Diagnosis of Mycobacteria tuberculosis

                                             Diagnosis of Tuberculosis

                                                  PKGhatak, MD

Tuberculosis is a very old disease. Egyptian mummies, some 4,000 years old, show evidence of tuberculosis (TB). However, TB in humans is still older. It can be traced back to 9,000 years ago in Atilt Yam, near Israel. Archaeologists in Israel found TB in the remains of a mother and child buried together there. The earliest written records, 3,500 years old, in India mention TB. In China, records show the presence of TB about 2,300 years ago

At one time, TB was causing havoc in Europe; 1 in every 7 deaths was due to TB. TB began to spread to the newly colonized countries in Africa and the Americas by the Europeans. It is now a disease of the poor countries of South Asia, Africa and the Western Pacific countries.

In South East Asia, the infection rate is 220 per 100,000 population, in Africa, the rate is 397 per 100,000 and inthe  Western Pacific, the rate is 400 per 100,000. And the mortality rates are 32, 37 and 90 per 100,000 infections per year, respectively.

The World Health Organization (WHO) declared TB to be a Global Health Emergency (GHE) in 1993. It is estimated that 1.45 million people died from TB, and 10 million new cases appeared in 2018. With help from the WHO and several NOGs, the mortality rates began to decline. Since this COVID-19 pandemic broke out, concerns are expressed that the fight to contain TB will fall behind and there will be a resurgence of TB cases and deaths.

History of progress in detecting TB.

In 1882, the great German scientist Robert Koch discovered the TB bacillus. His associate, Paul Ehrlich, developed a staining method, with a stain containing Alum hematoxylin to demonstrate the stained bacteria under a viewing microscope. Franz Ziech modified the stain by using carboxylic acid as the mordant. Now the TB bacillus is called Acid Fast bacillus (AFB).

Culture of TB.

The tuberculosis bacillus is grown on a solid medium known as the Lowenstein-Jensen medium, which has traditionally been used for this purpose. However, this method is quite slow, as this organism needs 4 to 6 weeks to grow. A faster result can now be obtained using Middlebrook liquid medium.

Skin test for TB infection:

Robert Koch extracted a bacterial protein and called it Tuberculin. He used it as a drug for the treatment of TB infections but it failed to cure TB. Tuberculin was modified by von Piquet and used as a skin test for the diagnosis of TB. Then, finally, Florence Seibert developed a purified protein derivative (PPD) and standardized the skin test. The PPD skin test is still in use today in most developing countries to identify new infections, either latent or active cases.

Mycobacterium tuberculosis (MTB).

There are 70 different mycobacteria, and only a handful of mycobacteria cause human diseases. Mycobacterium tuberculosis (MTB) causes devastating pulmonary disease and death in humans.

Another mycobacterium, Mycobacterium leprae, causes leprosy.

In clinical medicine, Mycobacterial diseases are discussed using the classification introduced by Runyon.

He divided Mycobacteria into these classes.

1. Slow-Growing – takes 4 to 6 weeks to grow in the lab. Then it was subdivided according to the pigment produced by the growing colony of bacteria under light or dark conditions.

  A. Photochromogen, these colonies produce yellow-orange pigment when          grown in the lab under light

  B. Scotochromogen, the colony produces pigment both in the dark and under light.

  C. Non-chromogens produce no pigment.

2. Rapid Growers - takes only 5 days to grow in the lab. They do not produce pigment.

Mycobacterium tuberculosis (MTB) is a slow-growing bacterium and the colony produces no pigment but looks green when grown in Lowenstein-Jensen media containing malachite green. The appearance of the colony is rough and dry. The bacteria produce the enzyme Catalase and a vitamin-niacin-that helps in the diagnosis from the other slow growers.

Why MTB infection is unique.

When any bacteria invade humans, the Macrophages are mobilized and macrophages engulf the bacteria and digest them completely. But MTB remains active inside the macrophages due to higher oxygen content bound to organic iron and MTB continues to multiply. The macrophages carry them to regional lymph nodes, where the MTB continues to multiply and eventually MTB kills the macrophages. The MTB may escape into the bloodstream and then infect the kidneys, Liver, Spleen, Pelvic organs, Peritoneum, Brain, Bone marrow, Vertebrae and other tissues.

Mode of Infection of MTB.

MTB infects people from person to person via ambient air and or droplets, like the spread of COVID. But MTB requires more prolonged contact in close quarters. The infectivity is low, about 10 - 15 people in 12 months.

The primary site of infection is the lung, particularly the apical parts of the lung, where oxygen content is higher.  From the primary location, the disease spread to the regional lymph nodes in the lung. These two lesions taken together are known as the Primary complex. In 85 to 90 % of cases of primary infections are controlled by cellular immunity. But the MTB bacteria remain alive with the macrophages throughout the entire life of the patient. In 5 to 10 % of primary Tbc cases, the disease continues to progress locally and destroys lung tissues, blood vessels and forms lung cavities. Once blood vessels are breached, the MTB spreads near and far to other organs. Pleural effusion, pericardial effusion, miliary spread and TB meningitis usually follow.

Tonsils may also act as the primary focus, specially, in children and regional cervical lymph nodes infection produces cervical adenitis.

Bovine TB.

Dairy products may contain living mycobacteria in the milk of infected cows.  In countries where people use unpasteurized milk or do not boil milk before consumption, the bacteria infect the terminal Ilium and or cecum. In due time progress to TB enteritis, ascites and peritonitis.

In South Asia, bovine TB is present but kept in check because they use boiled milk.

Diagnostic Criteria:

The MTB must be demonstrated from infected tissue, either by stained smears or by culture. Culture takes 4 to 6 weeks. That is a long period to wait for the treatment to start.

Tissue Biopsy:

The pathological features of TB infections are caseating granulomas. The necrosis is seen in the center of the granuloma, surrounded by mononuclear cells and at the periphery, a ring of Langhan giant cells is present. Pathologists describe these as caseating granulomas. Demonstration of AFB in the lesion is necessary for a definite diagnosis.

Alternately, the detection of MTB-specific antigen from the infected tissue, which is not shared by other mycobacteria, will satisfy that need. The genome of MTB is now known. Various tests are designed to achieve that goal.

Immunity to MTB is cellular – the T- T-lymphocytes carry that task. For that reason, no MTB-specific antibody test is possible.

Interferon gamma Release (IGRA) assays.

It is known that T-lymphocytes of infected patients when re-exposed to MTB antigen, the T- lymphocytic secrete Interferon gamma. This test has become the gold standard and has replaced the TB skin test to a great extent. The test is known as the Interferon gamma release assay (IGRA).

The sensitivity and specificity of the IGRA test increase by more than 10 times if T-cells are obtained from the infected trusses like CSF fluid in meningitis, pleural fluid in pleural effusion, and bronchial lavage fluid from the infected lungs.

Nucleic Acid Amplification Test (NAAT).

This test is basically similar to the RT-PCR test for identifying pathogenic organisms like COVID-19, malaria and other diseases. It is specially useful in lesions where only a few MTB are present like pleural fluid, CSF or skinny needle aspiration biopsies.

LAM urine test.

In Pulmonary TB patients, several MTB antigens are present in urine. A heat-stable Lipoarabinomannan (LAM), a glycolipid constituent of MTB, is released from active infection in the blood and is filtered out in the urine. It is detected by ELISA using polyclonal antibodies.

A new method of collecting sputum samples from the gut in MTB patients (Enterotest).

A method known as Enterotest involved swallowing a weighted gel capsule containing a coiled nylon string. One end of the string is protruding through a hole in the capsule, and the other end is held at the mouth and taped to the cheek. The capsule is carried down to the duodenum by the intestinal peristalsis. The capsule is kept in place for 4 hours; the capsule dissolves and the string collects swallowed sputum containing MTB. The string is retrieved and the sample is processed for the presence of AFB. It has the advantage over induced sputum – no technicians and equipment are needed, and the chance of the spread of MTB from vigorous cough produced by the induced method is eliminated.

Where Diagnostic Difficulty Remains.

Children.

Suspected Pulmonary tuberculosis in children is a special situation. Small children cannot expectorate sputum, instead, they swallow coughed-up secretions. It is not easy to obtain adequate samples from the stomachs of the children.

Until the development of the IGRA assay, the TB skin test was mostly relied upon. The administration of the test appears simple, but experienced technicians are essential for the tests. Reading of the induration at 72 hours after the skin test must be adhered to. Interpretation as positive or negative is discouraged; instead, the actual measurement of induration at 90 degrees from the needle insertion should be recorded and kept for future reference.

In most advanced countries, the TB skin test is abandoned and IGRA is now the standard test.

Smear negative MTB.

Identifying the Acid Fast Bacillus (AFB) on smears is quick and 100 % specific. Cultures are planted at the same time as the smears are examined. But cultures take 6 weeks to grow. Many initial negative smear cases turn out positive on culture. This is a problem for poor countries where more advanced methods of TB culture are not available. In children and adults with minimal lung lesions, the cases where sputum production is scant are the main reasons for negative smear tests. In military TB, TB meningitis and TB lymphadenitis, sputum cultures are useless.

Latent Tuberculosis.

Soon after a person is infected with MTB, usually in the apical areas of the lungs, inflammatory reactions begin and in many cases, the progression of TB does not happen. Often, the patients are not aware of the infection. Only by contact tracing, infected people are identified. In developing countries, contact tracing is not done and these people are lost. When those people move to other countries, the public health policy mandates TB skin tests and then they are identified. The IGRA test is currently used.  It should be remembered that the skin test or IGRA test cannot differentiate active TB from inactive TB; it simply means a previous infection with MTB.

The skin test and IGRA tests are not foolproof tests. In HIV/AIDS infections, the use of immunosuppressant drugs, Organ transplant patients, Diabetics, Malnutrition and concurrent Malignancy and Chemotherapy may result in false negative tests. In such situations, Nucleic Acid Amplification Test (NAAT) is indicated. However, NAAT is not sufficiently specific (about 85%), but 100 % sensitive. Modification of NAAT is in progress presently.

Nonpulmonary MTB.

Cervical Lymphadenitis in children and young adults is a usual presentation. The IGRA test is positive in these cases. Skinny Needle Aspiration Biopsy tissues subjected to the IGRA test produce a much higher degree of accuracy and sensitivity. If the IGRA test is negative, then NAAT should be performed.

These methods are equally applicable to all non-pulmonary TB infections.

Non-MTB Mycobacteria infections (NTM).

Pulmonary infection by Mycobacterium avium intracellulare in immunocompromised individuals is a serious health problem. The IGRA test for MTB will be negative, but the IGRA test with the antigen M. avian intracellularly will yield a positive result. The final confirmation requires bacterial culture.

BCG vaccination.

BCG vaccination reduced the incidence of pulmonary TB infections by 50 % and reduced 65% of meningitis and 80 % of disseminated TB in developing countries. BCG vaccination poses a problem for future TB skin tests because BBG vaccine antigen cross-reacts with the Tuberculin antigen. This problem can be avoided by the IGRA test.

Before BCG vaccination, the common reason for a false-positive skin test was

1. In countries where non-MTBs are common, skin tests yield 10 mm or more induration. 2. Newborns vaccinated with BCG retain positive skin test for 5 years, older children and adults retain the reactivity for 10 years.

To increase specificity, positive and negative controls are used. As mentioned before, the sensitivity of the test is poor in immunocompromised patients.

Drug Resistance MTB.

MTB resistant to Isonazid is not unusual in developing countries but MTB is also developing resistance to Rifampin.  MTB resistant to both these two drugs is growing and now spreading to advanced countries. It has become an urgent public health issue.

The usual method of detecting drug resistant TB takes 6 weeks. That is a waste of valuable time. Now, more rapid and more sensitive methods are available.

The microscopic observation drug susceptibility assay.

This method involves a direct inoculation of samples into wells on a tissue culture plate containing a liquid growth medium. Some wells contain Isoniazid, others Rifampin. The growth is determined by visual inspection using an inverted microscope. If a TB colony, in the form of cords, is observed, then MTB is resistant to that specific drug. The turnaround time of this test is 7 to 14 days. The CSF can be tested by this method and has greatly improved early detection of TB meningitis and improved survival.

This principle is now extended to many other drugs, including 2^nd line TB drugs. This method is sensitive 100 % to Isoniazid, 97 % to Rifampin, and 99% to multiple drug resistance.

Molecular Testing for detecting the drug resistance gene.

Mutation of the katG and rpoB genes in the MTB genome produces drug resistance. DNA probe and DNA sequence of MTB genes are available. The results are obtained in hours and the test is highly sensitive and specific, but expensive.

Recent advances in detecting MTB.

Line Probe Assays (LPAs).

LPAs are molecular tests. Currently, three such test kits are available for the rapid detection of MTB and drug resistance. The test is based on the targeted amplification of a specific fragment of MTB by the PCR technique.

Recovery of MTB from peripheral blood.

In this test, anticoagulated blood samples are lysed and then centrifuged. The sediment is inoculated in Westbrooks liquid medium. Valuable time is saved using this method and also increases the positive detection rates. It is a useful test for disseminating MTB infection.

Bacteriophage test for MTB.

A suitable bacteriophage is used in an agar plate containing MTB colonies. A non-pathogenic mycobacterium is used as a control. Central clearing of the colony indicates the presence of MTB. The turnaround time is 2 days. The test can detect as low as 100 MTB in a sample.

Immunodiagnostic Tests.

It can detect past or current TB infections. However, this test is limited in application because of its poor sensitivity and cross-reaction to non-MTB.

Antibody tests against MTB.

The results so far are not consistent. WHO does not recommend this method for detecting Pulmonary and extrapulmonary TB infection.

MTB is an ancient disease but continues to be a worldwide problem and the problem is magnified due to the prevalence of HIV/AIDS and these two entities coexist. Now, the COVID-19 pandemic has put an extra burden on poor countries. It is feared that the MTB new cases and the number of deaths will increase. The use of monoclonal antibodies in treating cancers, autoimmune diseases and organ transplants has taken the center stage in the treatment. The resurgence of TBs in these groups is increasing because of suppressed immunity. Latent TB poses a problem and MTB in association with diabetes mellitus is on the rise.

Many new diagnostic tools are available in advanced countries but their use is limited in areas where MTB is a serious health issue because of a lack of funding.

Developing a Blood Test to Differentiate Between Active and Latent TB

Thomas Glück, MD, reviewing Zhao H et al. Infection 2025 Mar 17

In a pilot study, measurement of IL-8, IL-18, and IL-33 in TB-antigen–stimulated whole-blood assays distinguished active TB from latent infection with >85% accuracy.

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