MYCOBACTERİUM
The Mycobacterium was found through out the world up to 4000 years ago, The origin of Mycobacterium tuberculosis, the causative agent of TB, were initially found in soil & that some species evolved to live in mammals, The domestication of cattle would have allowed the passage of Mycobacterial pathogen from domesticated livestock to human. The disease peaked in Europe in fist half of the 19th century, and it is estimated that one quarter Europeans died of TB. world wide (10 million) new cauese of TB & 3 million deaths from TB. annually.HISTORY
The morbidity & mortality rates due to TB steadily dropped during 20th century in the developed world, because of better public health practices & widespread use of the Mycobacterium bovis BCG (Bacille Calmette‐Guerin) vaccine, as well as development of antibiotics in the 1950. Mycobacterium tuberculosis cause Tuberculosis (very important human pathogen). Mycobacterium leprae cause Leprosy (Hansen’s disease). In 1882, the microbiologist Robert Koch discovered the tubercle bacillus, at a time when one of every seven deaths in Europe was caused by TB.
Mycobacteria
Mycobacteria are aerobic, acid-fast bacilli (rods).They are neither gram-positive nor gram-negative (i.e., they are stained poorly by the dyes used in Gram stain).The term acid-fast refers to an organism’s ability to retain the carbolfuchsin stain despite subsequent treatment with an ethanol–hydrochloric acid mixture.The high lipid content (approximately 60%) of their cell wall makes mycobacteria acid-fast.The major pathogens are Mycobacterium tuberculosis, the cause of tuberculosis & Mycobacterium leprae, the cause of leprosy.Mycobacteria
Auramine phenol fluorescent staining techniqueZiehl-Neelsen Stained
Mycobacteria
Mycobacteria can be classified as in the following: I- Mycobacterium tuberculosis complex: 1- Mycobacterium tuberculosis. 2- Mycobacterium bovis. 3- Mycobacterium africanum. II- Mycobacterium leprae: they are strict pathogens cause leprosy and cannot be cultured in vitro.M. bovis BCG vaccine M. avium-intracellulare AIDS patient
Mycobacteria
Mycobacteria Other than Tuberculosis (MOTT) or Tuberculoid bacilli (Atypical Mycobacteria): The MOTT are classified into four groups according to their rate of growth and whether they produce pigment under certain conditions (Atypical mycobacteria, can cause tuberculosis-like disease but are less frequent pathogens.) Group I: Contains the photochromogens, which produce pigment in the light. This group grows very slowly and includes the species Mycobacterium kansasii and Mycobacterium marinum. Group II: includes Mycobacterium scrofulaceum, which is a slow growing atypical Mycobacterium species that produces pigment both in the light and in the dark.
Mycobacteria
Group III: Contains the slow-growing nonphotochromogens, including Mycobacterium avium intracellulare complex and Mycobacterium ulcerans. Group IV: grow rapidly but do not produce any pigment. This grouping includes three potential pathogens - Mycobacterium abscessus - Mycobacterium fortuitum - Mycobacterium chelonaeMycobacterium tuberculosis (Tuberculosis)
• It is acid-fast bacillus (rod), An obligate aerobe, most commonly infects the lungs, Mycobacterium tuberculosis grows very slowly, taking up to 6 weeks for visible growth, so the generation time 12-18 hours, The colonies that form lump together due to their hydrophobic lipid nature, resulting in clumped colonies on agar and floating blobs on liquidmedia.M. tuberculosis is relatively resistant to acids and alkalis. NaOH is used to concentrate clinical specimens; it destroys unwanted bacteria, human cells, and mucus but not the organism.Mycobacterium tuberculosis (Tuberculosis)
M. tuberculosis is resistant to dehydration and therefore survives in dried expectorated sputum; this property may be important in its transmission by aerosol.Mode of Transmission
By inhalation of droplet nuclei (< 5–10 μm size) directly from cases of pulmonary tuberculosisLess frequently transmitted by:Ingestion (rare): Swallowing of sputum (in infants) or unpasteurized milk.Laboratory accident Mycobacterium bovisalso causes tuberculosis in humans. Is found in cow’s milk, which, unless pasteurized, can cause gastrointestinal tuberculosis in humans.Mycobacterium tuberculosis (Tuberculosis)
• Epidemiology Patients with active pulmonary tuberculosis shed large numbers of organisms by coughing, creating aerosol droplet nuclei. The organisms can remain viable as droplet nuclei suspended in room air for at least 30 minutes. The principal mode of contagion is person-to-person transmission by inhalation of the aerosol. A single infected person can pass the organism to numerous people in an exposed group, such as a family, classroom, or hospital ward without proper isolation.Mycobacterium bovis also causes tuberculosis in humans. M. bovis is found in cow’s milk, which, unless pasteurized, can cause gastrointestinal tuberculosis in humans.Pathogenesis of Tuberculosis Mycobacterium tuberculosis primarily affects the lung but can also cause disease in almost any other tissue. No mycobacteria produce toxins 1- Facultative intracellular growth With the first exposure (usually by inhalation into the lungs ), the host has no specific immunity. The inhaled bacteria cause a local infiltration of neutrophils and macrophages. Due to the various virulence factors, the phagocytosed bacteria are not destroyed. They multiply and survive in the macrophages. The bacteria cruise through the lymphatics and blood to set up camp in distant sites. This period of facultative intracellular existence is usually short-lived because the host rapidly acquires its prime defense against the acid-fast : cell-mediated immunity.
Pathogenesis of Tuberculosis 2- Cell-mediated immunity Some of the macrophages succeed in phagocytosing and breaking up the invading bacteria. These macrophages then run toward a local lymph node and present parts of the bacteria to T-helper cells. The sensitized T-cells then multiply and enter the circulation in search of Mycobacterium tuberculosis. When the T-cells encounter their antigenic target, they release lymphokines that serve to attract macrophages and activate them when they arrive. These activated macrophages can now destroy the bacteria. It is during this stage that the macrophage attack actually results in local destruction and necrosis of the lung tissue.
Pathogenesis of Tuberculosis 2- Cell-mediated immunity The necrosed tissue looks like a granular creamy cheese and is called caseous necrosis. This soft caseous center is surrounded by macrophages, multinucleated giant cells, fibroblasts, and collagen deposits, and it frequently calcifies. Following induction of cell-mediated immunity against Mycobacterium tuberculosis , any additional exposure to this organism will result in a localized delayed-type hypersensitivity reaction (type IV hypersensitivity).
Pathogenesis of TuberculosisTwo types lesions:Exudative lesions: which consist of an acute inflammatory response and occur chiefly in the lungs at the initial site of infection.Granulomatous lesions: which consist of a central area of giant cells containing tubercle bacilli surrounded by a zone of epithelioid cells. These giant cells, called Langhans’ giant cells, are an important pathologic finding in tuberculous lesions. A tubercle is a granuloma surrounded by fibrous tissue that has undergone central caseation necrosis. Tubercles heal by fibrosis and calcification.
A. Acid-fast stain of sputum from apatient with tuberculosis. B. Typical growth pattern showing “cording (that is, growing in strings).
Virulence factors No spore, no flagellum, no exotoxin, no endotoxin, no invasive enzyme Capsule:polysaccharide;CR3;enzyme; protect, Lipid, Heat-shock protein/Tuberculin , protein: antigenicity, old tuberculin; associate with wax D can cause hypersensitivity and form tubercle Mycobacterium tuberculosis Can infect and cause disease in many different body locations such as: Meninges, Brain, Bone, Kidney and Essentially any organ (lung primary target).
Mycobacterium tuberculosis (Tuberculosis)
• There is one class of lipid that only acid-fast organismshave and that is involved in mycobacterial virulence mycosides.Mycolic acid: is a large fatty acid.Mycoside: is a mycolic acid bound to a carbohydrate,forming a glycolipid.Cord factor: is a mycoside formed by the union of 2 mycolic acids with a disaccharide (trehalose). This mycoside is only found in virulent strains of Mycobacterium tuberculosis.Sulfatides: are mycosides that resemble cord factor with sulfates attached to the disaccharide. They inhibit the phagosome from fusing with the lysosome that contains bacteriocidal enzymes.Wax D: is a complicated mycoside that acts as an adjuvant.Mike (Mycosides). He is WAXING (Wax D) his Surfboard (Sulfatides) and has his surfboard CORD (Cord Factor) attached to his leg ( so as not to lose his)
Spectrum of Disease:- Primary pulmonary TB: Tuberculosis (TB) is classified as pulmonary (80%) and extrapulmonary forms (20%). It is classified into Primary PTB and Post-primary/secondary PTB Organisms replicate in naive alveolar macrophages, killing the macrophages until CMI (Cell-Mediated Immunity) is set up (Ghon focus), Macrophages transport the bacilli to the regional lymph node (Ghon complex) Organisms that are walled off within the Ghon complex remain viable unless treated.
2 ) Reactivational TB Symptoms can include fever, hemoptysis, night sweats, weight loss, Erosion of granulomas into airways (high oxygen) later in life under conditions of reduced T-cell immunity can lead to mycobacterial replication and disease symptoms, Complex disease with the potential of infecting any organ system, May disseminate (miliary TB): kidneys, GIT, brain, spine (Pott disease)
Latent TB Infection (LTBI)Person:Not illNot contagious Normal chest x-rayUsually the tuberculin skin test is positiveGerms:Sleeping but still aliveSurrounded (walled off) by body’s immune system
Active TB Disease Person: Most often feels sick Contagious (before pills started) Usually have a positive tuberculin skin test Chest X-ray is often abnormal (with pulmonary TB) Germs Awake and multiplying Cause damage to the lungs
EPTB results from hematogenous dissemination of tubercle bacilli to various organs. Though EPTB constitutes about 15 to 20% of all cases of TB, in HIV-positive patients the frequency is much higher accounting for 20–50% of all cases of tuberculosis.The sites commonly involved (in order of frequency) are:Tuberculous lymphadenitis is the MC form (35% of all EPTB cases). The most common sites are posterior cervical and supraclavicular lymph nodes.Pleural tuberculosis accounts for 20% of all EPTB cases. It presents as pleural effusion.Tuberculosis of the upper airways involving larynx, pharynx, and epiglottis.Genitourinary tuberculosis:Renal tuberculosisGenital tuberculosis: In female patients, fallopian tubes and the endometrium are commonly involved causing infertility. In male, epididymis is the MC site. Extrapulmonary Tuberculosis (EPTB)
Skeletal tuberculosis: Weight-bearing joints such as spine (Pott’s disease or tuberculous spondylitis is most common), hips and knees are commonly affected.Tuberculosis of CNS occurs commonly in children. Tuberculous meningitis and tuberculoma are the common forms.Gastrointestinal tuberculosis: Terminal ileum and cecum are the MC sites involved. Transmission is due to swallowing of sputum, hematogenous spread, or ingestion of cow’s milk contaminated with M.bovis.Tuberculous pericarditis.Tuberculosis skin lesions.HIV-associated tuberculosis: TB is the MC opportunistic diseases among HIV-infected persons. Worldwide, TB affects 70–80% of HIV infected individuals, EPTB being more common than PTB.Disseminated tuberculosis: Hematogenous spread of tubercle bacilli results in the formation of granulomatous lesions resembling millet seeds in various organs. It is more common in HIV infected people. Extrapulmonary Tuberculosis (EPTB)
Tuberculin Skin Test Tuberculin is a mixture known as Purified Protein Derivatives (PPD) from TB bacilli. It is a test for delayed type hypersensitivity. Positive reaction, reddening and thickening (> 5mm) at the site of injection after 2-3 days, indicates cellular immunity to tubercle bacilli. PPD = Purified Protein Derivative from M. tuberculosis
PPD Tuberculosis Skin Test Criteria
Interpretations of the Mantoux tuberculin skin test.Specimen collection:In pulmonary TB- sputum (2 specimens-spot and early morning), gastric aspirate (in children).In Extrapulmonary Tuberculosis (EPTB) specimens vary depending on the site involved.Digestion, decontamination and concentration of specimen:Petroff’s method (4% NaOH)NALC (N-acetyl-L-cysteine) + 2% NaOH.Acid-fast staining by Ziehl Neelsen (ZN) technique:Other staining methods:Auramine phenol technique: It is a fluorescent staining technique. Laboratory Diagnosis
Conventional culture media: Lowenstein Jensen (LJ): M.tuberculosis produces rough, tough and buff colonies. (Best Diagnostic Methode Sputum Culture)Automated culture methods: These systems monitor the growth continuously and detect growth faster (2-3 weeks); however, they are expensiveExample include BACTEC, MGIT and BACT/AlertBACTEC MGIT (Mycobacteria Growth indicator Tube)—Uses an oxygen sensitive fluorescent compound to detect:Mycobacterial growth.Resistance to first line anti-tubercular drug.Serology:Antigen detection—LAM and antigen-5 detection by ELISAAntibody detection—not useful in endemic area. Laboratory Diagnosis
Laboratory Diagnosis
The Best criteria for tuberculosis diagnosis by WHO: Sputum examination of symptomatic patients by Zn stainZiehl-Neelsen–stained (B) Auramine-rhodamine–stained
Mycobacteria Growth Indicator Tube (MGIT)Molecular Diagnosis
1- PCR detecting IS6110 gene. 2- Line probe assay: Detects drug resistance from samples. 3- Gene Expert: It detects growth and resistance to rifampicin. It takes very less time (2 hours). It is a cartridge based nucleic acid amplification technique (based on real time PCR). Pleural biopsy is better specimen than pleural fluid for Gene Expert.The two major drugs used to treat tuberculosis:- 1- First Line Drugs:- Isoniazid (INH), Rifampicine (RMP), Pyrazinamide (PZA), Ethambutol (EMB). 2- Second-line drugs:- Are more toxic or less effective (or both) Kanamycin, Capreomycin, Ethionamide, Cycloserine, Ofloxacin Ciprofloxacin.
Tretment
Multidrug Resistant Tuberculosis (MDR-TB) MDR-TB is defined as resistance to isoniazid and rifampicin with or without resistance to other first line drugs. DOTS (Directly Observed Treatment Short Course) DOTS-Plus refers to a DOTS service with additional elements for drug resistant TB. Extensively Drug Resistant Tuberculosis (XDR-TB)
Tretment
Extensively Drug Resistant Tuberculosis (XDR-TB) Definition: These are MDR-TB cases that are also resistant to: Fluoroquinolones (ofloxacin/levofloxacin) and At least one injectable aminoglycosides (kanamycin, amikacin or capreomycin). Epidemiology: In USA, 3% of MDR-TB cases have been found to be XDR. Treatment of XDR-TB is extremely difficult. XDR-TB has a very rapidly progressing clinical course with high mortality. So the XDR-TB is resistant to ( INH, RMP, Amikacin & Ofloxcin)
Tretment
BCG vaccine was developed by Calmette and Guerin (1921). They attenuated the strain by serial sub culturing in glycerol bile potato medium for 230 times over a period of 13 years. BCG Strain: Live attenuated M.bovis. Administration of BCG: Dose and strength: 0.1 ml containing 0.1 mgTU, Site: It is given above insertion of left deltoid Route: By intradermal route by using a 26 gauge tuberculin syringe.
Prophylaxis by BCG Vaccine (Bacillus Calmette Guerin)
Duration of immunity lasts for 15–20 years,Efficacy: Many trials have shown that BCG has a variable efficacy of 0–80%. Prophylaxis by BCG Vaccine (Bacillus Calmette Guerin)
Mycobacterium leprae
Hansen’s Dısease (Leprosy) caused by Mycobacterium leprae Hansen’s disease is a chronic, slowly progressive, granulomatous disease involving ectodermally derived tissue such as the skin and peripheral nerves. The disease is usually limited to the cooler parts of the body such as the skin, nose It rarely affects internal organs such as the brain, liver, spleen, kidneys, and bones. It has a specific predilection for peripheral nerves.Distinguishing Features Acid fast rods (seen in punch biopsy) Weakly gram-positive, Obligate intracellular parasite not artificial media (cannot be cultured in vitro), Optimal growth at less than body temperature. Reservoir: human mucosa, skin, and nerves Transmission: nasal discharge from untreated lepromatous leprosy patients
Pathogenesis: obligate intracellular parasite; cooler parts of body, e.g., skin, mucous membranes, and peripheral nerves.Disease(s): leprosy: a continuum of disease, which usually starts out with an indeterminate stage called “borderline”.Diagnosis Punch biopsy or nasal scrapings; acid fast stain Lepromin skin test is positive in the tuberculoid but not in the lepromatous form No culturesTreatment: multiple-drug therapy with dapsone and rifampin,with clofazimine added for lepromatous.Prevention: dapsone for close family contacts.