1) Tuberculosis (TB) is commonly diagnosed through direct microscopy, culture, immunodiagnostic tests, molecular tests, and histopathology using samples from sputum, BAL, CSF, tissues, and other body fluids. 2) Direct microscopy has low sensitivity but is quick, while culture has higher sensitivity and allows drug susceptibility testing but takes 1-2 weeks for results. Newer liquid culture systems can provide results in only a few days. 3) Molecular tests like PCR and interferon-gamma release assays provide rapid results within hours and are also used for diagnosis, but many have high costs.

1. Concise Laboratory Diagnosis of
Tuberculosis Infection (TB)
Dr Mostafa Mahmoud, MD, Ph D
Consultant Microbiologist
Assist. Prof. of Medical Microbiology & Immunology

2. Pulmonary, 70%
Extrapulmonary, 21%
Both, 9%
Pleural, 18%
Lymphatic, 42%
Bone/joint, 11% Genitourinary, 5%
Meningeal, 6%
Other, 12%
TB Cases by Form of Disease,
United States, CDC, 2005 Peritoneal, 6%
Clinical Presentation of TB: Extrapulmonary
 Incidence/site may vary  TB can involve any organ
 More common in HIV/TB (co-infection)

3. Introduction
 80% of all TB cases worldwide are from 22 high-
burden countries (India, China, Indonesia ,
Nigeria, Bangladesh, South Africa, Pakistan,
Ethiopia, Philippines, Congo, Vietnam, Tanzania,
Brazil, Uganda, Thailand, Mozambique, Myanmar,
Afghanistan, Cambodia) arranged in a descending
order by the number of cases.

4. Lab Diagnosis of TB.
1- Direct Microscopy Smearing (Staining) (AFB stains and
Fluorescent)
2- Culture: (Solid and liquid media also for measurement of
drug susceptibility testing DST for resistance).
3- Gamma interferon Release Assays ( Spot test and gold????)
4- Molecular testing (PCR and other NAA)
5- Serology
6- Histopathology.

5. Samples
 Type of sample:
Sputum (resp. sample), BAL
CSF (spinal/para-spinal/intra-cerebral),
gastric washings (Aspirate),
lymph nodes (tissues),
urine, faeces, blood

6. SPECIMEN QUALITY
 Now 2 specimens are used; before three specimens on
three or 2 successive days
 Early morning specimens (First thing out!)
 Adequate specimen volume (>7 ml)
 Sputum not saliva (epithelial cells > 25/ LPF = Saliva)
 Condition of specimen (pH, etc.)
 Appropriate specimen container
 Complete and accurate information
 Proper packaging (i.e. US Postal Service)
 “Prompt” delivery to the Lab.
 Proper documentation.

7. Transfer to laboratory
 Within 24 h (or 1 working day, max 48h)
Minimise overgrowth
Maintain AFB character
 Potentially infected clinical sample
Routine procedure

8. 1- Direct AFB Slide Examination
 Least Sensitive AFB Test (20% to 80%) increase
with advance of the TB infection.
 Requires 100,000 AFB/ml
 Requires an adequate specimen > 5 ml
 Negative smear – Does not rule out AFB
 Positive smear – AFB present (viability?)
 AFB Positive = Mycobacterium spp., etc.
 Help support TB diagnosis
 Quantitation: Positive/Positive Few/ Negative

9. Formats:
1. Conventional Microscopy with sensitivity of 32 to 94%.
2 techniques:-
i- Hot AFB staining (Ziehl-Neelsen) Method the most common
ii- Cold AFB staining (Kynon staining)
2- Fluorescent Microscopy with sensitivy of 52% to 97% by
Auramine staining (more sensitive and less cumbersome (low
power) than other 2 previous ones. However, less specific and
needs confirmation by Z-N staining).
- LED microscopy in now recommended to replace the
fluorescent microscope in all situations.
- Concentrated smears are more sensitive than direct one.

10. Ziehl- Neelsen Stain

11. Steps of Ziehl- Neelsen Staining

12. Auramine stained smear

13. Smear positive AFB by Ziehl-Neelsen

14. Recording Sputum Smear Microscopy Results
Number of Acid-
fast Bacilli (AFB)
# of Oil Immersion
Fields Examine
Reported as:
No AFB Per 100 fields No AFB seen
(No AFB per 100 fields)
1-9 AFB Per 100 fields Scanty, record exact figure
(1-9 AFB per 100 fields)
10-99 AFB Per 100 fields 1+ (10-99 AFB per 100 fields)
1-10 AFB Per field 2+ (1-10 AFB per field in 50
fields)
More than 10 AFB Per field 3+ (>10 AFB per field in 20
fields)

15. 2- Culture methods: solid versus liquid
Solid: on Lowenstein- Jensin (LJ) slopes. 7H11 Middlebrooks slopes or
plates. On LJ media it takes 12 weeks incubation.
Liquid: Early attempts high contamination. Recommended by WHO
on liquid Middlebrooks (7H9) broth media
Automated liquid systems -Bactec MIGT 960; in common use nowadays
– Bactec 460TB; old and use radioactive C 14
- ESP blood culture system
More Sensitive and Rapid.
Contamination with non-tuberculous MB can give false results.

16. Solid LJ media

17. Middlebrooks media liquid and solid

18. Automated Bactec MGIT 960

21. Advantages of AFB Culture and Isolation
 More sensitive than slide Microscopy (80%-90%)
 Requires only 10 AFB/ ml of specimen
 AFB Growth in 1-2 weeks (Bactec)
 Genetic Probes and HPLC Rapid ID
 Fewer AFB/ml – Delayed growth
 “AFB Growth” required for other tests e.g. species
identification and drug susceptibility.
 Mixed cultures require subcultures

22. 3- Gamma interferon Release Assays
(Immunodiagnostic) tests
 Used for detection (after in vitro stimulation) of either:
 Interferon γ (QuantiFERON-TB Gold)
 Activated specific T-cells (T-SPOT.TB)
Standard under development
Which patients? Mostly for latent TB
How long should it take? Around 1 day
Who provides it?
What do the results mean and who interprets them?

23. Explanation of immunodiagnostic assays:

24. Interferon gamma release assays

25. Quantiferon and T-Spot TB kits

26. Advantages of gamma interferon release assay:
 One visit test
 Not affected by vaccination with BCG.
 Can diagnose latent and active disease.

27.  1- From mycobacterial cultures for species identification:
i- Nucleic Acid hybridization (Gen-probe) for 16S rRNA
from culture isolates
ii- Line Probe assays (e.g. Hain Strips) it is reverse
hybridization.
iii- Fluorescence in situ hybridization (FISH) using peptide
nucleic acid (PNA) (TB PNA FISH) sensitivity 84-97%.
iv- PCR with or without sequencing
v- Xpert MTB/RIF (Cepheid) new fully automated but
costly technique for identification and DST.
4- Molecular diagnosis of Mycobacterial

28. vi- Other NAAT for typing: e.g. LCR,
Spoliotyping, RFLP etc.
2- Directly from clinical specimens:
 MTD (Gen-probe) or Amplicor (Roche )
for MTB
PCR

29. Advantages of molecular test:
More rapid results within hours.
 high specificity 98% - 100 %. However,
Less specific giving false positive and
negative results in clinical samples.
Disadvantages:
 High costs.

30. GenXpert System Cepheid

32. 5- Serology
 For detection of either antigens or antibodies.
 Little value in laboratory diagnosis.
 Variability and cross-reactivity in results i.e. low
specificity and sensitivity.
 Not distinguishing active from latent infection
 Formats:
- ELISA (82 sensitivity and 86% specificity).
- Detection of lipoarabinomannan (LAM) antigen

33. Recent Technologies:
 1- MALDI TOF (Matrix Assisted Laser Desorption Ionization
Time of Flight MS)
 Very rapid technique (< 1 hour)
 2- GenXpert (Cepheid) System
 Rapid
 Simple
 All reagent in one cartridge
 Can be used in general lab no need for spate rooms
 Used for clinical specimens
 Gives specie identification and drug susceptibility.

34. What test formats available in MOH
 1- Ziehl-Neelsen Stain
 2- Kynon Cold Stain
 3- GenXpert (Cepheid)
 4- Roche Amplicor PCR and sequencing

35. References:
 Imperial College Healthcare (NHS)
 Recent advances in the laboratory diagnosis of tuberculosis, Rommy Teran1, Jacobus H. de Waard2
 Parson LM et al. 2011. Laboratory Diagnosis of Tuberculosis in Resource-Poor Countries: Challenges and
Opportunities. CLINICAL MICROBIOLOGY REVIEWS, Apr. 2011, p. 314–350.
 Anochie PI et al.
 Recent advances in the diagnosis of Mycobacterium tuberculosis; Review. www.germs.ro • GERMS 2(3) •
September 2012 • page 110