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1. Laboratory Diagnosis
Of
Tuberculosis
Tarun Prudvi B
MBBS 3RD PROFESSSIONAL

2. A complete medical evaluation for tuberculosis
(TB) must include a medical history, a physical
examination, a chest X-ray and microbiological
examination (of sputum or some other appropriate
sample). It may also include a tuberculin skin test,
other scans and X-rays, surgical biopsy
Tuberculosis is diagnosed by finding Mycobacterium
tuberculosis bacteria in a clinical specimen taken from the
patient. While other investigations may strongly suggest
tuberculosis as the diagnosis, they cannot confirm it.

3. Diagnosis // MEDICAL HISTORY
Productive prolonged cough of three or more weeks, chest pain, and haemoptysis.
Systemic symptoms
Low grade remittent fever, chills, night sweats,
Appetite loss, weight loss, easy fatigability, and
Production of sputum that starts out mucoid but changes to purulent.
Other parts of the medical history include prior TB exposure, infection or disease;
past TB treatment;
demographic risk factors for TB;
and medical conditions that increase risk for TB disease such as HIV infection.
• Tuberculosis should be suspected when a pneumonia-like illness has
persisted longer than three weeks, or when a respiratory illness in an
otherwise healthy individual does not respond to regular antibiotics.

4. A definitive diagnosis of tuberculosis can only be made by culturing
Mycobacterium tuberculosis organisms from a specimen taken from the patient
(most often sputum, but may also include pus, CSF, biopsied tissue, etc.).
A diagnosis made other than by culture may only be classified as "probable" or
"presumed".
For a diagnosis negating the possibility of tuberculosis infection, most protocols
require that two separate cultures both test negative
Diagnosis // Microbiological studies

5. SAMPLE SOURCES // Sputum
Sputum smears and cultures should be done for acid-fast bacilli if the
patient is producing sputum.
In cases where there is no spontaneous sputum production, a sample
can be induced, usually by nebulized inhalation of a saline or saline
with bronchodilator solution.
A comparative study found that inducing three sputum samples is
more sensitive than three gastric washings

6. Alternative sample sources
Gastric washings,
Laryngeal swab,
Bronchoscopy (with bronchoalveolar lavage, bronchial washings,
and/or transbronchial biopsy),
FNAC (transtracheal or transbronchial).
In some cases, a more invasive technique is necessary, including tissue
biopsy during mediastinoscopy or thoracoscopy

7. Concentration methods
Petroff’s method
N acetyl cysteine is used, NaOH kills contaminating bacteria.Sputum is incubated w/ 4% sodium hydroxide at 37 degrees with
frequent shaking till it becomes clear. Then centrifuged at
3000rpm for 20 min and sediment neutralized with N/10 HCL.
NALC combined with 2% NaOH

8. Microscopy
Ziehl-Neelsen staining
After smears are dried then stained using Ziehl-Neelsen
technique and are observed under oil immersion for the
presence of acid fast bacilli

9. Auramine rhodamine
Smears are stained with Auramine phenol or Auramine fluorescent
dyes and examined under ultraviolet illumination.
Seen as bright rods against dark background.

10. AFB Report as per RNTCP Guidelines:
Result Grading No. of fields
>10/field positive 3+ 20
1-10/fiield Positive 2+ 50
10-99/field Positive 1+ 100
1-9/field
No bacilli
Positive
Negative
Scanty 100
1000

11. Culture
Solid media: Lowenstein-Jensen (LJ) medium.
Colonies are dry, rough, raised,
irregular with wrinkled surface.
They are creamy white or buff
colored.
They may take 3-8 weeks to develop.
Liquid media: middle brooke 7H10/7H11

12. Automated systems
Radiometric BACTEC 460 TB method:
This system detects the presence of mycobacteria based on their
metabolism rather than visible growth.
When the 14C labelled substrate present in the medium is
metabolised, 14Co2 is produced and measured by the BACTEC system
instrument and reported in terms of the growth index(GI) value.
The BACTEC system is also useful in the identification of M.
tuberculosis using a specific inhibitor, para-nitro-a-acetyl-amino-B-
hydroxypropiophenone.
Using the same system, drug susceptibility tests can also be
performed for all the anti-tuberculosis drugs when sufficient GI is
observed.
Mycobacteria in clinical samples can be detected in half the time
compared to conventional culture methods

13. MGIT 960 (Mycobacteria Growth Indicator Tube):
Growth detection is based on the AFB metabolic O2 utilization.
MB/BacT system:
This is a non-radiometric continuous monitoring system with
computerised database management. The system is based on
colorimetric detection of CO2.
ESP culture system II:
This is a fully automated continuous monitoring system based on the
detection of pressure changes within the headspace above the broth
culture medium in a sealed bottle, i.e. either gas production or gas
consumption due to microbial growth.
A special detection algorithm is present in this system for the detection
of very slowly growing mycobacteria.

14. Resistant testing.
Phenotypic methods
Absolute concentration method,
Resistance ratio method
Proportion method.
Recently developed phenotypic methods are E-test (commercially available
as AB BIODISK), micro-well alamar blue assay and micro plate tetrazolium
reduction assay, mycolic acid index susceptibility testing.
Microscopic observation of broth cultures for drug susceptibility assay,
micro-colony detection, Pha B assay and luciferase reporter phage assay,
etc., are also being developed37.

15. Important genotypic methods include Automated DNA sequencing,
PCR SSCP, PCR HDF, Line probe assay or LiPA (solid phase hybridisation
assay).
Mycobacteria usually acquire resistance either by alteration of the
drug target by mutation or by titration of drug through over production
of the target. MDR-TB usually results from an accumulation of
individual target genes

16. Demonstration of Mycobacteria Directly from
Clinical Samples
Genotypic methods:
Nucleic-acid Amplification Assays (NAA)
PCR
Phenotypic methods:
FAST Plaque TB

17. Serological Diagnosis
stages or types of TB infection Antigen (epitope)
Contacts and tuberculin convertors 38 KDa (TB 71,72), 14 KDa (TB 68)
Radiologically healed or less extensive
disease
14 KDa (TB 68
Radiologically extensive disease 38 KDa (TB 71, 72)
Miliary TB LAM (ML 34)
TB meningitis adults LAM (ML 34)
children 17 KDa (HBT2)
Human immuno-deficiency virus (HIV)
infection-
38 KDa, LAM, 17KDa
It is important to remember that different antibody specificities are stimulated in various stages or
types of TB infection

18. Tests.
Sandwich ELISA,
inhibition ELISA,
latex agglutination and
reverse passive haemagglutination tests are various methods used
for their detection
Newer tests are the TB STAT-PAK, enzyme Immuno-assay for detection of
anti-mycobacterial superoxide dismutase antibody, and the Insta test TB

19. Test to detect latent infection.
• The Heaf test was used in the United Kingdom until 2005, and is
graded on a four point scale. The Mantoux test is now used.
• The equivalent Mantoux test positive levels done with 10 TU PPD (0.1
ml 100 TU/ml, 1:1000) are
• 0–4 mm induration (Heaf 0 to 1)
• 5–14 mm induration (Heaf 2)
• Greater than 15 mm induration (Heaf 3 to 5)

22. IFN-gamma produced by T lymphocytes in whole blood after
stimulation with PPDs obtained from M. tuberculosis, M. avium, and
M. bovis- QauntiFERON-Gold test for TB.

23. RNTCP PROTOCOL

26. Radiography
• In active pulmonary TB, infiltrates or consolidations and/or cavities are often seen in the
upper lungs with or without mediastinal or hilar lymphadenopathy or pleural effusions (
tuberculous pleurisy).
• However, lesions may appear anywhere in the lungs. In disseminated TB a pattern of
many tiny nodules throughout the lung fields is common - the so-called miliary TB.
• In HIV and other immunosuppressed persons, any abnormality may indicate TB or the
chest X-ray may even appear entirely normal.
• Abnormalities on chest radiographs may be suggestive of, but are not
necessarily diagnostic of, TB. However, chest radiographs may be used to rule
out the possibility of pulmonary TB in a person who has a positive reaction to
the tuberculin skin test and no symptoms of the disease.
• Cavitation or consolidation of the apexes of the upper lobes of the lung or the tree-in-
bud sign may be visible on an affected patient's chest X-ray.
• The tree-in-bud sign may appear on the chest CTs of some patients affected by
tuberculosis, but it is not specific to tuberculosis

27. Diffuse bilateral, largely upper lobe,
consolidation and pulmonary
infiltrates. Suggestion of small area of
cavitation at the left lung apex

28. left upper lobe cavitation

32. STOP TB