloramphenicol, although they may be active in-vitro, are not clinically effective. The ability of Legionella to survive and multiply within macrophages may explain why antibiotics that penetrate tissue are effective in-vivo even though other antibiotics that are active in-vitro are not useful clinically. Also, most Legionella produce a beta-lactamase that is most active on the cephalosporin group of antibiotics. Clearly, more controlled clinical trials will be needed before questions concerning the efficacy of antibiotic therapy can be resolved.
Although a primary mode of transmission seems to be inhalation, the source and nature of infective Legionella remains to be clearly established. There is a tentative relationship between legionellosis cases and aerosolized water. Therefore, preventive measures recommended at this time are adequate disinfection of aerosol-producing equipment. Cooling towers and evaporative condensers heavily contaminated with Legionella should be treated with effective levels of an appropriate chemical biocide. Recirculating water systems may also be treated with suitable ultraviolet light systems. Equipment heavily contaminated with organic matter may not be effectively treated by chlorination, because the organic material will neutralize the free chlorine. Therefore, free chlorine levels must be monitored during disinfection procedures when calcium hypochlorite is used.
One problem that prevents more effective preventative measures is the lack of an efficient, cost-effective method to monitor the numbers of viable Legionella in water samples that have a high background bacterial flora. It is usually difficult to isolate Legionella from such samples by direct plating on CYE agar, because the other bacteria in the sample frequently overgrow or inhibit Legionella growth. The guinea pig intraperitoneal injection method is currently used, but it is time consuming and rather expensive. A more efficient isolation method is clearly needed.
Other preventive measures such as vaccination are being investigated. Immunization of guinea pigs has prevented subsequent infection, but more research will be required before the suitability of a vaccine can be established.
The author wishes to thank John J. Bozzola, PhD, Department of Microbiology at The Medical College of Pennsylvania, Philadelphia, for his contribution of electron micrographs to this chapter; critical reading of the manuscript by Susan B. Dillon of the same department is gratefully acknowledged. Portions of the work presented were supported in part by The Whitaker Foundation and by The Biomedical Research Support Grant Program of The Public Health Service, National Institutes of Health.
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