1. Bacteriophage based test for the rapid detection & Antibiotic sensitivity test of TB Viro102: Bacteriophages & Phage Therapy 3 Credit hours NUST Centre of Virology & Immunology

2. Tuberculosis (TB) is an infectious disease caused by bacteria Mycobacterium tuberculosis.Mycobacterium tuberculosis TB most commonly affects the lungs but also can involve almost any organ of the body. MTB is an obligate aerobic bacterium, therefore it needs oxygen to survive. The World Health Organization declared TB a global emergency in 1993. Since then, antibiotic resistant TB has been reported in 104 countries.

3. Tuberculosis combines one of the slowest division rates among bacteria with a hardy cell wall defense system. Both of these factors stretch treatment into a multiple month process, creating a massive window for human error in the form of incorrect or missed dosages. This slow pace of infection increases the possibility of evolution based antimicrobial resistance by giving tuberculosis bacteria time to mutate.

4. Cell Wall of MTB The cell wall of MTB is one of the major determining factors of its virulence. The structure of the cell wall has three major components: 1.mycolic acids, 2.cord factors, and 3.Wax-D. The mycolic acid molecules are of primary interest due to their deadly qualities. Mycolic acids create a lipid shield, which protects against cationic proteins, lysozyme, and oxygen radicals of phagocytosis.

5. Cell wall is impervious to gram staining due to the high content of lipids, especially mycolic acid. This characteristic places MTB in a category of bacterium known as acid fast bacteria, whose acid rich cell walls retain a red dye used for staining, despite attempts at decolorization ( Acid Fast Staining). The full genome of MTB was sequenced in 1998. Large amount of coding is dedicated to the genesis and lysis of lipids, compared to other bacteria. This explained why over 60 percent of the MTB cell wall is composed of lipids.

6. Antimicrobial Resistance The waxy, hydrophobic cell wall of MTB gives it the ability to survive long exposure to substances such as acids, detergents, oxidative bursts, and antibiotics. In fact, the typical “short” treatment of MTB involves a four antibiotic treatment for two months and then a two antibiotic treatment for an additional four months. The antibiotics involved are isoniazid, rifampicin, pyrazinamide, and ethambutol.

7. Isoniazid, are targeted at the synthesis of mycolic acids. Two types of drug resistant MTB strains are currently recognized. 1.Multi drug resistant tuberculosis (MDR TB) is resistant to at least two of the four first-line drugs listed above. 2.Extensively drug resistant tuberculosis (XDR TB) is defined as resistant to isoniazid, rifampin and also to fluoroquinolone and at least one of three injectable second line drugs.

8. How bacteriophage can be used? Principle Portions of decontaminated sputum incubated Bacteriophage (Actiphage) infect viable TB bacilli in sputum. A potent virucide (Virusol) destroys all bacteriophage that have not infected TB bacilli. Bacteriophage replicating in TB bacilli survive and are detected as plaques after plating with non pathogenic mycobacterial Sensor cells

12. Specimen processing Sputum or blood collection Decontaminate by NaOH method. Suspend pellet in 1.5ml Response Medium Plus. Centrifuge at minimum of 2000g for 20 mins Decant supernatant. Suspend in 1ml Response Medium Plus Add 0.5ml test suspension RIF- 0.5ml Response Medium Plus RIF+ 0.5ml Response Medium Plus Incubate both vessels for 18-24 hours. (commence with

13. Incubate overnight at 37ºC Thanks