2 Important Human Pathogens Mycobacterium tuberculosisMycobacterium leprae (uncommon)
3 Mycobacterium tuberculosis complex M. tuberculosis: Human TBM. caprae: Goat, Vet surgeonsM. bovix: cattle and other mammalsM. microti: Voles and other small animals/ not humanM. africanum: Africa in btw Human and bovine types
5 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 oxigenFacultative intracellular parasite, usually of macrophagesSlow generation time, hours,Straight or slightly curved rode (3x0.3 mM)Single in clinical samples-Serpentine cords in culture
6 Mycobacterium tuberculosis. Acid-fast stain NOTE: single growth of virulent strains
7 Acid-Fast (Kinyoun) Stain of Mycobacterium NOTE: cord growth (serpentine arrangement) of virulent strains
8 Lipid-Rich Cell Wall of Mycobacterium Mycolic acidsCMN Group: Unusual cell wall lipids (mycolic acids,etc.)(Purified Protein Derivative)Corynebacterium, Mycobacterium, Nocardia
9 Mycobacterium Description Grow in wide range of enriched mediathe common for clinical isolation.Löwenstein-Jensen (LJ) medium is an egg based mediumMiddlebrook's medium (MTB) is an agar based medium
10 Photochromogenic Mycobacterium kansasii on Middlebrook Agar NOTE: Mycobacteria pathogenic for humans can be differentiated (Runyon Groups) by:speed of growth (all are slower than most other pathogens) and byproduction of chromogenic pigments (in light, in dark, or none)
11 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 tuberculosisPrimary infection by M. tuberculosis of non-immune alveolar macrophages with unrestrained proliferation within the infected macrophages forming initial lesion or Ghon focusPrimary complex (PC): Hilar lymph nodes with GF
12 Pathogenesis of Tuberculosis H. bovis PC formed in the tosil, cervical nodes or ileocaecal region and mesentric lymph nodes.Prosector’s warts: primary focus on the skin
13 Pathogenesis of Tuberculosis Dissemination of infected macrophages through the draining lymphatics into the circulationDevelopment 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.
14 Diagram of a GranulomaNOTE: 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
15 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 mechanismsActivation by IF- of Mycobacterium tuberculosis killing by macrophages
16 Pathogenesis of Tuberculosis Minority of cases Foci progresses toProgressive primary lesions, meningitis, pleurisy and kidney, spine (pott’s disease), bone, joint)Miliary tuberculosis: focus ruptures into blood(table 18.2)
19 Pathogenesis of Tuberculosis 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)
20 Pathogenesis of Tuberculosis Reactivation of M. tuberculosis infection with partial immunity produces high tissue concentrations of mycobacterial antigens that provoke an intense mononuclear cell response (type 4 hyper- sensitivity reaction)
21 Pathogenesis of Tuberculosis Dense mononuclear cell infiltrates damage tissue due to release of active oxygen radicals and lysosomal neutral proteasesTissue damage occurs as caseation necrosis that progresses to liquefaction necrosis in the absence of tuberculosis drug treatment
22 Clinical Features of Tuberculosis Cough one of the earliest signs with production of sputum as tissue necrosis progressesDyspnea a later symptom indicating extensive involvement of pulmonary parenchymaFever and weight loss reflecting systemic actions of IL-1 and TNF- (cachectin) secreted by activated macrophages
32 Mycobacterium lepraeMycobacterium leprae is the causative agent of the disease, leprosy (Hanson's Disease).gram-positive, aerobic rod , not so strongly Acid fastThe bacilli are typically found within Mf in clumps.Surface lipid, peptidoglycolipid1 (PGL-1) is a unique carbohydrate antigenic determinant.PCR specific primers
33 Mycobacterium lepraeM. 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 bacilli/gm diseased tissue (Skin test reagent –Leprosin A)Mice footpads 106 (antileprosy drugs)
39 Clinical Pathology of Leprosy Early, Indeterminate LeprosySlight pandermal perineurovascular and peri-appendageal chronic inflammation.Without demonstrating bacilli:Diagnosis can only be presumptiveLepromatous 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.
40 Clinical Pathology of Leprosy Tuberculoid (TT) leprosyThe lesions are scanty, dry, erythematous, hypo-pigmented papules or plaques with sharply definededges.Anesthesia is prominent (except on the face).The number of lesions ranges from 1 to 5, and thelesions heal rapidly on chemotherapy.Primary TT leprosy has large epitheloid cells arrangedin compact granulomas along with neurovascularbundles with dense peripheral lymphocyte accumulationLanghans’ giant cells are typically absent.Dermal nerve may be absent or surrounded and erodedby dense lymphocyte cuffs. Bacilli are rarely found.
41 Clinical Pathology of Leprosy Borderline tuberculoid (BT) leprosyThe lesions are asymmetrical and may be scanty.Dry, hairless plaques with central hypopigmentation.Nerve enlargement is usually found and the lesionsare usually anesthetic.Granulomas with peripheral lymphocytes follow theneurovascular bundles and infiltrate sweat glands anderector pili muscles.Langhans’ giant cell are variable in number and arenot large in size.Typical nerve erosion and destruction.Granulomas along superficial vascular plexus frequently
42 Clinical Pathology of Leprosy Midborderline (BB) leprosyThe lesions are irregularly dispersed and shapederythematous plaques with punched-out centers.Dermal edema is prominent in the lesions.Macrophages are activated to epitheloid cellsBorderline lepromatous (BL) leprosyLess numerous and less symmetrical.Often display some central dimples.The lymphocytes are more prominent in BL andthere is a tendency for some activation of macrophagesto form poorly to moderately defined granulomas.Perineural fibroblast proliferation forming an typical“onion skin” in cross section.
43 Clinical Pathology of Leprosy Infiltrative-nodular type LLThe 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 theepidermis by a narrow grenz zone of normal collagen.The macrophages have abundant eosinophilic cytoplasmand contain mixed solid and fragmented bacilli .Lymphocyte infiltration is not prominent, but there maybe many plasma cells
44 Clinical Pathology of Leprosy Hitoid type LLThe occurrence of well-demarcated cutaneous andsubcutaneous nodules resembling dermatofibromas.It frequently follows incomplete chemotherapy oracquired drug resistance, leading to bacterial relapse.The highest loads of bacilli.The majority are solid-staining, arranged in clumpslike sheaves of wheat.Macrophage reaction is unusual.
45 Clinical Pathology of Leprosy Lepromatous leprosy -- with antimycobacterial therapyDegenerate bacilli accumulate in the macrophages(the so-called lepra cells or Virchow cells), whichhave foamy or vacuolated cytoplasm, resemblingxanthoma cellsFite stain reveals that the bacilli are fragmented orgranular and disposed in large basophilic clumpscalled globi especially in very chronic lesions.The nerves in the skin may contain considerablenumbers of leprosy bacilli, but remain well-preserved for a long time and slowly become fibrotic.
46 Clinical Pathology of Leprosy Lepromatous leprosy -- with antimycobacterial therapyWhen lepromatous leprosy is treated, the bacterialdebris to be cleared by host macrophages.The M lepra antigen may persist longer and can bedemonstrated by immunocytochemical stains even when no bacilli are evident.
47 (Jopling’s type 1 reaction) CMI Leprosy ReactionThe 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) CMIType 1 reactions are common in TT, BB and BL patients, but are not rare in LL or BL patients.Reaction induces increased intraneural inflammationand edema, which is damaging.At worst, there is a caseous necrosis of large peripheralnerves resulting from upgrading reactions.
48 Leprosy ReactionDelay-Type Hypersensitivity ReactionEdema within and about the granulomas and proliferation of fibrocytes in the dermis.In upgrading reactions, the granuloma becomes moreepitheloid and Langhans’ giant cells are larger.There may be erosion of granulomas into the lowerepidermis and fibrinoid necrosis with granulomas andeven within dermal nerves.In downgrading reactions, necrosis is much lesscommon and the density of bacilli increases.
49 Leprosy Reaction Ag-Ab complex Type 2 reaction (Erythema nodosum leprosum, ENL)Ag-Ab complexENL occurs most commonly in LL, less in BL.Tender, red plaques and nodules together with areas oferythema and occasionally also purpura and vesicles.It is accompanied by fever, malaise, arthralgia andleukocytosis.This is the only type of reactional leprosy that respondsto treatment with thalidomide.Polymorph neutrophils may be scanty or so abundant asto form a dermal abscess with ulcerationFoamy macrophage containing fragmented bacilli ormycobacterial debris.A necrotising vasculitis in some cases of ENL.
50 Leprosy Reaction Occur exclusively in diffuse lepromatous leprosy. Lucio ReactionOccur exclusively in diffuse lepromatous leprosy.It usually occurs in patients who have received eitherno treatment or inadequate treatment.The lesions consist of barely palpable, hemorrhagic,sharply marginated, irregular plaques.There may be repeated attacks or continuousappearance of new lesions for years.Endothelial proliferation leading to luminal obliterationwith thrombosis in the medium-sized vessels of thedermis and subcutis.Dense aggregates of bacilli are found in the walls andthe endothelium of vessels.
56 ENL necroticans improved 2 weeks after Thalidomide administration
57 Treatment WHO: the combination of dapsone (bacteriostatic) 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 monthlyand clofazimine (bacteriostatic) 50mg QD and 300mgmonthly for a routine duration of 2 years.20% relapse rate within 8 years after completion ofthis regimen.
58 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 onebacillusOther Methods of Diagnosis Antibodies directly against phenolic glycolipid I orlipoarabinomannan. Polymerase chain reaction (PCR).
64 Description of aerobic Actinomycets Gram-positive branching filaments that sporulate or fragment: the aerobicActinomycetes (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
65 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
66 Taxonomy of the Aerobic Actinomycetes: Pathogenic Genera NocardiaActinomaduraStreptomycesRhodococcusGordoniaTsukamurellaTropheryma whipplei (Non-cultivable)
67 Aerobic Actinomycetes: Natural Habitats Nocardia species and other aerobicActinomycetes ubiquitous in soil and primarily responsible for decomposition of organic plant matterRhodococcus species present in the intestinal bacterial flora of grazing herbivores especially horsesStreptomyces species (>3,000) widely distributed in soil
68 Anaerobic Actinomyces: Natural Habitats Anaerobic Actinomyces speciesare normal inhabitants of themucous membranes of humansand animals
69 Aerobic Actinomycetes: Modes of Infection Nocardia infection acquired by inhalation of or direct skin inoculation (traumatic) by environmental organismsRhodococcus 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
70 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 barefootedGordonia and Tsukamurella species are closely related to Rhodococcus, and are soil organisms considered opportunistic pathogens
71 Anaerobic Actinomyces: Modes of Infection Actinomyces invades normallysterile tissue from endogenousmucous membrane sites ofnormal colonization
72 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
73 Aerobic Actinomycetes: Types of Infectious Disease Nocardia asteroides complex: N. asteroides sensu stricto type VI, N. abscessus, N. farcinica, and N. nova, major cause of pulmonary infectionN. otitidiscavarium infrequent cause of systemic infectionN. brasiliensis inoculated into subcutaneous tissue of foot produces actinomycotic mycetomas
74 Aerobic Actinomycetes: Types of Infectious Disease 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
75 Aerobic Actinomycetes: Types of Infectious Disease 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
76 Aerobic Actinomycetes: Types of Infectious Disease 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.
77 Aerobic Actinomycetes: Types of Infectious Disease Whipple’s disease: diarrhea, weight loss, lymphadenopathy, fever, and arthralgiaTypical histopathology is presence of PAS-positive foamy macrophages infiltrating the lamina propria of the small intestineCaused by intracellular infection of macrophages by Tropheryma whipplei (non-cultivable, diagnosis by typical histopathology combined with PCR)
78 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:
79 Model illustrating the pathophysiology of Whipple's disease Model illustrating the pathophysiology of Whipple's disease. DC, dendritic cells
80 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.
81 Aerobic Actinomycetes: Identification Nocardia and Rhodococcus (potentially pathogenic) and Streptomyces (less frequently pathogenic) obligate aerobesNocardia asteroides complex organisms thin ( m) filaments up to 20 m in length demonstrating beaded gram-positivityRhodococcus equi gram-positive coccobacilli
82 Aerobic Actinomycetes: Identification 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
84 Aerobic Actinomycetes: Identification 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 lysozymeUrease activityDecomposition of the substrates casein, tyrosine, xanthine, and hypoxanthine
86 Anaerobic Actinomyces: Identification Actinomyces israelii anaerobic with clinical strains varying from obligate anaerobes to microaerophilicA. israelii definitively identified by detection using gas liquid chromato- graphy (GLC) of acetic and lactic acid as end products of carbohydrate metabolism