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Direct susceptibility tests of M. avium complex
156
Letter
to the Editor
Does this represent a change from chronic granulomatous, non-necrotising disease, to a more typically tuberculous necrotising response (Koch phenomenon)? The probability that it does is increased by the reports of the dire consequences of treating pulmonary disease with vitamin D in the pre-chemotherapy era. This caused liquefaction of caseum with rapid dissemination of disease throughout the lungs [II]. However it is not yet certain that these effects are attributable to the direct action of vitamin D on macrophages. Receptors for it also exist in some T-lymphocytes, and direct effects of 1,25-(OH)* D3 on T-cell function have been reported [12]. It is also possible that the metabolites modulate antigen-presenting functions, However liquefaction of caseum is likely to be due to release of enzymes from macrophages, and I have argued elsewhere that the Koch phenomenon may represent the triggering of secretory activities of activated macrophages 1131. We are now investigating the possibility that the interactions shown in the figure may cause development of very readily triggered macrophage populations. Department of Microbiology, Middlesex Hospital Medical London, W. I.
Graham
Rook
School,
References 1 Davies, 2
3
4
5
6 7 8
9 10 11 12 13
P. D. 0. (1985). A possible link between vitamin D deficiency and impaired host defence to Mycobacterium tuberculosis. Tubercle, 66, 301. Amento, E. P., Bhalla, A. K., Kurnick, J. T., Kradin, I?. L., Clemens, T. L., Holick, M. F., Krane, S. M. (1984). I,25 dihydroxy vitamin D3 induces maturation of the human monocyte cell line U937, and in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor. Journal of Clinical Investigation, 73, 731. Mangelsdorf, D. J., Koeffler, H. P., Donaldson, C. A., Pike, J. W., Haussler, M. R. (1984). I,25 dihydroxy vitamin D3induced differentiation in a human promyelocytic leukaemia cell line (HL60): receptor-mediated maturation to macrophage-like cells. Journal of Cell Biology, 96, 391. Rook, G. A. W., Steele, J., Fraher, L., Barker, S., Karmali. Ft., O’Riordan, J., Stanford, J. L. (1986). Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology, 57, 159. Koeffler, H. P., Reichel, H., Bishop, J. E., Norman, A. W. (1985). Gamma interferon stimulates production of 1,25dihydroxyvitamin D3 by normal human macrophages. Biochemical & Biophysical Research Communications, 127, 596. Abassi, A. A., Chemplavil, J. K., Farah, S., Muller, B. F., Arnstein, A. R. (1979). Hypercalcaemia in active pulmonary tuberculosis. Annals of Internal Medicine, 90, 324. Narang, N. K., Gupta, R. C., Jain, M. K. (1984). Role of vitamin D in pulmonary tuberculosis. Journal of the Association of Physicians of India, 32, 185. Epstein, S., Sterin, P. H., Bell, N. H., Dowdeswell, I,, Turner, R. T. (1984). Evidence for abnormal regulation of circulating 1,25-dihydroxy vitamin D in patients with pulmonary tuberculosis and normal calcium metabolism. Calcified Tissue International, 36, 541. Dowling, G. S., Prosser Thomas, E. W. (1946). Treatment of Lupus vulgaris with calciferol. Lancer, i, 919. Macrae, D. E. (1947). Calciferol treatment of Lupus vulgaris. British Journal of Dermatology, 59, 333. Brincourt, J. (1967). Le calciferol a-t-il une action liquefiante sur le caseum? Poumon-Couer, 23, 841. Bhalla, A. K., Amento, E. P., Serog, B., Glimcher, L. H. (1984). 1,25-dihydroxy vitamin D3 inhibits antigen-induced T cell activation. Journal of Immunology, 133, 1748. Stanford, J. L., Rook, G. A. W. (1983). Environmental mycobacteria and immunisation with BCG. In: Medical Microbiology vol. 2, lmmunisation against bacterial disease. (C.S.F. Easmon & J. Jeljaszewicz eds.), pp. 43-69. Academic Press.
Direct
Susceptibility
Tests
of M. awium Complex
Susceptibility testing of isolates in cases of Mycobacterium avium complex (MAC) infection is only rarely helpful in formulating therapy for these patients [Il. It is also very expensive:
Letters
to the Editor
157
proper testing requires two or three dilutions of the micro-organism for each of up to 10 drugs. Susceptibility tests should be performed, therefore, only if one is fairly sure that the organism isolated is the one causing the disease. This restriction is true, of course, for any susceptibility test procedure; but the role of MAC in an infection can be especially difficult to determine, even for the experienced clinician. One reason is that MAC are common environmental organisms [2] which can colonize tissue without producing disease [31. A disadvantage of the indirect MAC susceptibility test is that MAC have high mutation rates. Clinical isolates, which grow as transparent colonies, tend to change rapidly to forms than that grow as opaque colonies [4]. These opaque mutants are often more susceptible the original isolate to the action of cycloserine, ethionamide, capriomycin, ethambutol, kanamycin; and certain other drugs. This difference between direct and indirect test results is illustrated in the Table. The cultures were random transfers of isolates from five patients. The procedures were a modification of the proportion method: we prepared two or three IO-fold dilutions of the specimen (direct test) or cultural suspension (indirect test) and counted the number of colonies seen on 7HlO agar slants with and without the drugs. The percentage of resistance was determined as: [(no. of colonies on media containing drugs)/(no. of colonies on control media)]xlOO. To ensure that we are working only with unaltered isolates, we have instituted a policy in our laboratory of performing only direct susceptibility tests (except for specimens from immuno-compromised patients)-and of performing these tests only if the sputum is smear-positive. We can thus determine the drug resistance patterns of the organisms known to be present in the patient’s sputum. If the original isolate was only a contaminant or colonizer and not of diagnostic significance, it will probably not be present in a specimen collected 4-8 weeks after the original specimen was collected-or at least not in sufficient numbers to be demonstrable on smear-and will not produce growth for the direct test. Except in patients with AIDS and other serious immune deficiencies, disease due to these mycobacteria tends to be only slowly progressive. Isolates from AIDS patients are immediately studied with the indirect susceptibility test. In most other cases the delay in obtaining direct drug susceptibility results with a second specimen can do little harm. Table.
Comparison
of direct Spec. No.
1170 2706 2738 6762 6855
and
Jest
Direct Indirect Direct Indirect Direct Indirect Direct Indirect Direct Indirect
indirect
susceptibility
test
Resistant
results.
Percent
of Colonies
to Drug
CAP*
CYC
ETHl
EMB
KANA
100 1.0 100 100 100 1.0 100 5.0 100 100
50 0.01 0.1 0.8 0.4 Sens 0.1 Sens 100 Sens
100 100 100 100 100 100 0.1 Sens 100 0.1
75 1.0 100 100 25 1 .o 100 3.0 100 100
100 1.0 100 10 100 50 100 0.3 50 1.0
‘Abbreviations: CAP=Capreomycin; CYC=cycloserine; ETHI=ethionamide; ethambutol; KANA=kanamycin; Sens=Below detectable resistance.
EMB=
drug
158
Letters
to the Editor
With this policy we can maintain needed, as accurately as present State of New York, Department of Health,
costs at a minimum technology allows.
and still provide test results, when
Howard Albany,
Gruft
N. Y. References
Iseman,
M. D., Corpe,
Ft. F., O’Brien,
R. J., Rosenzweig,
D. Y., Wolinsky,
E. (1985).
Disease
due to Mycobacterium
avium-intracellulare. Chest, 87, Supplement 2, 139s. Gruft, H., Falkinham, J. 0. III, Parker, B. C. (1981). Recent experience in the epidemiology of disease caused by atypical mycobacteria. Review of infectious Disease, 3, 990. Ahn, C. J., McLarty, J. W., Ahn, S. S., Ahn, S. I., Hurst, G. A. (1982). Diagnostic criteria for pulmonary disease kansasii and Mycobacterium intracellulare. American Review of Respiratory caused by Mycobacterium Disease, 125, 388. McCarthy, C. (1970). Spontaneous and induced mutation in Mycobacterium avium. infection and Immunity, 2. 223.
Letter
to the Editor
Does this represent a change from chronic granulomatous, non-necrotising disease, to a more typically tuberculous necrotising response (Koch phenomenon)? The probability that it does is increased by the reports of the dire consequences of treating pulmonary disease with vitamin D in the pre-chemotherapy era. This caused liquefaction of caseum with rapid dissemination of disease throughout the lungs [II]. However it is not yet certain that these effects are attributable to the direct action of vitamin D on macrophages. Receptors for it also exist in some T-lymphocytes, and direct effects of 1,25-(OH)* D3 on T-cell function have been reported [12]. It is also possible that the metabolites modulate antigen-presenting functions, However liquefaction of caseum is likely to be due to release of enzymes from macrophages, and I have argued elsewhere that the Koch phenomenon may represent the triggering of secretory activities of activated macrophages 1131. We are now investigating the possibility that the interactions shown in the figure may cause development of very readily triggered macrophage populations. Department of Microbiology, Middlesex Hospital Medical London, W. I.
Graham
Rook
School,
References 1 Davies, 2
3
4
5
6 7 8
9 10 11 12 13
P. D. 0. (1985). A possible link between vitamin D deficiency and impaired host defence to Mycobacterium tuberculosis. Tubercle, 66, 301. Amento, E. P., Bhalla, A. K., Kurnick, J. T., Kradin, I?. L., Clemens, T. L., Holick, M. F., Krane, S. M. (1984). I,25 dihydroxy vitamin D3 induces maturation of the human monocyte cell line U937, and in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor. Journal of Clinical Investigation, 73, 731. Mangelsdorf, D. J., Koeffler, H. P., Donaldson, C. A., Pike, J. W., Haussler, M. R. (1984). I,25 dihydroxy vitamin D3induced differentiation in a human promyelocytic leukaemia cell line (HL60): receptor-mediated maturation to macrophage-like cells. Journal of Cell Biology, 96, 391. Rook, G. A. W., Steele, J., Fraher, L., Barker, S., Karmali. Ft., O’Riordan, J., Stanford, J. L. (1986). Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology, 57, 159. Koeffler, H. P., Reichel, H., Bishop, J. E., Norman, A. W. (1985). Gamma interferon stimulates production of 1,25dihydroxyvitamin D3 by normal human macrophages. Biochemical & Biophysical Research Communications, 127, 596. Abassi, A. A., Chemplavil, J. K., Farah, S., Muller, B. F., Arnstein, A. R. (1979). Hypercalcaemia in active pulmonary tuberculosis. Annals of Internal Medicine, 90, 324. Narang, N. K., Gupta, R. C., Jain, M. K. (1984). Role of vitamin D in pulmonary tuberculosis. Journal of the Association of Physicians of India, 32, 185. Epstein, S., Sterin, P. H., Bell, N. H., Dowdeswell, I,, Turner, R. T. (1984). Evidence for abnormal regulation of circulating 1,25-dihydroxy vitamin D in patients with pulmonary tuberculosis and normal calcium metabolism. Calcified Tissue International, 36, 541. Dowling, G. S., Prosser Thomas, E. W. (1946). Treatment of Lupus vulgaris with calciferol. Lancer, i, 919. Macrae, D. E. (1947). Calciferol treatment of Lupus vulgaris. British Journal of Dermatology, 59, 333. Brincourt, J. (1967). Le calciferol a-t-il une action liquefiante sur le caseum? Poumon-Couer, 23, 841. Bhalla, A. K., Amento, E. P., Serog, B., Glimcher, L. H. (1984). 1,25-dihydroxy vitamin D3 inhibits antigen-induced T cell activation. Journal of Immunology, 133, 1748. Stanford, J. L., Rook, G. A. W. (1983). Environmental mycobacteria and immunisation with BCG. In: Medical Microbiology vol. 2, lmmunisation against bacterial disease. (C.S.F. Easmon & J. Jeljaszewicz eds.), pp. 43-69. Academic Press.
Direct
Susceptibility
Tests
of M. awium Complex
Susceptibility testing of isolates in cases of Mycobacterium avium complex (MAC) infection is only rarely helpful in formulating therapy for these patients [Il. It is also very expensive:
Letters
to the Editor
157
proper testing requires two or three dilutions of the micro-organism for each of up to 10 drugs. Susceptibility tests should be performed, therefore, only if one is fairly sure that the organism isolated is the one causing the disease. This restriction is true, of course, for any susceptibility test procedure; but the role of MAC in an infection can be especially difficult to determine, even for the experienced clinician. One reason is that MAC are common environmental organisms [2] which can colonize tissue without producing disease [31. A disadvantage of the indirect MAC susceptibility test is that MAC have high mutation rates. Clinical isolates, which grow as transparent colonies, tend to change rapidly to forms than that grow as opaque colonies [4]. These opaque mutants are often more susceptible the original isolate to the action of cycloserine, ethionamide, capriomycin, ethambutol, kanamycin; and certain other drugs. This difference between direct and indirect test results is illustrated in the Table. The cultures were random transfers of isolates from five patients. The procedures were a modification of the proportion method: we prepared two or three IO-fold dilutions of the specimen (direct test) or cultural suspension (indirect test) and counted the number of colonies seen on 7HlO agar slants with and without the drugs. The percentage of resistance was determined as: [(no. of colonies on media containing drugs)/(no. of colonies on control media)]xlOO. To ensure that we are working only with unaltered isolates, we have instituted a policy in our laboratory of performing only direct susceptibility tests (except for specimens from immuno-compromised patients)-and of performing these tests only if the sputum is smear-positive. We can thus determine the drug resistance patterns of the organisms known to be present in the patient’s sputum. If the original isolate was only a contaminant or colonizer and not of diagnostic significance, it will probably not be present in a specimen collected 4-8 weeks after the original specimen was collected-or at least not in sufficient numbers to be demonstrable on smear-and will not produce growth for the direct test. Except in patients with AIDS and other serious immune deficiencies, disease due to these mycobacteria tends to be only slowly progressive. Isolates from AIDS patients are immediately studied with the indirect susceptibility test. In most other cases the delay in obtaining direct drug susceptibility results with a second specimen can do little harm. Table.
Comparison
of direct Spec. No.
1170 2706 2738 6762 6855
and
Jest
Direct Indirect Direct Indirect Direct Indirect Direct Indirect Direct Indirect
indirect
susceptibility
test
Resistant
results.
Percent
of Colonies
to Drug
CAP*
CYC
ETHl
EMB
KANA
100 1.0 100 100 100 1.0 100 5.0 100 100
50 0.01 0.1 0.8 0.4 Sens 0.1 Sens 100 Sens
100 100 100 100 100 100 0.1 Sens 100 0.1
75 1.0 100 100 25 1 .o 100 3.0 100 100
100 1.0 100 10 100 50 100 0.3 50 1.0
‘Abbreviations: CAP=Capreomycin; CYC=cycloserine; ETHI=ethionamide; ethambutol; KANA=kanamycin; Sens=Below detectable resistance.
EMB=
drug
158
Letters
to the Editor
With this policy we can maintain needed, as accurately as present State of New York, Department of Health,
costs at a minimum technology allows.
and still provide test results, when
Howard Albany,
Gruft
N. Y. References
Iseman,
M. D., Corpe,
Ft. F., O’Brien,
R. J., Rosenzweig,
D. Y., Wolinsky,
E. (1985).
Disease
due to Mycobacterium
avium-intracellulare. Chest, 87, Supplement 2, 139s. Gruft, H., Falkinham, J. 0. III, Parker, B. C. (1981). Recent experience in the epidemiology of disease caused by atypical mycobacteria. Review of infectious Disease, 3, 990. Ahn, C. J., McLarty, J. W., Ahn, S. S., Ahn, S. I., Hurst, G. A. (1982). Diagnostic criteria for pulmonary disease kansasii and Mycobacterium intracellulare. American Review of Respiratory caused by Mycobacterium Disease, 125, 388. McCarthy, C. (1970). Spontaneous and induced mutation in Mycobacterium avium. infection and Immunity, 2. 223.