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MYCOBACTERIA OF
MEDICAL IMPORTANCE
Prof M.I.N. Matee, PhDDepartment of Microbiology and
Immunology,MUCHS
MYCOBACTERIA OF
MEDICAL IMPORTANCE
Prof M.I.N. Matee, PhDDepartment of Microbiology and
Immunology,MUCHS
Mycobacteria: overview Introduction and Definitions Epidemiology of Mycobacterioses TB: Pathogenesis and clinical
presentation TB: Diagnosis TB: Treatment TB: Prevention Conclusions
MYCOBACTERIUMMYCOBACTERIUM
THIS GENUS IS COMPOSED OF: Strictly aerobic, acid-fast rods, does notstain well (gram stain indeterminant),DNA has high g+c content, unique cell wall,Mycolic acid carbon chain length > c60Relatively slow growth (two groups)
A. RAPID GROWERS (Visible colonies in <5 days)B. SLOW GROWERS (Visible colonies in > 5 days)TYPE SPECIES: Mycobacterium tuberculosis
TB: Epidemiology transmitted in respiratory aerosols Occasionally from animals (e.g. milk) With pasteurization, most TB now
due to human - human transmission 1/3 world’s population carry M.
tuberculosis: not infectious 8 million cases / year: contagious 3 million deaths / year
Burden of TB in TanzaniaBurden of TB in Tanzania
Tanzania is among the 22 countries with highest TB burden despite :- implementing DOTS strategy since early 1980’s Having national coverage of DOTS Having a well functioning TB control programme High Government and International commitment to
TB control in the country Notification rate total TB cases for 2002 was
about 188/100,000 Underlying course is the HIV/AIDS epidemic in
the country TB is 3rd leading cause of adult mortality
25 - 4950 - 99100 - 300
0 - 9
10 - 24
300No estimate
Estimated Global TB incidence rates, 2000
The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.
Global Tuberculosis Control. WHO Report 2002. WHO/CDS/TB/2002.295
Africa >300 /100,000
Prevalence of HIV among TB patientsPrevalence of HIV among TB patients
WHO supported surveys to establish prevalence of HIV among SM+ TB cases in 2 phases: 1991-1993; 1994-1998
Total of 6,084 and 10,612 TB cases surveyed in phase 1 and 2 respectively
TB/HIV co-infection in phase 1 was 32% (range 14%-59%) and 44% in phase 2 (range 21%-77%)
HIV accounted for 60% of increase in Smear positive TB notification rate from 54/100,000 in phase 1 to 74/100,000 in phase 2.
NTLP - MOHNTLP - MOH
HIV prevalence in tuberculosis patients in Tanzania
0%
10%
20%
30%
40%
50%
60%
70%
15-24 25-34 35-44 45-54 55+
Age group
Per
cen
t
Male 1991-93
Male 1994-98
Female 1991-93
Female 1994-98
Tanzania: TB Cases - trends, 1979 to 2001
0
10000
20000
30000
40000
50000
60000
1979 1983 1987 1991 1995 2001
Relapse (2.6% - 2.9%)
EPTB (11.2% - 20.0 %)
Smear- (20.8% - 33.6%)
Smear+ (65.2% - 40.0%)
Correlation Between Extent of HIV-Induced Immuno-Suppression and Clinical Manifestation of Tuberculosis
Duration of HIV infection
Me
dia
n C
D4
ce
ll co
un
t /
mm
3
0
100
200
300
400
500
De Cock KM, et al. J Am Med Assoc 1992;268:1581-7
Pulmonary tuberculosis
Lymphatic, serous tuberculosis
Tuberculous meningitis
Disseminated tuberculosis
Impact of HIV on the Epidemiology of Tuberculosis
5%
10%
35%
13%
0
10
20
30
40
50
60
70
80Cure rate
Failure rate
Mortality rate
Mortality rate in + HIV
Mortality rate in - HIV
Impact HIV in treatment outcomesHigh Mortality in HIV Co-infected patients
van den Broek & Mfinanga, 1998NTLP reports 2003
THE GENUS MYCOBACTERIUM CAN BE DIVIDED INTO FOUR BROAD GROUPSTHE GENUS MYCOBACTERIUM CAN BE DIVIDED INTO FOUR BROAD GROUPS
1. THE TUBERCULOSIS COMPLEX
2. SLOW GROWING MYCOBACTERIA OTHER THAN TUBERCULOSIS (MOTT)
3. RAPIDLY GROWING MYCOBACTERIA
4. MYCOBACTERIUM LEPRAE
THE TUBERCULOSIS COMPLEXTHE TUBERCULOSIS COMPLEX
(Organisms that resemble M. tuberculosis;Causing a similar type of disease in humans)
1. M. tuberculosis
2. M. bovis
3. M. africanum
THE RUNYON GROUPING:
An older idea that
Remains useful for differentiating mott
THE RUNYON GROUPING:
An older idea that
Remains useful for differentiating mott
RUNYON GROUPS I,II,III: Slow growing mycobacterium Visible colonies on solid media After more than 5 days incubation
RUNYON GROUPS I,II,III: Slow growing mycobacterium Visible colonies on solid media After more than 5 days incubation
GROUP I: PHOTOCHROMOGENS Produce pigment When grown in the light but not the darkEXAMPLES: M. kansasii; M. marinum; M. simiae
GROUP II: SCOTOCHROMOGENS Pigment when grown in both light and darkEXAMPLES: M. scrofulaceum; M. szulgai; M. xenopi
GROUP III: NONCHROMOGENSNo pigment when grown in both light and darkEXAMPLES: M. avium-intracellulare; M. genevense
M. ulcerans and M. leprae do not fit in this scheme
RUNYON GROUP IV:Rapidly growing mycobacterium
Visible colonies on solid medium
In less than 5 days incubation
RUNYON GROUP IV:Rapidly growing mycobacterium
Visible colonies on solid medium
In less than 5 days incubation
EXAMPLES:M. fortuitum; M. chelonae; M. abscessus
Robert Koch 1843-1910
MYCOBACTERIUM TUBERCULOSISMYCOBACTERIUM TUBERCULOSIS
Prototypic facultative intracellular pathogenPrototypic facultative intracellular pathogen
MYCOBACTERIUM TUBERCULOSISMYCOBACTERIUM TUBERCULOSIS
Prototypic facultative intracellular pathogenPrototypic facultative intracellular pathogen
Primary human pathogen
Transmitted from human to human
Closely related to M. bovis
It is an intracellular pathogen (typically inside alveolar
macrophages).
This organism does not secrete an identifiable exotoxin.
Host immunity to M tuberculosis appears to be cell
mediated (CMI).
CELL ASSOCIATED TOXINS MAY BE IMPORTANTCELL ASSOCIATED TOXINS MAY BE IMPORTANT VIRULENCE FACTORS FOR MANY MYCOBACTERIAVIRULENCE FACTORS FOR MANY MYCOBACTERIA
CELL ASSOCIATED TOXINS MAY BE IMPORTANTCELL ASSOCIATED TOXINS MAY BE IMPORTANT VIRULENCE FACTORS FOR MANY MYCOBACTERIAVIRULENCE FACTORS FOR MANY MYCOBACTERIA
The glycolipids, such as trehalose dimycolates, of Mycobacterium tuberculosis and related organisms appear to be related to their virulence.
Bacterial cell wall structure
Gram +Gram -
Mycobacterium
Lipid Peptido- Lipid + Porins Mycolate Acyl LAM arabino-Bilayer glycan LPS Lipids Lipo-arabino galactan mannin
Biochemical properties Niacin (+) Nitrate reduction(+) (–) for catalase at 680 C Tween hydrolysis, Arylsulfatase production, Tellurite reduction, Salt tolerance, pyrazinamidase (pyr) production
Reaction to physical and chemical agents
Resistant to chemical agents – malachite green, penicillins
In liquid media – grow in clumps – hydrophobic
Resistant to acids and alkalines – helps to decontaminate and concentrate samples
Constituents of tubercle bacilli Lipids – mycolic acids – long chain fatty
acids C78-C90) –responsible for acid fastness
Lipids linked to proteins and polysaccharides
Muramly dipeptide
Surpentine cords – cord factor – trehalose-6,6-dimycolate
Virulence factors (M. tb.)
Cord factor – is a glycolipid, trehalose 6,6’ dimycolate,
t is responsible for the serpentine growth (filaments or cords)
It is toxic to leukocytes, antichemotactic, interferes with mitochondrial function in mice and plays a role in the development of granulomatous lesions
Iron capturing ability – required for survival inside phagocytes
Sulfolipids prevent phagosome-lysosome fusion so that the organisms are not exposed to lysosomal enzymes (is important in intracellular survival)
Tuberculosis – primary infection route
Tuberculosis in humans
A key issue is to understand why individuals infected with M. tuberculosis experience different clinical outcomes
INTRACELLULAR pathogen (facultative extracellular)
ExposedInfected (2 billion, 8 million new
cases per year)
Primary TB
Latent TB Reactivation
30%
80-90%
5-10%
5-10%
Clearance
70%
Death
(2 million)
Pathogenesis
Inhalation of droplets (1-5 µm) Reach alveoli Disease due to proliferation and host
interaction Resistance and hypersensitivity
influence disease
T helper Cell Differentiation
Th0
Th2
Th1
• Type 1 response
• immunity to mycobacteria
• inflammation• rheumatoid arthritis, diabetes
• Type 2 response
• IgE antibody responses
• Immunity to some parasites
• allergic diseases
IFNTNF
IL4IL13IL10
Pathology Two principal lesions:
Exudative type – resembles bacterial pneumonia Acute inflammatory reaction Edema Later, monocytes Heals by fibrosis May develop to productive type Tuberculin test is positive
Productive type Chronic granuloma, consisting of three
zones: A central large area –
multinucleate giant cells with bacilli A mid zone area – epitheliod cells Peripheral zone – fibroblasts,
lymphocytes and monocytes Later the central area – caseation
necrosis, and peripheral area becomes fibrous → turbecle May break into a bronchus Or heal by fibrosis and calcification
Large caseating tubercle Miliary tubercles
HUMAN LUNGHUMAN LUNG HUMAN LUNGHUMAN LUNG
TUBERCULOSISTUBERCULOSIS
Spread of organisms in the host Tubercle spreads by direct extension to
lymphatics → lymph nodes → blood stream → various organs
Blood stream involvement also due to erosion of a vein
If contents of tubercle are swallowed could be passed to stomach and intestine
Tuberculosis – hematogenous extension
Extrapulmonary Lymph nodes
Liver Bones
Primary and reaction TB When host has first contact with TB bacilli
the following features will occur: 1. an exudative lesion develops and rapidly
spreads to lymphatics and regional lymph noded – Ghon complex
2. the lymph node undergo massive caesation, which ussually calcifies
3. tuberculin test becomes positive Primary TB may occur on any part of
the lung but more frequently on the base
Reactivation TB
Caused by bacilli that have survived in the primary lesion
Chronic tissue necrosis Formation of tubercles, caesation and
fibrosis Almost always begins at the apex of
the lung
M. tuberculosis
Symptoms of TB Disease
Prolonged cough (may produce sputum)* Chest pain* Hemoptysis* Fever Chills Night sweats Fatigue Loss of appetite Weight loss/failure to gain weight*commonly seen in cases of pulmonary TB
Clinical Manifestations of TB
Organ specific pneumonia: cough, sputum +/-
blood scrofula: swollen lymph nodes meningitis: headache, obtundation miliary TB: no obvious source genitourinary: sterile pyuria
1. Evidence of infectiona. Chest x-ray - hilar lymphadenopathy
calcification of primary focus/LNb. Delayed hypersensitivity response to purified protein
derivative (PPD) MANTOUX /HEAF TEST
2. Evidence of active diseasea. Sputum for AFB positive
3. Evidence of active diseasea. Indirect evidence of infection (Mantoux)
b. Direct evidence of infection PCR / culture
c. Histo-pathological evidence
DIAGNOSIS
Chest Radiograph
Abnormalities often seen in apical
or posterior segments of upper
lobe or superior segments of lower lobe
May have unusual appearance in
HIV-positive persons
Cannot confirm diagnosis of TB
Arrow points to cavity in patient's right upper lobe.
Diagnosis of tuberculosis
Tuberculin test intradermal injection of PPD (5 TU) Induration, edema, erythema Read after 48-72 h Positive: induration≥ 10 mm It is DTH
Administering Tuberculin Skin Test
•Inject intradermally 0.1 ml of 5TU PPD tuberculin
•Produce wheal 6 mm to 10 mm in diameter
•Do not recap, bend, or breakneedles, or remove needles from syringes
Reading the Tuberculin Skin Test
•Read reaction 48-72 hours after injection
•Measure only induration
•Record reaction in millimeters
Factors that affect the PPD Reaction
Type of Reaction Possible CauseFalse-positive Nontuberculous mycobacteria BCG vaccination
AnergyFalse-negative Recent TB infection Very young age (< 6 months old) Live-virus vaccination Overwhelming TB disease
Kinyon – uses a higher content of phenol (organic solvent) in the carbol fuchsin primary stain to allow penetration of the stain without the need to apply heat. Also uses acid alcohol to destain and methylene blue as the counterstain.
Auramine-rhodamine fluorochrome (a fluorescent stain) requires a fluorescent microscope, but allows one to scan the slide on high dry so that slides may be read much faster Stain with auramine-rhodamine for 10
minutes (phenol in the solution allows for penetration)
Destain with acid alcohol Counterstain with acridine orange A positive result is a bright yellow fluorescence.
Mycobacteria: auramine stain
Nucleic Acid HybridizationNucleic Acid Hybridization
Ability of DNA strands from one organism to hybridize with the DNA strands of another organism
Southern blotting
DNA fingerprintingDNA fingerprinting
Steps:1. Digest DNA into smaller fragments2. Separate fragments by size3. Visualize bands4. Each band corresponds to 1 fragment size5. Unique pattern of bands identifies donor of DNA
Mycobacteria colonies on LJ medium
MGIT System Middlebrook media
Fluorescent compound on bottom that is quenched by oxygen in the tube.
As mycobacteria consume oxygen, fluorescence can be detected.
Most of MAC detected in nine days while MTB by 14 days.
Follow up with acid fast stain.
http://labmed.ucsf.edu/CP/SFGH/Microbiology/images/MAI.jpeg
Tuberculosis and the Immune Reconstitution Inflammatory Syndrome (IRIS)
Names
Immune reconstitution inflammatory syndrome (IRIS)
Immune restoration disease (IRD) Paradoxical reactions
Pathogenesis
Increased lymphoproliferative response to mycobacterium antigens in vitro
Restoration of cutaneous response to Tuberculin
Associated with TNFA, IL6
Risk factors for TB/IRIS
Starting ARV’s within 6 weeks of TB treatment
Disseminated, extra-pulmonary disease
Low base line CD4 count Rise in CD4 % Fall in viral load High bacillary burden?
Types of TB IRIS
Patient unknown to have TB at the start of HAART
Patient on TB treatment before or at the start of HAART
Timing of IRIS Mean of 15 days after starting HAART
Up to months (years)
Syndrome lasts for 10-40+ days
Prevention and control of tuberculosis Prevention and control of tuberculosis
1. Prompt and effective treatment of patients with
active TB
2. Careful follow-up of contacts – tuberculin test, X-
rays etc
3. Prophylaxis of asymptomatics, tuberculin
positives
4. Correct underlying cause of immune suppression
5. Immunization with live-attenuated tubercle bacilli
e.g. BCG
6. Eradication of TB in cattle and pasteurization of
milk
OTHER IMPORTANT MYCOBACTERIAOTHER IMPORTANT MYCOBACTERIA1. Mycobacterium avium-intracelluare complex• causes TB like disease in birds • opportunistic pathogen in humans • Very prominent cause of disease in AIDS
patients
• has been decreased following HAART• • Not easily transmitted
• Difficult to treat ( drug of choice is rifabutin)
Mycobacterium ulcerans
Causes buruli ulcer Infection limited to fatty tissue
beneath dermis Does not grow above 33oc
Mycobacterium marinum
Extrapulmonary ulcerative lesions Growth of organism restricted to 340c
Disease called “swimming pool granuloma”
Does not respond well to therapy
Mycobacterium kansasii
• Pulmonary and disseminated disease similar to tuberculosis (organisms do not produce niacin)
•does not respond well to antimicrobials •(no response to anti-tuberculosis therapy)
• opportunistic pathogen• Runyon group I (photochromogen)
Mycobacterium scrofulaceum
Causes scrofula (cervical lymphadentis)
drug resistant
Runyon group II (scotochromogen)
Mycobacterium fortuitum complex
causes chronic abscesses(often wound associated)
can be confused with M. tuberculosis
often drug resistant
rapidly growing (Runyon group IV)
HANSEN’S DISEASE (Leprosy) caused by M. leprae
Hansen’s disease is a chronic, slowly progressive
Granulomatous
Disease involve ectodermally derived tissues e.g. skin
and peripheral nerves.
Usually limited to the cooler parts of the body e.g. skin,
nose and upper respiratory tract.
It rarely affects internal organs such as the brain, liver,
spleen, kidneys, and bones.
It has a specific predilection for peripheral nerves.
Mycobacterium leprae is an acid-fast rod that grows in macrophages, nerve cells, the
foot padsof thymectimized mice, and the nine banded armadillo.
It has never been grown in artificial medium,
an obligate intracellular pathogen.
has a generation time of 12 to 14 days,
the incubation period for onset of disease is prolonged,
therapy must also be prolonged (perhaps the lifetime of the individual).
There are 3 forms of the disease: A. Lepromatous:
Most progressive many acid fast bacteria in bundles called globi.
No well formed granulomas (T-cell deficiency) High serum antibody levels Low Th1 response to M. leprae Th2 response-->humoral immunity (not protective)
B. Tuberculoid: (Less contagious) Few bacteria present with multiple granulomas.
Organisms present at low levels Low infectivity Strong Th1 response to M. leprae antigens
C. borderline: Has mixed characteristics of both lepromatous and tuberculoid forms
HANSEN’S DISEASE is treatable
with outpatient therapy.
DRUGS OF CHOICE: SULFONES (i.e. Dapsone) PHENAZIDES (Lampren) RIFAMPIN CLOFAZAMINE
~ 500,000 cases Worldwide
Treatment – Dapsone (1940s-1970s) Rifampicin (1970s-) Multidrug Therapy- Dapsone, Rifampicin, Clofazamine