1. Mycobacteria İ. Çağatay Acuner M.D., Clinical Microbiologist, Associate Professor Department of Microbiology Faculty of Medicine, Yeditepe University, Istanbul cagatay.acuner@yeditepe.edu.tr

2. Mycobacteria Family Mycobacteriaceae Genus Mycobacterium ≈200 species MTC NTM, MOTT

3. Mycobacterium widespread in environment and in animals major human pathogens are M. tuberculosis and M. leprae, importance of other species (e.g. M. avium complex) is increasing in AIDS and other immunocompromised patients aerobic rods with a Gram-positive cell wall structure, but stain with difficulty because of the long-chain fatty acids (mycolic acids) in the cell wall acid fastness can be demonstrated by resistance to decolorization by mineral acid and alcohol (Ziehl-Neelsen stain) Mycobacteria grow more slowly than many other bacteria can be divided into: rapid growers (form visible colonies within ca. 3-7 days) slow growers (form visible colonies only after ca. 2 weeks to 2 months' incubation) Laboratory identification: staining and microscopic examination of specimens for acid-fast rods is important because of the time required for culture results all species except M. leprae can be grown in artificial culture but they require complex media Nucleic acid methods are available for identification

4. Mycobacterium Diseases M. tuberculosis causes tuberculosis in humans and animals M. leprae is restricted to humans and causes leprosy Mycobacteria other than tuberculosis (MOTT) are associated with a range of conditions; usually in immunocompromised hosts M. avium-intracellulare (M. avium complex) has important associations with AIDS patients in the USA in Africa M. tuberculosis is more common Transmission droplet spread aided by ability of organisms to survive in the environment (M. tuberculosis, M. leprae) social and environmental factors and genetic predisposition all have a role leprosy requires close and prolonged contact for spread Pathogenesis M. tuberculosis and M. leprae are intracellular parasites surviving within macrophages Treatment and prevention prolonged treatment with combinations of antimycobacterial drugs Bacille Calmette-Guérin (BCG) vaccination Isoniazid (or rifampin and pyrazinamide) prophylaxis pasteurization of milk and improvement of living conditions

5. Diagnosis of Tuberculosis (Tbc) – Clinical diagnosis (cough, night sweats, weight loss, etc.) + Radiological imaging – CT Microbiology laboratory – IGRAs (IFN-g RA; Interferon-gamma Release Assay) (Quantiferon) (six weeks after contact) – In Ehrlich-Ziehl-Neelsen (EZN) staining, acid-fast /resistant staining bacilli (ARB) (45%–80%) – Culture (still gold standard, but it takes 6-8 weeks) – Nucleic acid testing (NAT) Lower-Respiratory Tract Infections: Tuberculosis

6. Lower-Respiratory Tract Infection: Tuberculosis

7. Classification of Smear Smear Result Infectiousness of Patient 4+Strongly positive Probably very infectious 3+Strongly positive Probably very infectious 2+Moderately positiveProbably infectious 1+Moderately positiveProbably infectious Actual number of AFB seen (no plus sign) Weakly positiveProbably infectious No AFB seenNegative May not be infectious

8. Mycobacteria; Grow slowly than most of the bacteria pathogenic for humans The rapid growing species form colonies in 2-3 days Whereas most pathogenic mycobacteria require 2-6 weeks of incubation in Lowenstein- Jensen medium. This is reflected to the report of microbiology laboratory (6-8 weeks) Lower-Respiratory Tract Infection: Tuberculosis

9. Medical History and symptoms of pulmonary TB disease – Cough lasting 3 or more weeks – Chest pain – Coughing up sputum or blood Chest X-Ray – When a person has TB disease in lungs, the chest x-ray usually appears abnormal. – It may show: Infiltrates (collections of fluid and cells in lung tissue) Cavities (hollow spaces within lung) Bacteriologic Examination – Specimen Collection For pulmonary TB, specimens can be collected by: – Sputum sample – Induced sputum sample – Bronchoscopy – Gastric washing Lower-Respiratory Tract Infection: Tuberculosis TB patient coughing up sputum in a sputum collection booth

10. Bacteriologic Examination Sputum sample collection; – Easiest and least expensive method is to have patient cough into sterile container – HCWs should coach and instruct patient – Should have at least 3 sputum specimens examined – Collected in 8-24 hour intervals – At least one early morning specimen – Extrapulmonary TB Specimens other than sputum may be obtained Depends on part of body affected For example: – Urine samples for TB disease of kidneys – Fluid samples from area around spine for TB meningitis – Examination of AFB Smears Specimens are smeared onto glass slide and stained AFB are mycobacteria that remain stained after being washed in acid solution Lower-Respiratory Tract Infection: Tuberculosis AFB smear

11. Bacteriologic Examination – Culturing: Determines if specimen contains M. tuberculosis Confirms diagnosis of TB disease All specimens should be cultured – Identification of grown colonies : Step 1: Detect growth of mycobacteria – Solid media: 3 - 6 weeks – Liquid media: 4 - 14 days Step 2: Identify organism that has grown – Nucleic acid probes: 2 - 4 hours – Biochemical tests: 6 - 12 weeks – Drug Susceptibility Testing: Conducted when patient is first found to have positive culture for TB Determines which drugs kill tubercle bacilli Tubercle bacilli killed by a particular drug are susceptible to that drug Tubercle bacilli that grow in presence of a particular drug are resistant to that drug Lower-Respiratory Tract Infection: Tuberculosis Colonies of M. tuberculosis growing on Lowenstein- Jensen culture medium

12. Bacteriologic Examination – Types of Drug-Resistant TB; Lower-Respiratory Tract Infections: Tuberculosis

13. Mantoux Tuberculin Skin Test: – TST is administered by injection – Tuberculin is made from proteins derived from inactive tubercle bacilli – Most people who have TB infection will have a reaction at injection site – 0.1 ml of 5 tuberculin units of liquid tuberculin are injected between the layers of skin on forearm – Forearm should be examined within 48 - 72 hours by HCW – Reaction is an area of induration (swelling) around injection site – Induration is measured in millimeters – Erythema (redness) is not measured Lower-Respiratory Tract Infection: Tuberculosis

14. Quantiferon: – advised instead of TST – high spesificity – not affected by vaccine IGRA-How it Works; – blood samples are mixed with antigens and incubated for 16 - 24 hours – if infected with M. tuberculosis, blood cells will recognize antigens and release interferon gamma (IFN-γ) in response – results are based on the amount of IFN-γ released in response to antigens and control substances IGRA-Interpreting Results; – test results are based on IFN-γ concentrations – laboratories can use software provided by manufacturer to calculate results – results are sent to requesting clinician IGRA-Report of Results; Lower-Respiratory Tract Infection: Tuberculosis

15. Diagnostic performance of TST versus Quantiferon: – TST: sensitivity %90, spesificity problem. – Becomes positive in 8-12 weeks – Quantiferon: Becomes positive 8-10 weeks – Guidelines for Using the QuantiFERON®-TB, Gold Test for Detecting Mycobacterium tuberculosis Infection, United States Sensitivity 83-93 % Spesificity 80-95 % Indeterminate result: 1.3 % IGRA-Advantages : – Requires single patient visit to conduct test – Results can be available in 24 hours – Does not cause booster phenomenon – Less likely to have incorrect reading of results as compared to TST – BCG vaccination does not affect results IGRA-Disadvantages and Limitations : – Blood samples must be processed within 12 hours for some IGRAs – Errors in running and interpreting test can decrease accuracy – Limited data on its use in certain populations – Limited data on its use to determine who is at risk for developing TB disease Lower-Respiratory Tract Infection: Tuberculosis

16. NAT testing: – TBC PCR RESULT: POSITIVE – The positive predictive value of FDA-approved NAA Tests for TB is >95% in AFB smear-positive cases – If the NAAT result is negative and the AFB smear result is positive, a test for inhibitors should be performed and an additional specimen should be tested with NAA. – Sputum specimens (3%–7%) might contain inhibitors that prevent or reduce amplification and cause false-negative NAA results – Currently available NAA tests are not sufficiently sensitive (detecting 50%–80% of AFB smear- negative, culture-positive pulmonary TB cases) to exclude the diagnosis of TB in AFB smear-negative patients suspected to have TB Lower-Respiratory Tract Infection: Tuberculosis Melting Peak: Standard