concise discussion about the available lab diagnosis of TB in developing and developed countries.

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