Lab diagnosis of
pulmonary and extra
pulmonary
tuberculosis
Dr. Mohammed Faraaz Khan
Second year postgraduate
student
Department of microbiology
Introduction
Tuberculosis is a chronic granulomatous disease caused
by the bacterium Mycobacterium tuberculosis.
The pulmonary form affects the lungs whereas extra
pulmonary tuberculosis refers to the disease involving
other organs most commonly the pleura, lymph nodes,
kidneys, spine etc.
Tuberculosis affects 10 million people annually and
kills 1.5 million every year.
Half of all people with tuberculosis live in 8 countries
namely, Bangladesh, China, India, Indonesia, Nigeria,
Pakistan, Philippines and South Africa.
Tuberculosis is also the leading cause of death in HIV
infection.
Specimen collection
Respiratory specimens: Early morning sputum samples to
be collected by expectoration or ultrasonic nebulization.
24 hour sputum collections are discouraged.
For culture, three consecutive early morning sputum
samples are to be collected on successive 24 hour periods.
In paediatric patients unable to expectorate, a gastric
aspirate is ideal.
Extra pulmonary specimens
Pleural TB: Pleural fluid collected by a pleural tap.
TB lymphadenitis: Fine needle aspiration cytology or excision
biopsy of the suspected lymph node.
TB meningitis: CSF collected by a lumbar puncture.
Genital TB: Endometrial biopsy by dilatation and curettage. If
biopsy is contraindicated as in a nulligravida, menstrual blood on
the second day of bleeding can be collected into a sterile
container.
Renal TB: Early morning clean catch midstream urine samples
on three consecutive days of at least 40 ml each. 24 hour
collections are discouraged.
Microscopy
Two types of stains are in use:
Carbol fuchsin based staining procedures; Ziehl Neelsen
method (hot) and Kinyouns modification (cold).
Fluorescent stains such as Auramine and Rhodamine O.
At least 10,000 bacilli must be present per ml of sputum.
Pros and cons
ZN staining is cheaper. Fluorescent microscopy is
Sensitivity is lower than expensive.
fluorescent microscopy. Higher sensitivity than ZN
Increased time for staining.
screening. Faster screening time.
Sensitivity is poor in extra Non specific fluorescence
pulmonary tuberculosis. may be confusing.
Mycobacterial culture
Egg based media: Lowenstein Jensen medium, Dorset’s
medium, Petragnini medium and American Thoracic Society
medium.
Agar based media: Middlebrook 7H9 (Liquid), 7H10 and
7H11 media.
Culture on traditional egg based media takes 6 to 8 weeks but
may occur faster on Middlebrook media.
Pros and cons
Egg based media take a Growth is faster in
long time to give growth. Middlebrook media.
Lower rates of Higher rates of
contamination. contamination.
Early presumptive Early presumptive
identification not possible. identification possible.
CO2 not required. CO2 is mandatory.
MGIT
MGIT stands for Mycobacterium Growth Indicator Tube.
Automated broth based culture system using Middlebrook 7H9
medium and a fluorescent compound.
Can be used for all clinical samples except blood and urine.
Mean time to detection is usually 10 days.
MB/BacT ALERT
Automated broth based blood culture system for mycobacteria
containing Middlebrook 7H9 broth in an atmosphere of CO2,
nitrogen and oxygen under vacuum.
Identification of growth
Conventional means by biochemical tests.
Automated methods such as MALDI-TOF.
High Performance Liquid Chromatography.
MPT 64 antigen detection.
Xpert MTB/RIF assay
Nucleic acid amplification assay which detects Mycobacterium
tuberculosis and rifampicin resistance simultaneously.
Results available within 2 hours.
Approved sample types are raw sputum and sediments from
concentrated sputum.
Has been used with variable success in testing other sample
types.
Mantoux test
Skin test utilizing purified protein derivative (PPD) from
tubercle bacilli.
Injection is given on the volar aspect of left forearm and read
after 48 to 72 hours.
IGRA
Interferon Gamma Release Assays are tests that utilize whole
blood to determine the presence of latent Tuberculosis.
They are superior to skin tests because of higher specificity
due to no cross reactions with non-tuberculous mycobacteria,
leprosy bacilli and the BCG vaccine.
They are of two types; TB-spot and Quantiferon Gold.
Antimicrobial susceptibility testing
Phenotypic methods: Proportion method.
Genotypic methods: Xpert/MTB RIF assay, Line Probe Assay.
