1. Mycobacterium

2. Learning Objectives At the end of this lecture, the student should be able to:
List the important characteristics of Mycobacteria
List the medically important genera of Mycobacteria
List the most important epidemiological properties of Mycobacteria
List the most important diagnostic tests in the diagnosis of Mycobacteria
List the main properties of the vaccine for tuberculosis

3. Mycobacterium Aerobic bacilli
The rods occasionally form branched filaments

4. Mycobacterium Cell wall is rich in lipids
Hydrofobic surface resistant to many disinfectants & stains
resistant to decolorizing
“acid-fast bacilli”
Grow slowly (dividing every h):
requiring as long as 6-8 weeks before growth is detected in laboratory

5. Mycobacterium Once stained,
the rods also cannot be decolorized with acid solutions;
hence the name acid-fast bacteria.

6. Other species of bacteria may also be acid-fast
Nocardia,
Rhodococcus,
Tsukamurella,
Gordonia
they stain less intensely (are partially acid-fast), and
their mycolic acids chains are shorter.

7. Mycobacterium Mycobacteria are a significant cause of
morbidity and
mortality,
particularly in countries with limited medical resources.
Currently, more than 130 species of mycobacteria have been described, many of which are associated with human disease

8. TUBERCULOSIS kills about 3 million people/year
infects almost 9 million others/year
wherever poverty, malnutrition and poor housing prevail.
It affects the apparently healthy as well as being a serious disease of the immunocompromised, as has become particularly obvious in patients with AIDS.
Tuberculosis is primarily a disease of the lungs, but may spread to other sites or proceed to a generalized infection ('miliary' tuberculosis).

9. Infection is acquired by inhalation of Mycobacterium tuberculosis
in aerosols and dust.
Air-borne transmission of tuberculosis is efficient because infected people cough up enormous numbers of mycobacteria,
projecting them into the environment,
where their waxy outer coat allows them to withstand drying and therefore survive for long periods of time in air and house dust.

10. Mycobacterium
Despite the abundance of mycobacterial species, the following few species or groups cause most human infections:
M. tuberculosis
M. avium complex
M. kansasii
M. fortuitum
M. chelonei
M. abscessus
M. leprae

11. Mycobacteria possess a complex, lipid-rich cell wall
This cell wall is responsible for many of the characteristic properties of the bacteria:
acid-fastness,
slow growth,
resistance to detergents,
resistance to common antibacterial antibiotics
antigenicity
clumping

12. Mycobacteria possess
The basic structure of the complex, lipid-rich cell wall
is of typical for gram-positive bacteria:
an inner plasma membrane overlaid with a thick peptidoglycan layer and
no outer membrane.
However, the mycobacterial cell wall structure is far more complex than that in other gram-positive bacteria.

13. The proteins in the cell wall:
biologically important antigens, stimulating the patient's cellular immune response to infection.
Extracted and partially purified preparations of these protein derivatives (purified protein derivatives, or PPDs) are used as skin test reagents to measure exposure to M. tuberculosis.
Similar preparations from other mycobacteria have been used as species-specific skin test reagents

14. Mycobacterium The mycobacterial cell wall is complex,
This group of organisms is fastidious,
Most mycobacteria grow slowly, dividing every 12 to 24 hours
Requiring as long as 8 weeks before growth is detected in laboratory cultures.

15. Mycobacterium Slow-growings Rapid-growings
3 to 8 weeks of incubation
Rapid-growings
> 3 days
Non-growing: Mycobacterium leprae

16. Classification Growth properties and Colonial morphology
are used for the preliminary classification of mycobacteria.
M. tuberculosis and closely related species in the M. tuberculosis complex are slow-growing bacteria.
The colonies of these mycobacteria are either nonpigmented or of a light tan color

17. Mycobacterium tuberculosis colonies grow on Löwenstein-Jensen agar medium fter 8 weeks of incubation

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19. Other species of mycobacteria
referred to as
atypical mycobacteria,
mycobacteria other than tuberculosis (MOTT) or
non-tuberculous mycobacteria (NTM)
also cause infection in the lungs

20. «Nontuberculous mycobacteria» or NTM
Classified originally by Runyon by their rate of growth and pigmentation
Pigmented mycobacteria produce intensely yellow carotenoids which may be stimulated by exposure to light (photochromogenic organisms) or
produced in the absence of light (scotochromogenic organisms).
Mycobacterium kansasii colonies grow on Middlebrook agar in one day after exposure to light.

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22. The Runyon classification scheme
consisted of four groups:
slow-growing photochromogens (e.g., M. kansasii, M. marinum),
slow-growing scotochromogens (e.g., M. gordonae-a commonly isolated nonpathogen),
slow-growing nonpigmented mycobacteria (e.g., M. avium, M. intracellulare),
rapidly growing mycobacteria (e.g., M. fortuitum, M. chelonae, and M. abscessus).
Currently used methods for the rapid detection and identification of mycobacteria have made this scheme less important.
Nonetheless, a pigmented or a rapidly growing mycobacterium should never be mistaken for M. tuberculosis.

23. M. tuberculosis Complex
M. tuberculosis Strictly pathogenic
M. leprae Strictly pathogenic
M. africanum Strictly pathogenic
M. bovis Strictly pathogenic
M. bovis (BCG strain) Rarely pathogenic
M.microti
M.canettii
M.pinnipedii

24. Slow-Growing Nontuberculous Mycobacteria
M. avium complex (MAC): Usually pathogenic
M. kansasii: Usually pathogenic

25. Rapidly Growing Nontuberculous Mycobacteria
M. abscessus: Sometimes pathogenic
M. chelonae: Sometimes pathogenic
M. fortuitum: Sometimes pathogenic

26. Mycobacterium tuberculosis/Pathogenesis
Virulence
Capable of intracellular growth in unactivated macrophages
Disease primarily from host response to infection
M. tuberculosis is an intracellular pathogen that is able to establish lifelong infection.

27. Mycobacterium tuberculosis/Diagnosis
Clinical diagnosis +
Radiographic evidence of pulmonary disease
Positive skin test reactivity
The laboratory detection of mycobacteria either with
-microscopy
-culture
-molecular methods

28. Laboratory diagnosis
Tuberculin skin test (TST) and IFN-γ release tests (IGRA) are sensitive markers for exposure to organism
Microscopy and culture are sensitive and specific
Direct detection by PCR.

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30. Mycobacterium leprae(Hansen disease )
Weakly Gram-positive, strongly acid-fast bacilli
Unable to be cultured on artificial media
Diagnosis made with specific skin test or acid-fast stain
Capable of intracellular growth
Leprosy disease: tuberculoid-lepromatous-intermediate forms

31. Biology, Virulence, and Disease
Lipid-rich cell wall
Disease primarily due to host response to infection
Tuberculoid (paucibacillary) and lepromatous (multibacillary) forms of leprosy

32. Diagnosis
Microscopy is sensitive for the lepromatous form but not the tuberculoid form
Nasal scrapings and biopsies of skin lesions should be stained by Ziehl-Neelsen or auramine stain to demonstrate acid-fast rods.
Skin testing is required to confirm tuberculoid leprosy
Culture is not useful

33. Mycobacterium avium Complex
In environment:
Water(fresh, brackish,ocean, drinking water)
Soil
Before AIDS epidemic
Transient colonizer in asymptomatic patients
Disease in patients with compromised pulmonary function
With AIDS, a new spectrum of disease
“The most common mycobacterial disease”
disseminated

34. Laboratory Diagnosis of Mycobacterial Disease
Detection
Skin test
Microscopy
Carbolfuchsin acid-fast stain
Fluorochrome acid-fast stain
Culture
Solid agar-based or egg-based media
Broth-based media

35. Acid-fast stains of Mycobacterium tuberculosis.
A, Stained with carbolfuchsin
B, Stained with the fluorescent dyes auramine

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37. Laboratory Diagnosis of Mycobacterial Disease
Identification
Morphologic properties
Biochemical reactions
Analysis of cell wall lipids
Nucleic acid probes
Nucleic acid sequencing

38. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis
Conventional tests for laboratory confirmation of TB include
acid-fast bacilli (AFB) smear microscopy(24 hours)
culture
Although rapid and inexpensive,
AFB smear microscopy is limited by its poor sensitivity
(45%–80% with culture-confirmed pulmonary TB cases)

39. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis
NAA tests can provide results within 24–48 hours.

40. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis
Compared with AFB smear microscopy,
the added value of NAA testing lies in its
greater positive predictive value (>95%)
with AFB smear-positive specimens in settings
in which nontuberculous mycobacteria
are common and
2) ability to confirm rapidly the presence of M. tuberculosis
in 50%–80% of AFB smear-negative, culture-positive specimens
Compared with culture, NAA tests
can detect the presence of M. tuberculosis bacteria in a specimen weeks earlier
than culture for 80%–90% of patients suspected to have pulmonary TB
whose TB is ultimately confirmed by culture

41. Culture remains the gold standard for laboratory confirmation of TB and is required for isolating bacteria for drug-susceptibility testing and genotyping.
Routinely collect respiratory specimens (e.g., sputum), process (liquefy, decontaminate, and concentrate), and test by AFB smear microscopy and culture as previously recommended . Specimen collection and microbiologic testing should not be delayed to await NAA test results.

42. The positive predictive value of FDA-approved NAA tests for TB is >95% in AFB smear-positive cases
If the NAA result is negative and the AFB smear result is positive, a test for inhibitors should be performed and an additional specimen should be tested with NAA. Sputum specimens (3%–7%) might contain inhibitors that prevent or reduce amplification and cause false-negative NAA results

43. Currently available NAA tests are not sufficiently sensitive (detecting 50%–80% of AFB smear-negative, culture-positive pulmonary TB cases) to exclude the diagnosis of TB in AFB smear-negative patients suspected to have TB

44. For culture Sputum: early morning specimen on three consecutive days
Bronchial aspirate,BAL
Gastric lavage
Urine:early morning specimen on three consecutive days
Sterile body fluids(CSF,peritoneal,pericardial,synovial)
Tissue
Abscess contents
Blood
Stool
Should be refrigirated until being processed!

45. IFN-γ release tests -quantiferon
On May 2, 2005, a new in vitro test, QuantiFERON®-TB Gold (QFT-G, manufactured by Cellestis Limited, Carnegie, Victoria, Australia), received final approval from the U.S. Food and Drug Administration (FDA) as an aid in diagnosing Mycobacterium tuberculosis infection, including both latent tuberculosis infection (LTBI) and tuberculosis (TB) disease.
This enzyme-linked immunosorbent assay (ELISA) test detects the release of interferon-gamma (IFN-g) in fresh heparinized whole blood from sensitized persons when it is incubated with mixtures of synthetic peptides simulating two proteins present in M. tuberculosis: early secretory antigenic target--6 (ESAT-6) and culture filtrate protein--10 (CFP-10). ESAT-6 and CFP-10 are secreted by all M. tuberculosis and pathogenic M. bovis strains.

46. IFN-γ release tests
QFT-G is expected to be more specific for M. tuberculosis than tests that use tuberculin purified protein derivative (PPD) as the antigen.

47. Tuberculin skin test and IFN-γ release tests are sensitive markers for exposure to organism

48. Tuberculin skin test
purified protein derivative (PPD) from the mycobacterial cell wall
In this test, a specific amount of the antigen (5 tuberculin units of PPD) is inoculated into the intradermal layer of the patient's skin.
Skin test reactivity (defined by the diameter of the area of induration) is measured 48 hours later.
A positive skin test indicates previous infection but not necessarily active disease
Becomes positive 4-6 weeks after infection.

49. Tuberculin skin test
Additionally, individuals from countries where vaccination with attenuated M. bovis (bacille Calmette-Guérin [BCG]) is widespread will have a positive skin test reaction.

50. Mycobacteria are innately resistant to most antibacterial agents,
specific antituberculous drugs have to be used
The number of strains resistant to the first-line antituberculous drugs has increased
multidrug resistant (MDR) TB
drug susceptibility assay

51. Tuberculosis
is prevented by improved social conditions, immunization and chemoprophylaxis
Immunization with a live attenuated BCG (bacille Calmette-Guérin) vaccine, has been used effectively in situations where tuberculosis is prevalent. Immunization, which confers positive skin test reactivity, does not prevent infection, but it allows the body to react quickly to limit proliferation of the organisms. In areas where there is a low prevalence of disease, immunization has been largely replaced by chemoprophylaxis.

52. BCG (bacille Calmette-Guérin) vaccine
ATTENUATED VACCINE
M.BOVIS IS ATTENUATED BY
Passage for 10 years in glycerol-bile-potato medium
BCG vaccination is now given to approximately 90% of the world's babies at birth, and protects against the most severe disseminated forms of tuberculosis such as TB meningitis in children, even if its effectiveness against pulmonary TB in adults is more variable

53. Susceptibility to physical and chemical agents
Survives weeks to months on inanimate objects protected from sunlight
Chlorine compounds
70% ethanol
2%glutaraldehyde
Peracetic acid
Hidrogen peroxide

54. Treatment and resistance
Multidrug therapy
Long term
Organism that become resistant to one drug will be inhibited by the other
Although therapy is given usually for months but the patient sputum becomes noninfectious within 2 to 3 weeks.