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Sreeja Nair Reg No: 118/Jan 2014 External Part Time PhD Scholar

Genotypic characterisation and drug resistance profile of Multi Drug resistant mycobacterium tuberculosis ( mdr-tb ) in a tertiary care hospital, kerala. Sreeja Nair Reg No: 118/Jan 2014 External Part Time PhD Scholar Yenepoya University. Guide. Co- Guide.

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Sreeja Nair Reg No: 118/Jan 2014 External Part Time PhD Scholar

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  1. Genotypic characterisation and drug resistance profile of Multi Drug resistant mycobacterium tuberculosis (mdr-tb) in a tertiary care hospital, kerala. Sreeja Nair Reg No: 118/Jan 2014 External Part Time PhD Scholar Yenepoya University.

  2. Guide Co- Guide Dr.VidyaPaiMD Professor and Head Dept of Microbiology, YenepoyaMedicalCollege, Mangalore. Dr.SeemaOommenMD,DNB Professor & Head Dept of Microbiology, Pushpagiri Institute of Medical Sciences and Research Centre Tiruvalla Kerala.

  3. Action taken against the comments of the panel of experts

  4. ( WHO Global TB report., 2015 ) ( RNTCP status report., 2016 )

  5. *Ref: (V. Makeshkumar et al ., 2014)

  6. *( RNTCP Manual of Standard Operating Procedures (SOPs)., 2009)

  7. INTRODUCTION • Tuberculosis (TB) is a major global health problem. • It causes ill-health among millions of people each year and ranks along with human immunodeficiency virus (HIV) as a leading cause of death worldwide. ( Global TB report., 2015)

  8. epidemiology

  9. GLOBAL TB INCIDENCE • In 2014, there were an estimated 9.6 million new TB cases. • 3.3% of new cases of Multi drug resistant (MDR) and 20% of previously treated cases have MDR-TB. • On an average, an estimated 9.7% of people with MDR-TB have Extensively drug-resistant TB (XDR-TB). ( Global TB report., 2015 )

  10. Indian Scenario • As estimated by WHO, one fourth of the global incident TB cases occur in India. • 2.2 million cases were estimated to have occurred in India. • MDR-TB burden among notified new pulmonary TB patients is 0.024 million ie, 2.2% . • MDR –TB among notified re-treatment pulmonary TB patients is 0.047 million ie, 15%. (RNTCP status report., 2016)

  11. Burden of TB in Kerala • In 2015, the total number of suspects examined in Kerala were 418895, out of which 14147 (3.3%) were diagnosed to have tuberculosis. • Of the 3653 samples examined by culture for MDR TB , 161 (4.4%) were found to be MDR TB. (RNTCP status report., 2016)

  12. Social relevance and uniqueness • Tuberculosis is a common disease among patients with compromised immunity. • The emergence of MDR-TB has aggravated the situation particularly in developing countries including India. • Proper identification of the etiology is essential for the development of appropriate control and preventive strategies .

  13. This study will determine the diversity of species and strains in tuberculosis human isolates including their epidemiological links using molecular assays. • May guide on appropriate measures by the tuberculosis control program.

  14. AIM & OBJECTIVES

  15. AIM • To study the genotypic characterization and drug resistance profile of Multi drug resistant Mycobacterium tuberculosis (MDR- TB) in a tertiary care hospital.

  16. Objectives • To isolate M.tuberculosis from clinical specimens. • To detect drug resistance of M.tuberculosis isolates by Mycobacterial Growth Indicator Tube (MGIT) for 1st line drugs. • Molecular detection of drug resistance by using PCR. • To detect specific mutations of drug resistant TB isolates by sequencing.

  17. Objectives contd....... • To correlate MDR-TB genotypes, patient demographic and social characteristics with clinical outcomes in order to identify factors associated in the treatment of MDR-TB. • To evaluate Mycobacterial Growth Indicator Tube(MGIT) in the detection of M. tuberculosis in comparison with Lowenstein-Jensensmedium.

  18. Methodology

  19. Research design, setting and duration • Research Design: Prospective • Study Setting: In vitro studyin the Microbiology laboratory of Pushpagiri Medical College. • Duration of Study: 3 years from November 2015- October 2018.

  20. Sample size The sample size is calculated using the formula: n=Z2(1-α)PQ d2 - Where the prevalence rate p is calculated from previous studies and is 4.4%. - d= allowable error is put as 2%. Sample size (n)= 404

  21. Inclusion criteria • All consecutive pulmonary (sputum, bronchial lavage, gastric lavage) specimens received the study period are to be included in the study. • Extra pulmonary specimens - biopsy specimens, aspirated fluids,urinesamples,pus will be included in the study.

  22. Exclusion criteria • Samples less than 2ml. • Pulmonary specimen consisting mainly of saliva. • Swabs • Samples preserved in formalin • Non tuberculousmycobacteria.

  23. Work plan

  24. Patients with symptoms suggestive of tuberculosis Appropriate samples to be collected (eg: respiratory specimen, lung biopsy, urine, aspirated pus etc) according to *RNTCP guidelines. AFB SMEAR (*ZN and Fluorescent staining) Smear positive Smear negative *RNTCP- Revised National Tuberculosis Control Programme *ZN staining- ZiehlNeelsens staining

  25. N-acetyl L-cysteine *NaOH Decontamination Culture ( *LJ Medium) Micro *MGIT Characteristic colonies Reading above cut off (> 12 GU) Smear • *NaOH- Sodium hydroxide • *LJ- Lowenstein Jensens Medium • *MGIT- Mycobacterium growth indicator tube

  26. Smear Confirmation by Para nitro benzoic acid test, Tuberculosis identification card test and other biochemical tests. Negative Positive M.tuberculosis complex Non tuberculousMycobacteria

  27. Micro MGIT * SIRE test *PCR for rifampicin resistance Positive Sensitive Resistant *MDR M.tuberculosis Genotyping will be done. *SIRE- Streptomycin, Isoniazid,Rifampicin,Ethambutol *PCR- Polymerase chain reaction *MDR- Multidrug resistant

  28. Progress so far • Collection of samples started from November 2015. • A total of 112 samples were collected, of which 49 were pulmonary and 63 were extra pulmonary specimens. • Of this 12 samples were found to be positive. • Drug susceptibility testing was performed on 8 isolates.

  29. Results • A total of 112 samples were collected during a period of 6 months from 31st Oct 2015 to 5th June 2016. • Of the 112, 49 (43.75%) were pulmonary and 63 (56.25%) extra pulmonary. • 63 were male patients and 49 were females. • Of the 112 samples, 12 (10.7%) were found to be culture positive.

  30. Results contd……… • Of the 8 isolates subjected to drug susceptibility test, 1 (12.5%) was a drug sensitive isolates (susceptible to all the anti-tuberculosis drugs) and 7 (87.5%) were resistant to at least one of the drugs. • 2 (25%) were MDR. • Non tuberculousmycobacteria – 10 • Contaminants - 2

  31. Table1: Drug resistance pattern of isolates.

  32. Statistics to be used • Sensitivity and specificity. • Positive Predictive Value (PPV) and Negative predictive value (NPV). • Predictive Value (PV) would be calculated using SPSS statistical software. • Comparison using Chi Square test and odds ratio.

  33. TIME LINE

  34. Proposed work for next six months • Reviewing the literature. • Sample collection, processing and storage • Procurement of DNA extraction kit • Procurement of PCR master mix • Procurement of primers • Standardization of PCR.

  35. references

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  39. Thank You

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