Phage Project Review Meeting © London School of Hygiene & Tropical Medicine
DFID Tuberculosis Knowledge Programme
Mycobacteriophage Replication Technology
Introduction
The purpose of this document is to disseminate progress in the development of 'low-cost' tools for diagnosis and drug susceptibility testing of tuberculosis. The work presented has been undertaken as part of the Department for International Development (DFID) funded TB Knowledge Programme at the London School of Hygiene & Tropical Medicine.
Summary of research findings
Bacteriophage-based tests may be used for the detection of resistance to anti-tuberculosis drugs and for detection of mycobacteria in clinical specimens.
When detecting resistance to rifampicin in TB cultures results may be obtained in 48 hours. Thus the technology is faster than phenotypic methods, but slower than molecular methods.
Bacteriophage tests show high sensitivity for the detection of resistance to rifampicin.
A multiwell plate format provides a simple and convenient method of screening TB isolates for resistance to rifampicn and streptomycin.
For detection of mycobacteria in clinical specimens results may be obtained in 48 hours, thus the technology is faster than culture, but slower than smear microscopy or molecular methods.
When tested in a reference laboratory in Zambia an 'in-house' phage replication test for diagnosis of pulmonary tuberculosis was found to provide similar diagnostic sensitivity to smear microscopy.
When tested in a reference laboratory in Zambia a commercial kit (FASTPlaque, Biotec Laboratories Ltd) was prone to microbiological contamination due to inadequate specimen decontamination. The kit was found to be less sensitive than smear microscopy.
It was concluded that bacteriophage-based tests currently offer no advantage for the diagnosis of pulmonary tuberculosis in Zambia.
The technology requires P3 microbiological facilities and is thus likely to be restricted to tertiary diagnostic centres.
The bacteriophages used are not specific for M. tuberculosis and false positives may occur due to detection of mycobacteria other than tuberculosis (MOTTS).
Published articles
(1). In-house phage amplification assay is a sound alternative for detecting rifampin-resistant tuberculosis in low resource settings. Símboli N, Takiff H, McNerney R, López B, Martin A, Palomino JC, Barrera L and Ritacco V. Antimicrobial Agents and Chemotherapy. 2005; 49(1): 425-427.
(2). Comparison of two bacteriophage tests and nucleic acid amplification for the rapid diagnosis of pulmonary tuberculosis in sub-Saharan Africa. Mbulo G, Kambashi B S, Kinkese J, Tembwe R, Shumba B, Godfrey-Faussett P and McNerney R. Int J Tuberc Lung Dis 2004; 8(11):1342-1347.
(3). Development of a Bacteriophage Phage Replication Assay for Diagnosis of Pulmonary Tuberculosis. McNerney R, Kambashi B S, Kinkese J, Tembwe R and Godfrey-Faussett P. J Clin Microbiol 2004; 42 (5): 2115-20
(4). Phage replication technology for diagnosis and susceptibility testing. McNerney R. In Mycobacterium tuberculosis. Parish T and Stoker N.G. 2001 (eds), Humana Press, Totowa, NY. Methods in Molecular Medicine Vol 54 chapter 10 pp145-154 ISBN 0-89603-776-2
(5). Micro-well phage replication assay for screening mycobacteria for resistance to rifampicin and streptomycin. McNerney R. In Antibiotic Resistance Methods and Protocols. Gillespie SH (ed) 2000 Humana Press Inc, Totowa, NY. Methods in Molecular Medicine Vol 48 pp21-30 ISBN 0-89603-777-0
(6). Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin. McNerney R, Kiepiela P, Bishop K S, Nye P and Stoker NG. Int J Tuberc Lung Dis. 2000; 4 (1) 69-75.
(7). TB: the return of the phage. A review of fifty years of mycobacteriophage research. McNerney R. Int J Tuberc Lung Dis. 1999; 3 (3) 179-184
(8). Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis. McNerney R., Wilson SM, Sidhu AM, Harley VS, Nye PM, Al Suwaidi Z, Parish T and Stoker NG. Research in Microbiology. 1998; 149: 487-495.
(9). Phages, old friends in the fight against TB. McNerney R. Biologist, 1997; 44 (4): 395-396
Background
Mycobacteriophages are viruses that infect mycobacteria. First discovered 50 years ago, there are now over 250 known mycobacteriophages (1) . Mycobacteriophage D29 was isolated from soil (2) and is a lytic phage which is able infect and replicate in the slow-growing pathogenic strains such as Mycobacterium tuberculosis and Mycobacterium ulcerans and fast-growing environmental strains such as Mycobacterium smegmatis.
Phages are only able to replicate in living bacteria. Following infection and replication D29 breaks down (lyses) the bacterial cell wall releasing a new generation of phage. Thus, following innoculation with D29 an increase in the number of phages indicates the presence of live mycobacteria. Phages are unable to replicate in the presence those drugs such as rifampicin that disrupt the mechanisms of replication of the host bacteria . However, in drug resistant strains replication can proceed (3) . Other drugs such as INH and ethambutol do not block phage replication directly. Susceptibility of mycobacteria to these drugs can be demonstrated by preincubation with critical concentrations of the drug when phages will be unable to replicate in those bacteria that have been killed by the drug. The utility of D29 for testing susceptibility of mycobacteria to anti-tuberculosis drugs was demonstrated in 1980 by David and colleagues (4). The diagram below illustrates the infection of a mycobacterium by a D29 phage.
To facilitate detection of progeny phage it is necessary to remove any phage remaining in the broth that have not infected a bacterium. Previous methods of phage removal were by absorption with neutralising antibodies or by chemical inactivation using reagents such as sulphuric acid (5) . The discovery that ferrous (iron II) compounds will inactivate D29 while not harming the mycobacteria or any phage replicating inside them has enabled the development of simple, highly sensitive methods for the detection of mycobacteria (6).
The principle is shown in the diagram below. D29 phages are incubated with the sample to allow infection. Before lysis occurs ferrous ammonium sulphate (FAS) is added to each sample. Following treatment with FAS the only phage remaining are those within host mycobacteria that have resulted from successful infection and replication.
Detection of phage is by plating in agar containing M. smegmatis (7). As infected bacteria lyse they release progeny phage which will infect adjacent M. smegmatis bacteria which will in turn lyse, eventually causing clear areas (plaques) to form within the growing M. smegmatis lawn. These plaques can be seen following an overnight incubation of the indicator plate. If samples are plated prior to lysis then each plaque represents an infected cfu. If plating is left until after lysis of host bacteria and the release of progeny phage there is an amplification of the number of plaques observed but the assay is less quantitative.
This simple technology does not require investment in specialist equipment other than that required for culture of M. tuberculosis and stocks of phages and indicator bacteria may be maintained 'in-house'. However, P3 safety facilities and good microbiological skills are required when handling M. tuberculosis in liquid culture.
D29 phage are not specific for the M. tuberculosis complex and will replicate in a wide range of mycobacteria. If required confirmation should be undertaken by biochemical or molecular methods. Riska and colleagues working with luciferase reporter phages have demonstrated that inhibition by NAP (p-nitro-alpha-acetylamino-beta-hydroxy propiophenone) may be incorporated in the phage assay as a confirmatory test for M. tuberculosis (8).
It should be noted by those interested in commercial exploitation of bacteriophage technology that it has been the subject of a number of patents. The London School of Hygiene & Tropical Medicine holds no patents relating to this technology.
References
(1) McNerney, R. 1999. TB: the return of the phage. A review of fifty years of mycobacteriophage research. Int J Tuberc Lung Dis. 3(3):179-84.
(2) Froman, S., D. W. Will, and E. Bogen. 1954. Bacteriophage active against virulent Mycobacterium tuberculosis. I. Isolation and activity. Am. J. Public Health. 44:1326-1333.
(3) Jones, W. D., Jr., and H. L. David. 1971. Inhibition by rifampin of mycobacteriophage D29 replication in its drug-resistant host, Mycobacterium smegmatis ATCC 607. Am Rev Respir Dis. 103(5):618-24.
(4) David, H. L., S. Clavel, F. Clement, and J. Moniz Pereira. 1980. Effects of antituberculosis and antileprosy drugs on mycobacteriophage D29 growth. Antimicrob Agents Chemother. 18(2):357-9.
(5) David, H. L., N. Rastogi, S. Clavel Seres, and F. Clement. 1986. Action of colistin (polymyxin E) on the lytic cycle of the mycobacteriophage D29 in Mycobacterium tuberculosis. Zentralbl Bakteriol Mikrobiol Hyg A. 262(3):321-34.
(6) McNerney, R., S. M. Wilson, A. M. Sidhu, V. S. Harley, Z. al Suwaidi, P. M. Nye, T. Parish, and N. G. Stoker. 1998. Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and, M. tuberculosis. Res Microbiol. 149(7):487-95.
(7) David, H. L., S. Clavel, and F. Clement. 1980. Adsorption and growth of the bacteriophage D29 in selected mycobacteria. Ann. Virol. (Inst. Pasteur). 131:167-184.
(8) Riska, P. F., W. R. Jacobs, Jr., B. R. Bloom, J. McKitrick, and J. Chan. 1997. Specific identification of Mycobacterium tuberculosis with the luciferase reporter mycobacteriophage: use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone. J Clin Microbiol. 35(12):3225-31.
DFID Tuberculosis Knowledge Programme, London School of Hygiene & Tropical Medicine, Clinical Research Unit, Keppel Street, London, WC1E 7HT
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