Mycobacterium

MTB,NTM

1. Dr. Sumesh Kumar Dash Department of Microbiology, IMS & SUM HOSPITAL MYCOBACTERIA

2. INTRODUCTION Mycobacteria are slender rods, weakly gram positive, obligatory aerobic, nonmotile, non-capsulated, non-sporing, usually having slow growth. They sometimes show branching, filamentous forms resembling fungal mycelium. They are acid fast bacilli .

3. HISTORY Lepra bacillus :- Hansen 1874 Tubercle bacillus :- Robert Koch 1882

4. CLASIFICATION Mycobacterium tuberculosis complex (MTB ) :- M.tuberculosis - MCC of TB in human M.bovis – Bovine tubercular bacilli Other – Less common pathogen. e.g : M. africanum, M. microti, M. canetti, M. caprae,M. pinnipeddi . Mycobacterium leprae Non-tubercular mycobacteria (NTM):- Mostly saprophytic in nature or may be found commensal of human or animal. Very few like M.kansasii , M.fortuitum , M.chelonae can cause human infection.

5. MYCOBACTERIUM TUBERCULOSIS

6. MORPHOLOGY M. tuberculosis is a straight or slightly curved rod whereas M.bovis is straight and short. 3 x 0.3 μm in size. They can occur singly, in pairs or as small clumps. They are gram positive, filamentous, club shaped and branching. They are acid fast: Resist decolorization by dilute acid due to high concentration of Mycolic acid present in cell wall.

7. PATHOGENESIS Source of infection Human: Pulmonary TB Bovine source : Consumption of unpasteurized infected milk Mode of transition Inhalation: Droplet infection Inoculation: Direct contact Ingestion: Swallowing of sputum, Infected milk

8. CLINICAL MANIFESTATION Pulmonary Tuberculosis Primary Secondary / Post-primary Extrapulmonary Tuberculosis HIV Associated Tuberculosis

9. Pulmonary Tuberculosis (PTB) PTB accounts for 80% of all cases of TB. FEATURES PRIMARY TB SECONDARY TB RESULT Initial exogeneous infection with bacilli Exogeneous reinfection Reactivation of latent reinfection AGE Children Adult Affected part Lower lobe Upper lobe Lesion Fibrotic ( Ghon focus) Calcified (Simon’s focus) C/F Asymptomatic or Present with fever, productive cough, chest pain, wt loss Lesions under go necrosis & tissue destruction leading to cavity formation. FATE Lesions heal spontaneously Necrotic lesion breaks leading to bronchogenic or hematogenous spread.

10. Extrapulmonary Tuberculosis (EPTB) EPTB results from hematogenous spread of bacilli to various organ. It is constitute 15 – 20%. Tuberculous lymphadenitis Pleural Tuberculosis Tuberculosis of upper airway Genitourinal Tuberculosis Skeletal Tuberculosis Tuberculosis of CNS Gastrointestinal Tuberculosis Tuberculous skin lesion

11. HIV Associated Tuberculosis TB is most common diseases in HIV infected patients as they have low CMI. TB occur 70-80% of HIV cases. EPTB is more common in these patients.

12. LABORATORY DIAGONOSIS Specimen collection In pulmonary TB: Sputum (2 specimens: spot and early morning), gastric aspirate (in children) In EPTB: Specimens vary depending on the site involved. Direct microscopy by acid-fast staining Ziehl-Neelsen (ZN) technique - long slender, beaded, less uniformly stained red color acid fast bacilli Kinyoun's cold acid fast staining ( M.leprae ) Fluorescent (Auramine)staining it is more sensitive and smears can be screened more rapidly than ZN stain.

13. Digestion, Decontamination and Concentration of specimen Modified Petroff's method (4% NaOH) NALC (N-acetyl-L-cysteine) +2% NaOH Conventional culture media ( 6-8 weeks ) Sold media - Lowenstein Jensen (LJ) medium (  Shows rough tough and buff colored colonies ) Liquid media - Kirdchner's medium Middlebrook 7H9 medium

14. Automated culture methods (3-4 week) MGIT System: Detects growth and resistance to antitubercular drugs (ATDs); with a turnaround time of 2-3 weeks BacT /ALERT: Detects growth Versatek system: Detects growth and resistance to ATDs. Culture identification MPT 64 antigen detection Biochemical tests: Niacin test. Sensitive to paranitrobenzoic acid Catalase peroxidase test Aryl sulphatase test

15. Molecular methods PCR CBNAAT Line probe assay

16. RNTCP Revised National Tuberculosis Control Program of India has given a guideline for grading of ZN stained sputum smear. However, grading is depend on quality of sputum. NO. OF AFB SEEN OIF TO BE SEEN GRADING RESULT No AFB in 100 OIF 100 Negative 1-9/100 OIF 100 Scanty Positive 10-99/100 OIF 100 1+ Positive 1-10/ OIF 50 2+ Positive >10/ OIF 20 3+ Positive

17. LATENT TUBERCULOSIS Latent TB  occurs when a person has the  TB  bacteria within their body, but the bacteria are present in very small numbers. They are kept under control by the body's immune system and do not cause any symptoms. Latent TB is diagnosed by demonstration of delayed type or type IV hypersensitivity reaction against tubercle bacilli antigen.

18. Traditionally, the tuberculin test has been in use for diagnosis of latent TB for >100 years. It was discovered by Von Pirquet in 1907. Antigens used : PPD (Purified Protein Derivative antigen) It is a purified preparation of the active tuberculoprotein. Dosage: It is expressed in tuberculin unit (TU). One TU is equal 0.00002 mg of PPD Procedure: 0.1 mL of PPD containing 1 TU is injected intradermally into flexor surface of forearm Tuberculin Test

19. Reading: It is taken after 48-72 hours. At the site of inoculation, an induration surrounded by erythema is produced. If the width of the induration is: ≥  10 mm : Positive (tuberculin reactors) 6-9 mm: Equivocal/doubtful reaction 5 mm: Negative reaction. Interpretation of result Adults : Positive tuberculin test in adults only indicates present or past exposure with tubercle bacilli but does not confirm the presence of active stage of the disease. Children: In children, positive test indicates acne infection and used as diagnostic marker.

20. False-positive: The test becomes positive after BCG vaccination (after 8-14 weeks) Nontuberculous mycobacteria infection. False-negative: Early or advanced TB, Miliary TB Decreased immunity (HIV-infected people)

21. TREATMENT ATDs are classified into 2 groups First-line drugs (DS-TB) Isoniazid (H) Rifampicin (R) Pyrazinamide (Z) Ethambutol (E) Streptomycin (S) Second-line drugs (DR-TB) Fluoroquinolones Aminoglycosides Macrolides Ethionamide & Prothionamide etc.

22. AIM OF TREATMENT Interrupt transmission by rendering patients non-infectious. Prevent morbidity and death by curing patients. Prevent the emergence of drug resistance. Prevent relapse.

23. STRATEGIES Multidrug therapy: Combination of more than one drug for rapid and effective killing of tubercle bacilli. Short course chemotherapy lasting for 6 months (or longer for DR-TB) Two phase chemotherapy: The short course is divided into Intensive phase: Aims at aggressive treatment with multiple ATDs that rapidly kill the bacilli making the smear negative, followed by: Continuation phase: Aims at killing the remaining dormant bacilli and prevents relapse.

24. DOTS strategy Directly Observed Treatment, Short course It is recommend by RNTCP and WHO. Here, the strategies used are: The entire treatment course is supervised to improve the patient's compliance Treatment response is also monitored by sputum smear microscopy at the end of each phase.

25. REGIMENS Standard regimen for DS-TB: 2 Regimen Doses : All drugs must be given in fixed dose combination (FDC) , should be taken orally, once a day. Regimens for DR-TB: The treatment for DR-TB is complex, consists of use of higher numbers of second-line agents, given for longer duration. CATEGORY DEFINATION INTENSIVE PHASE CONTINUTION PHASE CATEGORY-I New cases Received ATDs <1 month (2)HRZE (4)HRE CATEGORY-II Previously treated patients Treatment after failure Recurrent Treatment after loss to follow up (2)HRZES + (1)HRZE (5)HRE

26. FOLLOW-UP OF TREATMENT Patients should be followed up at scheduled intervals for the assessment of improvement in clinical and laboratory parameters. Clinical follow-up: Should be carried out at least monthly during treatment Laboratory follow-up: For PTB cases, sputum smear examination is done at the end of intensive phase . Sputum smear plus culture is done for every patient a t the end of treatment. Long term follow-up is carried out up to 2 years of completion of treatment Follow-up for MDR TB: Sputum smear and culture are performed every month during intensive phase and every 3 months during continuation phase.

27. DRUG RESISTANCE Mono resistance: Resistance to only one first-line ATDs. Poly resistance: Resistance to >1 first-line ATDs. Rifampicin resistance (RR): Resistance to Rifampicin with or without resistance to other ATDs. Multidrug-resistance tuberculosis (MDR-TB): Resistance to both Isoniazid & Rifampicin with or without resistance to other ATDs. Extensively drug-resistance tuberculosis (XDR-TB): These are MDR-TB (+) Fluoroquinolones (+) one 2 nd -line injectable agent(SLI) Pre-XDR-TB: MDR-TB (+) Fluoroquinolones (or) SLI

28. NONTUBERCULOS MYCOBACTERIUM

29. Non-tubercul o us mycobacteria have been classified into four groups by Runyon (1959), based on pigment production and rate of growth. RUNYON GROUP PROPERTY SPECIES Photochromogens Produce pigment only on light M.kansasii , M.marinunm Scotochromogen Produce pigment both in dark & light M.scrofulaceunm , M.gordonae Non-chromogens Do not produce pigment M. Avium-intracellulare complex (MAC) M. xenopi, M. Ulcerans Rapid growers Grow with in 1 week M.chelonae , M.fortuitum

30. CLINICAL MANIFESTRATION OF NTM DISEASE ORGANISM Pulmonary infection M. Avium-intracellulare complex , M. xenopi, M.kansasii Lymph node infection M. Avium-intracellulare complex Cutaneous infection M.chelonae , M.fortuitum – Injection abscess M.Marinunm – Swimming pool granuloma M. Ulcerans – Buruli ulcer Disseminated infection M. Avium-intracellulare complex , M.kansasii

31. LABORATORY DIAGONOSIS Specimen are collected as per infection and lesion. Microscopy is done by ZN staining. Culture on LJ medium. Pigment production. TREATMENT Just as tuberculosis, NTM infection are treated with multi drug therapy. MAC, M.kansasii , M.Marinunm need macrolide, ethambutol and rifamycin combine. Macrolide need to be given prophylactically to the indivisual infected with HIV.

32. THANK YOU