Tuberculosis and Diagnosis – Dr. Surya Kant

Tuberculosis and Its Diagnosis

Dr. Surya Kant

Tuberculosis (TB) has been known to mankind since ancient times. TB is one of the oldest recorded and main causes of death of human beings. Earlier this disease has been called by numerous names including consumption (because of the severe weight loss and the way the infection appeared to “consume” the patient), phthisis pulmonaris and the white plague (because of the extreme pallor seen among those infected). In the nineteenth century, TB was known as “the captain of all men of death”. It is still true to a large extent today 1- 4 . TB is curable and preventable but still kills approximately three people every minute 3. India is considered as the second-most populous country in the world with one-fourth of the global incident TB cases annually (TB India 2014). The numbers are over 90% in the developing world 5Even today after the development of advanced screening, diagnostic and treatment methods for the disease, a third of the world’s population has been exposed and is infected with the organism.

Tuberculosis in Ancient times
The organism causing TB – Mycobacterium tuberculosis (MTB) existed 15,000 to 20,000 years ago. It has been found in relics from ancient Egypt, India, and China. Among Egyptian mummies spinal tuberculosis, known as Pott’s disease has been detected by archaeologists. Evidence of TB of the cervical lymph nodes or lymph nodes of the neck termed scrofula, is found in the middle ages. It was termed as the “king’s evil”. Poorly ventilated and overcrowded housing, primitive sanitation, malnutrition and other risk factors led to the rise. The term White plague emerged around this time 6, 7

Discoveries pertinent to tuberculosis
The tubercle bacilli or the causative organism of TB was discovered by Robert Koch in 1882. He showed that the organism’s unique protein coat made it difficult to visualize earlier until a specific stain called the Zeihl Neelson (ZN) stain was discovered 8 The bacteria was called Koch’s bacillus and since it took up the red acidic dye, it was called AFB or acid fast bacilli. Koch was awarded the Nobel Prize in 1905 9. In 1895 Wilhelm Roentgen developed X- rays which further advanced diagnostics of TB. This allowed early diagnosis and isolation of infected individuals.The early symptoms of activeTB include;

  • Fatigue,
  • Recurrent fever,
  • Unusual weight loss
  • Breathlessness
  • Coughing,
  • Night sweats and
  • Sometime blood in cough.
Transmission TB is a disease that is spread through the air from one person to another. Although transmission of bacterium from infected person may not always result in disease. It mainly depends upon the number of droplet nuclei expelled from the infected person into the air.

Active TB means that the TB bacteria are growing and causing symptoms. If the lungs are infected with active TB, it is easy to spread the disease to others. TB spreads when a person who has active disease exhales air that contains TB-causing bacteria and another person inhales the bacteria from the air. These bacteria can float in the air for several hours 10.Coughing, sneezing, laughing, or singing releases more bacteria than breathing. If TB is present in organs other than the lungs (Extra pulmonary tuberculosis; EPTB), it does not spread easily (i.e., It is less contagious). The specific symptoms will depend on whether the infection is in the lungs or in another part of the body (EPTB). There are two types of TB:

TB disease: People with TB disease can spread the bacteria to others, feel sick, and can have symptoms including fever, night sweats, cough, and weight loss.

Latent TB infection: TB bacteria can live in the body without making a person sick. People with latent TB infection do not feel sick, do not have TB symptoms, and cannot spread TB bacteria to others. Some people with latent TB infection go on to develop the disease.

Who Should Get Tested for TB? TB tests are generally not needed for people with a low risk of infection with TB bacteria. Certain people should be tested for TB bacteria because they are more likely to get TB disease, including:
  • People who have spent time with someone who has TB disease
  • People with HIV infection or another medical problem that weakens the immune system
  • People who have symptoms of TB disease (fever, night sweats, cough, and weight loss)
  • People from a country where TB disease is common.
  • People who live or work somewhere TB disease is more common (homeless shelters, prison or some nursing homes)
  • People who use illegal drugs

Tuberculosis Diagnosis: How is TB Detected? TB is diagnosed by finding MTB bacteria in a clinical specimen taken from the patient 11. People suspected of having TB disease should be referred for a complete medical evaluation, which will include:
  • Medical history,
  • Physical examination,
  • Chest radiograph (X-ray) and scans
  • Test for TB infection (TB skin test or TB blood test),
  • Appropriate laboratory tests/ Microbiological examinations/ surgical biopsy.
Medical history: The medical history includes obtaining the symptoms of pulmonary TB: productive, prolonged cough of three or more weeks, chest pain, and hemoptysis.

Systemic symptoms include low grade remittent fever, chills, night sweats, appetite loss, weight loss, easy fatigability, and production of sputum that starts out mucoid but changes to purulent.

Other parts of the medical history include prior TB exposure, infection or disease; past TB treatment; demographic risk factors for TB; and medical conditions that increase risk for TB disease such as HIV infection. Depending on the sort of patient population surveyed, as few as 20%, or as many as 75% of PTB cases may be without symptoms .12

TB should be suspected when a pneumonia-like illness has persisted longer than three weeks, or when a respiratory illness in an otherwise healthy individual does not respond to regular antibiotics.

Physical examination:

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.

Chest X-ray and CT
TB creates cavities visible in x-rays in the patient’s lungs.Lesions may appear anywhere in the lungs:
  • In active PTB, infiltrates or consolidations and/or cavities are often seen in the upper lungswith or without mediastinal or hilar lymphadenopathy or pleural effusions (tuberculous pleurisy).
  • In disseminated TB a pattern of many tiny nodules throughout the lung fields is common – the so-called miliary TB.
  • In HIV and other immunosuppressedpersons, any abnormality may indicate TB or the chest X-ray may even appear entirely normal.
  • Abnormalities like development of lesions in the lungs are to be suspected in TB.
  • 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. These lesions are of different sizes, shape, density, and cavitations. Chest radiographs helps in the evaluation of Cavitary and non-cavitary areas in lungs and are according to American Thoracic Society (ATS), classified as mild, moderate and far advance
Computerized Tomography (CT scans) and Magnetic Resonance Imaging (MRI) have proved for useful for imaging TB lesions particularly in the brain and spine.

A variant of the chest X-Ray, Abreugraphy (from the name of its inventor, Dr. Manuel Dias de Abreu) was a small radiographic image, also called miniature mass radiography (MMR) or miniature chest radiograph. Though its resolution is limited (it doesn’t allow the diagnosis of lung cancer) it is sufficiently accurate for diagnosis of TB. Much less expensive than traditional X-Ray, MMR was quickly adopted and extensively utilized in some countries, in the 1950s.

Immunological Tests:
There are two kinds of tests that are used to determine if a person has been infected with TB bacteria: The tuberculin skin test and TB blood tests.  
Tuberculin Skin Test (TST)
The Mantoux tuberculin skin test is a test to check if a person has been infected with TB bacteria. Using a small needle, a health care provider injects a liquid (called tuberculin) into the skin of the lower part of the arm. When injected, a small, pale bump will appear. This is different from a Bacilli Calmette-Guerin (BCG) shot (a TB vaccine that many people living outside of the United States receive). The person given the TST must return within 2 or 3 days to have a trained health care worker look for a reaction on the arm where the liquid was injected. The health care worker will look for a raised, hard area or swelling, and if present, measure its size using a ruler. Redness by itself is not considered part of the reaction.

 Positive TST results mean; The TST result depends on the size of the raised, hard area or swelling. It also depends on the person’s risk of being infected with TB bacteria and the progression to TB disease if infected.
  • Positive TST: This means the person’s body was infected with TB bacteria. Additional tests are needed to determine if the person has latent TB infection or TB disease.
  • Negative TST: This means the person’s body did not react to the test, and that latent TB infection or TB disease is not likely.
Who can receive a TST?
Almost everyone can receive a TST, including infants, children, pregnant women, people living with HIV, and people who have had a BCG shot. People who had a severe reaction to a previous TST should not receive another TST. If a TST is positive, additional tests are needed.

TB Blood Tests Interferon Gamma Release Assay (IGRA) Interferon-γ (interferon-gamma) release assays (IGRAs) are new developments in TB infection testing. IGRAs are based on the ability of the MTB antigens for early secretory antigen target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) to stimulate host production of interferon-gamma. Because these antigens are not present in non-tuberculous mycobacteria or in any BCG vaccine variant, these tests can distinguish latent TB infection (LTBI). The TB blood test measures how your immune system reacts to the germs that cause TB. An IGRA is a blood test that can determine if a person has been infected with TB bacteria. An IGRA measures how strong a person’s immune system reacts to TB bacteria by testing the person’s blood in a laboratory. Two IGRAs are approved by the U.S. Food and Drug Administration (FDA):
  • Quanti FERON®-TB Gold In-Tube test (QFT-GIT)Lymphocytesfrom the patient’s blood are incubated with the antigens (12).If the patient has been exposed to tuberculosis before, T lymphocytes produce interferon γ in response. The enzyme-linked immunospot assay (ELISPOT) is another blood test that may replace the skin test for diagnosis .14-16
  • T-SPOT®.TB test (T-Spot)T-SPOT.TB,[17] a type of ELISPOT assay,[18] counts the number of activated T lymphocytes that secrete interferon γ.
 How does the IGRA work?
Blood is collected into special tubes using a needle. The blood is delivered to a laboratory as directed by the IGRA test instructions. The laboratory runs the test and reports the results to the health care provider. IGRA result mean
  • Positive IGRA: This means that the person has been infected with TB bacteria. Additional tests are needed to determine if the person has latent TB infection or TB disease. A health care worker will then provide treatment as needed.
  • Negative IGRA: This means that the person’s blood did not react to the test and that latent TB infection or TB disease is not likely.
Who can receive an IGRA?
Anyone can have an IGRA in place of a TST. This can be for any situation where a TST is recommended. In general, a person should have either a TST or an IGRA, but not both. IGRAs are the preferred method of TB infection testing for the following:
  • People who have received the BCG shot
  • People who have a difficult time returning for a second appointment to look at the TST after the test was given
For diagnosing latent TB, three systematic reviews of IGRAs concluded the tests noted excellent specificity for the tests to distinguish latent TB from prior vaccination  19. There is no problem with repeated IGRAs.

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

Adenosine deaminase
In 2007, a systematic review of Adenosine deaminase (ADA) by the NHS Health Technology Assessment Programme concluded. There is no evidence to support the use of ADA tests for the diagnosis of PTB. However, there is considerable evidence to support their use in pleural fluid samples for diagnosis of pleural TB and for TB meningitis.

Full blood count
Erythrocyte sedimentation rate is usually raised.

Microbiological Findings:
A definitive diagnosis of TB can only be made by culturing MTB organisms from a specimen taken from the patient (most often sputum, but may also include pus, CSF, biopsied tissue, etc. 20. 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 Sputum smears and cultures should be done for acid-fast bacilli (AFB) if the patient is producing sputum 3, 20.

Smear Microscopy
(i) Worldwide, for more than 100 years, the most common method used for diagnosing TB is sputum smear microscopy. It is simplest laboratory test detecting TB bacilli. It is inexpensive and relatively easy to perform and read and detect transmitters of tubercle bacilli. Results can be reported within hours of receipt of the sample and provides reliable epidemiological indicators needed for the evaluation of the National Tuberculosis Control Programme (NTCP).The diagnosis of TB largely depends on the microscopic demonstration of acid-fast bacilli (AFB) in sputum samples using Ziehl-Nielsen (ZN) stain described by two German doctors, Franz Ziehl and Friedrich Nielsen. It is cheap and fast, but the cases of non-pulmonary TB cannot be detected. Besides AFB other than MTBcould also be stained and the specificity of sputum is not 100%. Smear microscopy has for long been the gold standard of TB diagnosis and is employed under the DOTS (directly observed therapy) strategy for TB control, which is internationally recommended by the WHO (2014) 3.
  1. The preferred method for this is fluorescence microscopy (auramine-rhodamine staining), which is more sensitive than conventional ZN staining 21In cases where there is no spontaneous sputum production, a sample can be induced, usually by nebulized inhalation of a saline or saline with bronchodilator solution.
A variant of the chest X-Ray, Abreugraphy (from the name of its inventor, Dr. Manuel Dias de Abreu) was a small radiographic image, also called miniature mass radiography (MMR) or miniature chest radiograph. Though its resolution is limited (it doesn’t allow the diagnosis of lung cancer, for example) it is sufficiently accurate for diagnosis of TB.

Culture Method
A gold standard method for active TB, Culture based method is extremely sensitive if TB bacilli are present in the sputum sample. Many types of cultures are available.22
  • Traditionally, cultures have used the Lowenstein-Jensen(LJ), Kirchner, or Middle brook media (7H9, 7H10, and 7H11).
  • A culture of the AFB can distinguish the various forms of mycobacteria, although results from this may take four to eight weeks for a conclusive answer.
  • New automated systems that are faster include the MB/BacT, BACTEC- 9000, and the Mycobacterial Growth Indicator Tube (MGIT).
  • The Microscopic Observation Drug Susceptibility assayculture may be a faster and more accurate method The culture test is time –consuming as it takes about 2 to 6 weeks. It also need good laboratory facilities requiring expensive equipment and high maintenance are vital factors for culture based techniques.
Alternative sampling
In patients incapable of producing a sputum sample, common alternative sample sources for diagnosing pulmonary TB (PTB) include 12. Molecular Methods:
Global research efforts have paved the way for revolutionary, molecular based techniques for the diagnosis of TB that have opened the door to rapid TB detection for initiating timely treatment. During the past several years, molecular methods have been developed for direct detection, species identification, and drug susceptibility testing of mycobacteria 3, 24  These methods can potentially reduce the diagnostic time from weeks to hours. For direct detection of MTB from clinical specimens, several molecular assays are commercially available today. They have been shown useful for the routine diagnostic laboratory test. Molecular methods have also been applied for the detection of mutations that confer drug resistance in mycobacteria. All in all, the future of clinical Mycobacteriology appears to be heading toward direct detection. These include:
  • Polymerase Chain reaction (PCR)/DNA amplification techniques; Sometimes called “molecular photocopying”. It is a fast and expensive technique to amplify up to millions of copies from a very small quantity of target DNA.
  • DNA sequencing for species determination
  • RFLP analysis,
  • Line Probe Assay (LPA),
  • Luciferase Reporter gene which is employed to deal with epidemiological problems.
Nucleic acid Amplification (NAA) tests
These techniques include polymerase chain reaction (PCR), Amplicor MTB Test (Roche Diagnostic Systems Inc. New Jersey, USA),and the amplified MTB Direct Test (MTD, Gen- Probe)24.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:
  • Amplified mycobacterium tuberculosis direct test (MTD, Gen-Probe)
  • The Amplicor (Roche Diagnostics) is a DNA based test that amplifies and detects the presence of a specific ribosomal RNA of TB bacilli in a colorimetric reaction.
In a more recent The MTD test is based on the amplification of the ribosomal RNA of the TB bacilli but its detection is with a DNA probe, found that use of the MTD test reduces inappropriate TB therapy24, 25 These methods confirm the presence of MTB within 1-3 days. These methods are being used to identify MDR-TB as mutations in the DNA of MTB, which confer the drug resistance, have been discovered. Table 1: Different diagnostic tests for TB
1. Immunological Tests ·         Mantoux tuberculin skin Test (TST)  ·         Quanti- FERON-TB Test ·         Adenosine Deaminase (ADA) ·         Antibody in Lymphocyte Supernatant assay (ALS Assay) ·         (T-Spot) T-SPOT.TB  
2. Radiological Tests ·         Chest X-Rays  ·         Computerised tomography (CT) ·         Miniature mass radiography (MMR) or miniature chest radiograph ·         Magnetic Resonance Imaging (MRI)
3. Bacteriological Tests ·         AFB Smear  ·         AFB Culture (Solid culture, Liquid culture) ·         BACTEC- 9000 ·         Mycobacterial Growth Indicator Tube (MGIT). ·         Drug Susceptibility Testing (DST)
4. Molecular Tests ·         Gene expert(Rifampicin drug resistance only)  ·         Polymerase Chain Reaction (PCR) ·         Spoligotyping ·         Restriction Fragment Length Polymorphism (RFLP) Analysis ·         Line Probe Assay (LPA) ·         Luciferase Reporter gene
Choosing a TB Test:
Choosing which TB test to use should be done by the person’s health care provider. Factors in selecting which test to use include the reason for testing, test availability, and cost. Diagnosis of Latent TB Infection or TB Disease If a person is found to be infected with TB bacteria; other tests are needed to see if the person has TB disease. TB disease is treated by taking several drugs as recommended by a health care provider. If a person does not have TB disease, but has TB bacteria in the body, then latent TB infection is diagnosed. The decision about taking treatment for latent TB infection will be based on a person’s chances of developing TB disease.

How is TB treated?
Treatment for TB depends on whether a person has active TB disease or only TB infection. If you have become infected with TB, but do not have active TB disease, you may get preventive therapy. This treatment kills germs that are not doing any damage right now, but could so do in the future. The most common preventive therapy is a daily dose of Isoniazid (INH) for 6 to 9 months. If you have active TB disease you will probably be treated with a combination of several drugs for 6 to 12 months. The most common treatment for active TB is INH plus two to three other drugs including Rifampin, Pyrazinamide and Ethambutol. After a few weeks you can probably even return to normal activities.

It is very important that:
  • Continue to take the medicine correctly for the full length of treatment.
  • If you take the medicine incorrectly or stop taking it, you may become sick again.
  • The TB may be harder to treat if it has become drug resistant.
  • TB that is resistant to more than one drug, called multidrug-resistant TB(MDR TB) is very dangerous.
  • Extensively drug-resistant TB (XDR TB) is an even more dangerous version of MDR TB because so many of the most effective TB drugs do not work against it.
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Dr. Surya Kant Tripathi, MD (Gild Medalist)
Professor  Head, Department Of Respiratory Medicine
King George’s Medical University UP, Lucknow – 226003 (UP)