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 – MycobacteriaNotes from LectureUSTEMED ’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 acido ~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 cultureo Reproduces the disease in animalso 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 peptidoglycano arabinogalactan

MTb Physiology- Cultural characteristics:

o Strictly aerobico 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 pigmentso Biochemical tests (niacin, catalase test,

urease test, etc)

Antigenic Structure of Mycobacteria1. 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 fractionationo PPD-S: adopted by WHO for skin testing

3. Purified Antigens:o Recombinant DNA techniques & antigen

expression in E.colio Affinity purification of antigen preparations

using monoclonal antibody immunosorbent columns

4. Polysaccharides:o Protein-free polysaccharides

(arabinogalactans & arabinomannans)i. Immunogenicii. Give precipitin reactions with

antiseraiii. 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 componentsa. Wax D & muramyldipeptide (MDP)

peptidoglycans (cell wall) adjuvant activity induce a cell-mediated immune

response against the proteinb. 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 population

o TB meningitis: 466 cases and rate of 0.6/100,000 population

o Other forms of TB: 11,494 cases and rate of 14.7/100,000 population

- Transmissiono Inhalation of dried residues of droplets

containing tubercle bacillio Droplet nuclei (1 to 10 um) can reach

the alveoli and initiated infection- Tuberculous infection vs. Tuberculous disease:

Tuberculin

test

Clinicalsympto

msTuberculous 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 diseaseso reticuloendothelial diseaseo Diabetes mellituso Silicosiso HIV infection

Pathogenesis of TB

Clinical Manifestations- Primary Tuberculosis

o No previous contact with the organismso 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 formationo 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 and mononuclear cells

- Stages in the Pathogenesis of tuberculosis

- Reactivation of TuberculosisDue to:o Impairment in immune statuso Malnutritiono Alcoholismo Advanced ageo Severe stress o Immunosuppressive txo DM, AIDS

- Chronic Pulmonary Tuberculosiso Insidious onset of fever, fatigue,

anorexia, night sweats, and wastingo Cough and sputumo Hemptysis and chest pain

- Extrapulmonary Tuberculosiso 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-So Activity is expressed as tuberculin units

(TU)o Intradermal injection (48-72 hrs)

indurationo 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 matrialsCavity formationSpread 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 immunityo Cross-reaction may be observed with

other spp. of Mycobacteria

Laboratory Identification- Identification of M. tuberculosis in clinical

specimens:o Ziehl-Neelsen staino Kinyoun staino Fluorescent acid fast dye (auramine-

rhodamine) of sputum, bronchial washings, urine, spinal fluid sediment, biopsy material

M. 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 DNAo facilitates DNA finger printing of specific

strains

Laboratory Report of Acid Fast BacilliNumber of Bacilli

Report

0 No AFB seen1-2/300 fields Doubtful; request another

specimen1-9/100 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 culture

M. 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 Isoniazido Rifampino Ethambutolo Streptomycino Pyrazinamide

- Multidrug resistance (MDR)- Drugs used in the treatment of tuberculosis

Drug Daily Adult Dosage

Major Toxicity

First-line agents

IsoniazidRifampin

Pyrazinamide

EthambutolStreptomycin

300 mg orally or im600 mg orally or iv

1.5-2.5 g orally

15-25 ng/kg orally0.5-1 g im

Hepatitis, neurophathyHepatitis, flulike syndrome

Hepatitis, hyperuricemia

Optic neuritisVestibular 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

Colonies of M. tuberculosis (3 to 4 weeks old) on Middlebrook 7H11 agar. Colonies have a rough appearance and exhibit cording, exemplified by the

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

- BCG vaccinationo Useless after the patient has been

infected with tubercle bacilli

Mycobacterium bovis

- 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 TBo cross-reactive to PPDo sensitive to standard antituberculous

drugs such as rifampino habitat is tap water

- M. mariumo 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 childreno resistant to antituberculous drugso surgical excision of infected cervical

nodeso habitat are raw milk, soil, water, dairy

products- M. szulgai

o Cervical adenitis in childreno 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. ulceranso Indolent cutaneous and subcutaneous

infectionso Infections occur in tropical or temperate

climateso 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

Smooth, 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

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.

- 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, keratitis

Mycobacterium Leprae

Classification of Leprosy

- clinical significance:o chronic granulomatous condition of

peripheral nerves and mucocutaneous tissues, particularly the nasal mucosa

- laboratory identificationo cannot be cultured on artificial mediao can be grown in the footpads of mice

and armadilloo acid-fast bacilli from nasal mucosa and

other infeted areas in lepromatous leprosy

o histopath and clinical findings in tuberculoid leprosy

TuberculoidLeprosy

Leptromatous leprosy

Progression

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.

Arm of patient with lepromatous leprosy: multiple typical nodulesskin. 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 Thalidomideo TNF-α inhibitor

-fin-

audsmartinez@gmail.comustmedc3@yahoogroups.com

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.

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.