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LEGIONNAIRES' DISEASE CAUSED BY LEGIONELLA PNEUMOPHILA SEROGROUP 3
47 LEGIONNAIRES’ DISEASE CAUSED BY LEGIONELLA PNEUMOPHILA SEROGROUP 3
SIR,-Four serogroups of Legionella pneumophila have been differentiated by immunofluorescent antibody techniques,’ one of which, serogroup 3, has not previously been reported to cause human disease. Hitherto this serogroup was represented by a single strain, Bloomington-2, isolated from creek water near a building associated epidemiologically with an outbreak of legionnaires’ disease.2 However, the strain isolated from the air-conditioning cooling tower of this building, in which 19 of 21 of the confirmed cases stayed and which appears to have been responsible for the outbreak, was identified’ as L. pneumophila serogroup 1. We have studied the strain isolated from a renal-transplant recipient in Oxford3 and have found that it is related to serogroup 3, showing that all serogroups currently recognised can cause legionnaires’ disease.
The Oxford strain together with a single serum specimen taken from the patient 16 days after onset of the episode of acute pneumonia was made available to us for study. A formolised yolk-sac antigen for indirect fluorescent antibody tests4 was prepared from this strain, and also from representative strains of the four serogroups. The patient’s serum was tested against these five antigens by the indirect fluorescent-antibody (FA) method with the following titres:
Formolisedyolk-sac antigen Serogroup 1 Serogroup 2 Serogroup 3 Serogroup 4 Patient’s isolate
FA titre 16 32 256 16 256
Strain Pontiac-1
Togus-1 Bloomington-2 Los Angeles-1 Oxford
These results show this serum to be broadly reactive with the four serogroups, although the high titre obtained with the Bloomington-2 antigen might indicate the infecting strain to be most closely related to serogroup 3. In order to gain more conclusive evidence samples of the patient’s serum diluted 1:8 in phosphate-buffered saline were absorbed at 37°C for 30min with organisms of one of each of the four serogroups or of the Oxford strain. The bacteria were grown on supplemented Mueller-Hinton agar and then suspended in buffered formol of organisms (1%) saline. The optical density of the 10.0 in each used for absorption was adjusted to E 640 " nm case. The FA titres of the unabsorbed and absorbed serum samples were determined using Oxford and Bloomington-2 formolised yolk-sac antigens:
suspension =
Absorbing strain
Titre with Serogroup of absorbing strain Oxford antigen
None
Philadelphia-1 Togus-1 Bloomington-2 Los
Angeles-1
Oxford
..
1 2 3 4 ..
256 256 256 64 256
<16
strain removed all activity. Absorption with the serogroup 3 strain removed all activity against Bloomington-2 antigen but only part of the activity against the Oxford antigen. These results suggest that the major antibodies in the patient’s serum measured by the indirect FA method are directed partly against antigens common to the Bloomington-2 strain and partly to antigenic determinants of the patient’s strain not shared with this serogroup 3 strain. Absorption experiments on hyperimmune rabbit sera5 raised against these two strains confirmed that, while they are antigenically related, each possesses distinct as well as shared antigens. The existence of serological variants of L. pneumophila has important implications in laboratory diagnosis and the serological characterisation of additional isolates is necessary if cases of legionnaires’ disease due to as yet unknown serovariants of this organism are to be recognised. The use of improved media6 will no doubt facilitate the isolation of new strains from clinical specimens. It has been suggested7 that the use of a polyvalent antigen made from heat-killed L. pneumophila grown on solid media is advantageous for the initial screening of sera by the indirect FA method. A serogroup 1 formolised yolk-sac antigen is issued by this laboratory to diagnostic laboratories in England and Wales. Because titres of 16 or more with this highly specific antigen are rare in the absence of legionnaires’ disease,4.8 we recommend that sera with such titres, but which do not meet current diagnostic criteria, be tested with formolised yolk-sac antigens of serogroups 2, 3, and 4. Had the causative organism not been isolated from the Oxford patient legionnaires’ disease would still have been diagnosed retrospectively on the single serum specimen made available to us, even though the titre with the serogroup 1 antigen was only 16, as further testing with antigens of serogroups 2-4 in the Oxford Public Health Laboratory (Dr J. O’H. Tobin, personal communication) gave a diagnostic titre of 256 with the serogroup-3 antigen. Moreover, we have recently examined a small group of sera from patients with legionnaires’ disease and these reacted broadly with all four serogroups. Hence our evidence to date suggests that infections due to serogroups currently recognised will not be overlooked through screening by the indirect FA method using only serogroup 1 formolised
yolk-sac antigen. We thank Dr J. O’H. Tobin for the serum specimen from the patient. Standards Laboratory for Serological Central Public Health Laboratory, London NW9 5HT
sending
us
the Oxford strain and ’
Reagents,
A. G. TAYLOR T. G. HARRISON
Titre with
Bloomington-2 antigen 256 256 256
<16 256 <16
Absorption of the patient’s serum with strains of serogroups 1, 2, and 4 had no effect on the titre of the patient’s serum against either antigen while absorption with the patient’s own
BROMPTON COCKTAIL
SiR,—The best explanation I have been offered for the use of cocaine in the Brompton mixture, discussed in your editorial of June 9 (p. 1220), was historical. It was given to me by Dr W. N. Mann, a physician at Guy’s hospital. He suggested that the prime reason for the addition of cocaine to the Brompton mixture was for its local anxsthetic action, especially for cases of severe tuberculosis of the chest. These patients, I believe, may get secondary infection of the pharynx with ulceration, and swallowing causes a great deal of pain. Can anyone substantiate or refute this explanation? I have attempted to use the mixture in this way in one case of carcinoma of the tongue, but the response was negligible.
1. McKinney
RM, Thacker L, Harris PP, Lewallen BS, Hebert GA, Edelstein PH, Thomason BM. Four serogroups of legionnaires’ disease bacteria defined by direct immunofluorescence. Ann Intern Med 1979; 90: 621-24. 2. Morris GK, Patton, CM, Feeley JC, Scott EJ, Gorman G, Martin WT, Skaliy P, Mallison, GF, Politi BD, Mackel DC. Isolation of the legionnaires’ disease bacterium from
environmental
samples.
Ann Intern
Med
1979; 90: 664-66. 3. Fischer-Hoch S, Hudson MJ, Thompson MH. Identification of a clinical isolate as Legionella pneumophila by gas chromatography and mass spectrometry of cellular fatty acids. Lancet (in press). 4. Taylor AG, Harrison TG, Dighero MW, Bradstreet CMP. False positive reactions in the indirect fluorescent antibody test for legionnaires’ disease eliminated by the use of formolised yolk-sac antigen. Ann Intern Med 1979; 90: 686-89.
Greenwich District London SE10 9HE
Hospital,
N. J. MASTERS
Pittman B, Harris PP, Hebert GA, Thomason BM, Thacker L, Weaver RE. Detection of legionnaires’ disease bacteria by direct immunofluorescent staining. J Clin Microbiol 1978; 8: 329-38. 6. Greaves PG, Sharp G, Macrae AD. Isolation of Legionella pneumophila. Lancet 1979; i: 551-52. 7. Fallon RJ, Abraham WH. Detecting legionnaires’ disease. Lancet 5.
Cherry WB,
1978; ii: 1318. 8. Bartlett CLR. Sporadic Intern Med 1979; 90:
cases
of
592-95.
legionnaires’
disease in Great Britain. Ann
SIR,-Four serogroups of Legionella pneumophila have been differentiated by immunofluorescent antibody techniques,’ one of which, serogroup 3, has not previously been reported to cause human disease. Hitherto this serogroup was represented by a single strain, Bloomington-2, isolated from creek water near a building associated epidemiologically with an outbreak of legionnaires’ disease.2 However, the strain isolated from the air-conditioning cooling tower of this building, in which 19 of 21 of the confirmed cases stayed and which appears to have been responsible for the outbreak, was identified’ as L. pneumophila serogroup 1. We have studied the strain isolated from a renal-transplant recipient in Oxford3 and have found that it is related to serogroup 3, showing that all serogroups currently recognised can cause legionnaires’ disease.
The Oxford strain together with a single serum specimen taken from the patient 16 days after onset of the episode of acute pneumonia was made available to us for study. A formolised yolk-sac antigen for indirect fluorescent antibody tests4 was prepared from this strain, and also from representative strains of the four serogroups. The patient’s serum was tested against these five antigens by the indirect fluorescent-antibody (FA) method with the following titres:
Formolisedyolk-sac antigen Serogroup 1 Serogroup 2 Serogroup 3 Serogroup 4 Patient’s isolate
FA titre 16 32 256 16 256
Strain Pontiac-1
Togus-1 Bloomington-2 Los Angeles-1 Oxford
These results show this serum to be broadly reactive with the four serogroups, although the high titre obtained with the Bloomington-2 antigen might indicate the infecting strain to be most closely related to serogroup 3. In order to gain more conclusive evidence samples of the patient’s serum diluted 1:8 in phosphate-buffered saline were absorbed at 37°C for 30min with organisms of one of each of the four serogroups or of the Oxford strain. The bacteria were grown on supplemented Mueller-Hinton agar and then suspended in buffered formol of organisms (1%) saline. The optical density of the 10.0 in each used for absorption was adjusted to E 640 " nm case. The FA titres of the unabsorbed and absorbed serum samples were determined using Oxford and Bloomington-2 formolised yolk-sac antigens:
suspension =
Absorbing strain
Titre with Serogroup of absorbing strain Oxford antigen
None
Philadelphia-1 Togus-1 Bloomington-2 Los
Angeles-1
Oxford
..
1 2 3 4 ..
256 256 256 64 256
<16
strain removed all activity. Absorption with the serogroup 3 strain removed all activity against Bloomington-2 antigen but only part of the activity against the Oxford antigen. These results suggest that the major antibodies in the patient’s serum measured by the indirect FA method are directed partly against antigens common to the Bloomington-2 strain and partly to antigenic determinants of the patient’s strain not shared with this serogroup 3 strain. Absorption experiments on hyperimmune rabbit sera5 raised against these two strains confirmed that, while they are antigenically related, each possesses distinct as well as shared antigens. The existence of serological variants of L. pneumophila has important implications in laboratory diagnosis and the serological characterisation of additional isolates is necessary if cases of legionnaires’ disease due to as yet unknown serovariants of this organism are to be recognised. The use of improved media6 will no doubt facilitate the isolation of new strains from clinical specimens. It has been suggested7 that the use of a polyvalent antigen made from heat-killed L. pneumophila grown on solid media is advantageous for the initial screening of sera by the indirect FA method. A serogroup 1 formolised yolk-sac antigen is issued by this laboratory to diagnostic laboratories in England and Wales. Because titres of 16 or more with this highly specific antigen are rare in the absence of legionnaires’ disease,4.8 we recommend that sera with such titres, but which do not meet current diagnostic criteria, be tested with formolised yolk-sac antigens of serogroups 2, 3, and 4. Had the causative organism not been isolated from the Oxford patient legionnaires’ disease would still have been diagnosed retrospectively on the single serum specimen made available to us, even though the titre with the serogroup 1 antigen was only 16, as further testing with antigens of serogroups 2-4 in the Oxford Public Health Laboratory (Dr J. O’H. Tobin, personal communication) gave a diagnostic titre of 256 with the serogroup-3 antigen. Moreover, we have recently examined a small group of sera from patients with legionnaires’ disease and these reacted broadly with all four serogroups. Hence our evidence to date suggests that infections due to serogroups currently recognised will not be overlooked through screening by the indirect FA method using only serogroup 1 formolised
yolk-sac antigen. We thank Dr J. O’H. Tobin for the serum specimen from the patient. Standards Laboratory for Serological Central Public Health Laboratory, London NW9 5HT
sending
us
the Oxford strain and ’
Reagents,
A. G. TAYLOR T. G. HARRISON
Titre with
Bloomington-2 antigen 256 256 256
<16 256 <16
Absorption of the patient’s serum with strains of serogroups 1, 2, and 4 had no effect on the titre of the patient’s serum against either antigen while absorption with the patient’s own
BROMPTON COCKTAIL
SiR,—The best explanation I have been offered for the use of cocaine in the Brompton mixture, discussed in your editorial of June 9 (p. 1220), was historical. It was given to me by Dr W. N. Mann, a physician at Guy’s hospital. He suggested that the prime reason for the addition of cocaine to the Brompton mixture was for its local anxsthetic action, especially for cases of severe tuberculosis of the chest. These patients, I believe, may get secondary infection of the pharynx with ulceration, and swallowing causes a great deal of pain. Can anyone substantiate or refute this explanation? I have attempted to use the mixture in this way in one case of carcinoma of the tongue, but the response was negligible.
1. McKinney
RM, Thacker L, Harris PP, Lewallen BS, Hebert GA, Edelstein PH, Thomason BM. Four serogroups of legionnaires’ disease bacteria defined by direct immunofluorescence. Ann Intern Med 1979; 90: 621-24. 2. Morris GK, Patton, CM, Feeley JC, Scott EJ, Gorman G, Martin WT, Skaliy P, Mallison, GF, Politi BD, Mackel DC. Isolation of the legionnaires’ disease bacterium from
environmental
samples.
Ann Intern
Med
1979; 90: 664-66. 3. Fischer-Hoch S, Hudson MJ, Thompson MH. Identification of a clinical isolate as Legionella pneumophila by gas chromatography and mass spectrometry of cellular fatty acids. Lancet (in press). 4. Taylor AG, Harrison TG, Dighero MW, Bradstreet CMP. False positive reactions in the indirect fluorescent antibody test for legionnaires’ disease eliminated by the use of formolised yolk-sac antigen. Ann Intern Med 1979; 90: 686-89.
Greenwich District London SE10 9HE
Hospital,
N. J. MASTERS
Pittman B, Harris PP, Hebert GA, Thomason BM, Thacker L, Weaver RE. Detection of legionnaires’ disease bacteria by direct immunofluorescent staining. J Clin Microbiol 1978; 8: 329-38. 6. Greaves PG, Sharp G, Macrae AD. Isolation of Legionella pneumophila. Lancet 1979; i: 551-52. 7. Fallon RJ, Abraham WH. Detecting legionnaires’ disease. Lancet 5.
Cherry WB,
1978; ii: 1318. 8. Bartlett CLR. Sporadic Intern Med 1979; 90:
cases
of
592-95.
legionnaires’
disease in Great Britain. Ann