Clinical , diagnosis and prophylaxis part of Mycobacterium tuberculosis.

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1. Mycobacterium tuberculosis Yubraj Bhatta ( M.sc.MLT Clinical Biochemistry)

2. Clinical Manifestations : Tuberculosis (TB) is classified as: i ) Pulmonary tuberculosis and ii) Extrapulmonary tuberculosis. i ) Pulmonary Tuberculosis ( PTB) Pulmonary tuberculosi s (PTB) accounts for 80% of all cases of Tuberculosis (TB). They are of two types : Primary tuberculosis Secondary or post primary tuberculosis.

3. a)Primary tuberculosis It is the initial infection caused by tubercle bacilli into a host where the bacilli are engulfed by alveolar macrophages giving rise to tuberculosis pneumonia Affect the lower lobe or lower part of upper lobe ( ghon focus). The hilar lymphnode are also involved. The ghon focus together with enlarged hilar lymphnode constitutes the primary complex.

4. b) Secondary tuberculosis: Occur due to reactivation of latent infection or exogenous reinfection . Affect mostly upper part of lungs, the lesion undergo necrosis and tissue destruction leading to cavitations. Sign and symptoms are non specific and mainly consist of fever, night sweat, loss of weight, malaise etc. Cough eventually develops with initial non-productive cough and subsequently accompanied by production of purulent sputum . Extensive disease may lead to respiratory distress syndrome.

5. ii) Extrapulmonary Tuberculosis (EPTB) EPTB results from hematogenous dissemination of tubercle bacilli to various organs. EPTB constitutes about 15-20% of all cases of TB , whereas in HIV-positive patients, the frequency is much high er accounting for 20-50% of all cases of tuberculosis. Cont....

6. Sites commonly involved in EPTB are: Tuberculous lymphadenitis Pleural tuberculosis Tuberculosis of upper airway Genitourinary tuberculosis Skeletal tuberculosis Tuberculosis of Central nervous system(CNS) Gastrointestinal tuberculosis Tuberculosis pericarditis Tuberculous skin lesions Disseminated tuberculosis ( miliary )

7. Tuberculous lymphadenitis : It is the most common form, accounting for 35% of all EPTB cases. The most common on sites are posterior cervical and supra- clavicular lymph nodes. It presents as painless swelling in the neck region Cont..

8. Cont...EPTB b) Pleural tuberculosis : It accounts for 20% of all EPTB cases. It presents as pleural effusion. c) Tuberculosis of the upper airways- involving larynx, pharynx, and epiglottis. d) Genitourinary tuberculosis:  Renal tuberculosis In female patients, fallopian tubes and the endometrium are commonly involved causing infertility. In males , epididymis is the most common site.

9. Cont.. e) Skeletal tuberculosis: Weight-bearing joints eg : spine, hips and knees are commonly affected. With advanced disease, collapse of vertebral bodies results in kyphosis . f) Tuberculosis of CNS : Seen most commonly in children . Tuberculous meningitis is common forms.

10. Cont... g) Gastrointestinal tuberculosis : Terminal ileum and ceacum are the most common sites involved. The route of spread may be due to swallowing of sputum , hematogenous spread, or ingestion of cow's milk (unpasteurized) contaminated with M.bovis (in developing countries).

11. Cont... h) Tuberculous pericarditis : Occurs as direct extension from adjacent lymph nodes or following hematogenous spread. Seen mostly in elderly people, in countries with low TB prevalence. i ) Tuberculous skin lesions : Scrofuloderma : It is a skin condition caused by tuberculous involvement of the skin by direct extension Lupus vulgaris : Apple jelly nodules are formed over the face in females.

12. Cont.. j) Miliary or disseminated tuberculosis : Hematogenous spread of tubercle bacilli results in the formation of yellowish 1- 2 mm size granulomatous lesions resembling millet seeds (thus termed as miliary ) in various organs. It is more common in HIV-infected people.

13. Cont.. k) HIV-associated Tuberculosis: Tuberculosis is one of the most common opportunistic diseases among HIV infected persons due to low CMI Worldwide, TB occurs in 70-80% of HIV-infected individuals, EPTB being more common than PTB..

14. Laboratory diagnosis: Specimen collection : In pulmonary Tuberculosis: Sputum (2 specimens- spot and early morning), • In Extra pulmonary tuberculosis: Specimens vary depending on the site involved.

15. Digestion, Decontamination of Specimen: Sputum and specimens from non-sterile sites need prior treatment for digestion (to liquefy the thick pus cells and homogenization), decontamination (to inhibit the normal flora) and concentration (to increase the yield). Processing should be carried out in class-II biosafety cabinet . following methods are available. Petroff's method (4% NaOH / Sodium hydroxide): Sputum is thoroughly mixed with equal volumes of 4% NaOH , centrifuged and the sediment is neutralized with 8% HCI .

16. Cont.. ii) NALC (N-acetyl-L- cysteine ) + 2% NaOH : This is superior than Petroff's method for isolation. NALC liquefies the sputum and NaOH kills the normal flora. This method is more compatible with automated culture systems.

17. Direct microscopy by Acid-fast Staining Ziehl-Neelsen (ZN)Technique Smears (2 x 3 cm size) are prepared from the mucopurulent part of sputum or with the sediment obtained after concentration. • Procedure : Smear is covered with primary stain, strong carbol fuchsin (basic fuchsin and phenol mixture) for 5 minutes . I ntermittent heating of the slide is done till the fumes appear. Heating helps in better penetration of the stain; Decolorization is done with 20% sulphuric acid for 3 minu tes; Counter staining is done with methylene blue / malachite green for 1 minute . Slide is examined under oil immersion objective.

18. Cont. ZN stain Negative result: At least 100 oil immersion fields should be examined before giving a negative report. Positive result: M. tuberculosis appears as long slender, beaded, less uniformly stained, red colored acid fast Advantages : Smear microscopy is rapid , easy to perform and is cheaper . Disadvantages : Smear microscopy is less sensitive than culture as the detection limit of smear microscopy is 10,000 bacilli/ mL of sputum. It cannot determine the viability of bacilli.

19. Cont.. Reporting : Microscopy provides only presumptive diagnosis. If typical beaded appearance is seen then it should be reported acid-fast bacilli resembling M. tuberculosis are seen by smear microscopy by ZN stain. It is difficult to differentiate M. tuberculosis from saprophytic mycobacterium present in tap water or even as commensals in clinical samples such as gastric aspirate, and urine.

20. Kinyoun's / Cold Acid Fast Staining : It differs from Z-N staining in that: Heating is not required, 2) Phenol concentration in carbol fuchsin is increased , 3) Duration of carbol fuchsin staining is more.

21. Auramine Phenol Technique It is a fluorescence staining technique, uses auramine phenol as primary stain, Acid alcohol as decolorizer and Potassium permanganate as counter stain. • The bacilli appear bright green against dark background • Smears are screened by using 20 X objective, hence can be screened faster (2 min for 1OO fields). • It is widely used by RNTCP ( Reserved national tuberculosis control programme) in laboratories having higher sample load.

22. Culture Techniques: Culture Conditions Incubating condition: Tubercle bacilli are slow growing due to long generation time (10-15 hours). Hence, inoculated culture media are incubated at 37'C, for 6-8 weeks . Media are incubated either aerobically for M. tuberculosis Microaerophilic condition (5% 02) for M. bovis .

23. Culture Media: Egg-based media : Examples include Lowenstein-Jensen (LJ): It consists of coagulated hen's eggs, mineral salt solution. asparagine and malachite green Colony appear as rough, tough and buff colored. Petragnani and Dorset egg media: Other solid media: • Blood-based ( Tarshis mediun ) • Serum-based ( Loeffler mediun ) • Potato-based ( Pawlowsky medium) • Agar-based ( Middlebrook 7H10 and 7H11)

24. Liquid media: Not usually used for culture instead it is used for drug sensitivity testing and preparation of antigen and vaccine. E.g : i ) Middlebrook 7H9, ii) Dubos, iii) Proskauer , iv) Sula , and Sauton media. Advantage of culture : Culture is more sensitive than microscopy as it has a detection limit of just 10- 100 viable bacilli.

25. Automated culture methods: Several automated culture methods have been developed in last decade. They are rapidly gaining popularity. Many hospitals, nowadays prefer to use these systems as they have many advantages over the conventional LJ culture. BACTEC MGIT ( Mycobacteria growth indicator tube): Uses an oxygen sensitive fluorescent compound, dissolved in the broth. Initially, The large amount of dissolved oxygen quenches emissions from the compound and little fluorescence can be detected. Later, actively respiring microorganisms consume the oxygen and the absence of oxygen allows the fluorescence to be detected

26. Cont.. BAcT /Alert: Uses the principle of colorimetric detection of pH change in the medium, which occurs due to CO2 liberated by the growth of M. tuberculosis ESP system : Detects a change of pressure in the medium which occurs due to the production of CO2 by M. tuberculosis BACTEC 460: It was the first semi automated system developed. As it was based on the use of radioisotopes to detect growth, hence not in use currently

27. Biochemical Identification: Colony smears from LJ bottles are stained to demonstrate acid fast bacilli. Various biochemical tests are done to differentiate M. tuberculosis from other mycobateria . Tests to Differentiate M. tuberculosis from M. bovis : 1) Niacin test: Positive for M. tuberculosis However, this is negative for M. bovis . 2) Nitrate reduction test : Positive for M. tuberculosis and negative for M. bovis . 3) Pyrazinamidase test : M. tuberculosis produces pyrazinamidase , therefore, hydrolyses pyrazinamide . This test is negative for M. bovis . 4) TCH ( Thiophene caroboxylic acid hydrazide ) : M. tuberculosis is resistant to TCH, whereas M. bovis is sensitive .

28. Tests to Differentiate Tubercle Bacilli from NTM Catalase test: Tubercle bacilli (human and bovine) are weakly catalase positive where as Non- tuberculous mycobacreria (NTM) are strongly catalase positive. NOTE: I soniazid resistant strains lose their catalase activities. Arylsulfatase test: Positive for rapid grower group of NTM, but negative for tubercle bacilli . Tween 80 hydrolysis test: Positive for certain NTM, such as M. kansasii and M.marinun . M. tuberculosis shows variable results.

29. Serology: Antigen detection : Various formats such as capture ELISA, dip stick and latex agglutination tests are available for detection of specific M. tuberculosis antigens such as lipoarabinomannan and antigen-5 in sputum, urine and other samples. These tests are less popular because their sensitivity is low (40-50%). • Antibody detection: Methods are no longer recommended in endemic areas because of cross reactivity with other environmental mycobacteria and variable antibody response is seen.

30. Molecular Methods Advantages: • They take less time than culture • They are more sensitive than culture, can detect as low as 1 bacillus/ mL of specimens. This is very much useful for extrapulmonary samples. • They can also detect the drug resistance genes . • Used for epidemiological typing of scrairis .

31. Molecular methods available are: Gene expert : Identification of strains of Mycobacterium tuberculosis which are resistant to rifampicin drug can be identified by nucleic acid testing. Other methods include: Probe based identification (Line probe assay) Transcription mediated amplification (TMA) Strand displacement amplification (SDA) Nucleic acid sequence based amplification (NASBA) Ligase chain reaction (LCR).

32. Animal pathogenicity Testing Guinea pig pathogenicity test Both M.tuberculosis and M. bovis are pathogenic to guinea pigs. Intramuscular inoculation of the concentrated specimen produces progressive weight loss of the animal with positive tuberculin test (after 3-4 weeks) and death (after 6 weeks). Autopsy of the animal shows: • Caseous lesion at the site of inoculation. • Enlarged caseous inguinal lymph nodes . • Tubercles may be seen in spleen, lungs, liver or peritoneum, but kidneys are unaffected.

33. Cont.. Rabbit pathogenicity test : M. bovis is pathogenic, where as M. tuberculosis is not pathogenic to rabbit.

34. Diagnosis of EPTB Diagnosis of EPTB differs from PTB in the following aspects: • The EPTB specimens are paucibacillary (bacilli present in few numbers) hence smear microscopy is less sensitive. therefore, molecular methods are more useful in the diagnosis of EPTB. • Specimens are free of normal flora; hence, initial decontamination and concentration methods are not required. • Pleural fluid examination reveals elevated ADA (adenosine deaminase ) and interferon (IFN)- gamma levels.

35. Cont.. ln renal tuberculosis: Urinary excretion of bacilli is intermittent; hence 3-6 consecutive early morning urine samples are collected, centrifuged and the sediment is used for processing. Acid alcohol is used as decolourizer . In tuberculous meningitis : CSF examination shows cobweb coagulum on standing, elevated CSF pressure, raised protein and chloride levels; whereas glucose levels is decreased.

36. Typing of Mycobacteria Is useful for epidemiological studies in determining the relatedness between various isolates in the communities.

37. Diagnosis of Latent Tuberculosis Latent tuberculosis is diagnosed by demonstration of delayed or type IV hypersensitivity reaction against the tubercle bacilli antigens. Two methods are available, i ) Tuberculin test, ii) IFN- gamma release assay

38. Tuberculin test Procedure : Mantoux test: It is the most commonly employed mediod . 0.1 mL of PPD containing one TU(tuberculin unit) is injected intradermally into fexor surface of forearm. 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: i ) More than 0r equals to 10 mm: Positive (tuberculin reactors) ii) 6-9 mm: Equivocal/doubtful reaction iii) <5 mm: Negative reaction

39. Cont.. Tuberculin test 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. Hence it is only used as an epidemiological marker. Children : In children, positive test indicates active infection and used as diagnostic marker.

40. Cont.. False-positive : The test becomes positive after: BCG vaccination (after 8- 14 weeks) • Nontuberculous mycobacteria infection False-negative: The test may become negative in various conditions such as- early or advanced TB, miliary TB, decreased immunity

41. Interferon Gamma Release Assay(IGRA ) This uses highly specific M. tuberculosis antigens such as CFP10 (culture filterate protein) and ESAT6 (early secreted antigenic target-6); both coded by RD1 genes . Procedure : It is an in-vitro test. Sensitized T lymphocytes collected from suspected individuals, are exposed to ESAT-6/CFP-IO antigens , which leads to release of high level of IFN-gamma from the T lymphocytes. An ELISA formats is available commercially ( QuantiFERON -TB Gold assay). • Advantage : It is highly specific , there are no false positive conditions.

42. Anti-microbial sensitivity testing: Commonly used AST include the following: Absolute concentration method Resistance ratio method E- test Nitrate reduction test

43. Prophylaxis: Classified as following: General prophylaxis Immuno prophylaxis Chemo prophylaxis General prophylaxis : Vaccination of new born with BCG vaccine. Providing tuberculosis awareness programme to public Early diagnosis of tuberculosis and their treatment. Pasteurization of milk.

44. Immuno prophylaxis : BCG vaccine ( prepared from live attenuated strain of M.bovis 0.1 ml is given intradermally as soon as birth and if missed before one year of birth. Complication : Prolonged ulceration at the site of vaccination. Lymphadenitis Osteomyelitis cont...........

45. Cont... Immuno prophylaxis Contraindication BCG should not be given to patient suffering from following infection.. Eczema HIV Leukemia Lymphoma Patient under immunosupressive treatment.

46. Treatment ( chemopropylaxis ) Anti-tubercular drugs can be cassified into: First line drugs Isoniazid (H) Rifampicin (R) Pyrazinamide (Z) Ethambutol (E) Streptomycin (S) Second line drugs Ethionamide Quinolones  Ofloxacin and ciprofloxacin Aminoglycosides  Kenamycin , capremysin and amikacin Cycloserine Macrolides

47. DOTS : DOTS is recommend by RNTCP (Revised national tuberculosis control programme) and WHO (World Health Programme). Here, the strategies used are: i )The entire treatment course is supervised to improve the patient's compliance. ii) Treatment response is also monitored by periodic sputum smear microscopy.

48. References: Apurva shankar shastry Ananthanarayana 7 th edition Essential of medical microbiology.

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