1. Mycobacteria are acid-fast, aerobic bacteria that include pathogens like Mycobacterium tuberculosis, M. leprae, and M. bovis. They are classified based on growth rate and pigmentation. 2. M. tuberculosis is the cause of tuberculosis and can infect the lungs and other organs. Atypical mycobacteria like M. kansasii can also cause pulmonary infections. 3. Laboratory diagnosis of tuberculosis involves microscopy, culture, and drug sensitivity testing of respiratory specimens. Leprosy is diagnosed by microscopy of skin and nerve tissue samples.

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COURSE: MLSM 505
LECTURE TOPIC:
MYCOBACTERIA

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Characteristics:
The genus Mycobacterium consists of numerous
species some of which are highly infectious.
Generally, they are slender, slightly curved rods,
non motile, poorly Gram stained positive, acid fast
and aerobic
The acid fastness is due to their possession of thick
cell wall made up of waxes and lipids with high
content of mycolic acids..

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They grow on protein rich culture media.
Improved growth is achieved in the presence of
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CO2
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Type species: Mycobacteria tuberculosis (aka
Tubercle bacillus)
Classification: Classification into 2 major
groups is based on rate and temperature of
growth, and pigmentation:
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1. Rapid growers and slow growers
2. Pigmented(chromogenic) and non-pigmented
(non chromogenic).
The pigment is yellowish.
Other characteristics are also used to classify them
into typical and atypical Mycobacteria

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Rate of growth: Most grow between 6 – 8
weeks. M.ulcerans is the slowest grower
Temp. of growth: Most species will grow at
35 – 370C, others may grow at lower or higher
temperature e.g. M. chlonei and M. fortitum
grow at 30-32°C while M. xenopi can grow at
44°C.
Mycobacteria were once classified according
to Ruyon classification which some workers
adopted.

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Pigmentation: Photochromgens – these produce
colour in light (Ruyon group 1): M. kansasii, M.
marinum, M. simiae.
Scotochromogens – these produce colour in the
dark (Ruyon group 11): M. scrofulaceum, M.
szulgai.
Non-chromogens – non pigmented (Ruyon group
111): M. tuberculosis, M. bovis, M. fortuitum, M.
chelonei.
Rapid growers (Ruyon group IV): Mainly
saprophytic

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PATHOGENICITY
There are more than 50 separate species of
Mycobacteria:
Eighty percent of all diseases is respiratory.
However, lymph nodes, bone and other organs may
be involved.
M. tuberculosis: causes tuberculosis(Tb) which
may be pulmonary or non pulmonary.

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Pulmonary tuberculosis is a slowly progressive
respiratory disease.
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Complications of pulmonary Tb include pleural
effusion, lung collapse, acute miliary Tb,
meningitis.
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Non pulmonary Tb include renal and urogenital Tb,
miliary Tb and Tb meningitis.
The organism is carried through blood stream to
reach and infect various organs.
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M. africanum: causes a milder form of human
tuberculosis
M. bovis: causes cattle tuberculosis; humans can
be infected by close contact with the animals or by
ingestion of contaminated animal product.
The above three species are also referred to as M.
tuberculosis complex.
M. leprae: causes leprosy, a skin infection.

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ATYPICAL MYCOBACTERIA (MOTT=
Mycobacteria other than tuberculosis)
These may grow rapidly at 25°C or 45°C, several
species are pigmented. Usually acquired from soil,
water, birds or animals.
M. kansasii. M. szulgai. M. simiae, and M. xenopi:
all of these cause pulmonary infections.
M. avium-intracellulare and M. scrofulaceum:
cause cervical adenitis
.

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M. fortuitum, M. chelonei and M. marinum:
cause skin infection.
M. ulcerans: cause skin ulcers (Buruli ulcer).
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Tuberculosis is a major killer infectious disease
world wide.
•
It is symptomised by long lasting ( chronic )
cough which may be productive ( purulent or
muco-purulent). The sputum may be blood
stained (haemoptysis).
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Other signs include weight loss, fever, tiredness
and night sweat. HIV-aids patients are now most at
risk (co-infection).
And the multiple drug resistance (MDR) nature of
the organism makes it very difficult to eradicate.
Tuberculosis today causes more morbidity and
greater mortality than any other infectious disease
except HIV-AIDS. It can co-exist with HIV-
AIDS.
Good nutrition and a well ventilated place of abode
can minimize the chances of contracting TB.

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Pathogenesis and transmission of tuberculosis
Infection acquired by inhalation of M. tuberculosis
in aerosol from infected person and dust, goes to
the lungs; and from there spreads to other organs.
Source of infection is another human case –
proximity, sharing utensils etc.
The bacteria are intra cellular and so can survive
and multiply in the macrophages of lung tissues.
Skin infection is acquired by inoculation.

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Inhalation: M. tuberculosis, M. leprae
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Ingestion: M. bovis
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Inoculation: M. ulcerans
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Primary tuberculosis is when contracted by an
individual for the first time.
It results in local infection of the regional lymph
nodes.
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Initially a mild and asymptomatic condtion: about
90% may not proceed further, but a few others may
become serious and disseminated through the
lymphatics to cause infections such as miliary Tb,
bronchopneumonia, meningitis, bone and joints
Tb.
Local lesion on the periphery of the lung is called
Ghon focus.
Post primary Tb: Asymptomatic as latent
infection that could be re activated.

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LABORATORY DIAGNOSIS
Specimens: Sputum – preferably early morning
samples for three consecutive days; gastric lavage
from children, early morning urine specimens for
three consecutive days for renal/urogenital Tb.,
pus.
The new national algorithm recommends 3
samples produced as follows:
First: On the spot i.e. in the laboratory
Second: Early morning sputum from home.
Third: On the spot i.e. in the laboratory

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Microscopy:
Auramine stain – used for screening
•
ZN stain – used to confirm an auramine +ve
smear.
Always include a control smear for each batch of
staining.
Report as + when 3-9 AFBs are seen in the whole
smear, ++ when 10 or more AFBs are seen in the
whole smear and +++ when 10 or more AFBs are
seen in several fields.
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Culture:
Sputum may be homogenized and decontaminated
before culture.
This is to get rid of commensals in the specimens.
Inoculate a pair of LJ glycerol medium and
incubate for 6-8 weeks.
With the automated machine like the Bactec 460
TB machine, growth may appear within ten days.
.

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Other useful culture media for mycobacteria are LJ
pyruvate medium (M. bovis), Dorset egg medium for
maintenance of cultures, Middlebrook 7H10 and
Middlebrook 7H9 broth for identification and
cultivation
Growth:
Colonies are rough and buff coloured.
A slow growing AFB is suggestive of M.
tuberculosis.

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Identification:
Identify and confirm with standard identification
tests such as colonial morphology on glycerol and
pyruvate media, sensitivity to pyrazinamide and
niacin production.
Animal inoculation : M. tuberculosis and M. bovis
are pathogenic to laboratory animals. Guinea pig can
be inoculated to confirm the infection.
PCR: This new technique is very useful for the
diagnosis of mycobacterial infection but it is
expensive.

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Sensitivity Test:
Special methods are used to test the organism
against the relevant drugs. The common drugs used
for the treatment are isoniazid, Rifampicin,
Pyrazinamide(PAS), Streptomicin, and ethambutol.
M. tuberculosis: Hypersensitivity.
·
Tuberculin test: Test for present or past immune
response/hypersensitivity Mantoux test using PPD

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BCG( Bacillus of Calmette and Guerin) vaccine
confers immunity on recipient especially young
children. The vaccine is an attenuated strain of M.
bovis.
LEPROSY:
A chronic disease of skin and nerve caused by M.
leprae. Mainly in the tropics. Long incubation period
of 3 to 5 years.
Skin lesions present as nodules, thickened patches,
thickened peripheral nerves and anaesthesia with
muscle atrophy ulceration. Great disfigurement.

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There are 2 main forms of leprosy and some
others in between based on the delayed
hypersensitivity reaction to infection:
Tuberculoid: a localized form affecting mainly
the skin found in partially immune individuals.
Lepromatous: a generalized form that destroys
tissues, usually in non immune persons.
The infection is slow and subclinical, spread by
respiratory tract droplets, especially the
lepromatous form.

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Laboratory Diagnosis
Specimens: Tissue juice, nasal swab, skin scraping.
Skin smear is best from active part of skin lesions –
2 ear lobes, back of arms or front thighs above the
knees. Specimens are decontaminated before it is
concentrated by centrifugation.
Microscopy: Non motile, slightly curved. Acid fast
with 5% H2SO4 or 1% acid alcohol. In skin tissue,
the bacteria are packed together in large
numbers.

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Morphological index (MI)
The smear usually shows a mixture of well stained (
viable) and poorly stained (non viable) bacilli. The
MI is the percentage of viable organisms and it is
used to monitor treatment.
Culture
Does not grow on artificial medium.
Can be cultured on the foot pad of 9 banded
armadillo or mouse or rat.

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Treatment
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This is effected with the usual anti Tb drugs.
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Dapsone is a popular drug for the treatment of
leprosy. Side effects are however, an issue with
this drug.
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