Mycobacteria slides for lecture

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