بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم GENUS: MYCOBACTERIUM Prof. Khalifa Sifaw Ghenghesh

2. Obligate aerobe, Gram-positive rods • Acid fast • Complex cell wall lipids • include mycolic acids • protects vs. phagolysosomal components • Peptidoglycan, glycolipids • acid-fastness • Two major groups: • Slow growers: • Rapid growers:

3. Mycrobacterium tuberculosis • Non-motile, Non-sporing, non-capsulate rods • Grows on several enriched culture media: • Lowenstein-Jensen medium: • Whole egg, Glycerol, Asparagine, Mineral salts, Malachite green Mycobacterium bovis:

4. Lowenstein-Jensen Plate Culture Inoculated with 15 Strains of Mycobacterium Species

5. CULTURE CHARACTERISTICS • On primary isolation: • visible growth after up to 8 weeks • Colonies: • Buff colour, dry bread crumb-like appearance • Growth is eugonic (M. bovis = dysgonic) • Growth temperature: • 35-37oC • Obligate aerobe --------------------------------------------------------------------------- • Heat-sensitive • Susceptible to alcohol, glutaraldehyde and formaldehyde.

6. Some differential characteristics of tuberculle bacilli causing human disease ____________________________________________________ Species Atmospheric preference Nitratase TCH Pyrazinamide --------------------------------------------------------------------------------------- M. tuberculosis Aerobic + S S M. bovis Microaerophilic -- R R _______________________________________ TCH = thiophen-2-carboxylic acid hydrazide S = sensitive, R = resistant

7. THE DISEASE • Not highly contagious: • transmission with prolonged contact between susceptible and active case • usually transmitted by airborne droplets, must penetrate deep into respiratory tree • infection can be via other routes: • ingestion => infection through cervical or mesenteric LN

8. Virulence • Ability to Survive within Macrophages • Primary Tuberculosis • Post-Primary Tuberculosis

9. Stages of Primary Tuberculosis in Childhood ---------------------------------------------------------------------------------------- Stage Time (from onset) Characteristics ---------------------------------------------------------------------------------------- 1. 3-8 weeks Primary complex (PC) develops and tuberculin conversion occurs 2. 2-6 months Progressive healing of PC, possibility of pleural effusion 3 6-12 months Possibility of miliary or meningeal tuberculosis 4 1-3 years Possibility of bone or joint tuberculosis 5 3-5 years Possibilty of genito-urinary or chronic skin tuberculosis ----------------------------------------------------------------------------------------

10. Main differences between primary and post-primary tuberculosis in the non-immunocompromised patients --------------------------------------------------------------------------------- Characteristics Primary Post-primary --------------------------------------------------------------------------------- Local lesion Small Large Lymphatic involvement Yes Minimal Cavity formation Rare Frequent Tuberculin reactivity Negative (initially) Positive Infectivity Uncommon Usual (pulmonary cases) Site Any part of lung Apical region Local spread Uncommon Frequent ----------------------------------------------------------------------------------------

11. TUBERCULIN TEST • Tuberculin: a heat-concentrated filtrate of a broth in which tubercle bacilli had been grown. • Injection of tuberculin into the skin >> • Large, indurated reactions >>Post-Primary Tuberculosis. • No induration >> Protective immunity • Purified Protein Derivatives (PPD): • Mantoux Method (Intracutaneous) • Heaf Method (Spring-loaded gun) • Tine Tests (Disposable single tests)

12. Mycobacteria-Positive PPD

13. LABORATOY DIAGNOSIS 1. Specimen: • Pulmonary tuberculosis:> Sputum, Bronchial washings, Laryngeal swabs, and Early-morning gastric aspirates. • Homogenized tissue biopsies. • Examine after centrifugation:Deposits of CSF, Pleural fluid, Urine and other fluids

14. 2. Microscopy: • Ziehl-Neelsen Stain • Fluorescent dyes 3. Culture: • Decontamination: • Lowenstein Jensen medium 4. Nucleic Acid Methods:

15. Mycobacterium tuberculosis sputum smear (Ziehl-Neelsen stain)

16. Mycobacterium tuberculosis in a sputum smear (Ziehl-Neelsen stain)

17. Mycobacteria - Auramine Stained and Viewed with Fluorescence Microscopy.Acid Fast Bacilli Appear as Glowing Yellow Rods.

18. Histopathology of Tuberculosis, Endometrium. Ziehl-Neelsen Stain.

19. An anteroposterior X-ray of a patient diagnosed with advanced bilateral pulmonary tuberculosis.

20. TREATMENT Chemotherapy Recommended by International Union against Tuberculosis and Lung Disease (examples) ---------------------------------------------------------------------------------------------------------- Intial phase Continuation Regimen (2 months) phase (4 months) Drug Drug ---------------------------------------------------------------------------------------------------------- HRZ HR Standard HRZ H3R3 Intermittent/ when HRZ H2R2 supervision is indicated HRZE HR When there is a high HRZS HR incidence of initial drug resistance ---------------------------------------------------------------------------------------------------------- H=isoniazid, R=rifampicin, Z=pyrazinamide, E=ethambutol, S=streptomycin -

21. EPIDEMIOLOGY • Transmission: • Open Pulmonary Tuberculosis • Crowdness in homes and workplaces • Tuberculosis and AIDS • Tuberculosis in Developing Countries: • Africa

22. CONTROL • Early Detection and Treatment of Open Cases • Reducing Overcrowding • Vaccination: • Bacille Calmette-Guerin (BCG) • Not effective as control measure

23. Mycobacterium leprae • Leprosy (Hansen's disease) • A chronic intracellular infectious disease unique to man (with few exceptions). Usually not fatal. • Never been cultivated in vitro • Armadillos: • 1010 bacilli/gram of diseased tissue

25. M. leprae can be grown in mouse foot pads or the nine-banded armadillo (picture).

26. PATHOGENESIS • Schwan cell>> Nerve damage>>Anaesthesia and Muscle paralysis >> Gradual destruction of extremities > Nasal bones and eyes Immune reactions >> • Severe and permanent nerve damage

27. THE DISEASE • Manifestations of the disease depend on the resistance of the host. 1. Tuberculoid: host is highly resistant, clinical abnormalities limited to a few peripheral nerves and adjacent skin areas, tuberculoid granuloma 2. Lepromatous: host lacks resistance, all tissues affected, foam cell granuloma 3. Borderline:

28. The feet become subject to bone damage and deformity through unnoticed wounds and infection. Serious infections can lead to amputations.

29. Victims of leprosy often suffer amputations of fingertips and toes due to unfelt trauma. X-rays of different stages.

30. Leprosy affects facial nerves > loss of blinking reflex of the eye > dryness, ulceration, and blindness.

31. Lepromatous lesions on human back

32. LABORATORY DIAGNOSIS • Histological Examination of Skin Biopsies • Detection of Acid-Fast Bacilli: • In Nasal Discharges • Scrapings from Nasal Mucosa • Slit-Skin Smears • Superficial incisions in skin >> Bacillary Index (BI):

33. Bacillary Index (BI) • 1+ 1-10 bacilli/100 fields • 2+ 1-10 bacilli/10 fields • 3+ 1-10 bacilli/ field • 4+ 10-100 bacilli/ field • 5+ 100-1000 bacilli/ field • 6+ >1000 bacilli/ field • Morphological Index (MI):

34. TREATMENT WHO Recommendations for Multidrug Therapy ----------------------------------------------------------------------------------------------- Type of Drug Dose Frequency Total Leprosy (mg) Duration ----------------------------------------------------------------------------------------------- Paucibacillary Rifampicin 600 Monthly, Superv. 6 months Dapsone 100 Daily, unsuperv. Multibacillary Rifampicin 600 Monthly, Superv. >2 years Dapsone 100 Daily, Unsuperv. 300 Mothly, Superv. Clofazimine { +50 Daily, Unsuperv. -----------------------------------------------------------------------------------------------

35. EPIDEMIOLOGY • Transmission: • Nasal secretions of patients with lepromatous leprosy. • Skin Test: Limited diagnostic value • Lepromins > boiled-bacilli rich lepromatous lesions • Leprosins >ultrasonicates of tissue-free bacilli from lesions. • Two Types of Reaction: • Fernandez Reaction: sensitized individuals > 48h (leprosin) • Mitsuda Reaction: granulomatous swelling > ~3 weeks (lepromin)

36. بسم الله الرحمن الرحيم ACTINOMYCETES

37. Gram +ve,Filaments that Break Up Into Bacillary and Coccoid Forms. • Non-Motile, Non-Sporing, Non-Capsulated. • Free Living >> Soil

38. 1. ACTINOMYCES SPECIES • A. israelii • Actinomycosis >> Chronic Granulomatous Infection. • Formation of Sulphur granules: • 3 Forms: i. Cervicofacial ii. Thoracic iii. Abdominal • Predisposing Factors: Trauma, Poor Oral Hygiene.

39. A. israeliiGram stain showing diphtheroidal rods and short branching filaments.

40. Actinomycosis-organisms aspirated from the lung. Long, tortuous and branching organisms can easily be visualized using silver stain. Sulfur granules not seen in aspirations.

41. LABORATORY DIAGNOSIS i. Direct Examination: Sputum, Pus, etc.. >>Examined for Granules ii. Culture:Brain Heart Infusion Agar • TREATMENT • Penicillin >>Several Weeks

42. Sulfur granule from human actinomycosis tissue section (hematoxylin and eosin stain).

43. 2. NOCARDIA SPECIES • N. asteroides • Nocardiosis. • Aerobic. • Disease Begins as Pulmonary Infection > • > 50% of Patients are Immunocompromised. • Fatality Rate >>

44. LABORATORY DIAGNOSIS i. Direct Examination: • Sputum, Skin Lesions, Tissue Biopsies or Surgical Material>>Microscopically. • Observe:G+ve, Multiple Branched and Beaded Filaments. • > Partially Acid-Fast. • ii. Culture: • iii. Identification:Biochemically. TREATMENT • Sulphonamides, NA, TMP-SMX.

45. Nocardia asteroidesSilver stain showing the twisted masses of long filamentous organisms

46. Nocardia asteroidesAcid fast stain shows the pale red staining organisms in an area of necrosis

47. Differences between the genera Actinomyces and Nocardia ------------------------------------------------------------------ Actinomyces species Nocardia species ------------------------------------------------------------------ Facultative anaerobes Strict aerobes Grow at 35-37oC Wide temp range of growth Oral commensals Environmental saprophytes Non-acid-fast mycelia Usually weakly acid-fast Endogenous cause of Exogenous cause of disease Disease ------------------------------------------------------------------