1. Lived and died for victims of Hansen’s disease (leprosy)
Mycobacteriaceae Aerobic Gram-Positive Bacilli Form Filaments Stain Acid-fast
FATHER DAMIEN (1840 – 1889)
Lived and died for victims of Hansen’s disease (leprosy)
on Molokai, Hawaii

2. Mycobacterium: Genera
“fungus” “small rod”
Aerobic, fastidious, slow growing
Cell division hr.
Lab culture 1-2 months
Large lipid content in cell wall, resistant to
Disinfectants
Detergents
Common antibiotics
Lab basic stains
~100 species, many isolated in humans
Important human pathogens:
M. tuberculosis – “small swelling”
M. avian-intracellulare – “birds”; TB-like illness, common in AIDS patients
M. leprae – “scaly skin”; Hansen’s disease

3. Nontuberculosis Mycobacteria: Runyon Classification
Based on growth rate, pigmentation
Non-Runyon Group: M. tuberculosis, M. leprae
Group I: Slow-Growing Photochromagen
No pigment grown in dark
Photoactivated pigment upon exposure to light
Group II: Slow-Growing Scotochromogen
Yellow, orange pigments grown in light or dark
Pigment deepens in two weeks
Group III: Slow-Growing Nonphotochromagen
Produce white, yellow pigment
Pigment not intensify upon exposure to light
M. avian complex
Group IV: Rapid Grower
Colonies in seven days

4. Mycobacterium: Staining
G(+) bacilli, slender, branched filaments; stain poorly because of lipids (mycolic acid, waxD) in cell wall
Acid-fast stain
Presumptive diagnosis mycobacterial disease
Heating for stain penetration of high lipid cell wall
Stain penetrates, binds to mycolic acid, not remove with acid-alcohol treatment
Not easily decolorize, stain holds “fast”)

5. Acid-Fast Stain Ziehl-Neelsen stain Kinyon stain Fluorochrome stain
Carbol fuchsin - heat to penetrate
Acid-alcohol - decolorize
Methylene blue - counterstain
Kinyon stain
Carbol fuchsin – no heat, higher phenol allow penetration
Fluorochrome stain
Auramine-rhodamine – stain, phenol
Acridine orange – counterstain
UV microscope scan slide high dry, detect AFB by fluorescence
Read stained slide easier, faster

6. Mycobacterium: Lab Culture
Work under biosafety cabinet
Specimen of choice
Patient coughs up sputum from lung
NaOH digest, decontaminate organic debris, RT normal flora
N-acetyl-L-cystine to liquify
Enrich, selective media
Egg or agar based
Antimicrobial agents - malachite green, cyclohexamide, nalidixic acid
Lowenstein-Jensen medium – egg based, colonies days
Middlebrook 7H10, 7H11 medium – agar based, colonies days

7. Mycobacterium tuberculosis: Lab Culture
Solid media - rough, dry, granular, non-pigmented, buff color colony
Liquid media - contains Tween 80, albumin, faster growth
RT grow best 5-10% CO2 , 370 C
Skin lesion grow best, C
RT culture 6-8 weeks before discard as negative
Skin lesion culture 12 weeks
CDC desires more timely report, possible with new DNA amplification methods of ID

8. Mycobacterium: Lab ID Rate of growth Culture temperature
Pigmentation and photoreactivity
Biochemical testing: niacin production, nitrate reduction, catalase at 680 C, tween hydrolysis, arylsulfatase production, tellurite reduction, salt tolerance, pyrazinamidase production
Test of choice - DNA amplification; routinely done in PH lab, rapidly ID species

9. Mycobacterium tuberculosis
Human only natural reservoir
Worldwide - third of population infected
~2 billion people
~8 million new cases/year
~2-3 million deaths/year
USA ~10 million infected
Since 1985, dramatic increase number cases/year
Infections in homeless, drug and alcohol abusers, prisoners, AIDS patients
After 1992, now slowly decline due to increase PH prevention programs

10. M. tuberculosis: Virulence Factors
Cord factor – cell wall glycolipid
Serpentine growth (filaments, cords), grow in close parallel arrangement
Toxic to leukocytes, anti-chemotactic
Role in development of granulomatous lesions
Iron capturing ability – required for survival inside phagocytes
Sulfolipids - prevent phagosome-lysosome fusion (important in intracellular survival)
Tissue damage - no known bacterial toxin or enzyme implicated; host immune response thought responsible, by inflammation, cell-mediate immunity (CMI)

11. M. tuberculosis: Transmission
Close contact - person-to-person
Inhalation - infectious aerosols into alveolar spaces
Exposure to few organisms (10-200) may establish “infection” (elicit immune response, no disease)
Humans very susceptible to infection, but remarkably resistant to tuberculosis disease

12. Primary Tuberculosis
Exposure - bacilli reach alveoli, ingested by macrophage
MO multiply - cause chemotactic response, recruits macrophages, T cells
Enzymes, cytokines release - start inflammatory response, wall off MOs (tubercle formation)
Inflammatory response also causes lung damage
Small number MO - no tissue damage
Large number MO - CMI response results in tissue necrosis
Patient becomes PPD skin test(+)

13. Early Tubercle

14. Reactivation Tuberculosis
Few weeks macrophages die - release bacilli, form caseous center in tubercle
In healthy individuals - disease usually arrested, lesions calcified
Tubercle bacilli - may remain dormant in lesion; later reactivation of disease
Host defenses fail - mature tubercle form; caseous center enlarge, liquify to form tuberculous cavity where bacilli multiply outside macrophage

15. Mature Tubercle

16. Extrapulonary Tuberculosis
Tubercle ruptures - release bacilli; disseminate throughout lung, circulatory, lymphatic system
Miliary (Extrapulmonary) TB – spread to lymph nodes, pleura, many other organs:
Progressive form of disease
Weight loss, coughing with blood, loss of vigor (old name consumption)

17. Tuberculosis: Infection and Disease
In 3-6 weeks - patient’s CMI activated, bacteria replication stops
Within 2 years - 5% patients progress to active disease
Sometime later in life % patients develop active disease
AIDS patient, TB infected:
Due to M. avian
10% develop active disease within 1 year
2x more likely to spread, rapidly progress to death
Impaired CMI unable to arrest infection

18. M. tuberculosis: Treatment
Slow growth of MO, chronic infection require 6-9 months drug treatment
Combination of drugs to prevent emergence of resistant strains
Current recommended drugs: isoniazid (INH), ethambutol, pyrazinamide, rifampin
Drug resistance
1990 report multidrug-resistant M. tb (MDR-TB) in AIDS patients, homeless in N.Y., Miami
In developing countries, extensively drug-resistant TB (XDR-TB), resistance to second line drugs, potentially untreatable

19. M. tuberculosis: Prevention
BCG (bacille Calmette-Guerin) Vaccine
Attenuated M. bovis
Used where TB high
Reduce TB if vaccinated young age
Tuberculin skin test
PPD (purified protein derivative M. tb) injected under skin
Test host CMI response
Delayed-type hypersensitivity reaction (>10 mm induration), 48 hr., if previous or current infection; not necessarily active disease
Control disease
PH surveillance
Drug treatment and intervention
Case monitoring, prevent transmission

20. M. avium-intracellular Complex
Common in soil, water, food
Before HIV - transient colonization in patients with compromised pulmonary function (bronchitis, obstructed pulmonary disease, previous infection); ~pulmonary TB
After HIV - USA most common mycobacteria disease in AIDS patients
Infection disseminated - all organs, large number MO

21. M. avium-intracellular Complex
Transmission – ingestion of contaminated food or water, not person-to-person
Greatest risk for infection are immunocompromised
Multiply in localized lymph nodes, spreads to disseminated disease
Impair organ function due to replication MOs, host immune response
MO ubiquitous and control of exposure difficult

22. Mycobacterium leprae: Hansen’s Disease
“to peel” “leprosy”
~12 M cases worldwide (Africa, Asia, Latin America); rare USA
Reservoir of MO in armadillo
Does not grow in cell-free culture
Transmission by person-to-person, direct contact, inhale infectious aerosols
Requires prolonged, intimate contact for transmission
Two clinical forms of disease:
Tuberculoid
Lepromatous

23. Hansen’s Disease
Mycobacteria obligate intracellular parasites in histiocytes, Schwann cells, epitheloid structures - called Lepra cells
Incubation period 2-4 years
Clinical manifestations depend upon adequacy of host CMI response
Like TB - many infected, few develop clinical symptoms of disease
Subclinical/Tuberculoid – infection contained by CMI
Lepromatous - large number bacilli in sputum, nasal secretion, skin

24. Hansen’s Disease: Tuberculoid
Patient - strong CMI, weak antibody response
Lesions - skin, peripheral nerves, few in number; raised, erythematous margin, flat center
Nerve damage due to CMI response - loss sensation of touch, temperature; pain within lesion
Skin biopsy reveals many lymphocytic, epithelial cells, but no AFB
Infectivity, transmission low
Lepromin skin test (+)

25. Hansen’s Disease: Lepromatous
Patient - strong antibody response, defective CMI (“foamy” macrophage, few lymphocytes, numerous bacilli)
Involve all areas of skin; waxy, nodular appearance, may thicken and fold
Destruction of cutaneous nerves, eyebrows, eyelashes, nasal septum
Skin biopsy reveals lymphocytes, many Lepra cells packed with AFB
Infectivity high
Lepromin skin test is (-)

26. M. leprae: Treatment and Prevention
Tuberculoid form – rifampicin, dapsone; 6 months
Lepromatous – rifampicin, dapsone, clofazimine; minimum 1 year
Control by prompt recognition and treatment of infected patients
Lepromin skin test is similar to TB skin test

27. The Gifts of Civilization: Germs and Genocide In Hawaii
O. A. Bushnell, University of Hawaii Press. 1993
Hawaii isolated, difficult to reach by sea
1778 – “discovered” by Captain Cook
Estimate ~1 million Hawaiian inhabitants
1832 – census ~130,000
~30,000
Why decline?
Infectious diseases upon immune naive population i.e. STD, plague, cholera, TB, Hansen’s disease from Chinese immigrants, smallpox, chickenpox, measles, mumps, rubella;

28. FATHER DAMIEN (1840 – 1889)
The man who lived and died for the victims of Hansen’s disease (leprosy)

29. Damien Born in Belgium 1840
He was an ordinary boy, brother, and priest.
So what made him different?

30. Damien arrives Hawaiian Islands 1864
He agreed to do a job that no one else would do.
He lived and worked for a group of people who had been sent away from their homes to a remote Hawaiian island, Molokai

31. They were outcasts because they had Hansen’s Disease.

32. Hansen’s Disease A scaly skin disease.
A chronic disease caused by a bacteria.

33. In those days there was no cure for Hansen’s Disease.
The outside world did not like to think about this awful disease, and chose to forget the people who suffered from it.

34. Damien gave back these people hope and pride.

35. He loved them like a family and in the end he died as one of them, a victim of Hansen’s Disease.

36. Damien’s life and death forced people to face the problem of Hansen’s Disease.
He said to the world that work needed to be done – and quickly!

37. Today, a cure for Hansen’s Disease has been found.
We no longer have to fear the disease or the people who suffer from it.
In 2009, for Father Damien’s many contributions to society, he was Canonized Saint Damien by the Vatican.

38. Class Assignment Textbook Reading Key Terms
Chapter 26 Mycobacterium Tuberculosis
Omit: Clinical Significance and Differentiation of Nontuberculosis Mycobacterium
Key Terms
Learning Assessment Questions

39. Case Study 7: Mycobacterium
A 35-year-old man with a history of intravenous drug use entered the local health clinic with complaints of a dry, persistent cough; fever; malaise; and anorexia.
Over the preceding 4 weeks, he had lost 15 pounds and experienced chills and sweats.
A chest radiograph revealed patchy infiltrates throughout the lung fields.

40. Case Study 7: Mycobacterium
Because the patient had a nonproductive cough, sputum was induced and submitted for bacterial, fungal, and mycobacterial cultures, as well as examination for Pneumocystis organisms.
Blood cultures and serologic tests for HIV infection were performed.
The patient was found to be HIV positive.
The results of all cultures were negative after 2 days of incubation; however, cultures were positive for M. tuberculosis after an additional week of incubation.

41. Case Study 7: Questions
1. What is unique about the cell wall of mycobacteria, and what biologic effects can be attributed to the cell wall structure?
2. Why is M. tuberculosis more virulent in patients with HIV infection than in non-HIV-infected patients?
3. What is the definition of a positive skin test (PPD) result for M. tuberculosis?
4. Why do mycobacterial infections have to be treated with multiple drugs for 6 months or more?