Regular articleDetection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system
Introduction
M. tuberculosis infects one-third of the world’s people (Raviglione et al., 1995), and while generally curable, is becoming increasingly resistant to commonly used antibiotics (Espinal et al. 2001; Pablos-Mendez et al., 1998). This resistance compromises the otherwise successful outcomes expected with implementation of the DOTS (directly observed short course therapy) strategy for tuberculosis (TB) control (Espinal et al. 2000). In fact, use of “standard” therapy in the face of unsuspected drug resistance can lead to nosocomial spread of TB (Edlin et al., 1992), and high mortality rates, especially among those HIV co-infected (Turett et al., 1995). These complications can be prevented by prompt diagnosis and institution of appropriate therapy (Turett et al., 1995). As HIV spreads into parts of Asia where M. tuberculosis infection is already endemic, the incidence of TB disease, and drug-resistant TB is expected to increase (Espinal et al. 2001). Thus, inexpensive and rapid tests to identify M. tuberculosis and the most appropriate medicines to use in therapy, will be highly desirable.
Detecting TB, and identifying drug-resistant TB by traditional microbiologic methods, is complicated by the slow growth rate of the causative agent, M. tuberculosis Heifets and Cangelosi 1999, Heifets and Good 1994. Therefore, surrogates of mycobacterial growth, such as oxygen consumption from the media, have been harnessed to develop optimized phenotypic detection methods such as the MGIT (Mycobacterial Growth Indicator Tube) system, which allows detection of M. tuberculosis in about 2 weeks (Alcaide et al. 2000). This system is also being developed for antibiotic susceptibility testing (AST), requiring a median of 8 days in a manual format (Rusch-Gerdes et al., 1999), and 6.5 to 9.5 days in an automated format Bemer et al 2002, Tortoli et al 2002, but it is not yet approved for this indication, and the technology is expensive. While molecular methods hold promise for rapid detection of genotypic correlates of drug-resistance, their utility is compromised by the multiple resistance mechanisms for most drugs (Riska et al. 2000), and the expense of these tests for use in the developing world where TB is concentrated. We have developed a rapid phenotypic identification and susceptibility test for M. tuberculosis which is simple to perform and relatively inexpensive. It relies on a recombinant mycobacteriophage –the luciferase reporter phage (LRP) Carriere et al 1997, Jacobs et al 1993, Riska et al 1999. The LRP is a virus which specifically infects mycobacteria, and has been engineered to signal the presence of viable mycobacteria by its rapid production of the sensitive reporter protein, firefly luciferase (Fflux). This luciferase in the presence of the exogenously added substrate, luciferin and endogenous ATP produces quantifiable light, which can be detected with a luminometer, or with slightly less sensitivity but lower cost, with polaroid film (Riska et al., 1999).
We have previously shown that the LRP can specifically detect M. tuberculosis (Riska et al., 1997), with a sensitivity comparable to acid-fast smear (Carriere et al., 1997), and can further provide accurate susceptibility information for the major anti-tuberculous drugs Riska et al 1999, Riska and Jacobs 1998. In this work, we aim to extend these findings by i.) developing a new LRP, phAE142 which uses a potent promoter to drive luciferase expression and enhances sensitivity of detection; ii.) demonstrating the ability of phAE142 to detect mycobacteria directly from processed sputum pellets; iii.)comparing LRP with MGIT technologies for time to detection and species identification of Mtb; and iv.)determining susceptibility results from primary sputum isolates of Mtb using LRP.
Section snippets
Materials and methods
Mycobacterium smegmatis strains mc2155 Carriere et al 1997, Jacobs et al 1993, Riska and Jacobs 1998 and mc24502 (see below) used to propagate phages were grown in Middlebrook 7H9 media (Difco, Detroit, Mi) containing 0.2% glycerol, 0.5% bovine serum albumin (BSA), fraction V, 0.2% dextrose and 0.09% NaCl (designated MADC) with 0.05% Tween-80 and 150 μg/ml hygromycin (for mc24502).
Phage phAE142 was derived by replacing the promoter driving Fflux expression, from Phsp60 in phAE85 (Carriere et al.
Detection Testing Protocol
Each sputum pellet was thawed and distributed as shown in Fig. 1, as follows:
a.) 150μl into a MGIT 960 bottle (7 ml) supplemented with 700 μL MGIT- growth supplement and 160μl of 50X PANTA, and loaded onto a MGIT 960 machine for 6 weeks or until flagged as positive by machine software.
b.) 50μl into 24-well plates (Falcon 3047, BD, Lincoln Park, NJ) with 2 mL MADC media containing 0.5% glycerol, 0.1% casamino acids, and supplemented with 40 μL of 50X PANTA(BD). These plates were sealed with
TB complex Assignment and Antibiotic Susceptibility test (AST) Protocol
Mycobacteria from either LRP-positive MADC cultures or machine-positive MGIT tubes, off-loaded on a 5-day week schedule were used. MADC cultures were used directly, while 1.5 mL of MGIT media was centrifuged (5000 rpm, 5 min, room temperature) and resuspended in 600 μL MADC. Then, 100 μL aliquots of the culture were added to 5 μL of each antibiotic, or 10 μL NAP, or sterile water in 96 well plates. After 48 h. incubation, 5 μL of phAE142 was added to each well and light output was determined as
Sensitivity of detection of M. tuberculosis
In preliminary work with spiked sputum samples or clinical specimens using phAE85, it became apparent that M. tuberculosis could be detected in conventionally processed sputum immediately, but only if the starting inoculum had “numerous” acid fast bacilli, or > 107 CFU/ml (Hobby et al., 1973). However, by allowing a brief period of growth (1-7 days) in culture, light could be detected at a rate faster than the doubling time of the organism, suggesting improved infectability in the appropriate
Detection of M. tuberculosis in clinical samples:phae142 compared to mgit
Twenty freshly thawed processed tuberculous sputum pellets were titered on 7H10 plates, with the range of initial CFU/ml shown in Fig. 3A. One specimen failed to grow on 7H10 (limits of detection:100 CFU/ml), and no microbial contamination was seen. In contrast, one processed sputum pellet was initially contaminated, with heavy non-mycobacterial growth on BAP. Despite use of a common PANTA antibiotic stock, this contaminant was suppressed in MGIT media, allowing prompt detection, but it did
LRP phAE142 detection of M. tuberculosis from machine-positive mgit bottles
Preliminary data revealed that M. tuberculosis could be detected in positive MGIT bottles by LRP, especially if the specimen is washed and resuspended in MADC, thereby removing the detergent components in the MGIT media. Generally, optimal light is obtained only when LRP infection is performed the next day after washing, so we chose to wash and set up AST tests immediately upon detecting a MGIT 960 positive bottle, without prior screening for phage infectability. In practice, this meant the
LRP phAE142 used to assign isolates to the TB complex
In our clinical mycobacteriology laboratory, as in others (Alcaide et al. 2000), a positive MGIT bottle is confirmed to contain M. tuberculosis by the presence of cording on AFB smear, followed by a DNA-probe assay performed a median of 3 days later. By comparison, any clinical isolate producing light after LRP infection should be strongly considered to be M. tuberculosis, since other clinical mycobacteria strains are less efficiently infected by the TM4 phage family (Riska et al., 1997).
LRP phAE142 used for AST of primary clinical specimens and positive MGIT bottles
Once light was obtained from an MADC culture using LRP, or a MGIT bottle became positive, an AST assay for the 4 conventionally tested antibiotics was set up. The overall time to AST results tended to be shorter for the MADC cultures than for the MGIT bottles in most instances (with a median of 11 days for MADC cultures, vs. 13 days for MGIT (t test, p > 0.5). Nonetheless, all the noncontaminated MGIT cultures had AST results by 16 days, whereas some of the MADC cultures took longer for AST
Discussion
Early detection of M. tuberculosis and its drug susceptibility pattern is being increasingly recognized as an important component of global TB control (Espinal et al. 2000). We here describe a protocol using luciferase reporter phages which can perform this task in 1 to 2 weeks from primary specimens in a format adaptable to the developing world where TB is so prominent.
To optimize this system, we utilized the most sensitive phage developed to date for the detection of M. tuberculosis, phAE142,
Acknowledgements
PFR was supported by grant KO8 –AI01628 from the National Institute of Allergy and Infectious Disease of the National Institutes of Health. Partial support for this work came from NIH Contract Grant N01-AI45244 (Tuberculosis Research Unit).
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