Inhaled MTb bacilli
alveoli
Multiply in the pulmonary epithelium or macrophages
Bacilli are destroyed by immune system
Survivors
Extrapulmonary sites
Bacterial sulfolipids inhibit fusion of phagocytic vesicles with lysosojmes
2-4 weeks
blood MICROBIOLOGY LECTURE 8 – Mycobacteria
Notes from Lecture USTEMED ’07 Sec C - AsM Characteristics:
- aerobic, slightly curved or straight rods - 0.2 to 0.6 um wide; 1.0 to 10 um long - cell wall: multilayered complex lipids
- common properties with Corynebacterium and Nocardia:
o produce mycolic acid
o ~guanine + cytosine content (G+C) Mycobacterium tuberculosis
Tuberculosis
- An ancient disease
- Seen in stone age skeletons and early Egyptian mummies
- 1882 – Koch’s discovery - Koch’s postulates:
o An organism associated with clinical disease o Isolated it in pure culture
o Reproduces the disease in animals
o Recovered the bacillus in pure culture from the experimental animal
Morphology of MTb
- Slender, straight or slightly curved rod with rounded ends
- 0.3 to 0.6 um width x 1-4 um length
- true branching in old cultures and smears from caseous lymph nodes
- acid fast: lipid-barrier (mycolic acid) and carbolfushin dye complex
- hydrophobic surface layers trap the dye Mycobacterial Cell Wall
- 60% lipid including mycolic acids
- backbone: covalent structure consisting of 2 polymers covalently linked by phosphodiester bonds:
o peptidoglycan o arabinogalactan MTb Physiology
- Cultural characteristics: o Strictly aerobic
o Most spp. Grow slowly with generation times 8 to 24 hrs.
o Grow fairly on simple artificial media except M. leprae
- Species are differentiated by:
o Rate and optimal temperature of growth o Production of pigments
o Biochemical tests (niacin, catalase test, urease test, etc)
Antigenic Structure of Mycobacteria 1. Old tuberculin (OT)
o 1881 – described by Koch by boiling a 6-week old broth culture
o Heat–stable protein is the active component 2. Purified Protein Derivative (PPD)
o Partially purified preparation of OT prepared by ammonium sulfate fractionation
o PPD-S: adopted by WHO for skin testing 3. Purified Antigens:
o Recombinant DNA techniques & antigen expression in E.coli
o Affinity purification of antigen preparations using monoclonal antibody immunosorbent columns 4. Polysaccharides:
o
Protein-free polysaccharides (arabinogalactans & arabinomannans)i. Immunogenic
ii. Give precipitin reactions with antisera iii. Active in C’fixation &
hemmaglutination reactions 5. Phosphatidyl Inositol Mannosides (PIMS)
o Lipoteichoic acid-like polymers with a role in macrophage recognition
o Associated with cross protective immunity
6. Other immunoreactive components a. Wax D & muramyldipeptide (MDP)
peptidoglycans (cell wall) adjuvant activity
induce a cell-mediated immune response against the protein
b. Trehalose-6-6’-dimycolate cord factor
immunoreative properties adjuvant activity
elicits extensive pulmonary granulomas anti-tumor properties
c. Sulfatides (Trehalose 2’-sulfates esterified with fatty acids)
sulfur-containing glycolipids (sulfolipids) can replace cord factor as a component of
an oil-BCG cell wall or endotoxin preparation causing tumor regression Determinants of Pathogenicity
- Cord factor - Sulfatides Epidemiology of MTb
- TB is a global problem
- 8 to 10 million new cases worldwide - 3 million deaths worldwide
- Yearly decline in TB incidence ended in 1984 when the HIV infection increased in number
- Philippine statistics: FHSIS-DOH 2001
o
Respiratory TB: 6th leading cause of morbidity: 11-,841 cases and rate of 142.2/100,000 populationo
TB meningitis: 466 cases and rate of 0.6/100,000 populationo Other forms of TB: 11,494 cases and rate of 14.7/100,000 population
- Transmission
o Inhalation of dried residues of droplets containing tubercle bacilli
o Droplet nuclei (1 to 10 um) can reach the alveoli and initiated infection
- Tuberculous infection vs. Tuberculous disease: Tuberculin
test symptomsClinical
Tuberculous infection +
-Tuberculous disease + +
- risk factors for the development of Tuberculous disease:
o intrinsic characteristics of the individual – age, sex, body build, & genetic susceptibility
o use of adenocorticosteroids & immnunosuppressive agents o hematologic diseases o reticuloendothelial disease o Diabetes mellitus o Silicosis o HIV infection Pathogenesis of TB
Clinical Manifestations - Primary Tuberculosis
o No previous contact with the organisms o 95% of cases are arrested
o positive tuberculin test
o chest x-ray: pulmonary nodule & some fibrosis (Ghon complex)
o 10% develop clinical Tb later in life - Primary disease:
1. Initial phase o Primary Tb
– mild or asymptomatic and results in exudative lesions with PMN and fluid accumulation around the bacilli
cell-mediated immunity and hypersensitivity rxn 2. tubercle formation
o granulomatous lesion
central area of large, multinucleate giant cells (macrophage syncytia) with tubercle bacilli, an id-zone of pale epithelioid cells, and a peripheral collar of fibroblasts andmononuclear cells
- Stages in the Pathogenesis of tuberculosis
- Reactivation of Tuberculosis Due to:
o Impairment in immune status o Malnutrition o Alcoholism o Advanced age o Severe stress o Immunosuppressive tx o DM, AIDS
- Chronic Pulmonary Tuberculosis
o Insidious onset of fever, fatigue, anorexia, night sweats, and wasting
o Cough and sputum o Hemptysis and chest pain - Extrapulmonary Tuberculosis
o Miliary lesions in the bones, joints, GIT, meninges, lymph nodes and peritoneum o AIDS patients with Tb progress into severe
and unusual manifestations Tuberculin Test
- delayed hypersensitivey to M. tuberculosis protein antigens
- Mantoux test: PPD
o 0.1 mL of 5 TU of PPD-S
o Activity is expressed as tuberculin units (TU)
o
Intradermal injection (48-72 hrs)induration
o Positive reaction 4-6 weeks after initial contact with bacilli
- Tine Test: multiple puncture method o For screening only
- Interpretations of the Mantoux skin test for Tuberculosis
- Mantoux test (tuberculin skin test) Primary Tuberculosis Fibrosis & cacification Lesion arrest Viable, non-proliferating organisms Liver, spleen, kidneys, bone, meninges Miliary Tb Lesion breaks down Caseous matrials Cavity formation Spread of infection Bacilli is dispersed in the lymph and blood stream
M. Tb on Middlebrook 7H11 agar (with casein hydrolysates): cream colored, dry and wrinkled
Colonies of M. Tb on Middlebrook 7H11 agar viewed microscopically. Note the beginning of the cording characteristics
- Conditions affecting the tuberculin reaction:
o Negative – active TB in suppressed cell-mediated immunity
o Cross-reaction may be observed with other spp. of Mycobacteria
Laboratory Identification
- Identification of M. tuberculosis in clinical specimens: o Ziehl-Neelsen stain
o Kinyoun stain
o
Fluorescent acid fast dye (auramine-rhodamine) of sputum, bronchial washings, urine, spinal fluid sediment, biopsy materialM. tuberculosis stained with Kinyoun acid-fast stain
M. tuberculosis stained with fluorochrome stain
- Molecular techniques: 1. Amplified MTb direct test
o makes copies of 16S ribosomal RNA and detected by genetic probe
2. PCR
o amplifies small portion of target DNA o facilitates DNA finger printing of specific
strains
Laboratory Report of Acid Fast Bacilli Number of Bacilli Report
0 No AFB seen
1-2/300 fields Doubtful; request another specimen 1-9/100 fields +
1-9/10 fields ++
1-9/field +++
>9/field ++++
Culture of MTb
- Lowenstein –Jensen (L-J) medium o egg- potato- base media - Middlebrook 7H-10
o agar- base media - 5% to 10% CO2
- 3-6 weeks incubation
-
12 B vial for Bactec MTb 460- radiometric and semi-automated method of TB cultureM. Tuberculosis on L-J agar slant
M. Tuberculosis colonies on Lowenstein-Jenssen agar 8 weeks of incubation
Treatment of MTb
- Multidrug therapy: First line drugs o Isoniazid o Rifampin o Ethambutol o Streptomycin o Pyrazinamide - Multidrug resistance (MDR)
- Drugs used in the treatment of tuberculosis Drug Daily Adult Dosage Major Toxicity First-line agents Isoniazid Rifampin Pyrazinamide Ethambutol Streptomycin 300 mg orally or im 600 mg orally or iv 1.5-2.5 g orally 15-25 ng/kg orally 0.5-1 g im Hepatitis, neurophathy Hepatitis, flulike syndrome Hepatitis, hyperuricemia Optic neuritis Vestibular dysfunction, deafness, renal (rare) Second line agents Cycloserine Ethionamide Capreomycin Kanamycin 250-500 mg twice a day orally 250-500 mg twice a day orally 0.5-1g im, then 1 g 2-3 times a day Seizures, psychiatric symptoms, CNS dysfunction Nausea, vomiting, hepatitis psychiatric symptoms Deafness, vestibular dysfunction, renal damage Prevention of MTb - INH prophylaxis
o No protection to uninfected person after treatment is stopped
- BCG vaccination
o Useless after the patient has been infected with tubercle bacilli
Mycobacterium bovis
Colonies of M. tuberculosis (3 to 4 weeks old) on Middlebrook 7H11 agar. Colonies have a rough appearance and exhibit cording, exemplified by the darker areas
Nonphotochromogens – non-pigmented when grown in the dark (E) and after light exposure (F)
Photochromogens – unpigmented when grown in the dark (A) and develop pigment after light exposure (B)
Scotochromogens – pigmented in the dark © and does not intensify after exposure to light (D)
Different colony morphology seen on culture of one strain of M. avium complex
- causes clinical illness similar to that caused by M. tuberculosis
- milk is the common vehicle
- primary lesion in the cervical or intestinal lymph nodes
Mycobacteria other than Tuberculosis (MOTT) - non-tuberculous Mycobacteria (NTM)
o Runyon Classification of NTM:
Photochromogens – develop pigment following exposure to light Scotochromogens – develop pigment in the dark or light
Non-Photochromogens – non-pigmented regardless of grown in the dark or light
Rapid-growers – colonies of NTM that appear on solid media in less than 7 days
Photochromogens - M. kansasii
o Chronic pulmonary disease; extra-pulmonary diseases (cervical lymphadenitis & cutaneous disease)
o 3% of clinical illness known as TB o cross-reactive to PPD
o
sensitive to standard antituberculous drugs such as rifampino habitat is tap water - M. marium
o Cutaneous disease
o Natural reservoir is fresh water and saltwater as a result of contamination from infected fish and other marine life
M. kansasii colonies exposed to light
Scotochromogens - M. scofulaceum
o
chronic cervical adenitis in children o resistant to antituberculous drugs o surgical excision of infected cervical nodes o habitat are raw milk, soil, water, dairyproducts - M. szulgai
o Cervical adenitis in children o Habitat is water and soil
Scotochromogen M. gordonae with yellow colonies
Non-photochromogens
- M.avium-intracellulare complex (MAC or MAI) o In patients without AIDS:
Pulmonary infections in patients with preexisting pulmonary disease; cervical lymphadenitis; disseminated disease in immunocompromised, HIV-negative patients
o In patients with AIDS:
Disseminated disease;
environmental sources are natural water
- M. ulcerans
o Indolent cutaneous and subcutaneous infections
o Infections occur in tropical or temperate climates
o Has not been isolated from the environment
Rapid Growers - M. abscesus
o Disseminated disease in
immunocompromised patients, skin and soft tissue infections, pulmonary infections, postoperative infections
- M. fortuitum
o postoperative infections infections in breast augmentation andmedian sternotomy; skin and soft tissue infections
- M. chelonae
o
Skin and soft tissue infections, postoperative wound infections, keratitisSmooth, multilobated colonies of M. fortuitum on Lowenstein-Jenssen medium
Indeterminate leprosy: on the extensor suface of extremities. Lesion is single, faintly hypochromic macule with ill-defined borders; slightly erythematous with hypoesthetic areas in some parts. At times sensation is intact
Indeterminate leprosy in children: solitary, ill-defined,
Hypopigmented macules, partially anesthetic. May progress to either tuberculoid or lepromatous forms.
H & E stain: Epithelioid granuloma with few scattered lymphocytes and a clear subepidermal zone
Borderline leprosy: lesions are multiform, from borderline tuberculoid appearance to more infiltrated nodules and plaques, with some loss of sensation at the center.
BL: thick erythematous plaques on the nose , right cheek and chin with some lesions appearing nodular.
Borderline leprosy: Uniformly
and symmetrically distributed, infiltrated maculopapular lesions. No sensory impairment. Mycobacterium Leprae Classification of Leprosy - clinical significance:
o chronic granulomatous condition of peripheral nerves and mucocutaneous tissues, particularly the nasal mucosa
- laboratory identification
o cannot be cultured on artificial media o can be grown in the footpads of mice and
armadillo
o acid-fast bacilli from nasal mucosa and other infeted areas in lepromatous leprosy o histopath and clinical findings in tuberculoid
leprosy Tuberculoid
Leprosy Progression Leptromatous leprosy Of disease
Fite-Faraco stain: single AFB in a nerve of patient with indeterminate leprosy
Borderline leprosy: Lesions are inflamed and succulent with central clearing, producing a “punched out” appearance, or may appear as plaques or bands with peripheral edges fading into normal surrounding skin. Central sensory deficits if present.
Borderline leprosy: In Fite-Faraco stain AFB is present in moderate numbers seen within a nerve
Tuberculoid leprosy: small ,single, circinate lesion with pink, elevated, finely granular,well-defined border. Central hypopigmented macule was insensitive to touch & pain. Associated with enlarged peripheral nerves.
Arm of patient with lepromatous leprosy: multiple typical nodules skin. Central sensory deficits if present.
Lepromatous leprosy: Diffuse infiltration, madarosis, and
loss of eyelashes(diffuse lepromatosis)
H & E stain of skin section with active Lepromatous leprosy: highly vacuolated foam cells, normal appearing nerves, histiocytic granuloma.
Fite-Faraco stain in Lepromatous leprosy: enormous numbers of AFB, in huge clumps, termed globi.
Treatment and prevention - sulfones: Dapsone - Rifampin - Clofozamine
- For erythema nodosum leprosum: o Thalidomide
o TNF-α inhibitor
[email protected] [email protected]
Tuberculoid leprosy: H&E stain of an epithelioid granuloma with thick zone of lymphocytes destroying a nerve which is unrecognizable
Far advanced nodular lepromatous leprosy. Diffuse infiltration with nodules over the eyebrows, cheeks, ear lobes, nose, and chin.
