Diagnose of M. Tuberculosis in Microbiology Laboratory

A complete medical evaluation for diagnosis of M. Tuberculosis in microbiology laboratory.

1. Physical Examination:

A physical examination is done to assess the patient’s general health and find other factors which may affect the TB treatment plan. It cannot be used to confirm or rule out TB. However, certain findings are suggestive of TB. For example, blood in the sputum, significant weight loss and drenching night sweats may be due to TB.

Radiography:

Chest X-ray and CT:  

Tuberculosis creates cavities visible in x-rays like this one in the patient’s right upper lobe.

In active pulmonary TB, infiltrates or consolidations and/or cavities are often seen in the upper lungs with or without mediastinal or hilar lymphadenopathy or pleural effusions (tuberculous pleurisy). However, lesions may appear anywhere in the lungs.

In disseminated TB a pattern of many tiny nodules throughout the lung fields is common so it called miliary TB. In HIV and other immunosuppressed persons, any abnormality may indicate TB or the chest X-ray may even appear entirely normal.

Abnormalities on chest radiographs may be suggestive of, but are not necessarily diagnostic of, TB. However, chest radiographs may be used to rule out the possibility of pulmonary TB in a person who has a positive reaction to the tuberculin skin test and no symptoms of the disease.

Cavitation or consolidation of the apexes of the upper lobes of the lung or the tree-in- bud sign may be visible on an affected patient’s chest X-ray. The tree-in-bud sign may appear on the chest CTs of some patients affected by tuberculosis, but it is not specific to tuberculosis.

2. AFB Staining (Ziehl – Neelsen Method):

Acid Fast Bacteria Staining:

The great majority of bacteria are easily stained by used simple procedure however there are some exception. Some bacteria are surrounded by a covering composed of fatty acid rich lipid or waxy substance. This organism are not readily stained but when once stains with strong reagent like hot carbol fuchsine are able to retain the colour even after treatment with drastic decolourizing agent like 20% H₂SO₄.

They are called acid fast because the stained bacteria are resistance to decolourization with acid (20% sulphuric acid). In acid fast staining methelene blue used as counter stain. So nonacid fast organism decolourized by acid treatment and counter stained with methylene blue. But acid fast organism stained with fuchsine and appeared as red colour.

Procedure:

1. Heat fixed smear cover with carbol fuchsine solution and heated until the steam just begins to rise. Once steam rises set the stop watch for 5 minutes and continue heating the slide without allowing the liquid to boil (of stain drying add more carbolfuchsin).

2. Allow the slide to cool and wash gently with water until the water that runs off is colourless.

3. The smear wash with 20% H₂SO₄ or 3% hydrochloric acid in 95% alcohol (methylated sprit) for declourization until the smear become pale pink then the slide wash with tap water.

4. Pour methylene blue or malachite green solution over the slide and leave it for to sees. Then smear washed with water, drained the water dry and examined under microscope using oil immension len).

After step 3, primary decolourization with the H₂S0₄ film may be treated with 95% alcohol as a secondary decolourization two minutes. We know that tubercle bacilli are alcohol fast as well as acid fast. So identification of tubercle bacilli from other acid fast bacilli which may be present in the pathological sample is possible because other acid fast bacilli decolourized by alcohol treatment.

Result:

Acid fast bacteria were showing red colour white nonacid fast bacteria green or blue colour depending upon the counter stain.

To stain leprosy bacilli, which are less acid fast than tubercle bacilli that case 5% H₂S0₄ is used as decolourizing agent but all procedure are same as above e.g. of acid fast bacteria. Mycobacterium tuberculosis.

A definitive diagnosis of tuberculosis can only be made by culturing Mycobacterium tuberculosis organisms from a specimen taken from the patient (most often sputum, but may also include pus, CSF, biopsied tissue, etc.). A diagnosis made other than by culture may only be classified as “probable” or “presumed”. For a diagnosis negating the possibility of tuberculosis infection, most protocols require that two separate cultures both test negative.

3. Immunological Test:

ALS Assay:

Antibodies from Lymphocyte Secretion or Antibody in Lymphocyte Supernatant or ALS Assay is an immunological assay to detect active diseases like tuberculosis, cholera, typhoid etc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis of tuberculosis.

The principle is based on the secretion of antibody from in vivo activated plasma B cells found in blood circulation for a short period of time in response to TB- antigens during active TB infection rather than latent TB infection.

4. Tuberculin Skin Test:

Two tests are available – the Mantoux and Heaf tests.  

The Mantoux test for TB involves intradermally injecting PPD (Purified Protein Derivative) tuberculin and measuring the size of induration 48-72 hours later.

The Mantoux skin test is used in the United States and is endorsed by the American Thoracic Society and Centers for Disease Control and Prevention (CDC).

If a person has had a history of a positive tuberculin skin test, another skin test is not needed.

5. Heaf Test:

The Heaf test was used in the United Kingdom until 2005, and is graded on a four point scale. The Mantoux test is now used.

The equivalent Mantoux test positive levels done with 10 TU (0.1 ml 100 TU/ml, 1:1000) are:

(i) 0-4 mm induration (Heaf 0 to 1)

(ii) 5-14 mm induration (Heaf 2)

(iii) Greater than 15 mm induration (Heaf 3 to 5)

CDC Classification of Tuberculin Reaction:

An induration (palpable raised hardened area of skin) of more than 5-15 mm (depending upon the person’s risk factors) to 10 Mantoux units is considered a positive result, indicating TB infection.

i. 5 mm or more is positive in

ii. HIV-positive person

iii. Recent contacts of TB case

iv. Persons with nodular or fibrotic changes on CXR consistent with old healed TB

v. Patients with organ transplants and other immunosuppressed patients

vi. 10 mm or more is positive in

vii. Recent arrivals (less than 5 years) from high-prevalent countries

viii. Injection drug users

ix. Residents and employees of high-risk congregate settings (e.g., prisons, nursing homes, hospitals, homeless shelters, etc.)

x. Persons with clinical conditions that place them at high risk (e.g., diabetes, prolonged corticosteroid therapy, leukemia, end-stage renal disease, chronic malabsorptionsyndromes, low body weight, etc.)

xi. Children less than 4 years of age, or children and adolescents exposed to adults in high-risk categories

xii. 15 mm or more is positive in

a. Persons with no known risk factors for TB

b. (Note- Targeted skin testing programs should only be conducted among high- risk groups)

A tuberculin test conversion is defined as an increase of 10 mm or more within a 2-year period, regardless of age.

6. BCG Vaccine and Tuberculin Skin Test:

There is disagreement on the use of the Mantoux test on people who have been immunized with BGG.

Under the US recommendations, diagnosis and treatment of latent tuberculosis infection (LTBI) is considered for any BCG-vaccinated person whose skin test is 10 mm or greater, if any of these circumstances are present:

(i) Was in contact with another person with infectious TB.

(ii) Was born or has resided in a high TB prevalence country.

(iii) Is continually exposed to populations where TB prevalence is high.

Adenosine Deaminase:

In 2007, a systematic review of adenosine deaminase by the NHS Health Technology Assessment Programme concluded “There is no evidence to support the use of ADA tests for the diagnosis of pulmonary TB. However, there is considerable evidence to support their use in pleural fluid samples for diagnosis of pleural TB, where sensitivity was very high, and to a slightly lesser extent for TB meningitis. In both pleural TB and TB meningitis, ADA tests had higher sensitivity than any other tests.”

7. Nucleic Acid Amplification Tests (NAAT):

This is a heterogeneous group of tests that use either the polymerase chain reaction (PCR) technique or Transcription mediated amplification (TMA) or other forms of nucleic acid amplification methods to detect mycobacterial nucleic acid.

These tests vary in which nucleic acid sequence they detect and vary in their accuracy. The two most common commercially available tests are the amplified mycobacterium tuberculosis direct test (MTD, Gen-Probe) and Amplicor (Roche Diagnostics).