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Mycobacterial dormancy
Tubercle and Lung Disease (1995)76, 477479 © 1995PearsonProfessionalLtd
Tubercleand Lung Disease
Mycobacterial dormancy P. R. J. Gangadharam
Mycobacteriology Research Laboratories, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA
lar series conducted 20 years later in mice 8 and guinea pigs 9 soon after rifampin was introduced, formed the scientific basis for SCC of tuberculosis. The success of SCC regimens containing rifampin was explained in the studies performed by Mitchison, Grosset and others, who showed that rifampin could kill tubercle bacilli even with short periods of contact, whereas other powerful drugs such as isoniazid require about one generation time (24 hours). Based on these explanations, and the now established success of SCC, effective regimens for treating tuberculosis were developed. With such a potential for success came the 'proof of the pudding' feeling that nothing more need be learnt about tuberculosis chemotherapy, leading to the premature discontinuation of interest in the disease, and the drastic diminution of support from different agencies worldwide, particularly in the industrialized countries. The unfortunate result is now evident in the rapid increase in tuberculosis, both in industrialized countries, and in developing countries. The global AIDS epidemic is creating additional serious consequences. This re-emergence of tuberculosis has caused serious concern among health authorities and has stimulated those actively involved in the varied aspects of the disease. One fringe benefit of this reinforced interest is the introduction of new branches of science such as molecular biology, which have progressed rapidly in the short time (less than 10 years) of their involvement with mycobacteria. However, most of these studies have dealt with the subtle aspects of the mycobacterial genome, to identify the genes which code for resistance for antituberculosis drugs and to develop rapid methods for diagnosis of tuberculosis and detection of multiple drug resistant tubercle bacilli. 1°-12 Popular molecular biological techniques such as the polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) analysis, and restriction fragment length polymorphism (RFLP), have been used increasingly in these studies. On the other hand, very little concern has been given to understanding the basic mechanisms of chemotherapy.
One of the significant landmarks developed in the middle of the century in the treatment of tuberculosis is the introduction of powerful chemotherapeutic regimens. 1 However, treatment had to be for 18-24 months in order to achieve complete success, mainly due to the presence of dormant tubercle bacilli. It was soon realized that drug regimens even of moderate or low efficacy, e.g. isoniazid + thiacetazone, or isoniazid + PAS, can render the sputum negative for tubercle bacilli as rapidly as the powerful isoniazid and rifampin combinations. The main difference between the two groups is in the maintenance of sputum negativity once the treatment has been discontinued. With weak regimens, the treatment has to be prolonged for up to 1.5 to 2 years, mainly to eradicate the persisters which remain dormant for several months. On the other hand, with regimens containing rifampin, which can act on such bacilli even with a short period of contact, the duration of treatment could be shortened to 9 or even 6 months. Soon after rifampin was introduced, Canetti et al's optimism 2 was soon proven correct by several controlled clinical studies conducted initially by the East African/British Medical Research Council, and later by several other groups) ,4 It was established that the success of the short course chemotherapy (SCC) regimens lay in their capacity to attack the dormant tubercle bacilli, besides the rapid elimination of the multiplying organisms. The existence of dormant tubercle bacilli ('persisters') for long periods after chemotherapy was shown by McCune et al nearly 40 years ago, soon after the introduction of isoniazid. 5,6 Their elegant studies, called the 'Cornell model' after the academic institution where the work was done, could be considered the key studies on mycobacterial dormancy and their relevance to tuberculosis chemotherapy. 7 These studies, along with a simi-
Correspondence to: Professor Pattisapu R. J. Gangadharam, Professor of Medicine, Microbiology and Pathology, Director of Mycobacteriology Research, University of Illinois at Chicago, 835 S. Wolcott (M/C 790), Rm E709, Chicago, IL 60612, USA. 477
478 Tubercleand Lung Disease The paper entitled 'The bacterial DNA content of mouse organs in Cornell model of dormant tuberculosis' by de Wit et a113 in this issue of Tubercle and Lung Disease, is a good starting point in this direction. This paper and other relevant papers by the same group 14 are welcoming signs of an understanding of some of the subtle intricacies of the host-parasite interactions with respect to tuberculosis chemotherapy and, more importantly, mycobacterial dormancy. In the original Cornell model, 5,6 mice were infected with a heavy dose of a virulent strain of tubercle bacilli and the disease was allowed to progress for two weeks, at which time isoniazid and pyrazinamide was given in combination for several weeks. Treatment was discontinued, but lung and spleen cultures were performed at regular intervals. It was shown that the organs became negative for tubercle bacilli soon after the treatment had progressed and maintained their negative status for some time. If no further treatment was given, the bacilli reappeared in these tissues. The resurgence of growth could also be hastened by immunosuppressive agents such as corticosteroids. de Wit et ~113 have remained faithful to the details of the original Cornell model despite the changes in treatment regimens over the last 40 years. However, since their aim was to study the molecular basis of dormancy, they used modern molecular biological techniques to detect mycobacterial DNA in the animal tissues at several periods after discontinuation of chemotherapy. They also inoculated various volumes of the homogenized tissue suspensions into fresh mice to study the pathobiological outcome in the new animal host. A significant finding from this study is the detection of mycobacterial DNA at several time points after cessation of chemotherapy, even though the regular culture of the homogenized suspensions did not show any growth of the organisms in artificial media. Likewise, few animals inoculated with large volumes of the tissue homogenates showed evidence of disease. Interestingly, another study on the same lines, but with human material, presented by Hellyer et al at the May 1995 conference of the American Society for Microbiology, Is has demonstrated the persistence of Mycobacterium tuberculosis DNA in smear-negative and culture-negative specimens over 12 months after the initiation of chemotherapy and 6 months after conversion to smear and culture negativity. Unlike the study of de Wit et al ~3 which used the isoniazid-pyrazinamide combination to mimic the original Cornell model, Hellyer et al ~5 used the well established Arkansas SCC regimen containing rifampin. As in the mouse studies, the presence of DNA could not be considered indicative of live (dormant) or dead bacilli. Obviously it will be valuable to know whether the DNA seen in the mouse or human studies represents small numbers of live bacilli which may be able to multiply actively when the host immunity is compromised. Previous studies by Dhillon and Mitchison, 14using the same type of mouse model, have demonstrated that after
prolonged culture negativity, positive cultures were seen even after vaccination procedures. The DNA may therefore represent live bacilli, warranting further studies. For instance, serial quantitative DNA measurements indicating a 'fall and rise' situation, to cite the phrase coined by Prof. Mitchison, to enable detection of drug resistance in tubercle bacilli 16will indicate whether bacteria are still alive in this culture negative status. Alternatively, as has been suggested by de Wit et all3 one can consider using RNA amplification as an indication of mycobacterial viability. 17 Besides the importance in chemotherapy, mycobacterial dormancy has great relevance in pathogenesis of tuberculosis and can facilitate greater understanding of latency in tuberculosis and mechanisms of endogenous reactivation. Knowledge in this area is very sparse and only modest beginnings have been made in the last few years; a search for a 'dormancy' gene 18along the lines of the whiB gene 19 for sporulation of Streptomyces coelicolor is being pursued, although attempts to identify such genes in mycobacteria have so far been unsuccessful (Chater K, personal communication to Predich et al22). Similarly, attempts are being made to identify a putative rpoS homologue in M. tuberculosis and other slow growing mycobacteria, as a stationary phase sigma factor5 ° These studies are now being extended to characterize some major response/stationary phase sigma factors (e.g. mysB of M. smegmatis) in mycobacteria as functional equivalents of the rpoS gene. 21 It is not unrealistic to hope that future studies may enable us to assess the significance at the molecular level of tuberculin reaction and conversion. They may also help to distinguish between tuberculin skin test positivity due to BCG vaccination from that due to fresh infection with tubercle bacilli. In future work, in addition to the IS6110 insertion element used in the mouse and human studies discussed above, m5 other genetic elements which specifically identify M. tuberculosis and M. boris should be used as IS6110 occurs in all members of the M. tuberculosis complex. 22Already a specific PCRbased amplication method has been used to detect DNA fragments that are M. tuberculosis specific, using the gene encoding the MTP40 protein. 23 Likewise, inoculation of the DNA containing tissue homogenates into immune deficient mice e.g. SCID, nude, interferon knock-out mice, etc, will prove whether the relapse in tuberculosis will be more rapid and frequent in AIDS patients and whether SCC will be adequate in AIDS patients, as in normal individuals. At the other extreme, one can hypothesize that a 'Jurassic Park' type of situation might occur in some immune compromised individuals, wherein the mycobacterial DNA can transfer to other innocuous microorganisms present in the host and turn into violent and dangerous forms. Thus the studies reported by de Wit et al~3aa as well as the studies in human situations ~5 and the various attempts to identify and characterize the 'dormancy gene' will open up a wide new scope of investigations into molecular mechanisms of dormancy in relation to che-
Mycobacterial dormancy m o t h e r a p y a n d p a t h o g e n e s i s o f t u b e r c u l o s i s . It is h o p e d that this e n t h u s i a s m a n d interest, d e p e n d e n t o n the s u p p o r t a n d f i n a n c i a l r e s o u r c e s available to i n v e s t i g a t o r s w o r l d w i d e , will c o n t i n u e to g r o w p r e f e r a b l y n o t in a 'rise and fall' cycle, a n d a l l o w m a n y o f t h e s e i m p o r t a n t q u e s t i o n s to b e a n s w e r e d . I f i n t e r e s t and s u p p o r t d o fall off, the o u t c o m e will b e d i s a s t r o u s ; w e will, as Sir J o h n C r o f t o n stated a4 ' r e t u r n to a sad state o f i n c o m p e t e n c e ' w i t h the t o o l s a n d skills w e h a v e b e e n d e v e l o p i n g to c o n t r o l this c u r a b l e d i s e a s e .
Acknowledgement I am grateful to Dr Joseph H. Bates, Dr Kathleen D. Eisenach and Dr M. V. Reddy for valuable criticisms and suggestions in the preparation of this editorial. I am also thankful to Mrs Madhavi Paturi for her ungrudging secretarial assistance.
References 1. Toman K (ed) 1979. What were the main landmarks in the development of tuberculosis chemotherapy? In: 'Tuberculosis case-finding and chemotherapy questions and answers'. WHO, Geneva, p 75-76. 2. Canetti G, Le Lirzin M, Porven G, Rist N, Grumbach F. Some comparative aspects of rifampicin and isoniazid. Tubercle 1968; 49: 367-376. 3. East African/British Medical Research Council. Controlled clinical trials of four short-course (6 month) regimens of chemotherapy for the treatment of pulmonary tuberculosis. Lancet 1974; 2: 237. 4. Fox W. Whither short-course chemotherapy? Bull Int Union Tuberc 1981; 56: 135-155. 5. McCune R M, Tompsett R, McDermott W. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J Exp Med 1956; 104: 763-802. 6. McCnne R M, Feldmann F M, Lambert H P, McDermott W. Microbial persistence I. The capacity of tubercle bacilli to survive sterilisafion in mouse tissues. J Exp Med 1966; 123: 445-468. 7. Grosset J. The sterilizing value of rifampicin and pyrazinamide in experimental short-course chemotherapy. Bull Int Union Tuberc 1978; 53: 5-12. 8. Grumbach F, Rist N. Activit6 antituberculeuse experimentale de la rifampicine, d6riv6 de la rifamycine SV. Rev Tuberc Pneum 1967; 31: 749-762.
479
9. Fox W, Mitchison D A. Short-course chemotherapy for pulmonary tuberculosis. Am Rev Respir Dis 1975; 111: 325-353. 10. Cole S T, Smith D R 1994 Toward mapping and sequencing the genome of Mycobacterium tuberculosis. In: Bloom B R (ed) Tuberculosis: pathogenesis, protection and control. American Society for Microbiology Press, Washington DC, p 227-238. 11. Jacobs W R, Barletta R G, Udani R et al. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 1993; 260: 819-822. 12. Jacobs W R, Kalpana G V, Cirilo J D et al. Genetic systems for mycobacteria. Methods Euzymol 1991; 204: 537-555. 13. de Wit D, Wootton M, Dhillon J, Mitchison D A. The bacterial DNA content of mouse organs in the Cornell model of dormant tuberculosis. Tubercle Lung Dis 1995; 76: 554-561. 14. Dhillon J, Mitchison D A. Effect of vaccines in a murine model of dormant tuberculosis. Tubercle Lung Dis 1994; 75: 61-64. 15. Hellyer T J, Fletcher T W, Spears P A e t al. Strand displacement amplification (SDA) and the polymerase chain reaction (PCR) for monitoring the response to chemotherapy in patients with tuberculosis. ASM General Meeting, Washington DC, May 21-25, 1995, Abstract # U 97. 16. Mitchison D A. Sensitivity testing. In: Heaf F, Rusby N L (eds). Recent advances in respiratory tuberculosis. London: J & A Churchill, 1968: 160. 17. Van tier Vliet G, Schepers P, Schukkink R A F, Van Gemen B, Klatser P R. Assessment of mycobacterial viability by RNA amplification. Antimicrob Agents Chemother 1994; 38: 1959-1965. 18. Bishai W. Molecular genetics of latent tuberculosis infection. In symposium on 'Scientific advances in tuberculosis'. American Thoracic Society International Conference, Seattle, Washington, May 20-24, 1995 19. Davis N K, Chater K F. The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation. Mol Gen Genet 1992; 232: 351-358. 20. Welty D M, Small P L C. Identification of a putative rpos homologue from M. marinum, M. tuberculosis, M. ulcerans and M. hemophilum. American Society for Microbiology General Meeting, May 1994, Abstract No. U 25. 21. Eisenach K D, Cave M D, Bates J H, Crawford J T. Amplification of a repetitive sequence specific for Mycobacterium tuberculosis using polymerase chain reaction. J Infect Dis 1990; 16l: 977-981. 22. Predich M, Douldlan L, Nair G, Smith I. Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis. Mol. Microbiol. 1995; 15: 355-366. 23. Parra C A, Londono L P, Portillo P D, Patarroyo M E. Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene: identification of a species-specific sequence. Infection mad Immunity 1991; 59:3411-3417. 24. Sir John Crofton. In: Foreword to Clinical tuberculosis. PDO Davies (ed). London: Chapman and Hall, 1994: XV.
Tubercleand Lung Disease
Mycobacterial dormancy P. R. J. Gangadharam
Mycobacteriology Research Laboratories, Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, IL, USA
lar series conducted 20 years later in mice 8 and guinea pigs 9 soon after rifampin was introduced, formed the scientific basis for SCC of tuberculosis. The success of SCC regimens containing rifampin was explained in the studies performed by Mitchison, Grosset and others, who showed that rifampin could kill tubercle bacilli even with short periods of contact, whereas other powerful drugs such as isoniazid require about one generation time (24 hours). Based on these explanations, and the now established success of SCC, effective regimens for treating tuberculosis were developed. With such a potential for success came the 'proof of the pudding' feeling that nothing more need be learnt about tuberculosis chemotherapy, leading to the premature discontinuation of interest in the disease, and the drastic diminution of support from different agencies worldwide, particularly in the industrialized countries. The unfortunate result is now evident in the rapid increase in tuberculosis, both in industrialized countries, and in developing countries. The global AIDS epidemic is creating additional serious consequences. This re-emergence of tuberculosis has caused serious concern among health authorities and has stimulated those actively involved in the varied aspects of the disease. One fringe benefit of this reinforced interest is the introduction of new branches of science such as molecular biology, which have progressed rapidly in the short time (less than 10 years) of their involvement with mycobacteria. However, most of these studies have dealt with the subtle aspects of the mycobacterial genome, to identify the genes which code for resistance for antituberculosis drugs and to develop rapid methods for diagnosis of tuberculosis and detection of multiple drug resistant tubercle bacilli. 1°-12 Popular molecular biological techniques such as the polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) analysis, and restriction fragment length polymorphism (RFLP), have been used increasingly in these studies. On the other hand, very little concern has been given to understanding the basic mechanisms of chemotherapy.
One of the significant landmarks developed in the middle of the century in the treatment of tuberculosis is the introduction of powerful chemotherapeutic regimens. 1 However, treatment had to be for 18-24 months in order to achieve complete success, mainly due to the presence of dormant tubercle bacilli. It was soon realized that drug regimens even of moderate or low efficacy, e.g. isoniazid + thiacetazone, or isoniazid + PAS, can render the sputum negative for tubercle bacilli as rapidly as the powerful isoniazid and rifampin combinations. The main difference between the two groups is in the maintenance of sputum negativity once the treatment has been discontinued. With weak regimens, the treatment has to be prolonged for up to 1.5 to 2 years, mainly to eradicate the persisters which remain dormant for several months. On the other hand, with regimens containing rifampin, which can act on such bacilli even with a short period of contact, the duration of treatment could be shortened to 9 or even 6 months. Soon after rifampin was introduced, Canetti et al's optimism 2 was soon proven correct by several controlled clinical studies conducted initially by the East African/British Medical Research Council, and later by several other groups) ,4 It was established that the success of the short course chemotherapy (SCC) regimens lay in their capacity to attack the dormant tubercle bacilli, besides the rapid elimination of the multiplying organisms. The existence of dormant tubercle bacilli ('persisters') for long periods after chemotherapy was shown by McCune et al nearly 40 years ago, soon after the introduction of isoniazid. 5,6 Their elegant studies, called the 'Cornell model' after the academic institution where the work was done, could be considered the key studies on mycobacterial dormancy and their relevance to tuberculosis chemotherapy. 7 These studies, along with a simi-
Correspondence to: Professor Pattisapu R. J. Gangadharam, Professor of Medicine, Microbiology and Pathology, Director of Mycobacteriology Research, University of Illinois at Chicago, 835 S. Wolcott (M/C 790), Rm E709, Chicago, IL 60612, USA. 477
478 Tubercleand Lung Disease The paper entitled 'The bacterial DNA content of mouse organs in Cornell model of dormant tuberculosis' by de Wit et a113 in this issue of Tubercle and Lung Disease, is a good starting point in this direction. This paper and other relevant papers by the same group 14 are welcoming signs of an understanding of some of the subtle intricacies of the host-parasite interactions with respect to tuberculosis chemotherapy and, more importantly, mycobacterial dormancy. In the original Cornell model, 5,6 mice were infected with a heavy dose of a virulent strain of tubercle bacilli and the disease was allowed to progress for two weeks, at which time isoniazid and pyrazinamide was given in combination for several weeks. Treatment was discontinued, but lung and spleen cultures were performed at regular intervals. It was shown that the organs became negative for tubercle bacilli soon after the treatment had progressed and maintained their negative status for some time. If no further treatment was given, the bacilli reappeared in these tissues. The resurgence of growth could also be hastened by immunosuppressive agents such as corticosteroids. de Wit et ~113 have remained faithful to the details of the original Cornell model despite the changes in treatment regimens over the last 40 years. However, since their aim was to study the molecular basis of dormancy, they used modern molecular biological techniques to detect mycobacterial DNA in the animal tissues at several periods after discontinuation of chemotherapy. They also inoculated various volumes of the homogenized tissue suspensions into fresh mice to study the pathobiological outcome in the new animal host. A significant finding from this study is the detection of mycobacterial DNA at several time points after cessation of chemotherapy, even though the regular culture of the homogenized suspensions did not show any growth of the organisms in artificial media. Likewise, few animals inoculated with large volumes of the tissue homogenates showed evidence of disease. Interestingly, another study on the same lines, but with human material, presented by Hellyer et al at the May 1995 conference of the American Society for Microbiology, Is has demonstrated the persistence of Mycobacterium tuberculosis DNA in smear-negative and culture-negative specimens over 12 months after the initiation of chemotherapy and 6 months after conversion to smear and culture negativity. Unlike the study of de Wit et al ~3 which used the isoniazid-pyrazinamide combination to mimic the original Cornell model, Hellyer et al ~5 used the well established Arkansas SCC regimen containing rifampin. As in the mouse studies, the presence of DNA could not be considered indicative of live (dormant) or dead bacilli. Obviously it will be valuable to know whether the DNA seen in the mouse or human studies represents small numbers of live bacilli which may be able to multiply actively when the host immunity is compromised. Previous studies by Dhillon and Mitchison, 14using the same type of mouse model, have demonstrated that after
prolonged culture negativity, positive cultures were seen even after vaccination procedures. The DNA may therefore represent live bacilli, warranting further studies. For instance, serial quantitative DNA measurements indicating a 'fall and rise' situation, to cite the phrase coined by Prof. Mitchison, to enable detection of drug resistance in tubercle bacilli 16will indicate whether bacteria are still alive in this culture negative status. Alternatively, as has been suggested by de Wit et all3 one can consider using RNA amplification as an indication of mycobacterial viability. 17 Besides the importance in chemotherapy, mycobacterial dormancy has great relevance in pathogenesis of tuberculosis and can facilitate greater understanding of latency in tuberculosis and mechanisms of endogenous reactivation. Knowledge in this area is very sparse and only modest beginnings have been made in the last few years; a search for a 'dormancy' gene 18along the lines of the whiB gene 19 for sporulation of Streptomyces coelicolor is being pursued, although attempts to identify such genes in mycobacteria have so far been unsuccessful (Chater K, personal communication to Predich et al22). Similarly, attempts are being made to identify a putative rpoS homologue in M. tuberculosis and other slow growing mycobacteria, as a stationary phase sigma factor5 ° These studies are now being extended to characterize some major response/stationary phase sigma factors (e.g. mysB of M. smegmatis) in mycobacteria as functional equivalents of the rpoS gene. 21 It is not unrealistic to hope that future studies may enable us to assess the significance at the molecular level of tuberculin reaction and conversion. They may also help to distinguish between tuberculin skin test positivity due to BCG vaccination from that due to fresh infection with tubercle bacilli. In future work, in addition to the IS6110 insertion element used in the mouse and human studies discussed above, m5 other genetic elements which specifically identify M. tuberculosis and M. boris should be used as IS6110 occurs in all members of the M. tuberculosis complex. 22Already a specific PCRbased amplication method has been used to detect DNA fragments that are M. tuberculosis specific, using the gene encoding the MTP40 protein. 23 Likewise, inoculation of the DNA containing tissue homogenates into immune deficient mice e.g. SCID, nude, interferon knock-out mice, etc, will prove whether the relapse in tuberculosis will be more rapid and frequent in AIDS patients and whether SCC will be adequate in AIDS patients, as in normal individuals. At the other extreme, one can hypothesize that a 'Jurassic Park' type of situation might occur in some immune compromised individuals, wherein the mycobacterial DNA can transfer to other innocuous microorganisms present in the host and turn into violent and dangerous forms. Thus the studies reported by de Wit et al~3aa as well as the studies in human situations ~5 and the various attempts to identify and characterize the 'dormancy gene' will open up a wide new scope of investigations into molecular mechanisms of dormancy in relation to che-
Mycobacterial dormancy m o t h e r a p y a n d p a t h o g e n e s i s o f t u b e r c u l o s i s . It is h o p e d that this e n t h u s i a s m a n d interest, d e p e n d e n t o n the s u p p o r t a n d f i n a n c i a l r e s o u r c e s available to i n v e s t i g a t o r s w o r l d w i d e , will c o n t i n u e to g r o w p r e f e r a b l y n o t in a 'rise and fall' cycle, a n d a l l o w m a n y o f t h e s e i m p o r t a n t q u e s t i o n s to b e a n s w e r e d . I f i n t e r e s t and s u p p o r t d o fall off, the o u t c o m e will b e d i s a s t r o u s ; w e will, as Sir J o h n C r o f t o n stated a4 ' r e t u r n to a sad state o f i n c o m p e t e n c e ' w i t h the t o o l s a n d skills w e h a v e b e e n d e v e l o p i n g to c o n t r o l this c u r a b l e d i s e a s e .
Acknowledgement I am grateful to Dr Joseph H. Bates, Dr Kathleen D. Eisenach and Dr M. V. Reddy for valuable criticisms and suggestions in the preparation of this editorial. I am also thankful to Mrs Madhavi Paturi for her ungrudging secretarial assistance.
References 1. Toman K (ed) 1979. What were the main landmarks in the development of tuberculosis chemotherapy? In: 'Tuberculosis case-finding and chemotherapy questions and answers'. WHO, Geneva, p 75-76. 2. Canetti G, Le Lirzin M, Porven G, Rist N, Grumbach F. Some comparative aspects of rifampicin and isoniazid. Tubercle 1968; 49: 367-376. 3. East African/British Medical Research Council. Controlled clinical trials of four short-course (6 month) regimens of chemotherapy for the treatment of pulmonary tuberculosis. Lancet 1974; 2: 237. 4. Fox W. Whither short-course chemotherapy? Bull Int Union Tuberc 1981; 56: 135-155. 5. McCune R M, Tompsett R, McDermott W. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J Exp Med 1956; 104: 763-802. 6. McCnne R M, Feldmann F M, Lambert H P, McDermott W. Microbial persistence I. The capacity of tubercle bacilli to survive sterilisafion in mouse tissues. J Exp Med 1966; 123: 445-468. 7. Grosset J. The sterilizing value of rifampicin and pyrazinamide in experimental short-course chemotherapy. Bull Int Union Tuberc 1978; 53: 5-12. 8. Grumbach F, Rist N. Activit6 antituberculeuse experimentale de la rifampicine, d6riv6 de la rifamycine SV. Rev Tuberc Pneum 1967; 31: 749-762.
479
9. Fox W, Mitchison D A. Short-course chemotherapy for pulmonary tuberculosis. Am Rev Respir Dis 1975; 111: 325-353. 10. Cole S T, Smith D R 1994 Toward mapping and sequencing the genome of Mycobacterium tuberculosis. In: Bloom B R (ed) Tuberculosis: pathogenesis, protection and control. American Society for Microbiology Press, Washington DC, p 227-238. 11. Jacobs W R, Barletta R G, Udani R et al. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 1993; 260: 819-822. 12. Jacobs W R, Kalpana G V, Cirilo J D et al. Genetic systems for mycobacteria. Methods Euzymol 1991; 204: 537-555. 13. de Wit D, Wootton M, Dhillon J, Mitchison D A. The bacterial DNA content of mouse organs in the Cornell model of dormant tuberculosis. Tubercle Lung Dis 1995; 76: 554-561. 14. Dhillon J, Mitchison D A. Effect of vaccines in a murine model of dormant tuberculosis. Tubercle Lung Dis 1994; 75: 61-64. 15. Hellyer T J, Fletcher T W, Spears P A e t al. Strand displacement amplification (SDA) and the polymerase chain reaction (PCR) for monitoring the response to chemotherapy in patients with tuberculosis. ASM General Meeting, Washington DC, May 21-25, 1995, Abstract # U 97. 16. Mitchison D A. Sensitivity testing. In: Heaf F, Rusby N L (eds). Recent advances in respiratory tuberculosis. London: J & A Churchill, 1968: 160. 17. Van tier Vliet G, Schepers P, Schukkink R A F, Van Gemen B, Klatser P R. Assessment of mycobacterial viability by RNA amplification. Antimicrob Agents Chemother 1994; 38: 1959-1965. 18. Bishai W. Molecular genetics of latent tuberculosis infection. In symposium on 'Scientific advances in tuberculosis'. American Thoracic Society International Conference, Seattle, Washington, May 20-24, 1995 19. Davis N K, Chater K F. The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation. Mol Gen Genet 1992; 232: 351-358. 20. Welty D M, Small P L C. Identification of a putative rpos homologue from M. marinum, M. tuberculosis, M. ulcerans and M. hemophilum. American Society for Microbiology General Meeting, May 1994, Abstract No. U 25. 21. Eisenach K D, Cave M D, Bates J H, Crawford J T. Amplification of a repetitive sequence specific for Mycobacterium tuberculosis using polymerase chain reaction. J Infect Dis 1990; 16l: 977-981. 22. Predich M, Douldlan L, Nair G, Smith I. Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis. Mol. Microbiol. 1995; 15: 355-366. 23. Parra C A, Londono L P, Portillo P D, Patarroyo M E. Isolation, characterization, and molecular cloning of a specific Mycobacterium tuberculosis antigen gene: identification of a species-specific sequence. Infection mad Immunity 1991; 59:3411-3417. 24. Sir John Crofton. In: Foreword to Clinical tuberculosis. PDO Davies (ed). London: Chapman and Hall, 1994: XV.