Mohammad Aulia Herdiyana

1. Comparison Of Concentration Of Mycobacterium Tuberculosis Inoculation In Vertebral Body Defect As A Tuberculous Spondylitis Model In New Zealand Rabbits Mohammad Aulia Herdiyana

2. Introduction • Tuberculosis remains a major global health problem. • second most common cause of death from infectious disease. • Indonesia  fourth largest incidence* 2 3 1 4 5 *World Health Organization. Global Tuberculosis Report 2012

3. Introduction • Tuberculosis spondylitismost common bone and joint tuberculosis infection (50% of osteoarticular tuberculosis) • aggravated morbidity  potential neurological deficits* • **Rasouli MR, et al. Spinal Tuberculosis: Diagnosis and Management. Asian Spinal Journal. 2012

4. Introduction • TB spondylitis management strategy varies from chemotherapy alone, surgery for specific indications, until decompression / debridement routine. • Surgical treatment fusion is the goals that must be achieved to obtain a stable spine, in addition to cure infection, reduce deformity and relieve pain. • *Shrestha OP, et al. Bone and Joint Tuberculosis. UPOJ May 2010;

5. Introduction • Evaluation of 700 cases after distraction and fusion procedures, 35 cases (5%) did not have stable fusion of the spine  complications.* • Nine year follow up of spinal TB management: 18 % had unfavorable status**  Demands the development of new methods in the treatment to achieve more satisfactory results • *Sapardan S. Total Treatment of Tuberculosis of the Spine. A Rational Problem Solving Approach. Perpustakaan Universitas Indonesia. 2004 • **Park DW et al. Outcome and management of spinal tuberculosis according to severity of disease. Spine. 2007

6. Introduction • Strains of New Zealand white rabbits can be infected with the Mycobacterium tuberculosis bacteria using an aerosol system after 6-33 weeks of exposure *, or by skin inoculation ** • Infection of MRSA to the rabbit’s spine has also been successful*** • *Smith D, Wiegeshaus E, Balasubramanian. Animal Models for Experimental Tuberculosis. CID 2003. • ** Zhang G et al. Evaluation of mycobacteril virulence using rabbit skin liquefaction model. Virulence .2010. • *** Poelstra KA et al. A novel spinal implant infection model in rabbits. Spine. 2000.

7. Introduction • However, no studies have induce Mycobacterium tuberculosis to the spine  No animal model for tuberculous spondylitis currently available

8. Research Questions • Can Mycobacterium tuberculosis bacteria inoculated on New Zealand rabbit’s vertebral body produce tuberculosis infection? • Are there differences in the results of AFB smear examination, PCR, culture and histopathology examination of the rabbit’s vertebral body inoculated with Mycobacterium tuberculosis ofdifferent concentrations?

9. Hypothesis • Mycobacterium tuberculosis inoculated on New Zealand rabbit’s vertebral body will produce tuberculosis infection. • There are differences in the results of AFB smear, PCR, culture and histopathology examinations of the rabbit’s vertebral body inoculated with Mycobacterium tuberculosis ofdifferent concentrations.

10. Aim • Knowing the effects of Mycobacterium tuberculosis inoculation with different concentrations to the L1 vertebral body of New Zealand white rabbits. • Obtaining a tuberculosis spondylitis model in New Zealand white rabbits

11. Mycobacterium tuberculosis Theoretical Framework Bacterial factors Host factors Inoculation Environment factors Macrophages Bacteria lysis by macrophages Bacterial phagocytosis but not destroyed(latency) Bacteria develops; Caseous necrosis, granuloma Cured White blood cells é ESR é CRP é AFB+ Culture + PCR + Histopathology + Infection Cold abscess Vertebral body destruction Neural deficit Paraplegia Kyphosis

12. Mycobacterium tuberculosis Conceptual Framework Bacterial factor Bacterial load Host factor Inoculation Environment factors Infection AFB smear Culture PCR Histopathology

13. Methods

14. Methods

15. Methods Sample • Size: 6 each group • Inclusion criteria: • New Zealand white rabbits • weight 2000-3500 grams • no abnormalities in the spine

16. New Zealand white rabbits Study Protocol Exclusion criteria Vertebra L1 identification, drillingtomake vertebral bodydefect Group A Inoculation 106CFU/ml M tuberculosis Group B Inoculation 107 CFU/ml M tuberculosis Group C Inoculation 108 CFU/ml M tuberculosis Control group Normal saline inoculation 4 weeks inoculation, temperature 18-21 oC, humidity 15% AFB, PCR, culture and histopathology Euthanasia + Debridement

17. Inoculation Procedure

18. Debridement Procedure

19. AFB smear • Stain with carbolic fuchsin 0.3%, methylene blue 0,1% • fine red bacil, curved, alone, in pairs or in groups with a bluish background

20. Culture • MGIT • 2 weeks • Confirmed with AFB smear

21. PCR • DNA purification with QIAamp DNA and Blood Mini Kit (Qiagen) • Amplification • Electrophoresis Compare DNA sequence of sample with positive control

22. Histopathology • The preparation is considered positive when histopathology shows signs of tuberculosis infection induced tissue inflammation: caseous necrosis, infiltration of lymphocytes, Langerhans cells

23. Results • 24 white New Zealand rabbits, • 3 TB inoculation groups, 1 control, 6 rabbits each • One paralysis • Four week incubation period

24. Results p=0,110 p=0,161 p=0,425 * Shapiro-wilk Test

25. Results • AFB smear p=0,17 p=0,36 Post-hoc analysis

26. Results • Culture p=0,02 p=0,17 p=0,17 Post-hoc analysis

27. Results • PCR p=0,04 p=0,02 Post-hoc analysis

28. Results • Histopathology p=0,02 Post-hoc analysis

29. Discussion • Inoculation of Mycobacterium tuberculosis into the corpus vertebrae defects  success of inoculation: positive result on each type of examination • Success of inoculation depends on a variety of things, • Host, • Bacteria, • Environment • Technique

30. Discussion • AFB smear: 50 % positive • Culture: 44% positive • PCR: 83% positive • Histopathology: 88% positive

31. Conclusion • Mycobacterium tuberculosis inoculated on New Zealand rabbit vertebral body can produce tuberculosis infection. • There are differences in the results of AFB smear examination after inoculation of Mycobacterium tuberculosis of different concentrations , with concentrations of 108cfu / mL producing the best results . • There was no difference in the results of culture after inoculation of Mycobacterium tuberculosis of different concentrations . • There are no significant differences in PCR examination after inoculation of Mycobacterium tuberculosis of different concentrations. • There are no significant differences in histopathological examination after inoculation of Mycobacterium tuberculosis of different concentration. A minimum concentration of 107cfu / ml infection achieving good results.

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