Pathology of tuberculosis of lung

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Pathology of tuberculosis of lung
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Pathogenesis
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TB Pathogenesis

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

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Morphology of Granuloma

1. Rounded tight collection of chronic inflammatory
   cells.
2. Central Caseous necrosis.
3. Active macrophages - epithelioid cells.
4. Outer layer of lymphocytes fibroblasts.
5. Langhans giant cells joined epithelioid cells.

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Tuberculous Granuloma
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Types of tuberculosis

• 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 tuberculosis

• Definition Infection of an individual who has
  not been previously infected or immunised.
• 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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Primary tuberculosis

• GHONS COMPLEX( Primary complex)
• Pulmonary component (Ghons Focus)
• Lymphatic component
• Lymph node component Hilar
• 
  Tracheo-bronchial

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Fate of primary tuberculosis

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

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Secondary tuberculosis

• Definition 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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• 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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Tuberculous Granulomas
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Caseation Necrosis
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Epitheloid cells in Granuloma
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Fate of secondary tuberculosis

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

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1. How to define/diagnose TB?

• i. Microbiological
• Smear positive for AFB (one or more ?)
• Culture positive
• ii. Immunological / Molecular
• iii. Histological / Cytological
• iv. Clinical and radiological
• v. Empirical

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2. Classification of TB cases
Tuberculosis cases
Pulmonary
Extra-pulmonary
Smear positive
Smear negative
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Extrapulmonary TB
TB of organs other than lungs confirmed by
bacteriological Or Histopathological
confirmation Or Strong clinical evidence
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• 3. What Methods to employ for diagnosis of TB ?
• 4. What are levels of diagnosis?
• 5. How to interpret the tests?

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3. What Methods of Diagnosis of TB ? (Pulmonary
Extra-pulmonary)

• Clinical features
• Bacteriology
• Radiology
• Serology/immunology
• Genetic/ Molecular techniques

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CLINICAL SYMPTOMS

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

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SYNDROMIC DIAGNOSIS

• Pulmonary
• Primary Pulm Foci
• Secondary Lymph Nodes
• Infiltrates Pleural
• Fibrocavitary Miliary
• Broncho pneumonia
• Abscess/others
• Extra pulmonary
• Disseminated

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RADIOLOGICAL Characteristics

• I. Chest Upper Lobes
• Infiltrates/Exudates
• Multiple, thin walled
• II. Others Enlargement of organs
• Erosions/Effusions
• Caseations/collections

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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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• BACTEC System
• Radiometric method
• 14C labelledpalmiticacid added to liquid 7H12
  medium
• Detects MTB by metabolism rather than growth
• 14CO2produced detected by specialized eqpt
• Growth index(GI) measured
• Results available in 7-14days (87-96)
• Ramachandranet al, IndJ TB 2003

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

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• PCR
• Synthesis of d s-DNA by hybridizationof
  oligonucleotides to targets s-DNA
• Uses thermal cyclerto denature the target DNA
• Thermostablepolymerase for DNA amplification
• Repeated cycles by varying temp for
  primerannealing(70-72 C) and denaturation(94-96
  C)
• Amplified product are then detected by southern
  blotting and fluorescent/radiolabelledprobeshybrid
  ization

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• MTB/RIF test platform (GeneXpert, Cepheid)
  integrates sample processing and PCR in a
  disposable plastic cartridge containing all
  reagents required for bacterial lysis, nucleic
  acid extraction, amplification and amplicon
  detection
• 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, which provides results within 2
  hours

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• Xpert MTB/RIF
• Automated molecular test for Mycobacterium
  tuberculosis (MTB) and resistance to rifampin
  (RIF)
• Uses real-time polymerase-chain reaction (PCR)
  assay to amplify an MTB-specific sequence of the
  rpoB gene
• Probed with molecular beacons for mutations
  within the rifampin-resistance determining region
• Boehme CC. N Engl J Med. 2010 September
  9 363(11) 10051015

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Indirect Tests Markers

• Biochemical
• Adenosine Deaminase(ADA)
• Bromide Partition Test
• Gas Chromatography Fatty acids, alcohols etc.
• Immuno-diagnosis
• Skin test (Mantoux)
• Serodiagnosis Detection of Antibodies
• 3. Genetic/ molecular studies

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Tuberculin Skin Test (Mantoux)

• 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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Interferon-? release assays

• An alternative to the TST in the form of a new
  type of in-vitro T-cell-based assay
• 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
• Initial IGRAs used PPD as the stimulating antigen
  - newer assays use antigens specific to M
  tuberculosis, such as ESAT6 and CFP10

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IGRA in LTBI

• In the absence of a gold standard for diagnosis
  of LTBI, 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
  settings
• Less cross reactivity as a result of BCG
  vaccination
• than TST
• At least as sensitive as the TST for active
  tuberculosis
• Lancet Infect Dis 2007 7 428-438

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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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Genetic/Molecular techniques

• Detection of DNA specific base sequences
• DNA amplification and detection
• ? RNA Presence of multiplying bacteria

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Detection and identification of
mycobacteriadirectly from clinical samples

• Genotypic methods Polymerase chain reaction
  (PCR) and its variations
• Transcription mediated amplification (TMA) and
  nucleic acid amplification (NAA)
• The ligase chain reaction
• Strand displacement amplification (SDA)
• Nucleic acid sequence based amplification (NASBA)
• Branched DNA (b-DNA)
• Line probe assay (LiPA)
• Phenotypic Methods mycobacteriophage assay (FAST
  Plaque TBTM)

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Gene X-pert Test

• 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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Difficulties for 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
• Follow standard guidelines

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Diagnosis of Extra-pulmonary Tuberculosis

• Largely clinical and radiological.
• Supported by laboratory parameters and other TB
  markers.
• Invasive procedures frequently required to obtain
  tissue, fluids, etc. to look for T.b. and/or
  histo-cytological criteria.
• Therapeutic trial as a diagnostic modality should
  not be used.

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Levels of Diagnosis of TB

• Suggestive
• Clinical / Epidemiological, Radiological
• Presumptive / Possible
• Therapeutic response, Immunological Markers
• Definitive
• Demonstration of Mycobacterium tuberculosis (on
  smear / culture)

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DIAGNOSIS
Site Site Empirical Suggestive Definitive
1 Lymph Nodes Clinical FNAC (Granuloma) AFB on FNAC
2. Pl. Effusion Clinico-radiological Exudative A.D.A. P.C.R. Pl. biopsy AFB positivity
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 As above As above
7. Bones Joints Clinical Radiology FNAC Biopsy As above
8. Meningeal As above CSF (Biochem.) CSF PCR AFB
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MYCOBACTERIAL DEMONSTRATION

• Smear Easiest, quickest, gt 10000 AFB/ml
• Sensitivity 50-60 Specificity High
• Culture More sensitive 10 AFB/ml
• Traditional 6-8 wks, microscopic detection of
  colonies can hasten detection
• Septi Chek Biphasic High yield, 3 weeks
• Radiometric BACTEC 7-14 days
• Mycobact Growth Indicator Tubes (MGIT 960) AFB
  metabolic O2 utilization and subsequent
  intensification of an O2 quenched fluorescent
  dye
• MB/Bac T Colorimetric detection of CO2
• Others Animal pathogenicity

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Mantoux Value of different cut-offs for
diagnosis active pulmonary tuberculosis
5mm 10mm 15mm 20mm
Sensitivity Sensitivity Sensitivity Sensitivity Sensitivity
PTB 0.87 0.67 0.09 0.01
PTB- 0.66 0.33 0.16 0.08
EPT 0.81 0.75 0.43 0.18
Overall 0.81 0.62 0.20 0.09
Specificity 0.7068 0.8901 0.9738 0.9895
Gupta D et al. J Assoc Physicians India 2001
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IGRA in active TB
Clin Infect Dis 2007 44 747
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Genetic/Molecular techniques

• Detection of DNA specific base sequences
• DNA amplification and detection
• ? RNA Presence of multiplying bacteria

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Detection and identification of
mycobacteriadirectly from clinical samples

• Genotypic methods Polymerase chain reaction
  (PCR) and its variations
• Transcription mediated amplification (TMA) and
  nucleic acid amplification (NAA)
• The ligase chain reaction
• Strand displacement amplification (SDA)
• Nucleic acid sequence based amplification (NASBA)
• Branched DNA (b-DNA)
• Line probe assay (LiPA)
• Phenotypic Methods mycobacteriophage assay (FAST
  Plaque TBTM)

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Restriction fragment length polymorphisms (RFLP)

• Identify the specific strains of MTB by pattern
  of gene fragments (DNA Fingerprinting)
• Has shown that recent infection is responsible
  for upto 50 TB cases in both HIV negative and
  HIV infected
• Used to confirm that cluster of TB cases are
  linked by recent transmission especially during
  nosocomial outbreaks
• Halvir DV, Barnes
  PF. NEJM 1999

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Nucleic Acid Probes

• Detection of DNA specific base sequences
• DNA amplification and detection
• ? RNA Presence of multiplying bacteria

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LAB. DIAGNOSIS Issues

• Specimen Relevance
• Method of collection Contamination
• Processing Technique
• Standardization and Instrument and
  calibration procedure
• Normality assumptions Titres ?
• Clinical interpretation Disease ?

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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
• Mycobac Growth Indicator Tubes
• Others
• Animal pathogenicity
• Antimicrob sensitivity
• Luciferase Reporter Assay

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Indirect Tests Markers

• Biochemical LDH, Proteins
• Adenosine Deaminase
• Bromide Partition Test
• Gas Chromatography Fatty acids,
• alcohols etc.
• Immuno-diagnosis
• Skin test (Mantoux)
• Detection of Antibodies

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• Mycobacterial components
• Antigen detection
• Lipo arabinomannan
• Nucleic Acid Probes
• Polymerase Chain Reaction
• Ligase Chain Reaction

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• Absence of Evidence
• is Not the
• Evidence of Absence
• Carl Sagan

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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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Xpert in Diagnosis of PTB

• Pooled sensitivity in HIV Negative- 89 (95 CI
  81-94)
• Pooled sensitivity in PL-HIV 80 (95 CI 67 -
  88)
• PLHIV Smear ve, Culture ve 100 (82-100)
• PLHIV Smear ve , Culture ve- 43 (30-58)
• Fresh specimen gt Frozen

Cochrane Review 2013
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Xpert MTB/RIF for Rif Detection in PTB
Sp- 98
Sn- 94
Cochrane Review 2013
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Xpert in Diagnosis of PTB

• Systematic review and Meta-analysis
• Studies published up to October 2011
• 18 studies, 10,224 patients
• 15 reported on Dx of PTB,
• Pooled sn- 904 (95 CI 892914), sp- 984
  (980987).
• Sm ve- 750
• Sm ve- 987
• Similar to Cochrane review, however current G4
  version, launched in Dec 2011might differ in
  performance
• G4 version in Africa- Specificity of 99.5

Chang K, J Infect 2012 64 58088 Muhammad Osman
et al. JCM, Nov 2013
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Anticipated bene?ts

• Increase case detection, esp. smear-ve
• Reduction in time to diagnosis and treatment
• Reduced patient default during investigation
• Reduced morbidity, mortality, and tuberculosis
  transmission
• Increased detection and Rx of MDR TB
• Reduced need of culture
• Reduced biohazard

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Limitations

• Lack of a battery-operated system
• Annual calibration
• Limited range of operating temperatures
• Detects Dead bacilli, Not suitable for monitoring
  Rx response
• Rif resistance gt15 PPV- gt90
• Rif Resistance lt5 PPV lt70
• Does not detect INH resistance

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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AFB - Ziehl-Nielson stain
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Colony Morphology LJ Slant
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Mantoux test

• Infection with mycobacterium tuberculosis leads
  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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PPD Tuberculin Testing

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

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• (i) Induration less than 5 mm no exposure to
  tubercular bacilli.
• (ii) Induration between 5-9 mm this can be due
  to atypical mycobacteria or BCG vaccination. It
  may suggest infection in immunocompromised
  children such as HIV infection or other
  immunosupression
• (iii) Induation 10 mm or more an induration of
  10 mm or more at 48-72 hours in a child with
  symptoms of tuberculosis should be interpreted as
  tubercular disease

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PPD result after 72 hours
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Granuloma or giant cell is not pathagnomonic of
TB!

• Foreign body granuloma.
• Fat necrosis.
• Fungal infections.
• Sarcoidosis.
• Crohns disease.

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