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Culture media for tubercle bacilli during chemotherapy with isoniazid
TUBERCLE
170
July 1954
conversion was less likely to occur in technicians of the Edinburgh Royal Victoria and associated group of hospitals. patients with gross cavitation. (5) The clinical significance of isoniazid References resistance is confirmed by the increase in Bignall, J. R., Clegg, J. W., Crofton,J. W., Smith, B . J . D . , Holt, H. D., Mitehison, D. A., and Armltage, P. (195o) sputum positivity and the incidence of Brit. reed. J., I, 1224. clinical and radiographic deterioration after Coates, E. O., Jr., Steeken, ~,V., Wolinsky, E., and Brinkman, G. L. (1953) New Engl. 07. ~Ied., calvin, Io8x. the emergence of resistant organisms. Conalty, M. L. (x953) Irish 07. reed. Sci., 6th Series, July, ~67. (6) In 3 cases a reversion in the degree of Crofton, J., and Mitchison, D.A. (1948) Brit. reed. 07., 1I, resistance occurred after stopping the drug. IOO9. M. (z948) 07. Bact., Lvl, 63. The results of re-treatment with isoniazid Demercc, Dye, SV. E. (1953) Transactions of the 12th Conference on the Chemotherapy of Tuberculosis, Veterans Adsuggested that such reversion is unlikely to ministration, United States of America, p. 11 I. be of any clinical importance. Joiner, C. L., MacLean, K. S., Chalmers, D. G., Anderson, K., Collard, P., King, M. B., and Knox, R. (1953)
Acknowledgments
Lancet, I[, 15"2.
This work was generously supported by the Royal Victoria Hospital Tuberculosis Trust. We should also like to thank Drs N. W. Horne, I. W. B. Grant, and J. D. Ross for allowing us to investigate their patients, for making the radiographic assessments and for their keen interest in the work. Unstinting help was received from the registrars, house physicians, nursing staff and laboratory
Lotte, A., and Poussier, J. (i953) Re:,. Tuberc., xwz, I. Lumsden, E. G. S., and Swoboda, J. A. F. (1952) Tubercle, XXXIII~ ~22.
Medical Research Council (i952) Brit. reed. 07, iz, 735Medical Research Council (1953a) Lancet, H, 213. Medical Research Council (x953b) Brit. reed. 07, I, 52I. Mitchison, D. A. (1949) Lancet, H, 694. Petit, A. (1953) Schweiz. reed. IVschr., LXXXIH,754. Selikoff, I.J., Robitzek, E. H., and Ornstein, G. G. (1952) 07. Amer. rned. Ass., eL, 973United States Public tteahh Service Co-operative Investigation (x953) Transactlons.of the a2th Conference on the Chemotherapy of Tuberculosis, Veterans Administration, United States of America, p. 72-
Culture Media for Tubercle Bacilli During Chemotherapy with Isoniazjd By D. A. MITCHISON*
Lecturer in Bacteriology, PostgraduateMedical Schoolof London, Ducane Road, W.12 i n the Medical Research Council (I953b) report on isoniazid in combination with streptomycin or with p-aminosalicylic acid (PAS) in the treatment of pulmonary tuberculosis, a footnote gave details of discrepa:ncies in the results of isoniazid sensitivity tests carried out in one of the cooperating centres and in the reference laboratory. Cultures from 3 patients treated with isoniazid plus PAS were found to be resistant by the local laboratory, which used Lowenstein-Jensen medium with the addition of o.I25 per cent bovine albumin fraction V (Armour & Co.) and o-o6 per cent Tween 8o (Atlas Powder C o . ) ( L T A Medium), both for the isolation of tubercle bacilli and for the subsequent sensitivity tests. The reference laboratory, using a con-
ventional Lowenstein-Jensen formula (L Medium) found theseculturessensitive. Now Fisher (i952 , 1954) has suggested that isoniazid resistant tubercle bacilli r e q u i r e a growth factor, haemin, which is present in bovine albumin. Thus a possible explanation of the discrepancies between results in_ the reference laboratory and in the local centre was that resistant bacilli which would_ not grow on L medium might have been isolated on LTA medium and that the results of sensitivity tests using these two media might therefore have differed. T o investigate this possibility a series of specimens from patients who had received isoniazid plus PAS in the Medical Research Council trials were cultured on the two media. In some cases cultures were also set
*In receipt of a grant from the Medical Research Council.
July 1954
TUBERCLE
up on L medium plus haemin. Sensitivity tests using the L and L T A media were set up from positive cultures. Method
A single specimen of sputum or a laryngeal swab was obtained from each of 48 patients, who had started a I3-wcek course of isoniazid and PAS 8 to 28 weeks (avcrage 19 weeks) prcviously. After finishing this course the subsequent chemotherapy until the specimen was collected was as follows: isoniazid and PAS in 17 patients, streptomycin and isoniazi.d in Io paticnts, other combinations in xo patients and no chemotherapy in z i patients. Sputum was examined by.the routine fluorescence microscopy method (Norman and Jelks, i945) for acid-fast bacilli and was then shaken with 4 per cent N a O H at 37 ° C. and centrifuged. After washing with distilled watcr loopfuls of the deposit were inoculated on to 2 slopes of L medium, .'2 slopes of L T A medium and, with 7 sputa that were positive on direct smcar examination, on single slopes of L medium containing 20 vg. haemin per ml. Laryngeal swabs were treated with 6 per cent H 2 S O 4 and inoculated on to 2 slopes of L medium and 2 sIopes of L T A medium. Any growth on L medium was tested for its sensitivity to isoniazid by the Medical Research C o u n c i l (x953a) method using two tests in parallel; one on slopes of L medium and the other on slopes of L T A medium. A second similar pair of tests was set up from growth on L T A medium. The sensitivity of cultures on L medium plus haemin was tested on L T A medium only. In addition 8 strains, isolated in various laboratories, from similar patients who had received isoniazid plus PAS and a known isoniazid-resistant strain were tested for isoniazid sensitivity in parallel on L and L T A media. Results
As can be seen from Table ], the direct smears from 3 specimens showed acid-fast bacilli, but cultures on L and L T A media and, in the one specimen tested, on L plus
171
TABLE I . - RESULTS OF DIRECT SMEAR EXAMINATIONS AND CULTURE OF 4 8 SPUTA AND LARYNGEAL S%VABS FROM PATIENTS TREATED %VITIt ISONIAZID PLUS PAS
Specimen Sputum ,, ,, ,, Lar. swab.
Direct smear Neg. Pos. Neg. Pos.
Culture
No. of specimens
Neg.* Neg.* Pos.~" Pos.~ Neg.*
24 3 I II 9
Total 48 *Culture negative on all media tested. "~Culture positive on one or more media. haemin medium were negative. In addition there were fairly numerous acid-fast bacilli in smears from 2 more sputa but the cultures yielded only single colonies. Positive cultures were obtained from I2 out of the 48 specimens examined and xo of these I2 came from patients who had received isoniazid and PAS only. As is shown in Table II, there was "no significant TABLE I I . - RESULTS OF CULTURING 4 8 SPUTA AND LARYNGEAL S%VABS ON L a n d L T A MEDIA
No. of specimens ~¢edium positive L LTA
9 xo
Average period of incubation No. of till posit#e slopes (weeks) positive Average Range 17 x5
3.6 3"4
2-8 2-5
difference in the frequency or speed With which these cultures appeared on the L or the L T A media. The cultures from 5 specimens were positive on either L medium alone or on L T A medium alone, but in 3 of these there were single colonies on only one of the slopes inoculated and in a fourth contamination accounted for the discrepancy. As far as could be judged from the limited number of slopes used growth appeared on L medium plus haemin with the same frequency and rapidity. Thus with 4 specimens the slopes of L medium plus haemin yielded growth similar in amount to that found on L and L T A media. With 2 specimens there was growth on only one slope of L T A medium, but none on L
July
TUBERCLE
172
medium plus hacmin nor on L medium nor on the remaining slopes of L T A medium. Finally one specimen yielded no growth on any of the media. A total of 42 isoniazid sensitivity tests were set up from the 12 sputa that yielded positive cultures and a further 16 tests were set up f r o m the 8 cultures received from other laboratories. In all of these 58 tests less than 20 colonies grew on slopes containing 0"2 /~g. per ml. or more isoniazid; that is to say all the cultures were sensitive. It was noticeable that, where a few colonies occurred on slopes containing isoniazid, their numbers were very similar in the parallel tests carried out on L and L T A medium. The known resistant strain yielded confluent growth on slopes of both media containing 0-2 #g. per ml. isoniazid, but on the L slope containing I t~g. per ml. there were innumerable discrete colonies whereas on L T A medium containing I/zg. per mh there was no growth.
1954
Acknowledgments My thanks arc due to Miss Margaret Monk for technical assistance and to Drs C. S. Anderson, R. Blowers, Professor A. ~V. Downie, Drs W. J. Godden, P. Kidd, M. Laidlaw, G. Laws, J. T . A. Lloyd, P. H. Martin, R. Pilsworth, A. I. O. Prentice, I. A. Purdie, R. XV. Riddell, B. R. Sandiford, W. K. Taylor, Mr G. L. C. Usher, Drs A. T. Wallace and M. M. H. Whyte for supplying the sputum, laryngeal swabs and culturcs. References Fisher, M. W. (z95~) Amer. Rev. Tuber¢., LXW,626. Fisher, M. W. 0954) Amer. Rev. Tuberc. In press. Medical Research Council (t953a) Lancet, n, 213. Medical Research Council (1953b) Brit. ined. o7, n, xoo5. Norman, W. A., and Jelks, F. W. 0945) Bull. Inst. Med. Lab. Tech., II, 37.
Correspondence Radiology in Childhood Tuberculosis To tile Editor-'TUBERCLE'.
SIR,--In this article (TUBERCLE, May I954, Conclusions
The results of culturing 4 8 sputa and laryngeal swabs from patients with pulmonary tuberculosis who had received treatment with isoniazid and PAS indicate that the incorporation of albumin and Tween 80 or haemin in Lowenstein-Jensen medium did not increase the number of positive cultures obtained nor the speed at which they grew. In spite of incorporation of these substances in the medium used for sensitivity tests all of the I~2 positive cultures a n d : a further 8 cultures derived from similar patients were found to be sensitive to isoniazid. Thus it seems unlikely that the very small number of isoniazid-rcsistant strains obtained from patients treated with isoniazid plus PAS (Medical Research Council, I953 b) is due to the presence of resistant bacilli in their sputum which require the addition to Lowenstein-Jensen medium of specific growth factors present in bovine albumin, Tween 80 or as haemin for growth.
p. 1I3) Dr Grzybowski is really proposing a new classification of active pulmonary tuberculosis in children in five groups. The first three of these groups, ' simple primary tuberculosis', 'primary tuberculosis with lobar or segmental lesions' and 'pleural effusion' (which, as he states, arises in the majority of cases some three to six months after primary infection) are obviously intended to be varieties of benign primary tuberculosis. The fourth group ' miliary tuberculosis' is not stated in the article to have any relationship with the primary complex, but in the corresponding book (Bentley et al., x954), it is stated that it arises most frequently in children from one of the components of the primary complex. It would appear, therefore, that these four groups are regarded by the authors as varieties of primary tuberculous disease. The fifth group 'chronic pulmonary tuberculosis' is the name which the authors gave to the 'adult type of the disease' occurring in children. It is stated that 'it is of the utmost importance to differentiate between these cases and cases of primary tuberculosis'. "in other_words, the author is describing two types of pulmonary tuberculosis, which might correctly be called 'primary' and 'secondary' from the pathogenetic point of view, or 'acute' and 'chronic' from the pathological, clinical and radiological point of view. This
170
July 1954
conversion was less likely to occur in technicians of the Edinburgh Royal Victoria and associated group of hospitals. patients with gross cavitation. (5) The clinical significance of isoniazid References resistance is confirmed by the increase in Bignall, J. R., Clegg, J. W., Crofton,J. W., Smith, B . J . D . , Holt, H. D., Mitehison, D. A., and Armltage, P. (195o) sputum positivity and the incidence of Brit. reed. J., I, 1224. clinical and radiographic deterioration after Coates, E. O., Jr., Steeken, ~,V., Wolinsky, E., and Brinkman, G. L. (1953) New Engl. 07. ~Ied., calvin, Io8x. the emergence of resistant organisms. Conalty, M. L. (x953) Irish 07. reed. Sci., 6th Series, July, ~67. (6) In 3 cases a reversion in the degree of Crofton, J., and Mitchison, D.A. (1948) Brit. reed. 07., 1I, resistance occurred after stopping the drug. IOO9. M. (z948) 07. Bact., Lvl, 63. The results of re-treatment with isoniazid Demercc, Dye, SV. E. (1953) Transactions of the 12th Conference on the Chemotherapy of Tuberculosis, Veterans Adsuggested that such reversion is unlikely to ministration, United States of America, p. 11 I. be of any clinical importance. Joiner, C. L., MacLean, K. S., Chalmers, D. G., Anderson, K., Collard, P., King, M. B., and Knox, R. (1953)
Acknowledgments
Lancet, I[, 15"2.
This work was generously supported by the Royal Victoria Hospital Tuberculosis Trust. We should also like to thank Drs N. W. Horne, I. W. B. Grant, and J. D. Ross for allowing us to investigate their patients, for making the radiographic assessments and for their keen interest in the work. Unstinting help was received from the registrars, house physicians, nursing staff and laboratory
Lotte, A., and Poussier, J. (i953) Re:,. Tuberc., xwz, I. Lumsden, E. G. S., and Swoboda, J. A. F. (1952) Tubercle, XXXIII~ ~22.
Medical Research Council (i952) Brit. reed. 07, iz, 735Medical Research Council (1953a) Lancet, H, 213. Medical Research Council (x953b) Brit. reed. 07, I, 52I. Mitchison, D. A. (1949) Lancet, H, 694. Petit, A. (1953) Schweiz. reed. IVschr., LXXXIH,754. Selikoff, I.J., Robitzek, E. H., and Ornstein, G. G. (1952) 07. Amer. rned. Ass., eL, 973United States Public tteahh Service Co-operative Investigation (x953) Transactlons.of the a2th Conference on the Chemotherapy of Tuberculosis, Veterans Administration, United States of America, p. 72-
Culture Media for Tubercle Bacilli During Chemotherapy with Isoniazjd By D. A. MITCHISON*
Lecturer in Bacteriology, PostgraduateMedical Schoolof London, Ducane Road, W.12 i n the Medical Research Council (I953b) report on isoniazid in combination with streptomycin or with p-aminosalicylic acid (PAS) in the treatment of pulmonary tuberculosis, a footnote gave details of discrepa:ncies in the results of isoniazid sensitivity tests carried out in one of the cooperating centres and in the reference laboratory. Cultures from 3 patients treated with isoniazid plus PAS were found to be resistant by the local laboratory, which used Lowenstein-Jensen medium with the addition of o.I25 per cent bovine albumin fraction V (Armour & Co.) and o-o6 per cent Tween 8o (Atlas Powder C o . ) ( L T A Medium), both for the isolation of tubercle bacilli and for the subsequent sensitivity tests. The reference laboratory, using a con-
ventional Lowenstein-Jensen formula (L Medium) found theseculturessensitive. Now Fisher (i952 , 1954) has suggested that isoniazid resistant tubercle bacilli r e q u i r e a growth factor, haemin, which is present in bovine albumin. Thus a possible explanation of the discrepancies between results in_ the reference laboratory and in the local centre was that resistant bacilli which would_ not grow on L medium might have been isolated on LTA medium and that the results of sensitivity tests using these two media might therefore have differed. T o investigate this possibility a series of specimens from patients who had received isoniazid plus PAS in the Medical Research Council trials were cultured on the two media. In some cases cultures were also set
*In receipt of a grant from the Medical Research Council.
July 1954
TUBERCLE
up on L medium plus haemin. Sensitivity tests using the L and L T A media were set up from positive cultures. Method
A single specimen of sputum or a laryngeal swab was obtained from each of 48 patients, who had started a I3-wcek course of isoniazid and PAS 8 to 28 weeks (avcrage 19 weeks) prcviously. After finishing this course the subsequent chemotherapy until the specimen was collected was as follows: isoniazid and PAS in 17 patients, streptomycin and isoniazi.d in Io paticnts, other combinations in xo patients and no chemotherapy in z i patients. Sputum was examined by.the routine fluorescence microscopy method (Norman and Jelks, i945) for acid-fast bacilli and was then shaken with 4 per cent N a O H at 37 ° C. and centrifuged. After washing with distilled watcr loopfuls of the deposit were inoculated on to 2 slopes of L medium, .'2 slopes of L T A medium and, with 7 sputa that were positive on direct smcar examination, on single slopes of L medium containing 20 vg. haemin per ml. Laryngeal swabs were treated with 6 per cent H 2 S O 4 and inoculated on to 2 slopes of L medium and 2 sIopes of L T A medium. Any growth on L medium was tested for its sensitivity to isoniazid by the Medical Research C o u n c i l (x953a) method using two tests in parallel; one on slopes of L medium and the other on slopes of L T A medium. A second similar pair of tests was set up from growth on L T A medium. The sensitivity of cultures on L medium plus haemin was tested on L T A medium only. In addition 8 strains, isolated in various laboratories, from similar patients who had received isoniazid plus PAS and a known isoniazid-resistant strain were tested for isoniazid sensitivity in parallel on L and L T A media. Results
As can be seen from Table ], the direct smears from 3 specimens showed acid-fast bacilli, but cultures on L and L T A media and, in the one specimen tested, on L plus
171
TABLE I . - RESULTS OF DIRECT SMEAR EXAMINATIONS AND CULTURE OF 4 8 SPUTA AND LARYNGEAL S%VABS FROM PATIENTS TREATED %VITIt ISONIAZID PLUS PAS
Specimen Sputum ,, ,, ,, Lar. swab.
Direct smear Neg. Pos. Neg. Pos.
Culture
No. of specimens
Neg.* Neg.* Pos.~" Pos.~ Neg.*
24 3 I II 9
Total 48 *Culture negative on all media tested. "~Culture positive on one or more media. haemin medium were negative. In addition there were fairly numerous acid-fast bacilli in smears from 2 more sputa but the cultures yielded only single colonies. Positive cultures were obtained from I2 out of the 48 specimens examined and xo of these I2 came from patients who had received isoniazid and PAS only. As is shown in Table II, there was "no significant TABLE I I . - RESULTS OF CULTURING 4 8 SPUTA AND LARYNGEAL S%VABS ON L a n d L T A MEDIA
No. of specimens ~¢edium positive L LTA
9 xo
Average period of incubation No. of till posit#e slopes (weeks) positive Average Range 17 x5
3.6 3"4
2-8 2-5
difference in the frequency or speed With which these cultures appeared on the L or the L T A media. The cultures from 5 specimens were positive on either L medium alone or on L T A medium alone, but in 3 of these there were single colonies on only one of the slopes inoculated and in a fourth contamination accounted for the discrepancy. As far as could be judged from the limited number of slopes used growth appeared on L medium plus haemin with the same frequency and rapidity. Thus with 4 specimens the slopes of L medium plus haemin yielded growth similar in amount to that found on L and L T A media. With 2 specimens there was growth on only one slope of L T A medium, but none on L
July
TUBERCLE
172
medium plus hacmin nor on L medium nor on the remaining slopes of L T A medium. Finally one specimen yielded no growth on any of the media. A total of 42 isoniazid sensitivity tests were set up from the 12 sputa that yielded positive cultures and a further 16 tests were set up f r o m the 8 cultures received from other laboratories. In all of these 58 tests less than 20 colonies grew on slopes containing 0"2 /~g. per ml. or more isoniazid; that is to say all the cultures were sensitive. It was noticeable that, where a few colonies occurred on slopes containing isoniazid, their numbers were very similar in the parallel tests carried out on L and L T A medium. The known resistant strain yielded confluent growth on slopes of both media containing 0-2 #g. per ml. isoniazid, but on the L slope containing I t~g. per ml. there were innumerable discrete colonies whereas on L T A medium containing I/zg. per mh there was no growth.
1954
Acknowledgments My thanks arc due to Miss Margaret Monk for technical assistance and to Drs C. S. Anderson, R. Blowers, Professor A. ~V. Downie, Drs W. J. Godden, P. Kidd, M. Laidlaw, G. Laws, J. T . A. Lloyd, P. H. Martin, R. Pilsworth, A. I. O. Prentice, I. A. Purdie, R. XV. Riddell, B. R. Sandiford, W. K. Taylor, Mr G. L. C. Usher, Drs A. T. Wallace and M. M. H. Whyte for supplying the sputum, laryngeal swabs and culturcs. References Fisher, M. W. (z95~) Amer. Rev. Tuber¢., LXW,626. Fisher, M. W. 0954) Amer. Rev. Tuberc. In press. Medical Research Council (t953a) Lancet, n, 213. Medical Research Council (1953b) Brit. ined. o7, n, xoo5. Norman, W. A., and Jelks, F. W. 0945) Bull. Inst. Med. Lab. Tech., II, 37.
Correspondence Radiology in Childhood Tuberculosis To tile Editor-'TUBERCLE'.
SIR,--In this article (TUBERCLE, May I954, Conclusions
The results of culturing 4 8 sputa and laryngeal swabs from patients with pulmonary tuberculosis who had received treatment with isoniazid and PAS indicate that the incorporation of albumin and Tween 80 or haemin in Lowenstein-Jensen medium did not increase the number of positive cultures obtained nor the speed at which they grew. In spite of incorporation of these substances in the medium used for sensitivity tests all of the I~2 positive cultures a n d : a further 8 cultures derived from similar patients were found to be sensitive to isoniazid. Thus it seems unlikely that the very small number of isoniazid-rcsistant strains obtained from patients treated with isoniazid plus PAS (Medical Research Council, I953 b) is due to the presence of resistant bacilli in their sputum which require the addition to Lowenstein-Jensen medium of specific growth factors present in bovine albumin, Tween 80 or as haemin for growth.
p. 1I3) Dr Grzybowski is really proposing a new classification of active pulmonary tuberculosis in children in five groups. The first three of these groups, ' simple primary tuberculosis', 'primary tuberculosis with lobar or segmental lesions' and 'pleural effusion' (which, as he states, arises in the majority of cases some three to six months after primary infection) are obviously intended to be varieties of benign primary tuberculosis. The fourth group ' miliary tuberculosis' is not stated in the article to have any relationship with the primary complex, but in the corresponding book (Bentley et al., x954), it is stated that it arises most frequently in children from one of the components of the primary complex. It would appear, therefore, that these four groups are regarded by the authors as varieties of primary tuberculous disease. The fifth group 'chronic pulmonary tuberculosis' is the name which the authors gave to the 'adult type of the disease' occurring in children. It is stated that 'it is of the utmost importance to differentiate between these cases and cases of primary tuberculosis'. "in other_words, the author is describing two types of pulmonary tuberculosis, which might correctly be called 'primary' and 'secondary' from the pathogenetic point of view, or 'acute' and 'chronic' from the pathological, clinical and radiological point of view. This