Laboratory diagnosis of Tuberculosis gs

Seminar regarding TB pathogenesis and Lab diagnosis with advancements

1. Laboratory Diagnosis of
Tuberculosis(TB)
MODERATOR – DR. ASHOK
PANCHONIA
Speaker – Dr. Gaurav Shelgaonkar

3. Mycobacteria Tuberculosis
Non sporing, Non-capsulated. Weakly Gram Positive.
Strongly Acid Fast (due to mycolic acid in cell wall).
Lipid Rich Cell Wall (confers resistance to disinfectants,
detergents, common antibiotics and traditional stains).
Long Generation time.
Capable to grow intracellularly in unactivated alveolar
macrophages.
Primarily disease caused from host response to infection.
Humans are the only natural reservoir.
Person to person transmission, by infectious aerosols/droplets.

4. Lin P et al. J Immunol 2010
Clearance
Pulmonary TB
Low grade TB
“percolating
“
Dormant infection
Septic TB
Miliary TB
Extrapulmonary TB
LT B
I
a
c
tiv
e
T
B
Spectrum of M. tuberculosis infection

5. Magnitude of the Problem
Source: WHO Geneva; WHO Report 2008: Global Tuberculosis Control; Surveillance, Planning and Financing
Global annual incidence = 9.1 million
India annual incidence = 1.9 million
India is 17
th
among 22
High Burden
Countries (in terms of
TB incidence rate)

6. Evolution of TB Control in India
1950s-60s Important TB research at TRC and NTI
1962 National TB Control Program (NTCP)
1992 Program Review
 only 30% of patients diagnosed;
 of these, only 30% treated successfully
1993 RNTCP pilot began
1998 RNTCP scale-up
2001 450 million population covered
2004 >80% of country covered
2006 Entire country covered by RNTCP

7. From robbins 9
th
Edition
Pathogenesis

8. Pneumonia
Granuloma formation with fibrosis
Caseous necrosis
•Tissue becomes dry & amorphous (resembling cheese)
•Mixture of protein & fat (assimilated very slowly)
Calcification
•Ca
++
salts deposited
Cavity formation
•Center liquefies & empties into bronchi
Typical Progression of Pulmonary
Tuberculosis

9. From robbins 9
th
Edition

10. From robbins 9
th
Edition

11. From robbins 9
th
Edition
Primary Pulmonary Tuberculosis

14. From robbins 9
th
Edition
Secondary Pulmonary TB Miliary Tuberculosis

16. Haematology
Complete Blood Count is usually Normal.
Usually moderate Normochromic or slightly
hypochromic anemia.
Anemia may be there due to the chronic
debiliting natureof the disease.
 Megaloblastic Anemia (Macrocytes in blood) in
cases of abdominal TB with malabsorption.
Thrombocytosis may be seen.
ESR & CRP are Raised.

17. Tubercular Lymphadenitis
Needle aspiration(FNAC) is a good test for TB
lymphadenitis in HIV-infected persons
Can be done the same day in the health facility.
Has a low rate of adverse effects.
Has a high yield for diagnosing TB.

18. TB – Lymph Node
H&E MGG Stain

19. TB Meningitis
Subacute or chronic
Headache, fever, neck stiffness, decreasing mental
status
Lumbar puncture essential for diagnosis
CSF –
oclear or slightly turbid, forms fibrin coagulum on
standing.
oRaised white cell count (100-1000 lymp/ul)
oElevated protein (>45mg/dl),
oReduced glucose ( <50mg/dl)

20. Traditional Methods for
Diagnosis of Tuberculosis
PresumptiveDefinitive
1. Clinical
2. Radiological Isolation & Identification
3. AFB Microscopy of M. Tuberculosis
4. Tuberculin Test
5. Pathological

21. Mantoux Tuberculin Skin Test (TST)
0.1 ml of tuberculin purified protein derivative (-
PPD) into inner surface of forearm intradermally.
Read between 48-72 hrs.
 A positive tuberculin skin test result is supportive
evidence in the diagnosis of TB in areas of low
prevalence (or no vaccination); however, a negative
tuberculin skin test result may occur in
approximately one third of patients.

23. PPD Tuberculosis Skin Test Criteria

24. A Negative Test Result could result from the following:
(1) Anergy secondary to immunosuppression or malnourishment;
(2) Recent infection;
(3) Circulating mononuclear cells suppressing the specifically
sensitized circulating T-lymphocytes in the peripheral blood; or
(4) Sequestration of purified protein derivative specific reactive T-
lymphocyte.
However, results of a tuberculin skin test is repeated 6 to 8 weeks
later would usually be positive.
False positive in Atypical mycobacterial infection and previous
BCG vaccination.

25. Role of Radiography
Chest X-Ray (CXR) can support a diagnosis of PTB
Not used routinely for follow-up
PTB can exist with normal CXR
Must be interpreted with other information
History and exam
Sputum smear results
Also useful in diagnosing other types of TB,
especially in bones, joints, and spine

26. Chest X-Ray of Patient with Active
Pulmonary Tuberculosis

28. Sputum Examination
CASE FINDING TOOLS
Sputum examination -:- sputum smear examination by
direct microscopy is the method of choice.
Collection of sputum –
Day 1 Sample 1 - Patient provide an “on the spot” sample
Day 2 Sample 2 - Patient bring early morning sample.

30. Slide reporting
 The number of bacilli
seen in a smear reflects
disease severity &
patient infectivity.
The table shows the
standard method of
reporting using 100X
magnification (WHO).
Number of bacilliNumber of bacilli Result Result
reportedreported
NO AFB per 100 oil NO AFB per 100 oil
immersion fieldimmersion field
00
1-9 AFB per 100 oil 1-9 AFB per 100 oil
immersion fieldimmersion field
ScantyScanty
10-99 AFB per 100 oil 10-99 AFB per 100 oil
immersion fieldimmersion field
+ (1+)+ (1+)
1-10 AFB per oil 1-10 AFB per oil
immersion fieldimmersion field
+ + (2+) + + (2+)
>10 AFB per oil >10 AFB per oil
immersion field immersion field
+ + + (3+) + + + (3+)

31. Smear and Culture
Direct examination by Zeihl-Neelsen
staining requires bacillary densities of
5000-10,000/mL
Culture requires a minimum of 10 to 100
viable bacilli.

32. Lipid-Rich Cell Wall of Mycobacterium
Mycolic acids
CMN Group:
Unusual cell
wall lipids
(mycolic
acids,etc.)

35. Mycobacterium Tuberculosis Stained
with Fluorescent Dye
From Carl Zeiss microimaging GmbH (FluoLED)
Yellow bacilli with green background.

36. Mycobacterial Culture
Reasons to request mycobacterial culture:
•Patient previously on anti-TB treatment (Relapse,
Defaulter)
•Still smear-positive after intensive phase of treatment
or after finishing treatment
•Symptomatic and at high-risk of MDR-TB
•To test fluids potentially infected with M.
tuberculosis
•Investigation of patients who develop active PTB
during or after IPT.
•TB in health workers

37. Culture Based Methods
1.Liquid Culture (e.g., automated mycobacteria
growth indicator tube) – Faster and more sensitive
than solid media)
2.Microscopic Observation Drug Susceptibility
Testing (DST) – Yields fasterb culture and DST
results than do liquid or solid media and is
inexpensive(Requires Skilled Technician to interpret)
3.Thin Layer Agar Methodology (same as above)
4.Calorimetric DST methods using redox tetrazolium
slats, or a nitrate reductase assay – Lower cost, less
time (Potential Biohazard)

39. Eight Week Growth of Mycobacterium
tuberculosis on Lowenstein-Jensen Agar

41. DST performed on all cultures
Tests for isoniazid, rifampicin, ethambutol, and
streptomycin
If found to be multi-drug resistant, then send for
additional testing for susceptibility to second-line
medicines
TB Drug Susceptibility Testing
(DST)

42. BACTEC 460 TB System
(radiometric)
Developed in 1969 by Deland and Wagner.
Principle –
BACTEC 12B vial, utilize 14C labeled substrate (Palmitic acid).
On inoculation, mycobacteria, grow and release 14CO2.
The BACTEC instrument measures quantitatively the
radioactivity on a scale ranging from 0-999, as GI (Growth
Indicator).
The daiy increase in GI is proportional to growth in the medium.
DST (Drug Susceptibility Test) – When ATT is introduced in
the medium, reduced production of 14CO2 & decrease in GI.

44. New Approach in Diagnosis of TB
Replication of M. Tuberculosis
1.Antigen Detection Tests –
LAM ELISA Urinary Antigen Test(ELISA BASED TEST, detect
LAM, Antigen 85 – LipoArabinoMannon) still developing
Sputum Antigen Test
2. Microscopic Visualization of bacteria –
LED Microscopy
Bleach Microscopy
3. Culture based Detection Tests –
Microscopic observation drug susceptibility assays (MODS)
Thin Layer Agar
Phage based tests
Calorimetric media

45. Replication of M. Tuberculosis
4. Nucleic Acid Amplification Tests (NAATs)-
LAMP
GeneXpert MTB
Transrenal DNA detection
Genotype MTBDRPlus
(High Specificity and Positive Predictive Value)
5. Volatile Organic Compounds (VOC) detection-
E-nose
Biosensors
(Emitted from the infected cells & released in exhaled breath through nanomaterial
biosensors or Gas Chromatography)

46. Immune Response to M. Tuberculosis
I. Cellular Immune Response -
INF-Y release assays(IGRA)
Quanti-FELON TB gold
T-SPOT TB
Rd ESAT-6 skin test
II. Humoral Immnune Response –
Antibody Detection Tests
Serological tests
New Approach in Diagnosis of TB

47. Polymerase Chain Reaction
Polymerase chain reaction (PCR) is based on
amplification of mycobacterial DNA fragments.
It can detect as few as 10 mycobacteria.
Advantages of PCR include rapid diagnosis,
improved specificity and sensitivity, and no
requirement of intact immunity.

48. Molecular Beacon Assay(at MDL)
Target: DNA
Realtime PCR
PCR to amplify target sequences
At the same time, Molecular beacon probes are used to
detect INH and RIF resistance mutations.
2 MBs for INH (targeting katG & inhA)
3 MBs for RIF (targeting core of rpoB)

49. Real-Time PCR
2 components
PCR to amplify target sequences.
A system to monitor PCR product.
Fluorophore-labeled probes
An optical device to detect
fluorescence
Software to record data
No post-PCR manipulations
Fast
when PCR is done, results are ready
for interpretation.
No amplicon contaminations
iCycler
IQ5

50. What is a Molecular Beacon?
¬Loop (15-30 nt)
¬Stem (5-7 nt)
Fluorophore® ¬Quencher
Hair-pin structure

51. Molecular
Beacon (off)
Hybrid (Molecular Beacon - On)
Detection of Mutations with a Molecular Beacon
(Loop portion containing wildtype SQ)
Mutant Sequence
Wildtype Sequence
Amplicon
Heat
Light
+
Courtesy of Dr. Probert
Loop
QuencherFluorophore
Fluorophore

52. An Example of a Good MB
No mutations,
Susceptible
Mutant,
Resistant
R
F
U
Threshold

53. Causes of false-positive results include DNA
contamination or presence of nonviable
organisms
The disadvantages of PCR include high cost,
risk of contamination, and the technology
involved in the procedure does not permit routine
diagnostic use at present.

54. 55Limitations
Limited genes & sites are targeted.
•Some mutations are not detected.
Emerging resistance in mixed populations may not be
detected.
Some mutations do not confer resistance.
•Rare occurring, but lead to wrong interpretation.
•Silent mutation in rpoB: codon 514.
•Not a silent mutation but only cause little change in MIC.
Available for INH and RIF only.
New MBs for other drugs not developed yet.
Phenotypic drug susceptibility testing is still needed.

56. GeneXpert Automated System at
MYH, Indore Resp. Medicine
(TCD) Department and TB
Hospital

57. CBNAAT Report

58. Line Probe Assays
Target: DNA
Traditional PCR (not realtime)
Amplify target sequences.
Reverse hybridization
Amplicons hybridize to probes immobilized on membrane
(strip).
Colorimetric detection of captured amplicons on strip.
Observation of bands. One probe for one band.

59. 60Line Probes
Hybridization and colorimetric detection
Amplicons bind to probes Color reaction to form bands

60. 61
Conjugate ctrl
rpoB wild-type,
5 segments
4 rpoB mutations
rpoB universal ctrl
MTBDR by HAIN Lifescience
LiPA RIF.TB by
INNOGENETICS
katG universal ctrl
katG wild-type
2 katG mutations
Universal ctrl
MTBC
516
526
531
315
marker line
MTBC
More probes are added in MTBDRsl to detected 2
nd
-line drug R-mutations.

62. 63
Line Probes Features
Many controls; more objective
MTBDRsl (HAIN) added embB, gyrA, rrs (screen for
XDR).
Exact mutations are available for most prevalent
mutations only.
Some mutations are detected by lacking bands in wild-
type sequences.
Emerging resistance in mixed populations may not be
detected.
Phenotypic drug susceptibility testing is still needed.

63. Loop Mediated Isothermal
Amplification(LAMP)of DNA
Small Heating Device
Runs at High Temperature(avoids nonspecific amplifications)
Multiple primers sets (increased specificity and speed)
Direct from Sputum.
Closed System (No risk of contamination)
Minimal instrumentation
Fast (Less than 2hrs total)
Visual Detection(no instrumentation- Mg2P2O7 ppt - white)
Test Under Development still…….

65. Cytokine Assays
T- cell Interferon-Gamma Release Assay (IGRA)
INF- y produced by T-lymphocytes, is capable of
activating macrophages, increasing their bactericidal
capacity against M tuberculosis and is involved in
granuloma formation.
Elevated concentrations of INF-y in TB is related to
increased production at the disease site by effectors T
cells.
The sensitivity of an elevated level varies from 78 to
100% and specificity from 95 to 100%

66. IGRA is useful in targeted strategy for latent TB
infection(LTBI) detection in low TB incidence settings
More specific than Tuberculin Skin Test
Can’t distinguish active from treated TB or LTBI.
False positive results in-
Hematologic malignancies
Empyema.
Note :- Immunosuppressed patients (HIV or after renal
transplant) had INF- Y levels similar to immunocompetent
individuals retaining its efficacy as a diagnostic test

67. Methods for detection of IFN-y
Two new blood tests
1.T-SPOT.TB [Oxford Immunec] – directly count the no of
IFN-y secreting T cells.
2. QuantiFERON-TB Gold [Cellestis Limited] –
measures the concentration of IFNy secretion.
Both tests based on detection of IFN-y in blood have been found to be more accurate than the
tuberculin skin test in the diagnosis of latent TB infection. Future research should focus on
the potential efficacy of quantification of specifically activated lymphocytes in body fluid
and blood using IFN-Y release assay in the diagnosis of TB.

69. Immunodiagnosis
Serological tests are simple to perform & can be developed
into a rapid method for wide screening.
Immuno- chromatographic card test –
Sensitivity 80 % & specificity 88 %.
Principle - the test employs antihuman Ig-G, A, M antibody
rabbit dye conjugate highly purified antigen A60 from
Mycobacterium bovis stain BCG (cell culture), fixed in the
test line, & anti-rabbit antibodies in the control line. As the
sample flows through the absorbent pad, human
immunoglobulins are bound by the antihuman Ig- dye
conjugates to form an immunocomplex. This binds to the
A60 proteins in the test line & produce a red violet test
line, if the anti- Tb antibodies were present in the sample.
In control line excess conjugate react with the anti rabbit
antibodies forming second red violet line to demonstrate the
correct function of the reagent.

70. Cytokines:
Significantly higher levels of IL-6 have been
demonstrated in TB (A Potent
Biomarker/Biosignature).
Furthermore, the serum/pleural fluid IL-6 ratio was
significantly higher in TB.
IL-1b and Tumor necrosis factor(TNFa), produced
predominantly by mononuclear phagocytes, have been
shown to be present in TB.
Levels of soluble IL-2 receptors, IL-18,
immunosuppressive acidic protein, and IL-12p40 are all
significantly elevated in TB

71. Adenosine DeAminase (ADA) in
Pleural Fluid
ADA levels in Pleural fluid are measured by
colorimetric method.
Increased ADA levels (>36IU/L) observed in
Tubercular Pleural Effusion.
Quick test.
Adjunct test to help rule in or rule out tuberculosis in
pleural fluid.

73. Lysozyme
Lysozyme, a bacteriolytic enzyme, have been
found to be higher in patients with TB.
However, a fluid to serum lysozyme ratio > 1.2 has
been found to be a better tool for the diagnosis of
TB.

74. Conclusion
Newer Technologies offer a significant time savings. However
these tests have limitations –
1. Costly.
2. Complex & Cumbersome.
3. Only smear positive (50% of culture positives)
4. Recommended only in special cases.
5. Add-on tests.
Culture is still the Gold Standard.
Asrecommended by CDC/WHO
* Whenever possible, use liquid culture & DST.
* Rapid testing and reporting essential for TB control.

75. Progress in TB Diagnosis
Past Present
Koch discovered tubercle bacillus 133
yrs back
No major discovery
Except TB Genome, IS6110, BACTEC
460 (liquid media)
TB diagnosed by symptoms - prehistoricStill the same practice in many High
Bruden Countries (HBCs)
Tuberculin test - > 100yrs Still Commonly used
Egg based media –almost 100yrs Still most commonly used
AFB Smear for diagnosis – 133 yrs backStill the major diagnostic tool in many
countries
Radiological Diagnosis Still Important (X-ray, CT Scan)

76. TB CowBoys
Thank You!