Concise Laboratory Diagnosis ofTuberculosis Infection (TB)

Dr Mostafa Mahmoud, MD, Ph D Consultant Microbiologist

Assist. Prof. of Medical Microbiology & Immunology

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)

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.

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- Serology6- Histopathology.

SamplesType of sample:

Sputum (resp. sample), BALCSF (spinal/para-spinal/intra-cerebral), gastric washings (Aspirate), lymph nodes (tissues), urine, faeces, blood

SPECIMEN QUALITYNow 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 containerComplete and accurate informationProper packaging (i.e. US Postal Service)“Prompt” delivery to the Lab.Proper documentation.

Transfer to laboratory

Within 24 h (or 1 working day, max 48h)Minimise overgrowthMaintain AFB character

Potentially infected clinical sampleRoutine procedure

1- Direct AFB Slide ExaminationLeast Sensitive AFB Test (20% to 80%)

increase with advance of the TB infection.Requires 100,000 AFB/mlRequires an adequate specimen > 5 ml Negative smear – Does not rule out AFBPositive smear – AFB present (viability?)AFB Positive = Mycobacterium spp., etc.Help support TB diagnosisQuantitation: Positive/Positive Few/

Negative

Formats:1. Conventional Microscopy with sensitivity of 32 to 94%.

2 techniques:- i- Hot AFB staining (Ziehl-Neelsen) Method the most commonii- 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.

Ziehl- Neelsen Stain

Steps of Ziehl- Neelsen Staining

Auramine stained smear

Smear positive AFB by Ziehl-Neelsen

Recording Sputum Smear Microscopy ResultsNumber 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)

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.

Solid LJ media

Middlebrooks media liquid and solid

Automated Bactec MGIT 960

Advantages of AFB Culture and IsolationMore sensitive than slide Microscopy (80%-90%)Requires only 10 AFB/ ml of specimenAFB Growth in 1-2 weeks (Bactec)Genetic Probes and HPLC Rapid IDFewer 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 (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 developmentWhich patients? Mostly for latent TBHow long should it take? Around 1 day Who provides it?What do the results mean and who interprets them?

Explanation of immunodiagnostic assays:

Interferon gamma release assays

Quantiferon and T-Spot TB kits

Advantages of gamma interferon release assay:

One visit testNot 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 hybridization.

iii- Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) (TB PNA FISH) sensitivity 84-97%.

iv- PCR with or without sequencingv- 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 ) for MTBPCR

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.

GenXpert System Cepheid

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

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.

What test formats available in MOH

1- Ziehl-Neelsen Stain2- Kynon Cold Stain3- GenXpert (Cepheid)4- Roche Amplicor PCR and sequencing

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.

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