1. COURSE: MLSM 505 LECTURE TOPIC: MYCOBACTERIA

2. Characteristics: The genus Mycobacterium consists of numerous species some of which are highly infectious. Generally, they are slender, slightly curved rods, non motile, poorly Gram stained positive, acid fast and aerobic The acid fastness is due to their possession of thick cell wall made up of waxes and lipids with high content of mycolic acids..

3. They grow on protein rich culture media
They grow on protein rich culture media. Improved growth is achieved in the presence of
CO2
Type species: Mycobacteria tuberculosis (aka Tubercle bacillus)   Classification: Classification into 2 major groups is based on rate and temperature of growth, and pigmentation:

4. 1. Rapid growers and slow growers 2
1. Rapid growers and slow growers 2. Pigmented(chromogenic) and non-pigmented (non chromogenic). The pigment is yellowish. Other characteristics are also used to classify them into typical and atypical Mycobacteria

6. Rate of growth: Most grow between 6 – 8 weeks. M
Rate of growth: Most grow between 6 – 8 weeks. M.ulcerans is the slowest grower Temp. of growth: Most species will grow at 35 – 370C, others may grow at lower or higher temperature e.g. M. chlonei and M. fortitum grow at 30-32°C while M. xenopi can grow at 44°C. Mycobacteria were once classified according to Ruyon classification which some workers adopted.

7. Pigmentation: Photochromgens – these produce colour in light (Ruyon group 1): M. kansasii, M. marinum, M. simiae. Scotochromogens – these produce colour in the dark (Ruyon group 11): M. scrofulaceum, M. szulgai. Non-chromogens – non pigmented (Ruyon group 111): M. tuberculosis, M. bovis, M. fortuitum, M. chelonei. Rapid growers (Ruyon group IV): Mainly saprophytic

8. PATHOGENICITY There are more than 50 separate species of Mycobacteria: Eighty percent of all diseases is respiratory. However, lymph nodes, bone and other organs may be involved. M. tuberculosis: causes tuberculosis(Tb) which may be pulmonary or non pulmonary.

9. Pulmonary tuberculosis is a slowly progressive respiratory disease.
Complications of pulmonary Tb include pleural effusion, lung collapse, acute miliary Tb, meningitis.
Non pulmonary Tb include renal and urogenital Tb, miliary Tb and Tb meningitis.
The organism is carried through blood stream to reach and infect various organs.

10. M. africanum: causes a milder form of human tuberculosis. M
M. africanum: causes a milder form of human tuberculosis M. bovis: causes cattle tuberculosis; humans can be infected by close contact with the animals or by ingestion of contaminated animal product. The above three species are also referred to as M. tuberculosis complex. M. leprae: causes leprosy, a skin infection.

11. ATYPICAL MYCOBACTERIA (MOTT= Mycobacteria other than tuberculosis) These may grow rapidly at 25°C or 45°C, several species are pigmented. Usually acquired from soil, water, birds or animals. M. kansasii. M. szulgai. M. simiae, and M. xenopi: all of these cause pulmonary infections. M. avium-intracellulare and M. scrofulaceum: cause cervical adenitis .

12. M. fortuitum, M. chelonei and M. marinum: cause skin infection. M
M. fortuitum, M. chelonei and M. marinum: cause skin infection. M. ulcerans: cause skin ulcers (Buruli ulcer).
Tuberculosis is a major killer infectious disease world wide.
It is symptomised by long lasting ( chronic ) cough which may be productive ( purulent or muco-purulent). The sputum may be blood stained (haemoptysis).

13. Other signs include weight loss, fever, tiredness and night sweat
Other signs include weight loss, fever, tiredness and night sweat. HIV-aids patients are now most at risk (co-infection). And the multiple drug resistance (MDR) nature of the organism makes it very difficult to eradicate. Tuberculosis today causes more morbidity and greater mortality than any other infectious disease except HIV-AIDS. It can co-exist with HIV-AIDS. Good nutrition and a well ventilated place of abode can minimize the chances of contracting TB.

14. Pathogenesis and transmission of tuberculosis Infection acquired by inhalation of M. tuberculosis in aerosol from infected person and dust, goes to the lungs; and from there spreads to other organs. Source of infection is another human case – proximity, sharing utensils etc. The bacteria are intra cellular and so can survive and multiply in the macrophages of lung tissues. Skin infection is acquired by inoculation.

15. Inhalation: M. tuberculosis, M. leprae
Ingestion: M. bovis
Inoculation: M. ulcerans
Primary tuberculosis is when contracted by an individual for the first time.
It results in local infection of the regional lymph nodes.

16. Initially a mild and asymptomatic condtion: about 90% may not proceed further, but a few others may become serious and disseminated through the lymphatics to cause infections such as miliary Tb, bronchopneumonia, meningitis, bone and joints Tb. Local lesion on the periphery of the lung is called Ghon focus. Post primary Tb: Asymptomatic as latent infection that could be re activated.

17. LABORATORY DIAGNOSIS  Specimens: Sputum – preferably early morning samples for three consecutive days; gastric lavage from children, early morning urine specimens for three consecutive days for renal/urogenital Tb., pus.   The new national algorithm recommends 3 samples produced as follows: First: On the spot i.e. in the laboratory Second: Early morning sputum from home. Third: On the spot i.e. in the laboratory

18. Microscopy: Auramine stain – used for screening
ZN stain – used to confirm an auramine +ve smear.
Always include a control smear for each batch of staining.
Report as + when 3-9 AFBs are seen in the whole smear, ++ when 10 or more AFBs are seen in the whole smear and +++ when 10 or more AFBs are seen in several fields.

19. Culture: Sputum may be homogenized and decontaminated before culture
Culture: Sputum may be homogenized and decontaminated before culture. This is to get rid of commensals in the specimens. Inoculate a pair of LJ glycerol medium and incubate for 6-8 weeks. With the automated machine like the Bactec 460 TB machine, growth may appear within ten days. .

20. Other useful culture media for mycobacteria are LJ pyruvate medium (M
Other useful culture media for mycobacteria are LJ pyruvate medium (M. bovis), Dorset egg medium for maintenance of cultures, Middlebrook 7H10 and Middlebrook 7H9 broth for identification and cultivation Growth: Colonies are rough and buff coloured. A slow growing AFB is suggestive of M. tuberculosis.  

21. Identification: Identify and confirm with standard identification tests such as colonial morphology on glycerol and pyruvate media, sensitivity to pyrazinamide and niacin production. Animal inoculation : M. tuberculosis and M. bovis are pathogenic to laboratory animals. Guinea pig can be inoculated to confirm the infection. PCR: This new technique is very useful for the diagnosis of mycobacterial infection but it is expensive.  

22. Sensitivity Test: Special methods are used to test the organism against the relevant drugs. The common drugs used for the treatment are isoniazid, Rifampicin, Pyrazinamide(PAS), Streptomicin, and ethambutol. M. tuberculosis: Hypersensitivity. · Tuberculin test: Test for present or past immune response/hypersensitivity Mantoux test using PPD .

23. BCG( Bacillus of Calmette and Guerin) vaccine confers immunity on recipient especially young children. The vaccine is an attenuated strain of M. bovis. LEPROSY: A chronic disease of skin and nerve caused by M. leprae. Mainly in the tropics. Long incubation period of 3 to 5 years. Skin lesions present as nodules, thickened patches, thickened peripheral nerves and anaesthesia with muscle atrophy ulceration. Great disfigurement.

24. There are 2 main forms of leprosy and some others in between based on the delayed hypersensitivity reaction to infection: Tuberculoid: a localized form affecting mainly the skin found in partially immune individuals.  Lepromatous: a generalized form that destroys tissues, usually in non immune persons.   The infection is slow and subclinical, spread by respiratory tract droplets, especially the lepromatous form.

25. Laboratory Diagnosis   Specimens: Tissue juice, nasal swab, skin scraping. Skin smear is best from active part of skin lesions – 2 ear lobes, back of arms or front thighs above the knees. Specimens are decontaminated before it is concentrated by centrifugation.   Microscopy: Non motile, slightly curved. Acid fast with 5% H2SO4 or 1% acid alcohol. In skin tissue, the bacteria are packed together in large numbers.

26. Morphological index (MI) The smear usually shows a mixture of well stained ( viable) and poorly stained (non viable) bacilli. The MI is the percentage of viable organisms and it is used to monitor treatment.   Culture Does not grow on artificial medium. Can be cultured on the foot pad of 9 banded armadillo or mouse or rat.   .

27. Treatment
This is effected with the usual anti Tb drugs.
Dapsone is a popular drug for the treatment of leprosy. Side effects are however, an issue with this drug.