FUN2: 11:00-12:00 Scribe: Ryan O’Neill
Monday, November 07, 2008 Proof: Caitlin Cox
Dr. Benjamin Mycobacteriology Page 1 of 6
Note: 10:00-11:00 lecture was canceled and will be rescheduled for Monday, November 10th at 9:00 AM.
TB – tuberculosis, AFB – acid fast bacillus, BCG – Bacille-Calmette-Guerin, INH – Isoniazid, HPLC – high performance liquid chromatography
I. Introduction [S1]:
a. TB is one that is not transmitted very easily. You need prolonged exposure.
b. This slide is what TB looks like grown in the lab and is very different than all the other Mycobacteria that will be discussed today. (He said TB stain resembles a Chinese dragon.)
II. Mycobacteria sp. [S2]
a. Mycobacteria are acid fast. That means they actually don’t stain with a gram stain, only the acid fast stain.
b. This species is defined by its mycolic acids, which are long fatty acids (78-91 carbons long tails).
i. Clorox has the ability to kill TB.
c. Waxes give them the ability to float. When we try to centrifuge them in the lab, you have to centrifuge very hard to counteract this floating ability in order to cause separation.
d. Mycobacteria are obligate aerobes, meaning they must have oxygen to grow.
e. They are also very slow growing. They only divide once per day instead of once every 20 minutes like E. coli does. It takes days to weeks to form colonies.
i. Should be able to detect a tuberculosis test in 2 weeks if grown in a liquid media like on slide 1.
ii. It took 3 weeks when solid media was used.
III. Identification of Acid Fast Bacilli [S3]
a. Mycobacterium sp. are identified by the acid fast stain.
b. Mycobacteria have been around for a long time, and there are about 140 different named species.
c. Most of these are nonpathogenic and live in the environment.
d. TB is the only Mycobacteria that infects and regularly causes disease in normal people.
e. There are more than 40 different species of Mycobacteria that grows on AIDS patients, and it is very common for AIDS patients or other immunosuppressed patients to have one of these Mycobacteria.
IV. The Bacterial Cell Wall [S4]
a. Gram positive organisms
i. Thick peptidoglycan layer. One molecule covers the whole bacteria, all covalently linked.
ii. The crystal violet iodine crystals stays inside.
b. Gram negative organisms
i. Smaller peptidoglycan layer and an outer membrane.
ii. Gram stain is washed out when you take away the outer membrane due to the small peptidoglycan layer.
c. Mycobacterium
i. Have fatty acids & Lipoarabinomannan - LAM (has a lot of properties in immunosuppression and causes the organism to go into macrophages and live inside our white blood cells).
ii. Very different type of wall that retains the acid fast stain.
V. Obligate Pathogenic Mycobacteria [S5]
a. These are the organisms that, if you find in someone, will always cause a disease.
b. Mycobacterium tuberculosis was the first bacteria shown to cause disease in the 1880’s.
i. Was stained and seen and completed Koch’s postulates. They took an organism from people, grew it on media, put it into an animal, and saw it caused disease.
c. Mycobacterium leprae is the other type of Mycobacteria that always causes disease.
d. [S6] Graph:
i. This graph is the mean annual morbidity from pulmonary tuberculosis in England and Wales.
ii. 300 out of 100,000 were dying each year of TB.
iii. The epidemic started to decrease before we even discovered what is was (1882). This shows you that TB is a social disease. People that are well fed aren’t too closely associated with each other and don’t breathe the same air and spread TB. Poorer classes spread TB more easily.
iv. Tuberculin test didn’t change the rate of disease mortality.
v. Note BCG vaccination point on graph that has some utility in children.
1. They won’t develop the disease as rapidly if they have the BCG vaccination, but later on will become infected and may spread the disease as well as anyone.
vi. We don’t get BCG in the U.S. because we use a skin test. The U.S. doesn’t vaccinate people because our rate of TB is so low.
vii. Note Chemotherapy point on graph and the decrease that soon followed.
VI. Mycobacterium tuberculosis [S7]
a. Humans are the only natural host. But some elephants and zoo animals have been shown to be infected.
b. 1/3 of the world’s population is infected with tuberculosis, but most of these will never develop the disease and this is the most important thing in the lecture today. ***
i. ex: slide 6 showing that only 300 out of 100,000 infected died each year.
ii. There are 9.2 million cases of disease infected tuberculosis/year in the world, but only 1.7 million deaths each year.
c. [S8]: Graph – Tuberculosis in the U.S.
i. In 1980, we diagnosed almost 27,000 per year of TB disease.
ii. The public health service decided that TB was being cured and we didn’t need to worry about it anymore.
iii. Was flat for a long time and then increased around 1990 by 20%.
iv. There were a lot of AIDS patients (especially in NYC) infected and transporting TB amongst themselves.
v. NYC increased their budget from 4 million to 40 million and the resistant organisms went away very rapidly.
vi. Immigrants are largely positive for the TB skin test (remember 1/3 of the population is infected).
vii. Currently we are down to 13,299 cases in the U.S. with a 5% death rate.
d. [S9]: Graph – U.S. Tuberculosis Cases
i. Total number of people who develop TB each year in the U.S. in 1991 was very high and has decreased since then, but foreign born cases have stayed consistent over the past 16 years with a slight increase.
ii. Main point: there is more TB in foreign born people than there are in U.S. born
iii. Rate of TB: 2.1 per 100,000 in U.S. born and 20.7 per 100,000 in foreign born.
iv. It is very difficult to transmit TB so it’s not like the foreign born are giving it to the U.S. born.
e. [S10]: Graph – Alabama Tuberculosis Cases
i. Alabama is currently below the national average. In the past we have been above the national average.
ii. Once a person is diagnosed with the disease, a public health nurse will make sure the patient will take their medicine daily for 3x a week for 6 months. Those people who don’t make themselves available to take their medicine are put in cells where they can easily be located.
f. [S11]: Graph: Examples of high prevalence countries
i. Namibia
1. Only 1.8 million people live here, but there are more TB cases there than in U.S.
ii. Zambia
1. Has 11.5 million with 78,000 tuberculosis cases (6x that of U.S.)
VII. Transmission of Tuberculosis [S12]
a. TB needs to be transmitted by very small droplets that don’t get stuck in upper airways, so they can get cleared into the alveoli where it is phagocytized by a macrophage and can then grow.
b. These droplets settle slowly at 9mm/min in still air.
c. Once they settle out from the air and you get them on your hand, it is very difficult to get sick.
d. It takes 5-200 infected droplets to infect someone.
e. The average patient only exhales 1.25 infectious droplets/hour.
f. Occasionally some persons for an unknown reason will have a very high number of infectious droplets/hour.
g. [S13]: Graph
i. Overview of what has already been mentioned previously
ii. If you have fairly close contact with someone, being in a room for at least 8 hours, 50% of those people won’t even have the organisms in them afterwards. 50% or so will be affected, but 95% of those cases are latent TB, and 90% of those will never reactivate.
1. The other 5% is primary active TB and develop disease immediately.
iii. Immunosuppressed patients and children are high risk for developing primary active TB.
iv. BCG prevents this primary disease from occurring in children and only gives them a latent infection.
v. Within the first year or two, 5% will reactivate and after that your risk is 95% or so not having the disease.
h. [S14]: M. tuberculosis infection without disease
i. What happens if you are infected and don’t develop the disease?
ii. You inhale the infectious droplet and the organism goes to the middle lobe of the lung and gets into the alveolus and the macrophage takes it up and causes disease there. Often a tuberculoma is formed in their lung and will be there for the rest of their life. Then it goes to the higher lymph nodes and causes swelling. These organisms are then spread all over the body through lymph and blood of the latently infected patients and can reactivate in lots of different places, normally in the apices of the lungs (where there is the maximum amount of oxygen for organism to grow)
iii. They can reactivate in bones in the spine, kidneys, etc.
iv. Tubercle formation is the immune system walling it off. We have a granuloma and the organisms are alive in the center and will sit there and grow, but we have walled them off. You end up with inflammation and caseous necrosis, which is a cheesy material in the center of the lesion that can be huge and once someone reactivates, these often will be coughed out and the patients will have a hole in their lung.
v. Dystrophic calcification (Ghon complex) is the peripheral lesion where the organism first started, which calcifies and some of the high lymph nodes will calcify and you can see these on X-rays.
i. [S15]: Tuberculosis Prevention
i. How do you prevent becoming infected?
ii. Decease your exposure. Homeless persons have both low and high risks depending on how much they associate with other people, which results in a bell-shaped curve on who is at risk of the homeless community.
iii. Have a lot of air changes in the room. If you put these organisms in a room and the air isn’t circulating, they can move around in the air and be infectious for a long time.
iv. UV irradiation will kill the organisms. This decreases the circulating organisms and will eliminate them.
v. Offer chemoprophylaxis to someone who has a positive skin test. Offer them a single drug (INH) for 6-9 months and this decreases the risk of you ever developing the disease.
1. First you have to rule out the actual disease, before you give them these drugs which may result in resistant organisms.
vi. BCG vaccination causes a positive skin test. Sometimes this causes confusion amongst those people who actually have the disease and have the BCG vaccination. They may just assume that the positive test is just the BCG, but it is actually TB.
j. [S16]: Environmental Resistance of M. tuberculosis
i. Survives drying, susceptible to UV irradiation.
ii. [SQ]: If you have BCG, you don’t have TB…correct?
iii. [A]: Right, if you have been vaccinated your skin test will be positive, but you have been exposed and still have the organism.
iv. Is resistant to many disinfectants, but is susceptible to chlorine and phenols.
v. Pasteurization was developed to kill Mycobacterium bovis, a very close relative of TB in cow’s milk. The temperatures were derived how long it took to kill these organisms in cow’s milk.
vi. High Efficiency Particulate Air filters which will filter out all of the TB. Hoods in the lab have these filters.
VIII. Germicidal Ultraviolet Light [S17]
a. Can put UV light in air ducts and in upper room irradiation.
b. Also used in biological safety cabinets.
c. [S18]: UV Exposed Culture Plate
i. Experiment was done where TB was put on a plate and put the lid over a small portion of the plate and exposed it for one minute, except for where the lid covered them. This shows you another important thing about UV light, that plain plastic will prevent it from penetrating and being effective.
IX. Diagnosis of Tuberculosis [S19]
a. First thing you do is a AFB smear of the sputum.
b. Then a tuberculin skin test (Mantoux test – inject the culture filtrate so they grow TB, then filter the TB out and inject that into your skin to see if you have an immune response) and follow with a chest radiograph and finally an AFB culture.
c. [SQ]: What does AFB stand for?
d. [A]: Acid Fast Bacillus – defined by the stain
e. [S20]: Mantoux skin test
i. Inject 100 microliters of culture filtrate under the skin and then 48-72 hours and look at it.
ii. The way you get the millimeters of induration is not what is red, that Erythema, but looking for something that is hard. You take a ballpoint pen and draw on the skin until you run into a lump that stops the pen and you do that for 4 directions and that gives you the diameter of the induration.