Microbiology: Mycobacteriologypg. 1

Microbiology: Mycobacteriologypg. 1

Don Norris

1. Few organisms that cause disease are included in Mycobacteria, but they are still importand because it includes Tuberculosis. This is TB grown in the lab. Looks like a dragon.

2. Mycobacteria is defined by acid fast stain and is called acid fastbascilli (AFB-stain). The mycolic acids on the cell wallhas carbon chains that are 78-91 carbons long (waxy and fatty). There are also other less well defined waxes on the surface. They are obligate aerobes (need oxygen). This is important to note when thinking of where they grow in the body (highly oxygenated areas). They are slow growing. When you send a culture to the lab it takes about 8 weeks to knowthat nothing is growing. It takes about two or three weeks to see colonies. TB takes about 18 hours to double (E coli takes about 18 minutes).

3. Mycobacteria have been around for a long time (before animal life). There are about 100 named species. Before HIV came around there were very few mycobacteria that infected humans. Since then there are about 40 species that infect humans (immuno-suppressed people).

4. This is what the cell walls looks like. Gram-positive organisms have a very large petidoglycan layer (this is what makes them gram-positive). When you stain them the crystal violet is caught in the cell wall. Gram-negative has small peptidoglycan layer and the outer membrane (LPS endotoxin layer) outside of that. Mycobacteria have a small peptidoglycan layer, but are related to the gram-positive organisms genetically. They are gram-positive but give a negative stain because of the mycolic acids and waxes. They have abinogalactans on the outer surface. They have a very tough cell wall that makes it more resistant to many disinfectants we use.

5. Obligate pathogenic mycobacteria. Mycobacterium tuberculosis is the first bacteria shown to cause disease. Koch in 1880’s developed a postulate to distinguish organism-causing diseases. According to the postulate you must isolate the organism causing the disease from a person, grow it in vitro, and then put it into an animal and cause the disease in animal. Mycobacterium tuberculosis is the first organism that was tested this way.

Mycobacterium leprae causes leprosy. This does not grow in the lab.

6. Humans are the only natural host of mycobacterium tuberculosis. There are some other animals that can be infected, but it is not transmitted well in those animals. 1/3 of the world is infected with TB, but very few of them have disease. In the US 5% or less are infected with TB. There are about 9.2 million cases of TB each year in the world (have the disease). 1.7 million deaths per year.

7. Prehistory of TB. It was found in bison in Wyoming from 17,000 Years ago (BPE=before present era). Found in DNA found in bones. Also found in Germany. In Egypt people have Potts disease from mummies. In Potts diseaseTB infects the spine and the people walk with a curved back because of the destroyed vertebrae (kyphosis). 1300 there was a 8 year old boy who was sacrificed in the Andes. They found AFB smear positive and DNA in him.

8. This is what the epidemiology of TB looks like. This is from 1860 to 1950. Even before we knew what it was, we were controlling it pretty well. There is a decrease in mortality rate over time. When we found out what it was, the slope didn’t change much. The skin test didn’t change the slope either. We came out with chemotherapy between 1940 and 1950 (streptomyosin was first) that possibly decreased this slope. BCG (anti-TB vaccination) didn’t have much change. This is a social problem. Crowding and poor nutrition cause the disease to develop.

9. This is what has been happening since 1950 in the United States. TB started to come back, but really has been going back down since 1992. In the early 80’s AIDS patients were housed together, and TB broke out among them (a resistant strain broke out in New York City). A lot of effort and money was put into it, and since then we have had a steady decline. At this rate we will get to 1 death/ million cases. This is the goal. This lower line shows 646 deaths. At UAB . . . 2 deaths this year.

10. Another way to look at it is this bar graph. People born in other countries come to this country and reactivate at the same rate that they would if they had stayed at home. The US-born cases of TB are decreasing. We are having less and less people who have been infected for a long time (they are dying). 2.7 / 100,000 US-born people develop disease per year. The foreign born is slowly increasing in percent (23.6/100,000).

11. Here you can see that 60% of US cases are from people who are born overseas. However, we see very little transmission of the disease from people born overseas. You have to be around infected persons for larger periods of time in order to transmit disease (couple of hours). People who are born in highly infectious countries and move to US typically self-segregate with other from the same native area. There is little transmission to US-born population. The main possibility is if someone were to get sick and transmit at the hospital.

12. Alabama has a very good TB control program. We are below the national average. Part of the reason it is so low is we were not looking very hard for TB (expected to go up this year).

13. These are examples of high rate TB countries in Africa. Namibia has 1.8 million people buthas about the same number of cases as we do in the US (16,000). Namibia has 822/100,000 and US has 4.2/100,000. The death rate in Namibia is also high. Zambia is also high in each of these categories. These numbers are probably lower than actuallity.

14. Transmission of TB is by infectious droplets. They are really small droplets (1-3 bacteria per drop) and must get all the way down to the alveoli where the alveolar macrophages are to cause infection. If the droplet is too large, it will get caught earlier in the bronchial tree, coughed up and swallowed (this rarely causes disease). The droplets settle about 9mm/min in still air. In a room where someone has been coughing, it takes about 90 minutes for the organisms to settle out of the breathing space (assuming the air isn’t stirred up).

There was an outbreak in Gadsden. People were smoking crack in a bathroom of a convenient store. TB got into this population. They found that a lot of transmission was going on among these people. It was shut down because it was too convenient.

Infectious dose is 5-200 infectious droplets. Infected people are only making 1.25 droplets per hour. You must be around someone for about 8 hours in close space to get transmission (coughing required). Some produce much more (150-200/hour) allowing open-air transmission of TB (ex. Players on a softball team able to transmit to each other). Originally we thought this was very virulent strain, but turns out the factors causing the larger amount of droplet forming is caused by something else – maybe specific to individual patients(not a more virulent strain of TB).

15. This is an example of what happens in transmission. If you have close contact for a significant period of time, only 50% of those people will be infected. Genetics and luck both have to do with who is infected. Of the infected, 5% will have primary active TB (disease w/in 6-12 months) and 95% will have Latent TB. The immune state of the person affects who will develop primary active and latent TB (babies and HIV patients more susceptible). 5 of the 95% will have reactivation some time in their lifetime. 90 of 95% will never reactivate. If people have HIV there will be 5-10% reactivation per year.

16. Risk factors for disease caused by TB: 1)AIDS patients with CD4 < 400 are more likely to reactivate disease. He said,“Actually, 200 is probably a better number I probably should change that.” 2)Iatrogenic immunosuppression. THFa inhibitors are given to people with arthritis or other autoimmune diseases. These are really good at reactivating TB in someone with latent infection. If you are to go on one of these drugs, you must have a skin test to make sure you don’t have TB and get disease. Steroids are not quite as effective at reactivation of TB. 3) Young and Old don’t have as good immune system. 4) Alcoholism 5)Diabetes 6)Genetics can all increase ones likelihood of reactivation.

It seems that part of the reason we are having a down trend in TB is those who are susceptible are dying. Many people living in closely inhabited areas are becoming resistant to TB. People who live in tribal communities in the Amazon are exposed to TB and die within 6 months. When people have not been around it in the past, they are extremely susceptible.

17. We’ve all had a skin test. A culture filtrate is injected in the skin (intradural) and you wait 48 – 72 hours and measure the greatest diameter of the induration or hard part (not the red part). A group of Eskimos in Canada had a clear delineation between the infected and uninfected. Only about 33% uninfected and 67% infected. This is high infection rate. In Atlanta the readings had many that were in a gray area (between 10 and 5 mm). The uninfected rate was much higher (89.7%) but it was difficult to give positive or negative to a number of the tests. There is something that is possibly giving false positive test results. Because of this we would only give the test to people who have a possible exposure.

18. Significant Induration. 15 mm always indicates infection (the individual probably has live organisms inside them). 10mm means we worry about at risk populations (low income, minorities, IV drug users). These groups have a higher frequency of TB, and therefore the test is more likely to be a true positive than a false positive. Other higher risk populations: foreign born (from high prevalence countries), institution populations (prisons, nursing homes, mental institutions), and people with following conditions: silicosis (impaired lungs), diabetes mellitus, malignancies, immunosuppressive agents. 5mm could be a positive test if it is recent infection and they have certain conditions: HIV positive or high risk w/ unknown HIV status, have a chest x-ray with indications of infection, and people in close contact with known TB cases.

19. Type of disease. Childhood TB is a primary disease. Someone gets infected and develops the disease immediately (this can happen in adults as well). Adult disease is reactivation disease. You breathe an organism that goes to the center of the lung (center lobe). It develops and goes all over the body and it reactivates where there is high oxygen level (upper lung). Reactivation TB often is located in the apices of the lung or highly vascularized organs (ex. Kidney or bone). Reactivation disease shows up over one year after infection.

Acute tuberculosis pneumonia (AIDS) doesn’t look typical. People with AIDS usually don’t have the cavities because they don’t have the immune system to cause the necrosis that forms the cavities.

Miliary TB is when you have little immune system, but you have TB growing everywhere and you have tubercles/granulomatus tissues all over with organisms growing in them.

Cold abscess looks like it should be staph, but is slow growing and is cold. There is little inflammation. It can be TB or another mycobacteria.

Addison’s disease is an adrenal infection with TB.

20. This is what lungs look like withdifferent types of TB. You can see the caseous necrosis (white cheesy material-left). This will liquefy and you will end up with a cavity that forms (middle). Someone with a cavity has a lot of organism and they will cough it up. This is what is really infectious. You can also see the miliary TB (right) – caseous necrosis and tubercles all over. You don’t last long with either of these without treatment.

21. Review of TB infection. You inhale the infectious droplet that goes to the center part of the lung. It forms a tubercle in the center of the lung and then goes to the hilar and peribronchiolar lymph nodes. After that, lymphohematogen is spread all over the body. Likely, your immune system will start to catch up and you don’t even know you were infected. However, it leaves the Ghon complex which is the spot where the primary organism grew and the lymph node. This can be seen on x-ray and is used to test for previous TB infection after a positive skin test. 90% of the people infected will go through these steps and never develop the disease even though they have these organisms all over the body.

22. Diagnosis of TB. Skin test is the first thing and if it is positive you go on to these steps. AFB smear - if they have respiratory TB and cough up sputum you put it on a slide, stain it with carbol fuchsin acid-fast stain and if you see it, put them on treatment and wait for a culture to grow to test for sure. The chest radiograph can eliminate some of the non-TB mycobacteria that we grow.

23. Look at tuberculosis prevention. Environmental- decrease exposure: Avoid crowded conditions, have air changes in labs where TB research is done (6 air changes per hour-pretty windy). UV irradiation kills TB quickly. Chemoprophylaxis – after positive skin test and you are sure that they do not have the disease. If don’t have the disease, you only give one drug. If they have the disease, they have more organisms and probably have a resistant group so you don’t want to only give one drug because one of the groups may be resistant to that drug. You want to give at least four drugs to be sure.

There is a TB vaccination (BCG vaccine) used around world but not US. It doesn’t change the epidemiology of the disease. People still get infected and infect others. It does help to decrease the number of primary diseases, so it is given to babies. We don’t give it here because it causes a positive skin test. About 10 years after vaccination, you probably won’t get positive skin test anymore.

24. This is a smear of sputum. You can see white cells (PMNs). Here is one organism. Techs will look at these slides for over 5 minutes trying to see one organism to label it positive.

25. Environmental resistance of TB. TB survives drying. It has been fed to roaches and found in the roaches feces after a long time. It is susceptible to UV light. 2 hours in sunlight and it is dead. It is resistant to many disinfectants (less-caustic disinfectants don’t kill mycobacteria- Clorox and phenols do kill it). Pasteurization wasdeveloped to kill mycobacterium in cow’s milk (62 degrees for 30 min or 72 degrees for 15 sec). TB can be filtered out of the air with High Efficiency Particle Air-filter (HEPA) will take TB out (very reliable).

26. Germicidal UV light. It can be used in hoods and air ducts and in upper room irradiation in order to kill TB. (you can’t look at it so they point it up and away from view)

27. This is a plate I smeared TB on. I exposed half of one slide to UV and not to other side for just one minute, and you can see how UV killed it. TB very susceptible to UV light.

28. We do 4-5 thousand cultures per year and 5% of those grow mycobacteria. Only 1% of the total cultures are TB.

29. These are the diagnostic cultures. These had TB. We had 10-15 TB patients from our 5,000/yr. It took15 to 17 days to tell if there was any growth in the bottle. We ID growth within a week after we see growth. 60% of people with TB are found smear positive. 5 out of our 27 smears were positive for TB. This shows specificity and sensitivity of a smear. 2/3 is not really good.

30. These are organisms we have identified over the years. There is more mycobacterio avium than TB. This is commonly in AIDS patients. Now that AIDS patients are treated better, we see less avium than we used to. Rapid growers can cause a TB-like disease. The rest are pretty rare

31. This is what the smear looks like after concentration. You digest the sputum, centrifuge it and put it on a slide and you can see lots of organisms.

32. Culture TB. Digest the sputum, decontaminate it (kill other organisms), concentrate it, do a smear and cultivate it on solid media and liquid media (liquid is faster but other organisms grow with it). Then we do susceptibilities of all our bacteria (state law requires). Send it to state and they check for resistance.

33. This is the hood w/ TB set up and tubes with sputum inside of them. Bottles for culture, slides for smears.

34. Mucolytic agent is N-acetyl-L-cysteine. Sodium Hydroxide is Lye (pH of about 13 at 2%). We use this to kill all of the other organisms (mycobacteria survive Lye). Then,centrifuge for 30 minutes.

35. Solid media takes 21-26 days to see colonies of mycobacteria (agar based and egg based). Liquid media is much quicker (within 2 weeks). Some use C14 radioactive labeled tests. We use MB/BacT that looks for CO2 production. ESP looks for gas change (utilization of O2 leads to pressure change in tube). MIDGIT is an O2 utilization that quenches fluourescence medium in the bottom. All of these are used to detect growth.