Mycobacterium tuberculosis - Lab diagnosis, Sputum, Microscopy, Grading, Culture, PPD test

Laboratory diagnosis of Mycobacterium tuberculosis

The laboratory diagnosis of Mycobacterium tuberculosis begins with the collection of samples or by imaging.

Sample

• Sputum (collected early morning for 3 regular days)

• BAL

• bronchial/tracheal aspirates (in the case of extrapulmonary tuberculosis)

• urine

• gastric aspirates

• biopsy specimen

• CSF

• pericardial and pleural fluid bone marrow

• blood feces from immunocompromised patients

Things to remember

• the gastric aspirate was collected in cases of difficulty in sputum production (in case of child patients)

• stimulation of cough can be done by using an aerosol solution of propylene glycol in 10% sodium chloride

• CSF is collected for diagnosis of tubercular meningitis

• Urine is for genitourinary tuberculosis. Urine specimens are centrifuged at 300 rpm for 30 mins and sediments are used for culture and microscopy

*Acid-fast bacilli can infect almost any tissue organ

Collection of sputum

When sputum is collected, the following procedure must be followed

• The patient is provided with a clean, dry, wide-necked, leak-proof container and requested to produce a sputum specimen

• If the patient is young or in cases in which sputum was swallowed during the night, gastric lavage can be collected

• The container must be labeled and the request form must be filled out before being dispatched to the processing

Processing of sputum

After collection, the sputum must be processed as

• A smear (of sputum sample) is prepared on a slide for Ziehl-Neelson’s staining using any caseous particles and the most purulent materials. The slide is then allowed to dry in a safe place

• The slide is fixed by using 1-2 drops of 70% ethanol

• The slide is covered with filtered carbon fuschin stain

• The stain is heated until the vapor just begins to rise

• Allow the heated stain to remain on the slide for 5 mins

• Wash well with clean water

• Cover the smear with 3% v/v acid alcohol for 5 mins and wash well with clean water

• Cover the smear with malachite green for 1-2 mins then wash with clean water

• Blot dry the back of the slide and place it in the rack

• Observe under oil immersion at 100x (pleural fluid, peritoneal fluid, and other exudates are collected in containers with citrate to prevent coagulation.)

* specimens should be transported asap. In case of delay, specimens are refrigerated at 4°C but not more than overnight.

Transport medium: 1% cetyl Dyridenium Chloride (CPC) + 2% NaCl or Tri Sodium Phosphate

Microscopy

• microscopy is a sensitive and rapid procedure for the presumptive identification of Mycobacterium tuberculosis

• presumptive identification

• the cell wall of mycobacteria contains long-chain, multiply cross-linked fatty acids, called mycolic acids

• The mycolic acids and lipids in the mycobacterial cell wall account for its resistance to drying and harsh decontamination agents

• low sensitivity stains both alive and dead mycobacterium (requires 10⁴- 10⁵) bacilli per ml of sputum

• 3 types of staining procedures can be used:

  • Fluorochrome stain (most sensitive)

  • Ziehl-Neelsen stain / carbolfuchsin stain

  • Kinyoun stain (cold stain procedure)

• In Z-N stain, Mycobacterium spp. Appear red or red-blue, beaded appearance whereas non-mycobacteria appear blue

• A procedure similar to Z-N stain but no heat is used. Acid-fast bacilli appear purple to read, slightly curved, short or long rods (2-8 μm). They also may appear beaded or banded.

• cross-contamination of slides during the staining process and the use of water contaminated with saprophytic mycobacteria can lead to false-positive results.

Grading of ZN-stained smears for AFB

Culture

The culture of Mycobacterium tuberculosis can be done by:

• Media used:-

  Lowenstein-Jensen (LJ medium) = egg-potato based (solid media)

  Middlebrook 7H10 medium = serum / albumin-based (solid media)

  Mycobacteria Growth Indicator tube (MGIT) (liquid media)

• One solid medium and one liquid medium is considered a standard culture procedure but is usually impractical

• More sensitive than microscopy

• The advantage of liquid media is that bacilli grow in approximately 10 days, compared with 17 days or longer incubation time

• 10-100 AFB per ml of culture rather than Petri plate so as to reduce exposure of tubercle bacilli with air thus limiting lab-acquired infection

Process:

The procedure for culture of Mycobacterium tuberculosis are as follows:

• About 20 min before culturing, the specimen is decontaminated by mixing the specimen (sputum) with the volume of NaOH (40g/l). It is mixed at intervals to homogenize the sputum

• Using a sterile Pasteur pipette, 200 μl of well-mixed sputum sample is inoculated on a slope of LJ medium and the specimen is allowed to run down the slope.

• The inoculated medium is incubated at 35-37°C in 5-10% CO2 atmospheric and high humidity in a rack placed at an angle of 45°C to ensure the full length of the slope is covered.

• After 1 week the slope is placed in an upright position and further incubated for 5-10 weeks examining twice a week for growth.

• Cultures should not be discarded as negative until they have been observed for 12 weeks

• The screw cap of the LJ medium can be left loose for the first week of incubation to allow the evaporation of excess fluid and entry of CO2.

• Mycobacterium tuberculosis is indicated by rough, buff, and tough colonies

Antigen-protein detection

Detection of microbial products/components has been used to diagnose infection caused by Mycobacterium tuberculosis.

Eg: Tuberculostearic acid (a fatty acid that can be extracted from the mycobacterial cell wall) is detected by gas chromatography or mass spectrometry in specimens containing few mycobacteria

It can also be used in cases of tuberculous meningitis (CSF sample).

A host enzyme Adenosine deaminase is increasingly produced in patients with tuberculous pleural effusions. This enzyme detection is a form of diagnosis (sensitive= 98%, specificity = 96%)

Immunodiagnostic testing

Interferon-gamma release assays (eg: T-SPOT-TB, QuantiFERON – TB gold) are being used for the diagnosis of tuberculosis.

For immunodiagnostic testing, Mycobacterial Ag such as BCG, 5 and 6 kDa protein of Mycobacterium tuberculosis, 34 and 64 kDa protein of M. bovis is used

Advantages

• Do not cross-react with non-tuberculous mycobacterium

• are not affected by the BCG vaccine

• are not variable as the serologic tuberculin skin test

* T-SPOT-TB does not require a follow-up visit with a physician and it measures T-cells that have been activated by Mycobacterium tuberculosis Ag.

* Quanti-FERON gold (a type of ELISA) measures a component of CMI (cell-mediated immunity) response to M. tuberculosis to diagnose latent and active tuberculosis

Tuberculin skin test / purified protein derivative (PPD) test

After infection by Mycobacterium tuberculosis, the individual develops a delayed hypersensitivity to certain Ag of the microorganism that can be detected by the Tuberculin skin test / purified protein derivative (PPD) test.

Procedure:

The procedure for Tuberculin skin test / purified protein derivative (PPD) test includes:

• 0.1 ml culture extract of Mycobacterium tuberculosis i.e. PPD of tuberculin containing 5 TU (tuberculin units) is injected intracutaneously in one arm

• Option: 0.1ml of sterile normal saline is injected intracutaneously into another arm as a control

• Delayed type hypersensitivity reaction to PPD is observed after 48-72 hours and characterized by erythema (redness) and firmness

• Its firmness (induration) is measured and interpreted as:

  10 mm or above = positive

  5 mm or less = negative

  6-9 mm = equivocal

• Interpretation criteria are different in different patient populations (eg: immunosuppressed individuals – with AIDS)

• DTH (delayed-type hypersensitivity) is observed in patients who have had the BCG vaccine

• PPD test is not 100% specific or sensitive

• False-negative test (anergy) can be seen in conditions such as miliary tuberculosis, convalescence form of some viral infection like measles, lymphoreticular malignancy, severe malnutrition, impaired CMI, immunosuppressive therapy

*(If the test is negative using 5 TU, can be repeated using 10 TU or 100 TU)

Pigment production

If the culture of Mycobacterium tuberculosis is left in light (not direct sunlight) for 2 hours and then reincubated at 35-37°C, it produces yellow pigment.

Molecular methods

Commonly used molecular methods for Mycobacterium tuberculosis diagnosis are:

• PCR

• Restriction fragment length polymorphism (RFLP)

• DNA microarrays

TCH test

• TCH (Thiophene-2-carboxylic acid hydrazide) inhibits the growth of some mycobacteria

• This test is used to distinguish Mycobacterium tuberculosis (is not inhibited by TCH) from M. bovis (inhibited by TCH)

Other tests

• Neutral red test: positive

• Catalase test: Positive (weekly)

• Aryl sulfate test = Negative

• Nitrate reduction = strongly positive

• Amidase test = Positive

• Niacin test = Positive