3. • In Greek Mykes means mushroom/fungus,
• Fungus like bacteria forming pellicle when grown in liquid medium.
General properties:
• Non- motile, non-sporing (except M.marinum),
• Slender slightly curved or straight, 0.2 to 0.6 × 1 to 10 μm, some species may
display branching morphology.
• Strictly aerobic (some may grow in reduced oxygen concentration).
• Have unusual cell wall as they have high content of lipid (Mycolic acid), N-
glycolyl muramic acid in lieu of N-acetyl muramic acid. Because of this, do not
stain well with other ordinary stains and are weakly gram positive and Acid
Fast.
• Relatively slow growth with colonies being visible in 2 to 20 days in optimal
temperature. This is because of the hydrophobicity, organisms tend to clump, so
that nutrients are not easily allowed into the cell. Generation time may range
from approximately 20 hours to 36 hours
• Robert Koch isolated M. tuberculosis (Koch’s bacillus) in 1882.
• Hansen discovered M.leprae (Hansen’s bacillus) in 1868.
• Johne (in 1985 ) discovered M.paratuberculosis (Johne’s bacillus) as a
causative agent of Johne’s disease in cattle. M. paratuberculosis has also
been suspected as a cause of Crohn’s disease, an illness in human similar to
Johne’s disease in cattle
4. • The genus Mycobacterium contains over 100 well defined
species which includes causative agents of tuberculosis,
leprosy, chronic hypertrophic enteritis of cattle (Johne’s
disease) and saprophytes.
5. Classification
1. Strict pathogens
a. Mycobacterium tuberculosis complex (all species cause tuberculosis):
Bacteria of M. tuberculosis complex are always pathogenic to human. M.
tuberculosis (human type) , M. bovis (bovine type , also infectious to
human, including the vaccination strain bacillus Calmette Guerin), M.
africanum (human type), M.microti (murine type), M. pinnipedii
(primarily infects seals) M.caprie (cattle and human)and M.canetti (an
emerging disease in the Horn of Africa, natural reservoir, host range, and
mode of transmission of the organism are still unknown). All of these are
slow growers and non-pigmented.
b. Lepra bacilli: M. leprae (human type), M. microti (murine type)
c. Other animal pathogens: M.microti (murine type), M. paratuberculosis
(Cattle type)
6. 2. Atypical mycobacteria/ MOTT (free living)
Group I: Photochromogens
Group II: Scotochromogens
Group III: Non-chromogens
Group IV: Rapid growers
3. Saprophytic mycobacteria (non-pathogenic)
M. Smegmatis: present in smegma, thermophiles (grow at 52
degree C)
M. phlei: Present in grass
M. stercoris: Present in dung
M. thermoresistible: tropical evironment
7. M. Tuberculosis complex
• Initially in 1886, Koch’s bacillus referred to both human and
bovine bacillus. But later in 1970, the name M.bovis was
specified for bovine bacilli.
• Both cause human lesions like pulmonary tuberculosis.
• M.bovis is zoonotic and is excreted in milk of cows.
• M. tuberculosis and M. bovis are typical tubercle bacilli while
M.microti, M. canetti, M.africanum, M.caprae, and
M.pinnipedii also belong to this group because all of these
species are so closely related as shown by both antigenic and
DNA studies, that they all are sometimes regarded as variants
of a single species.
8. • Species of M. tuberculosis complex can be distinguished on some basis
like Niacin accumulation, Oxygen preference, Nitrate reduction, drug
sensitivity like Pyrazinamide, Cycloserine etc. Species identification is
required for epidemiologic and public health reasons only.
1. Morphology
• 0.5 to 10 μ long, slender, straight or slightly curved, non-motile, non-
sporing, non-capsulated, arranged singly or in groups , acid fast due to
mycolic acid (a long chain fatty acid) in cell wall, weakly gram positive due
to presence of high amount of lipids (upto 60% of cell wall).
• M. bovis are straighter, stouter and show more uniform staining.
2. Culture
• Organisms belonging to this group are considered slow growers
(generation time12-24 hours) and colonies are non-pigmented. Visible
colony usually appear in 2-3 weeks, optimum temperature 37 degree C,
pH 6.4- 7.0. They are fastidious and require enriched media like egg
based media, serine based media etc. for growth.
• Growth is enhanced by 5- 10%CO2.
• Both solid (for routine isolation) and liquid media (for drug sensitivity,
biochemical tests, Ag and vaccine preparation) are available.
9. A. Liquid media:
Bacteria grow faster, Eg. Middlebrook 7H9 ,7H12, 7H13 broth ,
where growth is enhanced by inclusion of biotin and catalase.
B. Solid media:
• Egg- based media, eg. Lowenstein-Jensen medium (L J
medium), Ogawa media, Petragnani, American Thoracic
Society Media, etc.
• The basic ingredient is inspissated egg.
• Each contains malachite green to suppress the growth of
Gram Positive bacteria.
10. • Rate of growth and pigmentation are the basis of categorizing mycobacteria.
• Rapid growers grow in less than 7 days
• Slow growers require more than 7 days
• Specimen is inoculated on Lowenstein – Jensen’s medium and incubated at 370C
for 2 – 8 weeks
• Colonies of Mycobacterium tuberculosis appear as buff coloured, dry, irregular
colonies with wrinkled surface and not easily emulsifiable (Buff, rough and tough
colonies)
• Colonies of Mycobacterium bovis appear creamy white to yellow colour with
smooth surface and are easily emulsifiable
M.Tuberculosis M.bovis
11. Components
LJ, modified Ogawaa Ogawa, modified
Kudohb
Monopotassium dihydrophosphate
(KH2PO4), anhydrous (buffer)
2.4 g 6 g 12 g
Magnesium sulfate (MgSO4 ·7H2O)
(buffer)
0.24 g –
Magnesium citrate (selective agent
of Mycobacteria)
0.6 g – 0.6 g
L-Asparagine (nitrogen source)
3.6 g –
Sodium glutamate (nitrogen source)
– 6 g 3 g
Distilled water up to
600 ml 600 ml 600 ml
Glycerol (ml) or pyruvate (carbon
source)
12 ml or 7.2 g 36 ml 24 ml
Egg homogenate (accelerating
factor, protein and fatty acid)
1000 ml 1200 ml 1200 ml
Malachite green (2%) (inhibitory to
contaminanats pH indicator)
20 ml 36 ml 24 ml
pH (about)
6.8 6.8 6.4
a This medium is cheaper than Löwenstein–Jensen because it is made without asparagine.
b Recommended media for direct inoculation of alkaline-decontaminated specimens.
Various modifications of these media with addition of antimicrobials, antifungal, RNA etc are available
to make the medium more selective.
12. • Agar-based media: Serum albumin agar media
such as Middlebrook 7H10 and 7H11
medium, are prepared from basal medium of
defined salts, vitamins, cofactors, glycerol,
malachite green and agar combined with
enrichment consisting oleic acid, bovine
albumin, glucose, etc.
13. Difference between M.tuberculosis and M. bovis
Characteristics M.Tuberculosis M.bovis
Morphology Long, slender, usually curved Short, stout, straight
Staining Barred or beaded Uniform
Growth on LJ medium Eugonic Dysgonic
Presence of glycerol in medium Enhances Inhibits
Presence of pyruvate in medium Inhibits Enhances
Colony characters Dry, rough, tough, raised and
wrinkled, creamy white or buff,
difficult to emulsify
Moist, smooth, flat,
white and friable
Biochemical reactions
Niacin test + -
Nitrate reduction + -
Animal pathogenicity
In guinea pig + (progressive and fatal disease + (similar to that of
M tuberculosis
In rabbit - or mild lesion + (generalized lesion)
14. 3. Staining
Acid fast in ZN stain due to presence of mycolic acid (N-
glycolyl muramic acid) and other cell wall superficial lipids
which comprise almost 60% of dry weight.
15. 4. Cell wall structure and components
Mycobacteria have a complex outer envelope having following distinct layers.
I. The innermost layer is the plasma membrane, which is a lipid bilayer
structure. Two proteins namely phosphatidylinositol mannosides (PIM,
a precursor of lipoarabinomannan), and lipoarabinomannan (LAM) are
inserted into it.
II. Next is the peptidoglycan layer, which determines the shape of the cell
and is similar to peptidoglycans of other gram-positive bacteria. It
contains repeating units of N-acetylglucosamine-(β1–4)-N-
glycolylmuramic acid cross-linked by tetra-peptides bridges.
III. About 10 percent of the N-glycolylmuramic acid residues are covalently
attached to a branched-chain polysaccharide, arabinogalactan, via
phosphodiester bonds.
IV. Distinct arabinose residues of the arabinogalactan molecules are
esterified to high-molecular-weight mycolic acids.
V. Finally, the outer surface of the mycobacterium is formed by the interlink
of medium-chain and short-chain lipids (mycosides) , glycolipids, and
peptidoglycolipids into the uneven hydrophobic layer of mycolic acids.
VI. Proteins (e.g. porins, transport proteins) are found throughout the
various layers.
17. 5. Antigens
i. Several components of the mycobacterial envelope are strongly
immunologically active. Tuberculins (old tuberculin): a crude
preparation of 6-8 week culture filtrate of M tuberculosis grown in 5%
glycerol media concentrated by evaporation and are highly antigenic.
Purified protein derivative, a purified preparation of the active
tuberculoprotein by precipitation with 50%ammonium sulphate.
ii. LAM: structurally similar to lipopolysachharide layer in Gram negative
bacteria
iii. Mycosides: heterogeneous group of biologically and immunologically
active medium- and short-chain lipids, present on the outer surface.
iv. Soluble antigens: These are the antigens found in the supernatant
following high speed centrifugation of a lysate of Mycobacteria. These
are: cytoplasmic and secreted proteins , and soluble carbohydrates. The
sharing of antigens by the various species accounts for the lack of
specificity of serological tests for tuberculosis.
18. 6. Virulent factors:
• No spore, no flagellum, no exotoxin, no endotoxin, no invasive
enzyme
a. Cell surface glycolipids: Permit intracellular survival of the bacteria.
Lipoarabinomannan (LAM) is a lipoglycan and a major virulent factor
with following functions.
i. It serves as a modulin with immunoregulatory and anti-
inflammatory effects.
ii. Inhibits T-cell proliferation and macrophage microbiocidal activity.
iii. Neutralizes cytotoxic oxygen free radicals produced by
macrophages.
19. b. Cording factor (glycolipid and
peptidoglycolipid derived mycolic acid, trehalose
dimycolate present on outer surface):
I. Causes bacilli to grow in culture in
serpentine cords (parallel arrangements of
bacilli)
II. Is anti-phagocytic and antigenic in nature.
III. Also responsible for inhibition of migration
of polymorphological leucocytes and elicits
granuloma formation.
c. Mutation is common (antibacterial resistance
can be easily achieved).
20. 4. Biochemical tests
i. Niacin accumulation test
• Niacin (nicotinic acid) is a precursor in biosynthesis of NAD and NADP.
• All species of Mycobacteria produce nicotinic acid. However, most
mycobacteria possess the enzyme (transphosphoribolysase) that
converts free niacin to niacin ribonucleotide. But 95% of M. tuberculosis
lack this enzyme . Due to this, large amount of Niacin accumulates in the
culture medium. Niacin is detected by addition of equal volumes of 10%
cyanogen bromide and 4% ethanolic aniline in aqueous extract of
culture.
• Reagent impregnated strips have eliminated the need to handle and
dispose of cyanogen bromide which is both caustic and toxic.
• Positive reaction – canary yellow
• M. tuberculosis – Positive
• M. bovis – Negative
• Positive Niacin test is also seen with M.chelonei and M.simiae.
Note: niacin test may be negative when performed on young culures
with few colonies. So it is recommended that the test be done in
cultures 3 to 4 weeks old.
21. ii. Nitrate reduction test
• M. tuberculosis produces an enzyme nitro reductase which reduces nitrate to
nitrite
• This is detected by colorimetric reaction.
•The reaction is detected by inoculating a nitrate broth with a loopful of bacterial
colony and alpha-naphthalamine and sulfanilic acid that will react with the
released NO2 to produce colour.
•Positive reaction – pink or red colour
•M. tuberculosis – Positive
•M. bovis – Negative
•M. kansasii, M. szulgai and M. fortuitum are also
positive.
22. iii. Resistance to 10µg/ml TCH (Thiophene - 2
- carboxylic acid hydrazide)
M. tuberculosis : resistant
M. bovis : susceptible
M. bovis
M. tuberculosis
Growth in presence of TCH
23. Iv. Arylsulfatase test: This test is positive in culture of atypical bacteria as they
form the enzyme arylsulphatase. Arylsulfatase breaks down
phenolphthalein disulfate into phenolphthalein (which forms a red color in
the presence of sodium bicarbonate) . 3 day arylsulfatase test is used to
identify potentially pathogenic rapid growers such as M. fortuitum and M.
chelonae. Slow growing M. marinum and M. szulgai are positive in the 14 day
arylsulfatase test.
M. Tuberculosis complex: Negative
v. Catalase test: Most of the atypical mycobacteria are strongly catalase
positive and peroxidase negative. M. tuberculosis and M.bovis, however, are
peroxidase positive and weakly catalase positive.
The reagent is prepared by mixing equal volume of 30% H2O2 and 0.2%
catechol in d/w. The reagent is added to 5ml test culture in phosphate buffer
(pH7) at 68 degree C in water bath and left for a few minutes.
Catalase production is indicated by bubble formation and peroxidase activity
is denoted by browning of colonies.
vi. Tellurite reduction test: The ability of Mycobacterial species to reduce
tellurite in 3 to 4 days is used to distinguish members of M. avium complex
from most other non-chromogenic species.
24. vii. Tween 80 hydrolysis test:
Tween 80 is the trade name of the detergent polyethylene derivative of sorbitan
mono-oleate. Some Mycobacterium species posses an enzyme-lipase, that splits
the compound and releases oleic acid and polyoxyethylated sorbitol.
Positive reactions occur in 1 to 4 days of incubation, and 10 days of incubation are
required for confirmation of negative reactions. The color change from orange
yellow to red is the positive test. Color change is not due to shift is pH but is due
to hydrolysis of Tween 80, which modifies the optical rotation of light passing
through the substrate.
Result: M. tuberculosis complex : negative
M. kansasii : Positive
Also useful to differentiate two similar appearing scotochromogens
M.gordonae (positive) and M.scrofulaceum(negative).
viii. Iron uptake: This is mainly used to identify M.fortuitum which when incubated
with 20% ferric ammonium citrate for upto 3 weeks on LJ media will turn into a
dark, rusty brown colour. M.tuberculosis complex: negative for iron uptake.
25. • Others
– Pyrazinamidase: M. tuberculosis : Positive, M. bovis: negative
– Urease: M.tuberculosis complex: positive
– 5% NaCl tolerance: M.tuberculosis complex: no growth
27. History:
One of the oldest infectious diseases (known at the time of Hippocrates in 400
BC and even in the Hindu Vedas termed rajayakshma), known to have caused
more suffering and death than any other infection..
The transmissible nature of TB was established by Villemin in 1868 by
inoculating rabbits with tuberculous material from humans and cattle. He also
established that scrofula (tuberculous lymphadenitis) and pulmonary TB were
manifestations of the same disease process.
In 1882, Robert Koch cultivated the bacillus on inspissated serum and
transmitted the disease to many animals of different species by inoculation with
pure cultures of the bacillus.
In addition to cultivating the causative organism, Koch succeeded in staining it
by treatment with an alkaline solution of methylene blue for 24 h.
Subsequently, Ehrlich improved the technique by using a solution of aniline
basic fuchsin followed by decolorization with a mineral acid, and it is this
technique, slightly modified by Ziehl and Neelsen whose names it bears, that is
still widely used today.
28. Epidemiology of disease
According to WHO (2015), 10.4 million people got infected and 1.8
million died from TB (including 0.4 million among HIV infected
people). Every year, more than eight million new cases occur.
Ninety-five percent of cases and 98 percent of deaths occur in low
income countries. It is a major cause of death of children, killing at
least 500 000 annually. In1993, the WHO took the unprecedented
step of declaring TB a ‘global emergency’.
Impact of HIV coexistence
HIV is the most important factor fueling a TB epidemic. The co-
existence of HIV and TB is known to be the most serious threat to
the human health and leads to a rapid development of AIDS as
explained by the following evidences:
• TNF-α and other immunological mediators released in TB lead to
transactivation of the HIV provirus and its subsequent replication.
• TB causes a CD4+ T-cell lymphopenia, which may synergize with
that induced by the HIV.
29. Mode of infection
• Direct inhalation of aerosolised bacilli contained in the
droplet usually of less than 5µm diameter of
expectorated sputum.
• Patients with sputum that is positive on direct
microscopic examination, and thus contains at least
5 000 bacilli/ ml, are the principal sources of infection.
• Only 10 % of non-immunocompromised infected
people eventually develop active/overt TB, 5 % within
the first 2 years following infection and remaining 5%
during lifetime.
• Infection also occurs infrequently by ingestion for
example, through infected milk or by inoculation in an
open wound.
30. Transmission of M. tuberculosis
Millions of tubercle bacilli in lungs (mainly in cavities)
Coughing projects droplet nuclei into the air that contain
tubercle bacilli.
One cough can release 3,000 droplet nuclei.
One sneeze can release tens of thousands of droplet nuclei
32. Primary tuberculosis
• Disease occurring in a person never previously exposed
to a tubercle bacillus.
• In endemic countries, it usually occurs in young children.
• The site of the initial infection is usually the lungs, but
sometimes it can be any organ such as tonsil, intestine or
skin. The bacilli engulfed by alveolar macrophages,
multiply and give rise to a sub-pleural focus of
tuberculous inflammation which is commonly located in
the lower lobe or lower part of the upper lobe to form
the initial lesion or Ghon focus, named after Anton
Ghon, an Austrian pathologist.
• Some bacilli are carried to the hilar lymphnodes through
macrophages, where additional foci of infection
develops. The Ghon focus, together with the enlarged
hilar lymph-nodes, form the primary complex.
33. This occurs for about 3-8 weeks from the time of infection and
is associated with the development of tuberculin
hypersensitivity. In the majority of the non-
immunocompromised cases, most of the cases are
asymptomatic and the lesion heals in about 4 months time
leaving a calcified nodule.
Careful search of lung tissue of adults may show healed
primary TB lesion. The bacilli in the lesion slowly die, however,
a few bacilli may survive even in the healed lesion and
remain latent for decades.
• In some people, particularly children under 3 years of age,
these foci progress to serious, even fatal, disease principally
involving the meninges, kidney, bones and pleurae. Foci
developing in the endothelium of major blood vessels may
rupture and give rise to widespread small granulomata, a
disease termed ‘miliary TB‘ (Latin: milium, a millet seed).
34.
35. Post-primary tuberculosis:
Post-primary TB develops in previously infected people either as a result of
progression of primary TB, endogenous reactivation of latent disease or of
exogenous re-infection. Reactivation usually occurs within 5 years after
primary infection.
The characteristic feature of such disease is extensive tissue necrosis. Very
large tumor-like lesions termed tuberculomas develop and, in common with
primary lesions, the conditions within them do not favor mycobacterial
growth. The necrotic tissue is softened and eventually liquefied by
macrophage-derived proteases and, if the lesion erodes , the liquefied
contents are discharged and a cavity is formed. In distinct contrast to closed
lesions, the well-oxygenated cavities are ideal environments for bacillary
replication, and their walls are lined by numerous bacilli.
Bacilli escaping from cavities may enter the sputum and be expectorated,
thereby infecting other people. They may also spread through the respiratory
tract and cause secondary lesions. On the other hand, spread of disease to
lymph nodes and more distant organs is uncommon in post-primary disease,
probably due to the obliteration of draining lymphatics and capillaries by the
tissue necrosis and subsequent deposition of scar tissue. The formation of
cavities and localization of the disease process characteristic of post-primary
TB are dependent on immune reactivity. Cavity formation is often limited or
absent in immunocompromised people, and dissemination of disease to many
organs frequently occurs in them.
36. Thus, TB in HIV-positive people with relatively limited immunosuppression
resembles that in HIV-negative people but, in the more profoundly
immunosuppressed, atypical forms of the disease are commonly seen
37. Pathogenesis and immunology
• Protective immune reactions and tissue necrotising hypersensitivity
(Delayed Type) are two aspects of immunlogy in TB.
• Both of these reactions are principally cell-mediated, relying on
macrophage activation and granuloma formation.
• Tubercle bacilli entering the tissues are taken up by macrophages. Entry
into macrophages is receptor mediated. Recent results showed that
heparin- or fibronectin-binding proteins, present on the bacterial surface,
play a role to facilitate their binding to epithelial cells or macrophages.
• Once inside the macrophage, the intracellular mycobacteria employ a
variety of survival strategies, which include:
(1) prevention of an oxidative burst in phagocytosing cells and inhibition of
phagosome-lysosome fusion.
(2) resistance to lysosomal enzymes and reactive oxygen intermediates (by
means of cell-wall lipids, including peptidoglycolipids (mycosides) and
LAM and secretion of superoxide dismutase; and
(3) escape from the phagosome into the cytoplasm.
38. • If the bacilli within macrophages are not destroyed, they replicate and kill the
cell. A local area of inflammation is thus established and more phagocytes are
attracted to the site by inflammatory mediators and cytokines.
• Some bacilli are transported, probably within phagocytes, to the regional
lymph nodes, where they are engulfed by antigen-presenting cells (APC).
Epitopes from mycobacteria lying within phagosomes within the APC are
presented on the cell surface by the major histocompatibility complex (MHC)
class II molecules to CD4+ helper T-cells.
• Helper T cells now undergo maturation and clonal proliferation into two
distinct cells, Th1 and Th2 which secrete cytokines like IFN-gamma,
interleukins 1 and 2, TNF-alpha, and others exerting different biological
effects.
• Th1 dependant cytokines activate macrophages resulting in protective
immunity and killing the organisms.
• Th2 mediated cytokines induce delayed type hypersensitivity, tissue
destruction, called tissue necrotizing hypersensitivity.
If the tubercle bacilli proliferate within the APC and escape from the phagosomes,
their epitopes are presented to CD8+ T-cells by MHC class I molecules. The
CD8+ lymphocyte population contains cytotoxic T-cells that are able to lyse any
cell presenting antigen in this manner.
39. Macrophage activation and granuloma formation
• A single human macrophage, though fully activated, is not capable of
killing tubercle bacilli.
• Hence aggregation of activated cells called granuloma, which is a
characteristic lesion of TB (and other chronic infections too) is formed. All
granulomas, regardless of cause, may contain additional cells and matrix
which include lymphocytes, neutrophils, eosinophils, multinucleated giant
cells (Langhan’s cells), fibroblasts and collagen (fibrosis).
• Microscopically, these activated macrophages resemble epithelial cells
and are thus termed epithelioid cells.
• Fusion of epitheloid cells is called multinucleate giant cell (Langhan’s cell)
• Entire granuloma kills bacteria more effectively than single macrophage.
The metabolically active macrophages in granuloma consume oxygen and
create hypoxia and anoxia leading to tissue necrosis producing cheesy
material (caseous material) and the formation of such substance is called
caseation.
• The anoxic and caseous centre creates an unfavorable environment and
many of the bacilli die out.
40. IMMUNOPATHOLOGY OF TB
M. tuberculosis
Macrophage
Class II MHC
Activated Macrophage
(Phagocytosis)
Bactericidal activity
T–Cell
Receptor
CD4+ T- Cell
CD8+ T- Cell
Delayed
Hypersensitivity
Class I MHC
Macrophage
Caseous Necrosis
41. Tubercle or granuloma formation in tuberculosis
ROI: Reactive Oxygen Intermedias
RNI: Reative Nitrogen Intermediaes
LT α : lymphotoxin α, also known as TNF beta
42.
43. When fully developed, tubercle/granuloma consists of 3 zones
i. A central area of large, multinucleated giant cells containing
tubercle bacilli.
ii. A mid zone of pale epitheloid cells, often arranged radially
iii. A peripheral zone of fibroblasts, eosinophils, neutrophils, lymphocytes and
monocytes
Later, peripheral fibrous tissue develops, and the central
area undergoes caseation necrosis.
It may subsequently heal by fibrosis or calcification
44. Characteristics Primary Postprimary
Local lesion Small Large
Cavity formation Rare Frequent
Lymphatic
involvement
Yes Minimal
Infectivity* Uncommon Usual
Local spread Uncommon Frequent
*Pulmonary cases
Differences between primary and post-
primary tuberculosis
45. Koch’s phenomenon
It explains the contrast between primary and secondary
tuberculosis.
When a normal guinea pig is injected with virulent tubercle bacilli,
the injection site heals quickly and there is no immediate visible
reaction. But after 10 – 14 days, a nodule appears at the site of
injection which ulcerates soon and persists till the animal dies due to
pulmonary tuberculosis after a few months. This is an example of
progressive tuberculosis. In this period, there is also presence of
regional lymphnode enlargement and caseous necrosis.
After 4 to 6 weeks of first injection, when the same animal is
injected with tubercle bacilli in different site, a dark indurated area
of about 1cm diameter appears at the site of injection in 1-2 days
which soon undergoes rapid necrosis to form shallow ulcer. This
ulcer heals up rapidly without involvement of regional lymph nodes
or tissue.
47. The highest priority for TB control is the
identification and cure of infectious cases i.e.
patients with sputum smears positive PTB.
Therefore, all patients with clinical features
suggestive of PTB must submit sputum for
diagnostic sputum smear microscopy.
Failure to diagnose TB may not only delay
appropriate therapy, but may lead to the spread
of TB in the community or the health care setting.
And the diagnosis of TB is based on the detection
of AFB in clinical specimens.
48. Specimen:
• Must be handled in a safe manner. M. tuberculosis has a low infective dose
for humans (infection rate of approx.50% with exposure to <10 bacilli).
• Specimens are collected in sterile, leak-proof, disposable, and
appropriately labeled containers and placed into bags to contain leakage
should it occur.
• CDC of US recommends BSL-2 practices for AFB smears and BSL-3 for
culture and other tests requiring bacteria propagation.
• Specimens should be sent promptly to the laboratory to avoid being
overgrown with organisms other than mycobacteria.
• If delay is unavoidable, specimens should be stored in the refrigerator.
Specimens sent through the post in warm weather should be packed in
dry ice or with an ice pack.
49. Specimen collection and transport:
1. Pulmonary specimens:
May be collected by following methods:
Spontaneously produced or induced Sputum, Gastric Lavage, Broncho
Alveolar Lavage, Transtracheal Aspiration, Laryngeal Swab.
• Sputum: To raise sputum, patients must be instructed to take a deep
breath, hold it momentarily, and then cough deeply and vigorously. Ten
milliliters of sputum should be collected. Saline-induced sputum
specimens is collected from children as young as 5 years of age.
• Gastric lavage: Used to collect sputum from patients who may have
swallowed sputum during the night. The procedure is limited to senile,
non-ambulatory patients, children younger than three years of age, and
patients who fail to produce sputum by saline induction.
50. 2. Urine specimen:
• About 2% to 3% of patients with pulmonary tuberculosis show urinary tract
involvement/ renal tuberculosis.
• Using clean-catch collection technique, early morning voided urine specimens
(40 mL minimum) in sterile containers should be submitted daily for at least 3
days.
• The 24-hour urine specimen is undesirable because of excessive dilution,
higher contamination, and difficulty in concentrating.
3. Faecal specimen:
• In patients with AIDS.
• Clinical utility of this practice, remains controversial
4. Tissue and body fluid: CSF for tuberculous meningitis,
5. Blood specimen: Immunocompromised patients particularly HIV can have
disseminated tuberculosis. Conventional methods are unacceptable,
specialized automated systems are available for growth of Mycobacterium
spp., including the Bactec MGIT 960 system (Becton-Dickinson, Franklin Lakes,
N.J.), and the BacT/ALERT 3D (Biomerieux,Durham, N.C.).
51. Specimen processing
• While processing the samples especially for cultures, specimen from the
sterile sites can be inoculated directly or after concentration into the
media.
• But when the sample like sputum that has organic debris, such as mucin,
tissue, serum,and other proteinaceous material contaminated with other
organisms. has to be cultured for tubercle bacilli, then lab must process
such specimen to kill or reduce contaminating bacteria, by a method
called decontamination, and also should dissolve the mucin that trap
tubercle bacilli by mucolysis/digestion and finally should lead to
concentration of the sample.
• The digestion-decontamination procedures must be as gentle as possible
in order to avoid killing of tubercle bacillus. Various methods are available
for this purpose.
52. a. Petroff’s method:
• Simple and widely used
• Sputum mixed with equal volume of 4% NaOH and incubated at 37 C for
30 min. Mixture is then frequently shaken till it gets completely liquefied.
NaOH acts as liquefying and also kills contaminating bacteria. The
mixture is then centrifuged for 30 min at 3,000rpm, neutralised with 8%
HCl in presence of a drop of phenol red indicator. The deposit is used for
culture and animal inoculation if needed.
b. Homogenization method:
• Specimen treated with equal volume of dilute acids such as 6%H2SO4
and then clearing the acids by repeated washing with sterile normal
saline.
54. Lab. diagnosis of pulmonary tuberculosis
A PTB suspect should submit three sputum samples (1st spot, 2nd early
morning, 3rd spot) for microscopy. Nowadays , a two sample concept is
being used. 1st early morning sample for microscopy, 2nd spot sample for
microscopy/PCR (gene xpert).
Secretions build up in the airways overnight. So an early morning sputum
sample is more likely to contain TB bacilli than one taken later in the day.
Sputum specimen must be free of food particles, residues and other
extraneous matter.
Saliva and nasal secretions are not to be collected nor should the patient
use oral antiseptics during the period of collection
55. 1. Sputum smears stained by Z-N stain
Advantage: - Cheap – rapid
- Easy to perform
- Sensitivity > 90%
- Specificity of 98%
Disadvantages:
- Sputum ( need to contain 5000-10000 AFB/ ml.)
- Young children, elderly & HIV infected persons may not produce
cavities & sputum containing AFB.
56. Interpretation of sputum stained by
Z N Stain (WHO )
More than 10 bacilli / field ------- +++
From 1 – 10 bacilli / field ------- ++
From 10 – 99 bacilli / 100 fields ----- +
From 1 -9 bacilli/100 fields ------ write the number
No bacilli seen in 300 fields ---------- negative
57. 2. Detecting AFB by fluorochrome stain using
fluorescence microscopy:
The smear may be stained by auramine-O dye. In this method
the TB bacilli are stained yellow against dark background &
easily visualized using florescent microscope.
Advantages:
- More sensitive
- Rapid
Disadvantages:
- Hazards of dye toxicity
- More expensive
- Must be confirmed by Z-N stain
58. a. Cultures on L J media
Lowenstein –Jensen medium is an egg based media with addition
of salts, glycerol, malachite green & various antibiotics
Advantages:
- Specificity about 99 %.
- More sensitive (lower no. of bacilli 10-100 / ml)
- Can differentiate between TB complex & NTM using
biochemical reactions.
- Source of bacteria for sensitivity tests.
Disadvantages:
- Slowly growing ( up to 8 weeks )
3. Culture of M. tuberculosis
59. b. Culture on Middlebrook Agar Base 7H10 Media
• Defined salts, Vitamins and Cofactors, Oleic acid, Albumin,
Catalase, Glycerol, Dextrose, Malachite Green (0.0025g/100
mL)
c. Culture on Middlebrook Agar Base 7H11 Medium
• Same composition as Middlebrook 7H10 except 0.1% casein
hydrolysate added for enhanced recovery of fastidious
isoniazid-resistant Mycobacterium tuberculosis
• Modified 7H11 contains carbenicillin, amphotericin B,
polymixin B, and trimethroprim to inhibit oropharyngeal
commensals.
60. d. Growth rate and growth at 25°C and 42°C
• Tubercle bacilli grow slowly compared to other non-
pathogenic acid-fast organisms, taking more than 7 days to
appear on culture media.
• M. tuberculosis complex do not grow at 25°C and 42°C.
• The optimum temperature for the growth of these
mycobacteria is 37°C.
61. e. Growth on medium containing p-nitrobenzoic acid (PNB)
• Para-nitrobenzoic acid has been used for the selective
screening of M. tuberculosis.
• Human and bovine type of tubercle bacilli can be
differentiated from all other mycobacteria in their inability to
grow in L-J medium containing PNB.
63. 5. Recent Methods for Diagnosis
a. BACTEC 460 ( rapid radiometric culture system)
Specimens are cultured in a liquid medium (Middle brook7H9
broth base )containing C14 – labeled palmitic acid & PANTA
antibiotic mixture.
Growing mycobacteria utilize the acid, releasing radioactive CO2
which is measured as growth index (GI) in the BACTEC instrument.
64. The PANTA antibiotic mixture
P ---- Polymyxin B
A ---- Amphotericin B
N ---- Nalidixic acid
T ---- Trimethoprim
A ---- Azlocillin
The antibiotic mixture inhibits the growth of contaminating
bacteria.
65. Advantages
- Rapid (mycobacteria can be detected within 12 days.)
- To determine drug susceptibility .
- To differentiate between TB complex & NTM by NAP test.
- This method is based on the selective growth inhibition of the
TB complex in the presence of p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP).
- Specificity is very high
Disadvantages:
- Expensive
- Hazards of using radioactive material.
66. b. Mycobacteria Growth Indicator Tube (MGIT)
• Tube contains modified Middlebrook 7H9 broth base with
OADC enrichment & PANTA antibiotic mixture.
• All types of clinical specimens, pulmonary as well as extra-
pulmonary can be cultured on this type of media.
67. The OADC supplement
O: Oleic acid ( Metabolic stimulant)
A: Albumin ( to bind toxic free fatty acid )
D: Dextrose (Energy source )
C: Catalase (Destroys toxic peroxides that may be present in the
medium).
68. Principle:
• A fluorescent compound (which is sensitive to O2 eg. an
oxygen quenched fluorochrome like Tris 4,7-diphenyl-1,10
phenonthroline ruthenium chloride pentahydrate) is
embedded in silicone on the bottom of the tube.
• The actively respiring microorganisms consume the oxygen &
allow the fluorescence to be observed using UV trans-
illuminator lamp.
69. MGIT 960 System
• The MGIT 960 system is an automated system that uses the
MGIT media & sensors to detect the fluorescence.
Advantages:
The system holds 960 plastic tubes which are continuously
monitored.
Early detection as the machine monitoring & reading the
tubes every hour.
70. c. Polymerase Chain Reaction (PCR)
Both conventional and real time PCR methods have been devised.
Nucleic acid probes & nucleic acid amplification tests in which polymerase
enzymes are used to amplify ( make many copies of specific DNA or
RNA sequences extracted from mycobacterial cells.
Advantages:
- Rapid procedure ( 3 – 4 hours)
- High sensitivity (1-10 bacilli / ml sputum)
Disadvantages:
Very expensive.
- Require specialist training & equipments.
- False positive results.
- Can not differentiate between living & dead bacilli.
71. d. FASTplaque TB Test
-Patient’ s sputum is mixed with myco-bacteriophage.
- A virucide (30 mM ferrous ammonium sulfate )is added which
destroys any phages outside the TB bacilli.
- Lysis of cells & release of phages after replication within the tubercle
bacilli.
- Non-pathogenic mycobacteria are added & the sample incorporated
in agar mixture( over night incubation)
- Zones of clearing indicate that patient’ s sputum contained viable M.
tuberculosis.
72. The MGIT 960 System
The MGIT 960 system is a non-radiometeric
automated system that uses the MGIT media
& sensors to detect the fluorescence.
Advantages:
-The system holds 960 plastic tubes which are
continuously monitored.
- Early detection as the machine monitoring &
reading the tubes every hour.
73. e. Interferon –γ Tests
An in vitro T-cell-based assays tests for
diagnosis of latent TB infection :
These whole-blood assays measure IFN-γ
production by previously sensitised
lymphocytes in response to M.tuberculosis-
specific protein antigens (Early Secreted
Antigen Target)ESAT-6 and (Culture Filtrate
Protein)CFP-10.
74. g. MycoDot antibody test
• It is dipstick test, manufactured by Mossman
associates. Mycodot uses purified lipoarabinomannans
(LAMs) as antigen (highly immunogenic
lipopolysaccharide found in the cell wall of all
mycobacteria).
• Mycodot has been designed for diagnostic use, i.e. it
detects anti-mycobacterial antibody levels likely to be
found in those with active disease, i.e. high levels.
• A mycodot test should therefore only be preformed on
those with suspected TB (it is not a screening test).
• A positive test indicates active TB.
75. h. AccuProbe
Acridinium ester-labeled DNA probes are utilized that
hybridize to Mycobacterium tuberculosis complex-specific 16S
rRNA.
Target 16S rRNA released by sonication of organisms from
culture.
Acridinium ester is chemiluminescent, and DNA probe-16S
rRNA hybrids emit light when acridinium ester hydrolyzed to
ground state by alkaline peroxidation
Chemiluminescence measured in a luminometer
Amount of light emitted proportional to amount of DNA-RNA
hybrids formed
Total time for AccuProbe test is 2 hours
76. Antimicrobial susceptibility testing
(AST)
• Standard conventional diffusion techniques not suitable
due to growth rate.
• CDC recommends in vitro susceptibility testing on all M.
tuberculosis isolates.
• Poor clinical outcomes are predicted with an agent when
more than 1% of bacilli in a test population are resistant.
• Susceptibility testing is used to determine the resistance
of strains isolated before the commencement of
treatment (initial or primary resistance); or to discover
whether resistance has arisen during treatment
(secondary resistance).
77. • Initial isolates of M.tuberculosis are tested against five
antimicrobials, which are referred to as primary (essential)
drugs. The five primary drugs are Isoniazid (H), Rifampicin
(R), Pyrazinamide (Z), Ethambutol (E), Streptomycin (S).
78. • Isoniazid kills 90% of the total population of bacilli during the first few days of
treatment. It is most effective against the metabolically active, continuously
growing bacilli. RIFAMPICIN can kill the semi-dormant bacilli which isoniazid
cannot. PYRAZINAMIDE kills bacilli in an acid environment inside cells, e.g.
macrophages.
79. • Multidrug-resistant M. tuberculosis (MDR-TB) are those bacilli
resistant to both isoniazid and rifampicin as these two drugs
are two primary components for the treatment .
• Second line drugs are used for treatment of MDR-TB. Second
line drugs are ethionamide, cycloserine, kanamycin,
capreomycin etc.
• Extensively drug resistant (XDR-TB) are those that are not
only resistant to rifampicin and isoniazid, but also to some
second line drugs.
80. Direct vs. Indirect susceptibility
testing
• Susceptibilities may be performed by either the
direct method or the indirect method.
• The direct method uses as inoculum, a smear
positive concentrate containing more than 50 AFB
/100 OIF; the indirect method uses a culture as the
inoculum source.
• Although, the direct testing provides earlier rapid
results, the results may be unsatisfactory because of
contamination or low numbers of colony-forming
units.
• So, direct method is less standardized
81. Conventional methods
• Different conventional methods used for
determination of antimicrobial susceptibility
pattern are
– resistance ratio
– proportion
– absolute concentration
– disc diffusion methods
82. a. Resistance ratio method (RR
method)
• The RR method is performed either on solid
media (egg or Middlebrook agar) or in liquid
media (Middlebrook or Dubos broth).
• This method compares the resistance (MIC) of
unknown strains of tubercle bacilli (test
organism) with that of a standard laboratory
strain H37Rv, or preferably three wild strains,
taking their modal resistance as control.
83. b. Proportion method (PR method)
• It is a quantitative test, in which several dilutions of
standardized inoculum are inoculated onto control
(drug free) and drug containing agar medium.
• The decrease of growth in drug-containing media and
drug-free media are compared.
• If growth at the critical concentration of a drug is more
than 1%, the isolate is considered clinically resistant.
• The critical concentration of a drug is the amount of
the drug required to prevent growth above the 1%
threshold of the test population of tubercle bacilli.
84. c. Absolute concentration method
• This method is used in the USA and parts of
Europe to determine test strain MICs. The
tests are usually performed on Middlebrook’s
7H10 medium.
85. d. Disc diffusion method
• This method is possible if the mycobacterium
is a “rapid grower”.
• Agar plates are flood-seeded with the test
organism, antibiotic discs are applied.
86. Modern methods
• Faster, more reliable, easier.
a. Bactec®
• Radiometric method is commonly used
• Uses proportion method principle
• Growth indicated by the rate and amount of CO2 produced
• Resistance: Growth rate at the critical concentration of a drug is > 1%
b. Bactec® MGIT system
c. Molecular methods: example, hybridization-based probe assay for
detection of rifampicin-resistant gene (rpoB) and its mutations leading to
rifampicin resistance. Molecular methods have also been developed for
identification of resistance to other drugs.
d. Luciferase-reporter mycobacteriophage test: only viable bacteria can
become infected, it is an assay based on chemiluminescence.
87. Other names of MOTT
• Nontuberculous Mycobacteria (NTM), Anonymous, Atypical,
Unclassified, Unknown, Tuberculoid, Environmental, Opportunistic
NTM are present everywhere in the environment and are also present as
normal flora in healthy human skin, GI tract, UR tract etc. Opportunistic
pathogens generally in immune suppression.
Risk factors for NTM infection are: lowered CMI, COPD
120 different species omnipresent in our environment.
Human pathogenicity varies for different species.
Infections acquired from an environmental reservoir of organisms.
Mycobacteria Other Than Tuberculosis (MOTT)
88. There are three typical forms of NTM infection
: a tuberculosis-like pattern observed mainly among older men with COPD
: presentation with nodules seen in slender older ladies known as the
‘Lady Windermere syndrome’.
: hypersensitivity associated with exposure to water-containing systems
such as hot tubs or baths.
Many patients, however, will have more varied or mixed presentations
that do not fit into these prototypic categories
Ernst Runyon in 1959, classified MOTT in four groups on the basis of
pigment production and growth rate.
89.
90. Runyon Classification of MOTT Organisms:
Pigment Production
Photochromogens (Runyon group I)
Produce non-pigmented colonies when grown in the dark and
pigmented colonies only after exposure to light.
Scotochromogens (Runyon group II)
Produce deep yellow to orange pigmented colonies when grown
in either the light or dark (some strains show increased pigment
production on continuous exposure to light).
Nonchromogens (Runyon group III)
Non-pigmented in both the light and dark or have only a pale
yellow, tan, or buff pigment that does not intensify with light
exposure .
91. Detection of Pigment Production
• Three LJ slants are inoculated with organism.
• Two slants are completely shielded from light with cardboard
tube or aluminum foil.
• When growth is detected in unshielded tube, growth is
examined in one of the shielded tubes.
• If colonies are not pigmented, then the tube is exposed to
light (100-W tungsten bulb for 5 hr) with cap loosened
(maximal oxygenation required for pigment production).
• Tube is reshielded and incubated for 24 hr.
• Colonies in the light-exposed tube and non-exposed tube are
compared for pigmentation.
92. Runyon Classification of MOTT Organisms:
Rate of Growth
• Rapid Growers (Rounyon group IV)
Mycobacteria forming colonies within 7 days are termed rapid
growers, those requiring longer periods are termed slow growers
(Runyon group I, II and III). Unlike other mycobacteria, they can be
grown on routine bacteriologic media.
Determination of growth rate
• Inoculate well isolated colony of organism to 7H9 broth containing
Tween 80
• Incubate broth for several days until medium is faintly turbid.
• Dilute broth 1:100, streak inoculate to Middlebrook 7H10 agar
plate. Observe cultures at 5 to 7 days and (if no growth) weekly
thereafter for visible colonies
93.
94. Group I: Photochromogens
• Slow grower (2-3 weeks). Grow at 25oC to 41oC.
• Pigment is chemically beta-carotene in nature.
• Mycobacterium kansasii: Causes pulmonary disease as well as
skin and lymphadenitis. Progression is slow.
Detection: yellow pigment, rapid hydrolysis of tween 80, Nitrate
reduction is strong, rapid catalase reaction, strong pyrazinamidase
activity. Confirmation can be done by AccuProbe test.
• M. marinum: Causes granulomatous skin disease ( swimming pool
granuloma). Optimal growth at lower temperature 30oC-33oC, Niacin
–/+ , nitrate –, Tween 80 + (24 hr) (97%).
95. Group II: Scotochromogens
• M. scrofulaceum: Causes chronic cervical lymphadenitis
(scrofula) in children. Growth at various temperature (25oC, 31oC,
37oC). Tween 80 –/+ (2%), nitrate reduction –/+ (5%), urease –/+
(31%)
• M. xenopi: optimal growth at 42oC, Tween 80 + (97%), nitrate
reduction –, urease –
• M. szulgai: scotochromogen at 37oC, photochromogen at 24oC,
Tween 80 –/+ (49%), nitrate reduction +, urease + (72%)
• Mycobacterium gordonae: detection by AccuProbe method
96. MOTT Organisms: Identificaton of
Nonchromogens
• Do not form pigment even on exposure to light.
• Mycobacterium avium-intercellulare complex (MAI complex): M.
intracellulare is closely relatd to M.avium. Pigmentation (-), growth
rate at 300 C (+), nitrate reduction (-), Tween 80 hydrolysis(+),
urease (-), Niacin (-). Infection by nonchromogens other than M.
avium complex is infrequent.
98. MOTT Organisms: Identificaton of Rapid
Growers
• Mycobacterium fortuitum group:
arylsulfatase + (97%), nitrate reduction +,
iron uptake +
• M. chelonae and M. abscessus: arylsulfatase
+ (95% and 100%), nitrate reduction –/+ (1%)
and –, iron uptake –
1Standard reactions: arylsulfatase, nitrate
reduction, iron uptake
99. Molecular Identification of MOTT
Organisms
• High performance liquid chromatography
(HPLC) analysis of cell wall mycolic acid esters
• 16S rRNA gene sequencing
100. Treatment of MOTT
Surgical treatment
Medical treatment:
• first-line drugs used for tuberculosis, macrolides,
streptomycin
• The duration of treatment should cover at least 12 months
following conversion to a negative culture.
• One example of regimen includes a rifamycin (rifampin or
rifabutin), ethambutol, and a macrolide administered for 18–
24 months, including 12 months of sputum culture negativity
