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Mycobacterium
1. Dr. Sumesh Kumar Dash
Department of Microbiology,
IMS & SUM HOSPITAL
MYCOBACTERIA
2. INTRODUCTION
• Mycobacteria are slender rods, weakly
gram positive, obligatory aerobic,
nonmotile, non-capsulated, non-sporing,
usually having slow growth.
• They sometimes show branching,
filamentous forms resembling fungal
mycelium.
• They are acid fast bacilli.
4. CLASIFICATION
• Mycobacterium tuberculosis complex (MTB):-
M.tuberculosis - MCC of TB in human
M.bovis – Bovine tubercular bacilli
Other – Less common pathogen.
e.g: M. africanum, M. microti, M. canetti,
M. caprae,M. pinnipeddi .
• Mycobacterium leprae
• Non-tubercular mycobacteria (NTM):- Mostly saprophytic
in nature or may be found commensal of human or animal.
Very few like M.kansasii, M.fortuitum, M.chelonae can cause
human infection.
6. MORPHOLOGY
• M. tuberculosis is a straight or slightly curved rod
whereas M.bovis is straight and short.
• 3 x 0.3 μm in size.
• They can occur singly, in pairs or as small
clumps.
• They are gram positive, filamentous, club shaped
and branching.
• They are acid fast: Resist decolorization by dilute
acid due to high concentration of Mycolic acid
present in cell wall.
7. PATHOGENESIS
Source of infection
1. Human: Pulmonary TB
2. Bovine source: Consumption of unpasteurized
infected milk
Mode of transition
1. Inhalation: Droplet infection
2. Inoculation: Direct contact
3. Ingestion: Swallowing of sputum, Infected milk
9. Pulmonary Tuberculosis (PTB)
• PTB accounts for 80% of all cases of TB.
FEATURES PRIMARY TB SECONDARY TB
RESULT Initial exogeneous infection with
bacilli
• Exogeneous reinfection
• Reactivation of latent
reinfection
AGE Children Adult
Affected part Lower lobe Upper lobe
Lesion Fibrotic
(Ghon focus)
Calcified
(Simon’s focus)
C/F Asymptomatic or Present with
fever, productive cough, chest
pain, wt loss
Lesions under go necrosis
& tissue destruction leading
to cavity formation.
FATE Lesions heal spontaneously Necrotic lesion breaks
leading to bronchogenic or
hematogenous spread.
10. Extrapulmonary Tuberculosis (EPTB)
EPTB results from hematogenous spread of
bacilli to various organ. It is constitute 15 – 20%.
• Tuberculous lymphadenitis
• Pleural Tuberculosis
• Tuberculosis of upper airway
• Genitourinal Tuberculosis
• Skeletal Tuberculosis
• Tuberculosis of CNS
• Gastrointestinal Tuberculosis
• Tuberculous skin lesion
11. HIV Associated Tuberculosis
• TB is most common diseases in HIV infected
patients as they have low CMI.
• TB occur 70-80% of HIV cases.
• EPTB is more common in these patients.
12. LABORATORY DIAGONOSIS
Specimen collection
• In pulmonary TB: Sputum (2 specimens: spot and early morning), gastric
aspirate (in children)
• In EPTB: Specimens vary depending on the site involved.
Direct microscopy by acid-fast staining
• Ziehl-Neelsen (ZN) technique - long slender, beaded, less uniformly
stained red color acid fast bacilli
• Kinyoun's cold acid fast staining (M.leprae)
• Fluorescent (Auramine)staining
it is more sensitive and
smears can be screened more
rapidly than ZN stain.
13. Digestion, Decontamination and Concentration of specimen
• Modified Petroff's method (4% NaOH)
• NALC (N-acetyl-L-cysteine) +2% NaOH
Conventional culture media (6-8 weeks)
• Sold media - Lowenstein Jensen (LJ) medium
( Shows rough tough and buff colored colonies)
• Liquid media - Kirdchner's medium
Middlebrook 7H9 medium
14. Automated culture methods (3-4 week)
• MGIT System: Detects growth and resistance to antitubercular
drugs (ATDs); with a turnaround time of 2-3 weeks
• BacT/ALERT: Detects growth
• Versatek system: Detects growth and resistance to ATDs.
Culture identification
• MPT 64 antigen detection
• Biochemical tests:
• Niacin test.
• Sensitive to paranitrobenzoic acid
• Catalase peroxidase test
• Aryl sulphatase test
16. RNTCP
• Revised National Tuberculosis Control Program of India has given a
guideline for grading of ZN stained sputum smear.
• However, grading is depend on quality of sputum.
NO. OF AFB SEEN OIF TO BE SEEN GRADING RESULT
No AFB in 100 OIF 100 0 Negative
1-9/100 OIF 100 Scanty Positive
10-99/100 OIF 100 1+ Positive
1-10/ OIF 50 2+ Positive
>10/ OIF 20 3+ Positive
17. LATENT TUBERCULOSIS
• Latent TB occurs when a person has the TB bacteria
within their body, but the bacteria are present in very
small numbers.
• They are kept under control by the body's immune
system and do not cause any symptoms.
• Latent TB is diagnosed by demonstration of delayed
type or type IV hypersensitivity reaction against
tubercle bacilli antigen.
18. Traditionally, the tuberculin test has been in use for diagnosis of latent
TB for >100 years. It was discovered by Von Pirquet in 1907.
Antigens used:
PPD (Purified Protein Derivative antigen) It is a purified preparation of
the active tuberculoprotein.
Dosage:
It is expressed in tuberculin unit (TU). One TU is equal 0.00002 mg of
PPD
Procedure:
0.1 mL of PPD containing 1 TU is injected intradermally into flexor
surface of forearm
Tuberculin Test
19. Reading:
It is taken after 48-72 hours. At the site of inoculation, an induration
surrounded by erythema is produced. If the width of the induration is:
• ≥ 10 mm: Positive (tuberculin reactors)
• 6-9 mm: Equivocal/doubtful reaction
• 5 mm: Negative reaction.
Interpretation of result
• Adults: Positive tuberculin test
in adults only indicates present
or past exposure with tubercle
bacilli but does not confirm the
presence of active stage of the
disease.
• Children: In children, positive
test indicates acne infection and
used as diagnostic marker.
20. • False-positive:
» The test becomes positive after BCG vaccination
(after 8-14 weeks)
» Nontuberculous mycobacteria infection.
• False-negative:
» Early or advanced TB,
» Miliary TB
» Decreased immunity (HIV-infected people)
•
21. TREATMENT
• ATDs are classified into 2 groups
First-line drugs (DS-TB)
• Isoniazid (H)
• Rifampicin (R)
• Pyrazinamide (Z)
• Ethambutol (E)
• Streptomycin (S)
Second-line drugs (DR-TB)
• Fluoroquinolones
• Aminoglycosides
• Macrolides
• Ethionamide & Prothionamide etc.
22. AIM OF TREATMENT
o Interrupt transmission by rendering patients non-
infectious.
o Prevent morbidity and death by curing patients.
o Prevent the emergence of drug resistance.
o Prevent relapse.
23. STRATEGIES
• Multidrug therapy: Combination of more than one drug for rapid
and effective killing of tubercle bacilli.
• Short course chemotherapy lasting for 6 months (or longer for DR-TB)
• Two phase chemotherapy: The short course is divided into
• Intensive phase: Aims at aggressive treatment with multiple
ATDs that rapidly kill the bacilli making the smear negative,
followed by:
• Continuation phase: Aims at killing the remaining dormant
bacilli and prevents relapse.
24. DOTS strategy
• Directly Observed Treatment, Short course
• It is recommend by RNTCP and WHO.
• Here, the strategies used are:
o The entire treatment course is supervised to improve the
patient's compliance
o Treatment response is also monitored by sputum smear
microscopy at the end of each phase.
25. REGIMENS
• Standard regimen for DS-TB: 2 Regimen
• Doses: All drugs must be given in fixed dose combination (FDC),
should be taken orally, once a day.
• Regimens for DR-TB: The treatment for DR-TB is complex, consists
of use of higher numbers of second-line agents, given for longer
duration.
CATEGORY DEFINATION INTENSIVE
PHASE
CONTINUTION
PHASE
CATEGORY-I New cases
Received ATDs <1 month
(2)HRZE (4)HRE
CATEGORY-II Previously treated patients
Treatment after failure
Recurrent
Treatment after loss to follow up
(2)HRZES
+
(1)HRZE
(5)HRE
26. FOLLOW-UP OF TREATMENT
Patients should be followed up at scheduled intervals for the
assessment of improvement in clinical and laboratory parameters.
• Clinical follow-up: Should be carried out at least monthly during
treatment
• Laboratory follow-up: For PTB cases, sputum smear examination is
done at the end of intensive phase. Sputum smear plus culture is
done for every patient at the end of treatment.
• Long term follow-up is carried out up to 2 years of completion of
treatment
• Follow-up for MDR TB: Sputum smear and culture are performed
every month during intensive phase and every 3 months during
continuation phase.
27. DRUG RESISTANCE
• Mono resistance: Resistance to only one first-line ATDs.
• Poly resistance: Resistance to >1 first-line ATDs.
• Rifampicin resistance (RR): Resistance to Rifampicin with or without
resistance to other ATDs.
• Multidrug-resistance tuberculosis (MDR-TB): Resistance to both
Isoniazid & Rifampicin with or without resistance to other ATDs.
• Extensively drug-resistance tuberculosis (XDR-TB): These are
MDR-TB (+) Fluoroquinolones (+) one 2nd-line injectable agent(SLI)
• Pre-XDR-TB: MDR-TB (+) Fluoroquinolones (or) SLI
29. Non-tuberculous mycobacteria have been classified into four
groups by Runyon (1959), based on pigment production and
rate of growth.
RUNYON GROUP PROPERTY SPECIES
Photochromogens Produce pigment only
on light
M.kansasii,
M.marinunm
Scotochromogen Produce pigment both
in dark & light
M.scrofulaceunm,
M.gordonae
Non-chromogens Do not produce
pigment
M. Avium-
intracellulare complex
(MAC)
M. xenopi,
M. Ulcerans
Rapid growers Grow with in 1 week M.chelonae,
M.fortuitum
30. CLINICAL MANIFESTRATION OF NTM
DISEASE ORGANISM
Pulmonary
infection
M. Avium-intracellulare complex,
M. xenopi, M.kansasii
Lymph node
infection
M. Avium-intracellulare complex
Cutaneous
infection
M.chelonae, M.fortuitum –
Injection abscess
M.Marinunm – Swimming pool
granuloma
M. Ulcerans – Buruli ulcer
Disseminated
infection
M. Avium-intracellulare complex,
M.kansasii
31. LABORATORY DIAGONOSIS
• Specimen are collected as per infection and lesion.
• Microscopy is done by ZN staining.
• Culture on LJ medium.
• Pigment production.
TREATMENT
• Just as tuberculosis, NTM infection are treated with multi drug
therapy.
• MAC, M.kansasii, M.Marinunm need macrolide, ethambutol and
rifamycin combine.
• Macrolide need to be given prophylactically to the indivisual
infected with HIV.
