Tuberculosis: Pathophysiology and Diagnosis

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TuberculosisPathophysiology and diagnosis
Dr. Aditya Jindal Interventional Pulmonologist
Intensivist Jindal Clinics SCO 21, Sec 20D,
Chandigarh DM Pulmonary and Critical Care
Medicine (PGI Chandigarh), FCCP
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Course of TB infection
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Types of TB

• Primary tuberculosis
• is a form of disease that develops in a
  previously unexposed and therefore unsensitized
  person
• Secondary tuberculosis
• is the pattern of disease that arises in
  previously sensitized or infected host.

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Primary TB

• Infection of an individual who has not been
  previously infected or immunized.
• The inhaled bacilli implant in the distal
  airspaces of lower part of upper lobe or upper
  part of lower lobe close to the pleura
• As sensitization develops, a gray-white
  inflammatory consolidation is formed? Ghon focus

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Sites of Primary TB

• Most common - Lungs
• Other sites Tonsils, adenoids
• Site of BCG
  vaccination
• GIT ileum, colon etc
• GUT

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Ghons complex

• Consists of 2 components
• Pulmonary component
• lesion in the lung (Ghon focus or primary focus)
• 1-2cm solitary area located peripherally in the
  subpleural focus in the lower part of upper lobe
  or upper part of lower lobe
• Micro the lung lesion show tuberculous granuloma
  with caseous necrosis
• Lymphatic component
• lymphatics draining lung lesion containing
  phagocytes with M. tuberculosis bacilli

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Ghon complex
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Fate of Primary complex

• Heal by fibrosis? calcification
• Progressive primary tuberculosis
• Primary miliary tuberculosis

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Secondary TB

• The infection of an individual who has been
  previously infected or sensitized
• The infection may be acquired from
• Endogenous source ? reactivation of dormant
  primary complex
• Exogenous source

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Pathological lesions of Secondary TB

• The initial lesion is a small focus of
  consolidation of lt2cm in diameter within 1 to 2cm
  of apical pleura
• Gross sharply circumscribed, firm, gray white to
  yellow with variable amount of central caseation
  necrosis
• Micro coalescent tuberculous granulomas with
  central caseation necrosis.

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Fate of secondary TB

• The lesion may heal with fibrous scarring and
  calcification
• Fibrocaseous tuberculosis (progressive pulmonary
  TB )
• Tuberculous caseous pneumonia
• Miliary tuberculosis

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Pathology of TB

• Granuloma formation is the hall-mark of pathology
  of TB
• Granuloma is a
• i. Rounded tight collection of chronic
  inflammatory cells
• ii. Central Caseous necrosis
• iii. Active macrophages - epithelioid
  cells
• iv. Outer layer of lymphocytes
  fibroblasts
• v. Langhans giant cells joined
  epithelioid cells

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TB Pathology

• Bacterial entry
• T Lymphocytes.
• Macrophages.
• Epithelioid cells.
• Proliferation.
• Central Necrosis.
• Giant cell formation.
• Fibrosis.

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Granuloma Histopathology
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Caseation necrosis
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Causes of granuloma formation

• Tuberculosis
• Other mycobacterial infections, Leprosy
• Bacterial infections Brucellosis
• Other infections Fungal, viral, protozoal
• Non-infectious causes
• - Sarcoidosis
• - Foreign bodies
• - Lymphomas

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Miliary TB

• Occurs when organisms drain through lymphatics
  into? lymphatic ducts? venous return on the right
  side of heart? pulmonary arteries
• Individual lesions are either microscopic or
  small, visible (2mm) foci of yellow-white
  consolidation scattered through the lung
  parenchyma (resembling millet seeds)
• Micro the lesion shows structure of granuloma
  with minute areas of caseous necrosis.

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Miliary TB
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Course of TB infection
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Tuberculosis

• Diagnosis

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• Clinical Features
• Sputum Examination
• Chest Radiology
• Bronchoscopy
• Mantoux test
• Indirect laboratory tests

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Clinical Symptoms

• Prolonged fever, malaise, weakness, wt. loss etc.
• Pulmonary Cough, sputum, haemoptysis
  persistent
• Lymphadenopathy, organ enlargement, others

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• Clinical Features
• Sputum Examination
• Mantoux test
• Chest Radiology
• Bronchoscopy
• Indirect laboratory tests

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Sputum examination

• Smear examination (Sputum, other secretions)
• Auramine- Rhodamine staining
• Culture of material/ tissues

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Myco-bacteria

• Myco (fungus like) Bacterium (bacteria)
• Ability to resist decolourization by a weak
  mineral acid after staining with an aryl-methane
  dyes (acid-fastness)
• Slender, straight or slightly curved, rod shaped
• Length 2-4 u, Breadth 0.2-0.8 u
• Occur singly, in pairs or small groups
• Long, filamentous, club-shaped (rarely branching)

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MYCOBACTERIAL DEMONSTRATION

• Smear Easiest, quickest
• Requires gt 10000 AFB/ml
• Sensitivity 50-60
  Specificity High
• Culture More sensitive 10 AFB/ml
• Traditional 6-8 wks
• Septi Chek Biphasic High
  yield
• Radiometric BACTEC
• Others
• Animal pathogenicity
• Antimicrobial sensitivity

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M tb in sputum smear
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• Rapid culture methods
• BACTEC system
• MycobactGrowth Indicator Tube(MGIT)
• MB/BacT system
• Septi-chek
• ESP culture system
• Microscopic observation of broth/slide cultures

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M tb Colonies on culture (LJ medium)
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• BACTEC System
• Radiometric method
• 14C labelled palmitic acid added to liquid 7H12
  medium
• Detects MTB by metabolism rather than growth
• 14CO2 produced detected
• Growth index(GI) measured
• Results available in 7-14 days (87-96)

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• MGIT
• Automated system
• Capable of analyzing 960 specimens
• Metabolism of MTB produces O2
• Fluorescence of dye with oxygen measured
• Results available in 7-14 days
• Cost effective for high load micro labs

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• Clinical Features
• Sputum Examination
• Chest Radiology
• Bronchoscopy
• Mantoux test
• Indirect laboratory tests

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Chest radiology

• I. Chest Upper Lobes/Diffuse miliary
• Infiltrates/Exudates/Fibrosis
• Multiple, thin walled cavities
• Lymphadenopathy,
  Pl.effusion
• II. Others Enlargement of organs
• Erosions/Effusions
• Caseations/collections

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Role of Chest X-ray

• No chest X-ray pattern is absolutely typical of
  TB
• 10-15 of culture-positive TB patients not
  diagnosed by X-ray
• 40 of patients diagnosed as having TB on the
  basis of x-ray alone do not have active TB

X-ray is unreliable for diagnosing and monitoring
treatment of tuberculosis
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• Clinical Features
• Sputum Examination
• Chest Radiology
• Bronchoscopy
• Mantoux test
• Indirect laboratory tests

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Role of bronchoscopy

• Valuable in early diagnosis of strongly suspected
  sputum-negative TB
• Diagnosis of endobronchial TB/miliary TB
• TBLB yield is greater (82) than BAL (26)
• TBNA has a role in mediastinal lymph nodal
  tuberculosis with negative sputum smears

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ESR?

• NO ROLE IN DIAGNOSIS

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• Clinical Features
• Sputum Examination
• Chest Radiology
• Bronchoscopy
• Mantoux test
• Indirect laboratory tests

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Tuberculin (Mantoux) Test

• Infection with mycobacterium tuberculosis leads
  to delayed hypersensitivity reaction which can be
  detected by Mantoux test
• About 2 to 4 weeks after infection
• Intracutaneous injection of purified protein
  derivative (PPD) of M.tuberculosis
• Induces a visible and palpable induration that
  peaks in 48 to 72 hours

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How to do the test?

• Sub cutaneous
• Weal formation
• Itching no scratch
• Read after 72 hours
• Induration size.
• 5-10-15mm

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Positive test
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Interpretation

• Induration less than 5 mm gt no exposure to
  tubercular bacilli
• Induration between 5-9 mm gt this can be due to
  atypical mycobacteria or BCG vaccination. It may
  suggest infection in immunocompromised children
  such as HIV infection or other immunosuppression
• Induation 10 mm or more gt in a child with
  symptoms of tuberculosis should be interpreted as
  tubercular disease

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Clinical significance

• Denotes infection
• Does not differentiate infection from active
  disease
• A strongly positive Mantoux can support a
  clinical diagnosis
• Better negative than positive predictive value
• Cut-off for a positive test?

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• Clinical Features
• Sputum Examination
• Chest Radiology
• Bronchoscopy
• Mantoux test
• Indirect laboratory tests

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Indirect Tests

• Biochemical tests
• LDH, Proteins
• Adenosine Deaminase
• Bromide Partition Test
• Gas Chromatography Fatty acids, alcohols etc
• Immuno-diagnosis tests
• Skin test (Mantoux)
• Detection of Antibodies (Tests banned)
• Genetic/ molecular studies
• Antigen detection
• Lipo arabinomannan
• Nucleic Acid Probes
• Ligase Chain Reaction
• Polymerase Chain Reaction
• Gene Xpert

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Serological Tests

• Low turn around time
• Limitation
• Low sensitivity in
• smear negative patients
• HIV positive cases,
• In disease -endemic countries with a
  high infection rate
• Poor standardization
• Banned in 2012.

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• Interferon-? release assays
• An alternative to the TST in the form of a new
  type of in-vitro T-cell-based assay
• (Test-tube TST)
• Gold IGRA
• Elispot T test
• T cells of individuals sensitized with
  tuberculosis antigens produce interferon-? when
  they re-encounter mycobacterial antigens
• High level of interferon-? production -
  presumptive of tuberculosis infection

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• IGRA in LTBI
• In the absence of a gold standard for diagnosis
  of Latent TBI, the sensitivity and specificity
  cannot be directly estimated
• IGRA have higher specificity than TST
• Better correlation with surrogate markers of
  exposure to M tuberculosis (in low-incidence
  setting countries)
• Less cross reactivity as a result of BCG
  vaccination than TST

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• PCR
• Synthesis of dsDNA by hybridization of
  oligonucleotides to targets s-DNA
• Uses thermal cycler to denature the target DNA
• Thermostable polymerase for DNA amplification
• Repeated cycles by varying temp for primer
  annealing (70-72 C) and denaturation (94-96 C)
• Amplified product are then detected by southern
  blotting and fluorescent/radiolabelled probes
  hybridization

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• Gene X-pert Test
• Detection and identification of
  mycobacteriadirectly from clinical samples
• Uses real-time polymerase-chain reaction (PCR)
  assay to amplify an MTB-specific sequence of the
  rpoB gene
• Cartridge based, PCR test for detection of
  mycobacteria and Rifampicin resistance
• Rapid test. Results within hours.
• Costly
• Continuous electric supply and temperature
  maintenance
• ? Field feasibility, sensitivity and specificity
  in India

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• Integrates sample processing and PCR
• Reagents required for bacterial lysis, nucleic
  acid extraction, amplification and amplicon
  detection are present in a disposable plastic
  cartridge
• Only manual step -- addition of a bactericidal
  buffer to sputum before transferring a defined
  volume to the cartridge
• MTB/RIF cartridge is then inserted into the
  GeneXpert device
• Results within 2 hours

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Diagnosis of Extra Pulmonary TB

• Sputum or other smears are often Negative
• These are difficult to use for diagnosis and
  start of treatment
• Follow up
• Monitoring
• End point
• Recurrence / Relapse
• Mostly clinico-radio-histo/cytological
• Invasive procedures frequently required to obtain
  tissue, fluids, etc. to look for T.b. and/or
  histo-cytological criteria.

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Difficulties of specimens testing for EPTB

• Specimen
• Relevance of a particular sample
• Method of collection
• Contamination/ inappropriate site
• Processing
• Technique, Standardization and calibration of
  Instrument/procedure
• Clinical interpretation
• Disease ?

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How to confirm the Diagnosis? LEVELS OF
DIAGNOSIS OF TB

• Suggestive
• Clinical/Epidemiological
• Radiological
• Presumptive/Possible
• Therapeutic response
• Immunological
• Markers
• Definitive
• Demonstration of Myco tuberculosis
  (smear/ culture)
• Histo/Cytological criteria

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EPT DIAGNOSIS
Site Site Empirical/ Suggestive Possible Definitive
1 Lymph Nodes Clinical FNAC (Granuloma) AFB on FNAC
2. Pl. Effusion Clinico-radiological Exudative A.D.A. Pl. biopsy AFB positivity P.C.R.
3. Pericarditis As above As above As above
4. Peritoneal As above As above As above
5. Intestinal Clinical Radiological Biopsy Granuloma AFB positivity
6. Genitourinary Endoscopy As above Biopsy Granuloma AFB positivity
7. Bones Joints Clinical Radiology FNAC Biopsy As above
8. Meningeal As above CSF (Biochem.) CSF PCR AFB
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Comparison of Various Diagnostic Tests for
Diagnosis of TB
Microscopy LED Microscopy GeneXpert MTB/RIF LAMP Solid Culture Liquid Culture
Threshold (CFU/ml) 10,000 - 131 (106-176) - 100 10-50
Turnaround time 1-2 days 1day 90 min - 4-8 week Days - 2 week
Sensitivity 50-60 10 gtthan ZN staining 90 88 Reference Reference
Specificity 98 94 Reference Reference
Technical expertise Required Required Minimal Required Required Required
Biosafety Better than Microscopy
Other Prone to contamination

• Boehme CC et al. Semin Respir Crit Care Med
  20133417 31.
• Lawn SD et al. Lancet Infect Dis 2013 13 34961.

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• Absence of evidence is not the
  evidence of absence
• Carl Sagan

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• THANK YOU