(nih/researchmatters/march2009/03092009tuberculosis.htm )

1. (http://www.nih.gov/researchmatters/march2009/03092009tuberculosis.htm)(http://www.nih.gov/researchmatters/march2009/03092009tuberculosis.htm) (http://www.biochem.wisc.edu/faculty/rayment/lab/gallery.aspx) Luciferase Reporter Mycobacteriophage (LRP) Assays for Diagnosis of Antibiotic Resistant Mycobacterium tuberculosis Clare Bruggeman

2. General TB Information • WHO reported 9.9 million incident cases of TB in 2008. • TB is treatable, yet it killed 2.3 million people in 2008. • TB diagnostic tools are old-fashioned. • Goal is to develop an inexpensive, rapid and accurate tool to diagnose TB, MDR TB and XDR TB.

3. Pulmonary Tuberculosis Primary Infection in the Lungs • Cough • Coughing up blood • Chest pain http://www.path.cam.ac.uk/partIB_pract/P09/

4. Symptoms of Tuberculosis TB in Other Organs • Kidney • Blood in urine • Brain • Headache and fever • Spine • Extreme Curvature of the spine http://www.merck.com/mmhe/sec17/ch193/ch193a.html http://www.path.cam.ac.uk/partIB_pract/P09/

5. Diagnosis and Treatment • Although TB is less common in developed countries, there are a few highly burdened countries that have 80% of the reported TB cases (WHO 2009 Report). • DOTS-Directly Observed Therapy • TB can be treated if patients adhere to a strict, multi-drug regimen. • 6-12 months of antibiotic treatment • Attempting to prevent the spread of multi-drug resistant strains.

6. Acid-Fast Stain (DOTS) Fails to identify 30-50% of active cases BACTEC and MGIT http://pathmicro.med.sc.edu/infectious%20disease/mycobacterial%20diseases.htm

7. Luciferase Reporter Mycobacteriophages 1. Find a mycobacteriophage that infects M. tuberculosis 2. Genetically engineer the mycobacteriophage to contain luciferase. 3. Infect the cultured sputum sample with the phage. 4. If viable mycobacteria are present, light is produced in the presence of D-luciferin. 5. If the light does not diminish when the mycobacteria are treated with an antibiotic, then the mycobacteria are resistant. 6. Measure Relative Light Units (RLU) with a luminometer. Banaiee et al. (2001)

8. Luciferase http://www.lifesci.ucsb.edu/~biolum/chem/

9. Construction of the mycobacteriophage Schematic of phAE39 and phAE40 DNA. Constructed in the E. coli cosmid pYUB216, then inserted into TM4 mycobacteriophage. ColE1 = ORI Ap = Amp (selectable marker for E. coli). Phsp60 = promoter Fflux = firefly luciferase (Jacobs 1993)

10. Banaiee et al. (2001) NTM = Nontuberculosis mycobacterium MTC = M. tuberculosis complex

11. Banaiee et al. (2001)

12. Banaiee et al. (2001)

13. Banaiee et al. (2001) Only really changing the detection method and maybe the culture media. Conclusions: • LRP has the potential to be the rapid and affordable method that developing countries need to diagnose active TB, MDR TB and XDR TB. • 76% of positive smears were identified with LRP • If contamination is minimized and both solid and liquid media methods are used, LRP efficiency increases to 97%. • 94% sensitivity and 100% specificity to MTC(agreement between BACTEC and LRP) • LRP determination of drug resistance was possible within 2-4 days Concern: • Phage resistant TB strains or phages with too broad of a host range.

14. Kumar et al. (2008) • Had evidence that a temperate phage (compared to a lytic phage) might have increased light output (i.e. better sensitivity). • Collected phage samples near a tuberculosis sanatorium in India • Che12 was a likely candidate (formed turbid plaques on a lawn of M. smegmatis).

15. Kumar et al. (2008) + - M. smegmatis M. tuberculosis

16. Kumar et al. (2008)

17. Kumar et al. (2008) • Conclusions • Che12 infects M. smegmatisand M. tuberculosis. • In the lab you can work with the safer M. smegmatis • Che12 integrates with the host genome. • phAETRC16 had increased and sustained light output • Che12 is the first true temperate phage that infects M. tuberculosis. • Hope that Che12 will be useful for assays and vaccine construction because of the sustained light production.

18. Final Conclusions “The LRP assay appears to be the most consistently accurate test” (Minion and Pai, 2010). But the method is still slow, requires non-contaminated samples, and uses up antibiotics during susceptibility tests.

19. References Minion, J., and M. Pai. 2010. “Bacteriophage assays for rifampicin resistance detection in Mycobacterium tuberculosis: updated meta-analysis.” Int J Tuberc Lung Dis14 (8): 941-951. WHO.int. 2010. The World Health Organization. 29 October 2010. <http://www.who.int/en >. CDC.gov. 2010. The Center for Disease Control and Prevention. 29 October 2010. <http://www.cdc.gov >. “Hypersensitivity.” 2010. Practical Pathology Class Website. Department of Pathology, University of Cambridge. 29 October 2010. <http://www.path.cam.ac.uk/partIB_pract/P09/ >. Kumar, V., P. Loganathan, G. Sivaramakrishnan, J. Kriakov, A. Dusthakeer, B. Subramanyam, J. Chan, W. Jacobs Jr. and N. Paranji Rama. 2008. “Characterization of temperate phage Che 12 and construction of a new tool for diagnosis of tuberculosis.” Tuberculosis88, 616-623. Banaiee, N., M. Bobadilla-del-Valle, S. Bardarov Jr., P. F. Riska, P. M. Small, A. Ponce-de-Leon, W. Jacobs Jr., G. F Hatfull and J. Sifuentes-Osornio. 2001. “Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis in Mexico.” J ClinMicrobiol39 (11), 3883-3888. Gali, N. J. Dominquez, S. Blanco, C. Prat, F. Alcaide, P. Coll, V. Ausina and the Mycobacteria Research Group of Barcelona. 2006. “Use of a mycobacteriophage-based assay for rapid assessment of susceptibilities of Mycobacterium tubersulosis isolates to isoniazid and influence of resistance level on assay performance.” J ClinMicrobiol44(1) 201-205. Jacobs, W. R. Jr, R. G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G. Sarkis, G. Hatfull, B. Bloom. 1993. “Rapid Assessment of Drug Susceptibilites of Mycobacterium tuberculosis by Means of Luciferase Reporter Gene.” Science, New Series 260 (5109) 819-22.