Mycobacterium. Mycobacterium tuberculosis Mycobacterium leprae (uncommon) Important Human Pathogens

Mycobacterium

Mycobacterium tuberculosis

Mycobacterium leprae (uncommon)

Important Human Pathogens

Mycobacterium tuberculosis complex

M. tuberculosis: Human TB

M. caprae: Goat, Vet surgeons

M. bovix: cattle and other mammals

M. microti: Voles and other small animals/ not human

M. africanum: Africa in btw Human and bovine types

Mycobacterium Description

M. Tuberculosis complex (tubercle bacilli)

Non-motile, non-sporing, non-capsulate.

Obligate aerobe: For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. M bovis like reduce oxigen

Facultative intracellular parasite, usually of macrophages

Slow generation time, 15-20 hours,

Straight or slightly curved rode (3x0.3 mM)

Single in clinical samples-Serpentine cords in culture

Mycobacterium tuberculosis. Acid-fast stain

NOTE: single growth of virulent strains

Acid-Fast (Kinyoun) Stain of Mycobacterium

NOTE: cord growth (serpentine arrangement) of virulent strains

Lipid-Rich Cell Wall of MycobacteriumMycolic acids

CMN Group: Unusual cell wall lipids (mycolic acids,etc.)

(Purified Protein Derivative)

Corynebacterium, Mycobacterium, Nocardia

Mycobacterium DescriptionGrow in wide range of enriched media

the common for clinical isolation.

Löwenstein-Jensen (LJ) medium is an egg based medium

Middlebrook's medium (MTB) is an agar based medium

NOTE: Mycobacteria pathogenic for humans can be differentiated (Runyon Groups) by: speed of growth (all are

slower than most other pathogens) and by

production of chromogenic pigments (in light, in dark, or none)

Photochromogenic Mycobacterium kansasii on Middlebrook Agar

Pathogenesis of Tuberculosis

• Inhalation of small (1-5 m) droplet nuclei containing M. tuberculosis expelled by coughing, sneezing, or talking of another individual with cavitary tuberculosis

• Primary infection by M. tuberculosis of non-immune alveolar macrophages with unrestrained proliferation within the infected macrophages forming initial lesion or Ghon focus

• Primary complex (PC): Hilar lymph nodes with GF


• H. bovis PC formed in the tosil, cervical nodes or ileocaecal region and mesentric lymph nodes.

• Prosector’s warts: primary focus on the skin


• Dissemination of infected macrophages through the draining lymphatics into the circulation

• Development within 3-8 weeks of a CD4+ T cell dependent cell-mediated immune response with granuloma formation and macrophage activation at sites of infection by IF- and calciriol.

Diagram of a Granuloma

NOTE: ultimately a fibrin layer develops around granuloma (fibrosis), further “walling off” the lesion.

Typical progression in pulmonary TB involves caseation, calcification and cavity formation.

Activated Macrophages

Immunity in Tuberculosis

• Antigen-specific activation of CD4+ T lymphocytes with secretion of IL-2, increased expression of IL-2 receptors, and secretion of IF-

• Antigen-driven clonal expansion of CD4+ T lymphocytes by IL-2 acting via autocrine and paracrine mechanisms

• Activation by IF- of Mycobacterium tuberculosis killing by macrophages


• Minority of cases Foci progresses to– Progressive primary lesions, meningitis, pleurisy

and kidney, spine (pott’s disease), bone, joint)–Miliary tuberculosis: focus ruptures into blood

• (table 18.2)


Post-primary

tuberculomas


• Active infection usually transformed into latent infection (exceptions: infants, AIDS)

• With decrement in T-cell dependent cell mediated immunity (years later) infection reactivated with development of tuberculosis (HIV infection, diabetes mellitus, renal disease, cancer, advanced age)


• Reactivation of M. tuberculosis infection with partial immunity produces high tissue concentrations of mycobacterial antigens that provoke an intense mononuclear cell response (type 4 hypersensitivity reaction)


• Dense mononuclear cell infiltrates damage tissue due to release of active oxygen radicals and lysosomal neutral proteases

• Tissue damage occurs as caseation necrosis that progresses to liquefaction necrosis in the absence of tuberculosis drug treatment

Clinical Features of Tuberculosis

• Cough one of the earliest signs with production of sputum as tissue necrosis progresses

• Dyspnea a later symptom indicating extensive involvement of pulmonary parenchyma

• Fever and weight loss reflecting systemic actions of IL-1 and TNF- (cachectin) secreted by activated macrophages

Clinical Features of Tuberculosis

• Apical cavitary lesions in upper lobes of lung by X-ray film of the chest

• Positive tuberculin skin test with PPD (purified protein derivative)

PPD Tuberculosis Skin Test Criteria

PPD = Purified Protein Derivative from M. tuberculosis

Chest X-Ray of Patient with Active Pulmonary Tuberculosis

Lab Diagnosis

• Specimen• Microscopy• Culture• Nucleic acid• Drug susceptibility test

TreatmentBCG (bacille Calmette-Guerin) = attenuated M. bovis

Mycobacterium leprae


Mycobacterium leprae is the causative agent of the disease, leprosy (Hanson's Disease).

gram-positive, aerobic rod , not so strongly Acid fast

The bacilli are typically found within M f in clumps.

Surface lipid, peptidoglycolipid1 (PGL-1) is a unique carbohydrate antigenic determinant.

PCR specific primers


M. leprae has never been grown in artificial culture, but will grow in the footpads of mice and in armadillos. The culture can take several weeks to mature.

Armadillos- 1010 bacilli/gm diseased tissue (Skin test reagent –Leprosin A)

Mice footpads 106 (antileprosy drugs)

http://www.who.int/lep/situation/PrevStart2007a.pdf

Pathogenesis

Principal target cells is Schwann cells

Nerve damage

anaesthesia and muscle paralysis

Repeat injuries and infection

Gradual destruction

Infiltration of skin & cutaneous nerves

Visible lesion wth pigmentary changes

Pathogenesis

First signes

Non specific skin lesion

Spontanuas healing

Disease progress

Clinical manifestation by the immune response of the patiant (Table 18:7)

Classification The dichotomy: multibacillary or paucibacillary The Ridley system: TT (polar tuberculoid) BT (borderline tuberculoid) BB (borderline/midborderline) BL (borderline lepromatous) LLs (subpolar lepromatous) LLp (polar lepromatous) The paucibacillary: TT and most BT. The multibacillary: BB, BL, LLs and LLp. Lepromin skin test: classification of diagnosed patients Positive: all TT and most BT.

Negative: others.

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Clinical Pathology of Leprosy Early, Indeterminate Leprosy Slight pandermal perineurovascular and peri-appendageal

chronic inflammation. Without demonstrating bacilli:

Diagnosis can only be presumptive

Lepromatous leprosy (LL) The lesions usually are numerous and symmetrically arranged. Three clinical types: macular, infiltrative-nodular and diffuse. A distinctive variant of LL: histoid type.

Clinical Pathology of Leprosy Tuberculoid (TT) leprosy The lesions are scanty, dry, erythematous, hypo- pigmented papules or plaques with sharply defined edges. Anesthesia is prominent (except on the face). The number of lesions ranges from 1 to 5, and the lesions heal rapidly on chemotherapy. Primary TT leprosy has large epitheloid cells arranged in compact granulomas along with neurovascular bundles with dense peripheral lymphocyte

accumulation Langhans’ giant cells are typically absent. Dermal nerve may be absent or surrounded and eroded by dense lymphocyte cuffs. Bacilli are rarely found.

Clinical Pathology of Leprosy Borderline tuberculoid (BT) leprosy The lesions are asymmetrical and may be scanty. Dry, hairless plaques with central hypopigmentation. Nerve enlargement is usually found and the lesions are usually anesthetic. Granulomas with peripheral lymphocytes follow the neurovascular bundles and infiltrate sweat glands and erector pili muscles. Langhans’ giant cell are variable in number and are not large in size. Typical nerve erosion and destruction. Granulomas along superficial vascular plexus frequently

Clinical Pathology of Leprosy Midborderline (BB) leprosy The lesions are irregularly dispersed and shaped erythematous plaques with punched-out centers. Dermal edema is prominent in the lesions. Macrophages are activated to epitheloid cells

Borderline lepromatous (BL) leprosy Less numerous and less symmetrical. Often display some central dimples. The lymphocytes are more prominent in BL and there is a tendency for some activation of macrophages to form poorly to moderately defined granulomas. Perineural fibroblast proliferation forming an typical “onion skin” in cross section.

Clinical Pathology of LeprosyInfiltrative-nodular type LL The classical and most common variety. The patients are not notably hypoesthetic disturbances of

sensation and nerve paralyses develop after large peripheral nerve involved.

Most common involved nerve: ulnar, radial and common peroneal nerves. Extensive cellular infiltrate with separated from the epidermis by a narrow grenz zone of normal collagen. The macrophages have abundant eosinophilic cytoplasm and contain mixed solid and fragmented bacilli . Lymphocyte infiltration is not prominent, but there may be many plasma cells

Clinical Pathology of Leprosy Hitoid type LL The occurrence of well-demarcated cutaneous and subcutaneous nodules resembling dermatofibromas. It frequently follows incomplete chemotherapy or acquired drug resistance, leading to bacterial relapse. The highest loads of bacilli. The majority are solid-staining, arranged in clumps like sheaves of wheat. Macrophage reaction is unusual.

Clinical Pathology of Leprosy Lepromatous leprosy -- with antimycobacterial therapy Degenerate bacilli accumulate in the macrophages (the so-called lepra cells or Virchow cells), which have foamy or vacuolated cytoplasm, resembling xanthoma cells Fite stain reveals that the bacilli are fragmented or granular and disposed in large basophilic clumps called globi especially in very chronic lesions. The nerves in the skin may contain considerable numbers of leprosy bacilli, but remain well- preserved for a long time and slowly become fibrotic.

Clinical Pathology of Leprosy Lepromatous leprosy -- with antimycobacterial therapy

When lepromatous leprosy is treated, the bacterial

debris to be cleared by host macrophages.

The M lepra antigen may persist longer and can be

demonstrated by immunocytochemical stains even when no bacilli are evident.

Leprosy Reaction The reactional status of leprosy are distinctive, tissue

destructive, inflammatory processes, putatively immunologically driven greatly increasing the morbidity of the disease.

Delay-Type Hypersensitivity Reaction (Jopling’s type 1 reaction) CMI Type 1 reactions are common in TT, BB and BL

patients, but are not rare in LL or BL patients. Reaction induces increased intraneural inflammation and edema, which is damaging. At worst, there is a caseous necrosis of large peripheral nerves resulting from upgrading reactions.

Leprosy Reaction Delay-Type Hypersensitivity Reaction

Edema within and about the granulomas and proliferation of fibrocytes in the dermis.

In upgrading reactions, the granuloma becomes more

epitheloid and Langhans’ giant cells are larger.

There may be erosion of granulomas into the lower

epidermis and fibrinoid necrosis with granulomas and

even within dermal nerves.

In downgrading reactions, necrosis is much less

common and the density of bacilli increases.

Leprosy Reaction Type 2 reaction (Erythema nodosum leprosum, ENL)

Ag-Ab complex ENL occurs most commonly in LL, less in BL. Tender, red plaques and nodules together with areas of erythema and occasionally also purpura and vesicles. It is accompanied by fever, malaise, arthralgia and leukocytosis. This is the only type of reactional leprosy that responds to treatment with thalidomide. Polymorph neutrophils may be scanty or so abundant as to form a dermal abscess with ulceration Foamy macrophage containing fragmented bacilli or mycobacterial debris. A necrotising vasculitis in some cases of ENL.

Leprosy Reaction Lucio Reaction Occur exclusively in diffuse lepromatous leprosy. It usually occurs in patients who have received either no treatment or inadequate treatment. The lesions consist of barely palpable, hemorrhagic, sharply marginated, irregular plaques. There may be repeated attacks or continuous appearance of new lesions for years. Endothelial proliferation leading to luminal obliteration with thrombosis in the medium-sized vessels of the dermis and subcutis. Dense aggregates of bacilli are found in the walls and the endothelium of vessels.

Type 1 leprosy reaction

TTS in Type 1 reaction (RR)

Type 2 reaction (ENL)

Type 2 reaction (ENL)

Type 2 Reaction in LL (ENL Necroticans)

ENL necroticans improved 2 weeks after Thalidomide administration

Treatment Paucibacillary disease (TT or BT) WHO: the combination of dapsone (bacteriostatic) 100mg QD and rifampin (bactericidal) 600mg

monthly for a duration of 6 months. Multibacillary disease (BB, BL, and LL) WHO: dapsone 100mg QD, rifampin 600 mg monthly and clofazimine (bacteriostatic) 50mg QD and 300mg monthly for a routine duration of 2 years. 20% relapse rate within 8 years after completion of this regimen.

Identification and Quantitation of Bacilli Acid-fast stain: weak. Modifications of the Ziehl-Neelsen method (collectively called Fite-Farraco stains) Bacilli are usually found in macrophage and nerves. Bacillary index (BI): the numbers of bacilli per oil- immersion field (OIF) or OIFs sought to find one bacillusOther Methods of Diagnosis Antibodies directly against phenolic glycolipid I or lipoarabinomannan. Polymerase chain reaction (PCR).

Lepromatous vs. Tuberculoid Leprosy

Lepromatous Leprosy (Early/Late Stages)

Lepromatous Leprosy Pre- and Post-Treatment

Chapter 19

Environmental mycobacteria

Home study

actinomyces nocardia and tropheryma

Description of aerobic Actinomycets

• Gram-positive branching filaments that sporulate or fragment: the aerobic

Actinomycetes (order Actinomycetales)• Aerobic Actinomycetes whose cell walls

contain mycolic acid: Nocardia species and Rhodococcus species (family Nocardiaceae)

• Aerobic Actinomycetes whose cell walls lack mycolic acid: Streptomyces species

Description of the Anaerobic Actinomyces

• Anaerobic non-sporulating gram-positive rods consist of two groups based on guanosine (G) plus cytosine (C) DNA content: Low mole percent (30-53%) and high mole percent (49-68%)

• Actinomyces species member of the high G+C group

Taxonomy of the Aerobic Actinomycetes: Pathogenic Genera

• Nocardia• Actinomadura• Streptomyces• Rhodococcus• Gordonia• Tsukamurella• Tropheryma whipplei (Non-cultivable)

Aerobic Actinomycetes: Natural Habitats

• Nocardia species and other aerobic

Actinomycetes ubiquitous in soil and primarily responsible for decomposition of organic plant matter

• Rhodococcus species present in the intestinal bacterial flora of grazing herbivores especially horses

• Streptomyces species (>3,000) widely distributed in soil

Anaerobic Actinomyces: Natural Habitats

Anaerobic Actinomyces species

are normal inhabitants of the

mucous membranes of humans

and animals

Aerobic Actinomycetes: Modes of Infection

• Nocardia infection acquired by inhalation of or direct skin inoculation (traumatic) by environmental organisms

• Rhodococcus infection due primarily to inhalation of organisms by animal handlers (horses, pigs, cattle)

• Streptomyces are soil organisms that can infect traumatic wounds especially of the feet

Aerobic Actinomycetes: Modes of Infection

• Actinomadura species (A. madurae, A. latina, A. pelletieri) produce subcutaneous infections in tropical and subtropical countries with those who walk barefooted

• Gordonia and Tsukamurella species are closely related to Rhodococcus, and are soil organisms considered opportunistic pathogens

Anaerobic Actinomyces: Modes of Infection

Actinomyces invades normally

sterile tissue from endogenous

mucous membrane sites of

normal colonization

Aerobic Actinomycetes: Types of Infectious Disease

• Nocardia a facultative intracellular parasite that infects human macrophages and inhibits the fusion of phagosomes containing organisms with lysosomes.

• Nocardia infections generally occur in immunocompromised patients or those with underlying pulmonary disease


• Nocardia asteroides complex: N. asteroides sensu stricto type VI, N. abscessus, N. farcinica, and N. nova, major cause of pulmonary infection

• N. otitidiscavarium infrequent cause of systemic infection

• N. brasiliensis inoculated into subcutaneous tissue of foot produces actinomycotic mycetomas


• Nocardial pneumonia occurs primarily in immunocompromised hosts and produces necrotizing pyogranuloma formation.

• Extrapulmonary dissemination (~50%) and metastatic brain abscess (~30%) complications of nocardial pneumonia.

• Actinomycotic mycetoma (pyogenic subcutaneous infection) causes local tissue destruction including bone


• Rhodococcus equi infects macrophages inhibiting phagolysosome fusion, and produces pulmonary disease with cavitation. Infection occurs in immunocompromised (especially HIV-infected) individuals who handle horses.

• R. equi disseminates to other organs including the brain and subcutaneous tissue


• Streptomyces (S. anulatus formerly S. griseus, and S. somaliensis) associated with actinomycotic mycetoma in warm climates.

• S. somaliensis a frequent cause of actinomycotic mycetomas of the head and neck.


• Whipple’s disease: diarrhea, weight loss, lymphadenopathy, fever, and arthralgia

• Typical histopathology is presence of PAS-positive foamy macrophages infiltrating the lamina propria of the small intestine

• Caused by intracellular infection of macrophages by Tropheryma whipplei (non-cultivable, diagnosis by typical histopathology combined with PCR)

Periodic acid-Schiff (PAS) Staining method used to detect high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), in tissues. The reaction of periodic acid selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent and creates a purple-magenta color. A suitable basic stain is often used as a counter stain.

Várvölgyi C et al. Ann Rheum Dis 2002;61:377-378

Model illustrating the pathophysiology of Whipple's disease. DC, dendritic cells

Anaerobic Actinomyces: Types of Infectious Disease

• Actinomyces israelii causes actinomycosis in which chronic granulomas become suppurative. Cervicofacial actinomycosis most common (~60%), followed by abdominal (20%) and pulmonary (15%).

• Tissue pus contains sulfur granules, a tangled mass of branching bacteria. Presence of sulfur granules establishes a diagnosis of actinomycosis.

Aerobic Actinomycetes: Identification

• Nocardia and Rhodococcus (potentially pathogenic) and Streptomyces (less frequently pathogenic) obligate aerobes

• Nocardia asteroides complex organisms thin (0.5-1.0 m) filaments up to 20 m in length demonstrating beaded gram-positivity

• Rhodococcus equi gram-positive coccobacilli


• Nocardia grows in a variety of media including blood and chocolate agars, Sabouraud’s dextrose agar without chloramphenicol, Lowenstein-Jensen slant, Middlebrook agar, and thioglycolate or trypticase soy broth.

• Growth is slow requiring 5-7 days up to 3 weeks for colony formation at 25o to 37oC.

• Growth in culture of Actinomadura and Streptomyces similar to Nocardia


• Nocardia and Rhodococcus are partially acid-fast positive by modified Kinyoun stain (1% H2SO4 used as decolorizing agent)

• Resistance or sensitivity of growth in glycerol broth to lysozyme

• Urease activity• Decomposition of the substrates casein,

tyrosine, xanthine, and hypoxanthine

Anaerobic Actinomyces: Identification

• Actinomyces israelii anaerobic with clinical strains varying from obligate anaerobes to microaerophilic

• A. israelii definitively identified by detection using gas liquid chromato- graphy (GLC) of acetic and lactic acid as end products of carbohydrate metabolism

Documents

Mycobacterium. Mycobacterium tuberculosis Mycobacterium leprae (uncommon) Important Human Pathogens