History and Metabolic Context

The Tuberculosis Disease

Tuberculosis is the second leading cause of death by infectious disease worldwide (Dutta and Karakousis 2014). It is caused by the bacterium Mycobacterium tuberculosis (MTB), and can be categorized as either latent or active (CDC Basic TB Facts). Although it can manifest itself in various ways such as in the bones and central nervous system, it is mainly a disease of the lungs (Smith 2003).  When first infecting its host, MTB gives rise to what is called latent tuberculosis (Dutta and Karakousis 2014). The disease itself is lying dormant during this time, as neither signs nor symptoms are present (Dutta and Karakousis 2014). However, the progression from the latent to the active form can occur years or even decades later (Dutta and Karakousis 2014). Only when the disease is in its active form can it be spread through the air via speaking, coughing, or sneezing (CDC Basic TB Facts).

Mycobacterium tuberculosis. Source: http://ktla.com/2015/12/13/350-infants-possibly-exposed-to-tuberculosis-by-employee-at-san-jose-hospital/
Mycobacterium tuberculosis. Source: http://ktla.com/2015/12/13/350-infants-possibly-exposed-to-tuberculosis-by-employee-at-san-jose-hospital/

Symptoms of Active Tuberculosis:

  • Fever
  • Chills
  • Chest pain
  • Severe coughing that lasts at least 3 weeks
  • Coughing up blood and sputum
  • Loss of appetite
  • Weight loss
  • Weakness and fatigue

(CDC Basic TB Facts)

Normal Immune Response

The immune response plays an important role in the body’s ability to fight infection. However, due to its ability to suppress immune response, Mycobacterium tuberculosis will survive (Wu et al. 2015). Normally, macrophages will engulf the bacterium in order to sequester and contain it once it has been detected (Guirado et al. 2015). This is regulated by interferon-gamma, which is mostly produced by natural killer cells (Schoenborn and Wilson 2007), and interleukin-12 (IL-12) (Gong et al. 1996). Production of interferon-gamma promotes type 1 helper T cell (Th1) response (Gong et al. 1996). This leads to the production of killer T cells, which will destroy the invading bacteria (Gong et al. 1996).

On the History of Tuberculosis

Although Robert Koch was the first microbiologist to successfully isolate the bacterium that causes tuberculosis in 1882, there is evidence that the disease has existed for thousands of years (Cambau and Drancourt 2014). The earliest recorded accounts of the symptoms associated with tuberculosis date back to seventh century B.C. (Smith 2003). It is thought that the disease spread to this area, which now consists of Turkey, Iraq, and Syria, from the migration of Indo-European cattle herders, who were exposed to the bacterium from infected cattle (Smith 2003). From there, the disease migrated with the cattle herders to Europe and Asia (Smith 2003).

With the increase in European populations came the increase in large urban centers, which became a breeding ground for tuberculosis in the 1500s and 1600s (Smith 2003). However, it wasn’t until the first half of the 19th century that the disease ran rampant, killing a quarter of the population (Smith 2003).

One of the first major investigations of the disease was conducted by the French military doctor Jean-Antoine Villemin (Cambau and Drancourt 2014). Since many 19th century Europeans believed that tuberculosis was inherited, congenital, or spontaneous, it was difficult to convince the public that the disease could be spread through the air, for instance by coughing (Cambau and Drancourt 2014). Thus, Villemin performed an experiment that would demonstrate that the disease could be spread from one person to another (Cambau and Drancourt 2014). He was able to transmit tuberculosis between a patient and a rabbit, a cow and a rabbit, and two rabbits (Cambau and Drancourt 2014).

Villemin’s work paved the way for Robert Koch’s discovery of Mycobacterium tuberculosis (Cambau and Drancourt 2014). Koch’s goal was to identify the presence of something foreign, which worked in the same way as a parasite (Cambau and Drancourt 2014). During the time of his work, staining was considered to be a innovative method developed by Karl Weighert (Cambau and Drancourt 2014). However, this staining technique did not work for Koch’s tuberculosis lesions (Cambau and Drancourt 2014). Instead, he used the methylene blue staining method, which was created only a year after Weighert’s staining method (Cambau and Drancourt 2014). Normal animal tissue was shown to stain brown, whereas the tuberculosis bacteria were shown to stain blue, making it easy to compare the two using microscopy (Cambau and Drancourt 2014). Koch described the bacteria he saw under the microscope to be rod-shaped bacilli that look similar to leprosy bacilli (Cambau and Drancourt 2014).

Koch wanted to further prove that the microbial hypothesis for the cause of tuberculosis is true, and so he set out to isolate the bacterium in pure culture, which he accomplished (Cambau and Drancourt 2014). Not only did he isolate the bacterium, he also proved it was an infectious agent of the tuberculosis (Cambau and Drancourt 2014). He observed the bacterium in tuberculosis lesions, cultured it outside of the tissue sample, and then inserted the now purified bacterium into normal tissue (Cambau and Drancourt 2014). The normal tissue had become a tuberculosis lesion (Cambau and Drancourt 2014). Taking his experiment one step further, Koch introduced sputum and lung tissue from either a tuberculosis patient or animal into guinea pigs (Cambau and Drancourt 2014). These guinea pigs developed tuberculosis lesions (Cambau and Drancourt 2014). For his big finish, Koch sequestered these now-infected guinea pigs with normal, healthy guinea pigs, and the healthy guinea pigs developed tuberculosis (Cambau and Drancourt 2014). These experiments not only proved that bacteria were the causative agents of tuberculosis, but also that tuberculosis could be spread through the air (Cambau and Drancourt 2014).

Diagnosing Tuberculosis

There are multiple tests used in order to diagnose tuberculosis. Due to the absence of symptoms in latent tuberculosis, a skin test is used to determine if infection has occurred (CDC Basic TB Facts). The patient is injected with a small amount of tuberculin in the forearm (CDC Basic TB Facts). Tuberculin comes from M. tuberculosis and is considered to be an antigen since it stimulates an immune response if the patient has been infected with tuberculosis (Ayubi et al. 2015). However, the results of the test are not apparent until 48-72 hours after injection (CDC Basic TB Facts).

However, the tuberculosis skin test often results in false positives due to the tuberculin antigen reacting against other mycobacterium that do not result in tuberculosis (Ayubi et al. 2015).  Thus, other diagnostic tests are run in order to rule out the possibility of infection in the case of false positives. One of the ways to rule out a false positive is to perform a chest x-ray (CDC Basic TB Facts). Although a chest x-ray cannot definitively show the presence of tuberculosis, the absence of lesions in the lungs can rule out the possibility of tuberculosis (CDC Basic TB Facts).

Tuberculosis Patient Chest X-Ray Showing Lung Lesion. Source: http://www.nursingceu.com/courses/386/index_nceu.html

One of the best ways to determine if a patient has tuberculosis, latent or active, is to obtain a sputum sample and look for acid-fast-bacilli (AFB), of which Mycobacterium tuberculosis is one (CDC Basic TB Facts). These particular bacteria cannot be stained easily using most staining methods because their cell wall contains waxy lipid-like components that prevent staining from occurring (Madison 2001). However, the stain will remain if the bacteria are first stained with a phenol dye, and are then treated with an acid-alcohol mixture (Madison 2001). The positive results of this test can be the catalyst for beginning treatment, and it is helpful for monitoring patient progress (Madison 2001).

Source: http://veterinarymicrobiology.in/acid-fast-staining/
Tuberculosis Acid-Fact Stain. Mycobacterium tuberculosis is stained pink. Source: http://veterinarymicrobiology.in/acid-fast-staining/

4 Replies to “History and Metabolic Context”

  1. Your pages show a very in-depth knowledge of immunology beyond the scope of an undergraduate student – very impressive! I was wondering if you could comment on the transition between latent TB and active TB. In other words, how does latent TB become active TB in a patient? There is another test commonly used to diagnose TB which is called the IFN-gamma release assay. Can you explain how it works and its significance? Well done overall. – Zachary Shuler, Rutgers Robert Wood Johnson Medical School

    1. Thank you for your feedback! I really appreciate it Zach! Looking back, I regret not addressing the transition from latent TB to active TB. Unfortunately I could not find a lot of information on it, but what I did find is quite interesting! Although the granuloma basically captures the bacterium, the granuloma’s structure can begin to break down. There are a few ways in which this occurs. There is either a decline in the host’s immune response, or the necessary immune signals cannot develop or be maintained. In the case of a decline in immune response due to genetics, mutations can arise is specific chemokines and cytokines, as well as their receptors, which disrupt critical immune response signaling. If immune signals cannot be developed or maintained, it is most likely due to co-infection with another disease, such as HIV. Also, the bacterium can undergo what is called “resuscitation.” Specific phospholipids and mycobacterial proteins have been shown to induce the activation of the bacterium from dormancy. A resuscitation promoting factor (Rpf) has been shown to revive growth in mycobacteria. The genes the code for Rpfs have been shown to be expressed during dormancy.

      There are four approved IFN-gamma release assays, which are QuantiFERON-TB test (QFT), QuantiFERON-TB Gold test (QFT-G), QuantiFERON-TB Gold In-Tube test (QFT-GIT), and T-spot. QFT measures IFN-gamma released by T cells in response to tuberculin-purified protein derivative (PPD) when compared with controls. QFT-G measures immune response to M. tuberculosis protein ESAT-6. QFT-GIT overcame the limitation of testing patient blood hours after collection by using special tubes to maintain control materials and antigens. T-spot uses peripheral blood mononuclear cells (PBMCs) to detect an increase in the number of cells that release IFN-gamma, which would indicate an active immune response. These tests are significant because they are seen as a better test for tuberculosis than the TB skin test. Patients do not have to return to get their test read 48-72 hours later, and the IFN-gamma release assays reduce the possibility for inaccuracies and bias, which are common in the reading of the skin test.

      Thanks again for your feedback! I hope I sufficiently answered your questions!

  2. Hey Nicole. It was really interesting to read your project because I myself have needed to be tested for TB in order to work at certain places and it’s really interesting to see the explanation behind the tests I had to undergo. I was wondering if you could expand on the identification of acid-fast-bacilli as a form of diagnosing TB. Specifically, you mention that these “bacteria cannot be stained easily using most staining methods because their cell wall contains waxy lipid-like components that prevent staining from occurring.” Could you use your research as well as our discussion of bacterial cell walls in class to further explain why the lipid-like components interfere with the staining process?

    1. Thanks for your feedback Steph! Basically, acid-fact bacilli cell walls differ from Gram-positive and Gram-negative, hence the reason for their own category. Above the layer of peptidoglycan is a thick layer composed mainly of the fatty acid mycolic acid. The high lipid content of mycolic acid gives the cell wall a very waxy texture, which actually acts as a seal, to some extent. This prevents the Gram stain from passing though. I hope this clarified why Gram staining won’t work, and why the lipid-like components interfere with the staining process!

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