Chapter 28 Mycobacterium Chapter 27 Nocardia Actinomyces
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Transcript of Chapter 28 Mycobacterium Chapter 27 Nocardia Actinomyces
Chapter 28 Mycobacterium
Chapter 27 Nocardia
Actinomyces
Microbiology 20115/11/2011Yu Chun-Keung
Tuberculosis
Tubercular decay has been found in the spines of Egyptian mummies. Pictured: Egyptian mummy in the British Museum [Source: Wikipedia]
One new case / 36 min
One death / 6 hours
Genus Mycobacterium ( 分枝桿菌屬 )Nonmotile, non-spore-forming aerobic bacilli
Ubiquitous presence, 130 species; 7 species cause most infections
Pathogenic Mycobacteria
M. tuberculosis (結核分枝桿菌 ) : airborne
M. leprae (痲瘋分枝桿菌 ) : close contact
M. avium complex : water/soil
M. kansasii : water/soil
M. fortuitum : water/soil
M. chelonae : water/soil
M. abscessus : water/soil
Cell Wall of Mycobacteria
G(+) bacteria
Inner cytoplasmic membrane
Anchor : proteins, phosphotidylinositol mannosides, lipoarabinomannan (LAM)
Thick peptidoglycan layer
Attach arabinogalactan (a branched polysaccharide) + mycolic acid (70-90 carbon)
No outer membrane
Cell Wall of Mycobacteria
Rich in lipids: responsible for many characteristic properties (acid-fastness, resistant to disinfectants and antibiotics, antigenicity, slow growth, clumping), 60% of dry weight.
Acid-fastness: mycolic acid -resistant to common laboratory stain. Once stained, cannot be decolorized with acid solutions
Polypeptides: transport proteins and porins; 15% dry weight; PPD (purified protein derivatives) - induce cell-mediated immunity.
Preliminary classification of mycobacteria by growth properties and colonial morphology
1. M. tuberculosis complex slow-growing, no pigmentation
2. Non-tuberculous mycobacteria (NTM)
Runyon group I slow-growing, yellow pigment (+) in light
Runyon group II slow-growing, yellow pigment (+) in dark
Runyon group III slow-growing, nonpigmented
Runyon group IV rapidly growing
A pigmented and / or a rapidly growing mycobacterium M. tuberculosis
1. Mycobacterium tuberculosis complex
Tuberculosis
M. tuberculosis (airborne/droplet)
M. bovis (unpasturized milk products)
M. tuberculosis - pathogenesisInhalation of aerosolized infectious particles to alveoli
Intracellular pathogen, infect inactivated alveolar macrophages, lifelong infections
Prevent phagosome-lysosome fusion by blocking early endosomal autoantigen 1 (EEA1)
Acquire nutrients from other intracellular vesicle
Resist killing by nitric oxide and superoxide anions
Induce protective immunity: macrophages release IL-12 and TNFα, and recruit T cells and NK cells; induce TH1 cell; secrete IFN-γ; activate macrophages; increase intracellular killing
Infected cells spread locally (lymph nodes) or hematogenous spread (bone marrow, spleen, kidneys, CNS), and attract macrophages and lymphocytes; formation of granuloma
Caseous necrosis ( 乾酪壞死 )
Granuloma ( 肉牙腫 )
Inner mass : alveolar macrophages, epithelioid cells, Langhans giant cells (fused epithelioid cells)
Dense wall : CD4, CD8, NK, T cells, macrophages
Caseous necrosis ( 乾酪壞死 )
Signs and symptomsMost infections are restricted to the lungs
Insidious at onset
Non-specific : malaise, weight loss, cough, night sweats, sputum (purulent, bloody = lung tissue destruction, cavity)
Primary focus is present in middle or lower lung fields.
Hematogenous spread : disseminated (miliary 粟粒狀 ) tuberculosis
Clinical diseaseIn most patients replication ceases within 3-6 wks after CMI is activated. PPD (+), x-ray (-)
~5% infected patients will progress to active disease within 2 years. PPD (+), x-ray
(+)
Other 5-10%, Latent PPD (+), x-ray (-), will develop disease sometime later in life. PPD (+), x-ray (+)
Progression to disease is related to infectious dose and the patient’s immune competence.
Pathogenesis
No toxins and enzymes
Histological signs (i.e., granuloma) are host immune responses to infection (DTH response)
Immunopathology results in tissue necrosis (cytokine toxicity, complement activation, ischemia, etc.)
Pathogenesis
Small antigenic burden + protective immunity: activated macrophages can penetrate small granulomas (< 3 mm) and kill all bacteria with minimal tissue damage.
But if many bacilli are present, cellular immune response (over-reactive, impaired) results in formation of large, necrotic or caseous granulomas encapsulated with fibrin, which protect bacteria from macrophage killing (latent), thus may be reactivated years later when patients’ immunologic responsiveness wanes.
EpidemiologyHumans - natural reservoir
Non-human primates and guinea pigs - experimental infection
Person-to-person contact Inhalation: small particles containing one to three tubercle bacilli can reach alveolar spaces and establish infection. Patients with tubercle bacilli in sputum infect 10-15
people/year.
Ingestion of unpasteurized milk products (M. bovis)
EpidemiologyOne third of the world’s population is infected.
9 million new cases and 2 million deaths annually.
Endemic area: Southeast Asia, sub-Saharan Africa, Eastern Europe
In USA, immigrants, homeless persons, drug and alcohol abusers, prisoners, HIV patients
World TB incidence. Cases per 100,000; Red = >300, orange = 200–300; yellow = 100–200; green 50–100 and grey <50. Data from WHO, 2006.
In most patients, the only evidence of infection :
• Radiographic evidence of calcification in lungs
• Lifelong positive tuberculin skin test to PPD
• Lab detection of mycobacteria : microscopy or culture.
Clinical Diagnosis of M. tuberculosis
[Source: Wikipedia]
Immunodiagnosis (for M. tuberculosis)
Tuberculin skin test
•5 unit PPD, intradermal; diameter of the area of induration after 48h
•Patient’s response to exposure to M. tuberculosis (a DTH response)
•Differentiate between infected and non-infected people.
[Source: Wikipedia]
Reactivity to PPD
10 mm (positive): 3-4 wks after infection; lifelong
acute disease or protective immunity = has been infected & develop immune response
False negative: anergy (eg. HIV infection), overwhelming TB
False positive: BCG vaccination, isoniazid treatment
In vitro IFN-γ release assays
Patient’s whole blood (contain sensitized T cells)
Stimulate with M. tuberculosis-specific antigens (i.e., ESAT-6, CFP-10)
Monitor IFN-γ production
More sensitive and specific alternative to PPD skin test
MicroscopyThe most rapid method to confirm mycobacterial disease - acid-fast stain
Auramine-rhodamine strain : Truant-fluorochrome method
Carbolfuchsin strain : Ziehl-Neelsen method or Kinyoun method
Specificity > 95% (mycolic acid)
Sensitivity relate to (1) respiratory specimens and (2) specimens with high bacterial count
Positive acid-fast stain = high infectivity
Cannot identify mycobacterial species Sputum specimens with acid-fast stain
“positive” : must differentiate between true infection with M. tuberculosis and transient colonization of other non-pathogenic spp.
CultureSpecimens: Pulmonary infection – abundant organisms in respiratory secretions Extrapulmonary infection – need repeated samplings
Most isolates grow slowly; thus obscured by rapidly growing bacteria
Specimen (sputum) decontaminated with 2% NaOH.
Egg-based medium: Löwenstein-Jensen Agar-based medium: Middle-brook
Broth culture of special formula : reduce culture time of most mycobacteria (from 3-4 wks to 10-14 days)
Microscopic observation drug susceptibility assay (MODS): 24-well plate with broth and
antimycobacterial drug Add specimen Mycobacteria growth is detected by an
inverted microscope Susceptibility test done simultaneously
Leprosy ( 癩病 ) (Hansen’s disease): a chronic infection that affects skin and peripheral nerves (Schwann cells and macrophages)
Since 1985, Global prevalence drops dramatically
Endemic in few countries in Asia, Africa and Latin American
Endemic in armadillos
2. Mycobacterium leprae
Nine-banded Armadillo (犰 )
Leprosy
Long incubation period > 20 years
Several clinical manifestations - depend on patient’s immune reaction to the bacilli
Tuberculoid leprosy ( 類結核型痲瘋病 )
Lepromatous leprosy ( 痲瘋結節型痲瘋病 )
Tuberculoid leprosy
Hypopigmented skin macula
Peripheral nerve damage with complete sensory loss
Lepromatous leprosy
Many erythematous macula, papules or nodules
Extensive disfiguring skin lesions (nasal cartilage, ears)
Tuberculoid leprosy
• Paucibacillary Hansen disease
• Strong cell-mediated immunity
• Granuloma in tissues with few bacilli; low infectivity
Lepromatous leprosy
• Multibacillary Hansen disease
• Strong humoral immunity
• Abundance bacilli in skin and peripheral nerves; infectious
Transmission
Person-to-person contact
Inhalation of infectious aerosols or skin contact with respiratory secretions and wound exudates
Diagnosis of M. lepare
Base on clinical disease
M. leprae cannot grow in cell-free cultures
Histopathology - presence of acid-fast bacteria in lesions
Skin test to lepromin Lepromin: derived from inactivated M.
leprae Confirmative test for tuberculoid leprosy No use for lepromatous leprosy (anergy)
3. M. avium complex (MAC)Common environmental isolate in soil, water, plants.
Developed after ingestion of contaminated food or water, no person-to-person spread
In immunocompetent patients:
M. avium subsp. intracellulareRecover from clinical specimens – mostly transient colonization (= infection, disease)
Three forms: Middle-age or older men with a history of smoking and underlying
pulmonary disease; Elderly, female nonsmokers (pneumonitis, bronchiectasis) Solitary pulmonary nodule
In HIV-infected patients:
• M. avium subsp. hominissuis
• The most common mycobacterial disease in USA, typically disseminated
• All organs are involved, especially in terminal stages with CD4 T counts < 10 cells/mm3
Tissue from a patient with AIDS who is infected with Mycbacterium avium complex
4. Slow-growing mycobacteria (3-8 weeks)
Present in soil and water
Opportunistic pathogens for immuno-compromised patients
Thus, isolation in specimens of immuno-competent patients mostly represents a transient colonization
No person-to-person spread (except M. bovis)
Slow-growing mycobacteria
M. bovis Pulmonary tuberculosis M. kansasii
M. scrofulaceum Lymphatic tissue infection
M. ulcerans Cutaneous infection M. marinum M. haemophilumNew species and spectrum of diseases are expending.
5. Rapidly growing mycobacteria (< 7 days)
M. fortuitum, M. chelonae, M. abscessus.
Relatively low virulence, susceptible to “conventional” antibacterial antibiotics
Infections establish in deep subQ tissues after introduced by trauma or iatrogenic infections.
No person-to-person spread
Incidence increases as invasive procedures increases.
Preliminary classification of mycobacteria by growth properties and colonial morphology
1. M. tuberculosis complex slow-growing, no pigmentation
2. Nontuberculous mycobacteria (NTM)
Runyon group I slow-growing, yellow pigment (+) in light
Runyon group II slow-growing, yellow pigment (+) in dark
Runyon group III slow-growing, nonpigmented
Runyon group IV rapidly growing
A pigmented and / or a rapidly growing mycobacterium M. tuberculosis
Preliminary identificationColonial morphological (pigmentation) and growth properties for preliminary identification is important.
Only M. tuberculosis are transmitted from person to person. Thus only these patients are needed to be isolated and close contacts given prophylactic antibiotics.
Guide empirical antimicrobial therapy
Definitive identificationBiochemical tests : production of nicain and reduction of nitrate (need > 3 weeks)
Chromatographic analysis of cell wall lipids
Species-specific nucleic acid probes for popular species
For species without specific probe: amplification of species-specific 16S ribosomal RNA gene or SecA gene followed by sequence analysis
Treatment (complex and controversial)
Slow-growing mycobacteria:
resistant to most common antibiotics, need multiple antimycobacterial agents for extended period (6-9 m)
Isoniazid (INH) + rifampin + pyrazinamide + ethambutol for 2 months;
then isoniazid + rifampin for 4 to 6 months
Treatment Rapidly growing mycobacteria: resistant to
commonly used antimycobacterial agents; susceptible to antibiotics - amikacin, imipenem, clarithromycin
M. avium complex Clarithromycin + ethambutol + rifabutin
M. leprae tuberculoid form: dapsone + rifampin for 6 m; lepromatous form: dapsone + rifampi + clofazimine for 12 m
Effective of TreatmentRight drug combination
Sufficient treatment time
Compliance
Otherwise drug resistant strains may develop
Multidrug-resistant M. tuberculosis (MDR-TB) : isoniazid and rifampin
Extensively drug-resistant (XDR)-TB : drugs for MDR-TB + second line drugs (kanamycin, amikacin, capreomycin); untreatable
Vaccination for M. tuberculosis
Attenuated M. bovis (BCG strain) (France 1921)
Effective in young people; less effective in adult
Cannot be used for immunocompromised patients (i.e., HIV patients)
Low skin test reactivity develops after vaccination – false postive
IFN-γ release assays are not affected by BCG.
Control
Elimination is highly unlikely
Active surveillance + prophylactic intervention + therapeutic intervention + case monitoring
Directly Observed Treatment, Short Course (DOTS) 短期直接觀察療法
送藥到口服藥入口吃了再走
Chapter 27 Nocardia
Gram (+), aerobic rod; form beaded filaments in tissues and cultures (similar to fungal hyphae).
Contain mycolic acid (50-62 carbon), thus stain weakly acid-fast (use a weak decolorizing solution of hydrochloric acid).
Epidemiology
Ubiquitous presence, widespread in soil and in nature, > 100 species.
Infections are exogenous.
Cause disease in immuno-suppressed patients (AIDS, leukemia, transplant recipients)
Transmission: unknown, no person-to person spread
Pathogenesis
Pathogenic strains
Resist phagocytic killing
Secrete catalase and superoxide dismutase
Replicate in macrophages
Cord factor (trehalose-6,6’-dimycolate): prevent phagosome-lysosome fusion
Clinical disease - nocardiosis
Lung and skin are primary sites of infections.
Necrosis and abscess formation, sinus tract
Highly predilection for hematogenous spread to CNS or skin
Clinical diseases Bronchopulmonary infections:
immunocompromised patient + pneumonia with cavitation + CNS or skin involvement
Brain abscess : develop in 1/3 of patients, single or multiple
Cutaneous infection: Primary (following trauma) or secondary (from a pulmonary site)
Mycetoma, lymphocutaneous infections, skin infections with abscess and cellulitis
Laboratory diagnosis
Specimen: sputum, infected tissues
Microscopic examination: Partially acid-fast, filamentous
Culture: Medium for Legionella (BCYE agar)
Aerial hyphae = hyphae protrude upward from the surface of a colony
T/P/CAntibiotic – amikacin + cephalosporin + sulfonamides for > 6 wks
Surgical debridement
Localized infections – good prognosis
Disseminated disease in immunocompromised patients – poor prognosis
Actinomyces
Greek words for “ray fungus”
Form filamentous hyphae in specimens or culture
G(+), anaerobic bacilli
Contain no mycolic acid, non-acid-fast
Pathogenesis
normal flora of the mouth, alimentary tract and vagina (but not skin)
low virulence potential, invade and cause disease when tissue is injured (trauma, surgery, or infection)
unlike Nocardia, actinomyces are pathogenic to normal hosts, all infection are derived endogenously, no person-to-person spread.
Actinomycosis
Chronic granulomatous lesions
Suppurative, abscess formation with sinus tracts
Discharge (wound exudates) contain sulfur granules (pigmented microcolonies of organisms + calcium phosphate)
Actinomycosis Cervicofacial (lumpy jaw)
Poor oral hygiene, invasive dental procedure, oral trauma
Patient with cervicofacial actinomycosis. Note the draining sinus tract.
Actinomycosis
Thoracic (lung): History of aspiration
Abdominal: GI surgery, bowel trauma
Pelvic: Intrauterine device user
CNS: Solitary brain abscess, hematogenous spread
Laboratory diagnosis
Confirmation is difficult (normal population on mucosal surface)
Sulfur granules for Gram’s stain
Anaerobic culture: grow slowly, >2 wks.
Colonies resemble top of a molar
Treatment
Surgical debridement of involved tissues
Prolonged administration of antibiotics – penicillin, erythromycin, clindamycin
NocardiaActinomyce
s
Mycolic acid 50-60 carbon No
Acid-fast stain
weak Negative
Present Ubiquitous Normal flora
Infection mode
Exogenous Endogenous
HostImmuno-
suppressed Immuno-
competent
ColonyAerial
hyphae
Resemble top of a molar