Concise Laboratory Diagnosis of
Tuberculosis Infection (TB)
Dr Mostafa Mahmoud, MD, Ph D
Assist. Prof. of Medical Microbiology & Immunology
Bone/joint, 11% Genitourinary, 5%
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)
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.
Lab Diagnosis of TB.
1- Direct Microscopy Smearing (Staining) (AFB stains and
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)
Type of sample:
Sputum (resp. sample), BAL
gastric washings (Aspirate),
lymph nodes (tissues),
urine, faeces, blood
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.
Transfer to laboratory
Within 24 h (or 1 working day, max 48h)
Maintain AFB character
Potentially infected clinical sample
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
1. Conventional Microscopy with sensitivity of 32 to 94%.
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.
Recording Sputum Smear Microscopy Results
Number of Acid-
fast Bacilli (AFB)
# of Oil Immersion
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
More than 10 AFB Per field 3+ (>10 AFB per field in 20
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.
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
3- Gamma interferon Release Assays
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?
Advantages of gamma interferon release assay:
One visit test
Not affected by vaccination with BCG.
Can diagnose latent and active disease.
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
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
vi- Other NAAT for typing: e.g. LCR,
Spoliotyping, RFLP etc.
2- Directly from clinical specimens:
MTD (Gen-probe) or Amplicor (Roche )
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.
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
- ELISA (82 sensitivity and 86% specificity).
- Detection of lipoarabinomannan (LAM) antigen
1- MALDI TOF (Matrix Assisted Laser Desorption Ionization
Time of Flight MS)
Very rapid technique (< 1 hour)
2- GenXpert (Cepheid) System
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.
What test formats available in MOH
1- Ziehl-Neelsen Stain
2- Kynon Cold Stain
3- GenXpert (Cepheid)
4- Roche Amplicor PCR and sequencing
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