Download Mycobacteria

CLASS: Hour 1
DATE: 10-20-10
PROFESSOR: Benjamin
Mycobacteriology
Scribe:Spencer Terry
Proof:Ashley Tate
Page 1 of 6
I.
Mycobacteriology [S1]
a. These are a rare group of bacteria that are very important.
II. Mycobacteria sp. [S2]
a. Mycobacteria are TB and a lot of its friends
b. They are acid fast bacilli, and this is described by the stain.
c. When you stain them, you can’t decolorize them with acid fast alcohol. Other bacteria will be decolorized.
d. The reason for this is that they have mycolic acids (long fatty acid chains of 78 to 91 carbons)
e. They also have lots of waxes on the surface that make them light and able to survive in dry conditions.
f. They are obligate aerobes (need oxygen to live)
g. Slow growing- where E. coli divides every 20 minutes or so, TB takes around 24 hours. 18 hours to double.
Takes weeks to create a colony on a plate.
III. Identification of Acid Fast Bacilli [S3]
a. First is the stain. Mycobacterium have been around a long time. They infected amoebas and such in the
environment, before infecting humans.
b. There are 100 different species that are named. More than 40 have been found to infect humans and cause
disease.
c. Many of these have been found since the AIDS epidemic due to suppressed immunities.
IV. The Bacterial Cell Wall [S4]
a. Gram-positive organisms have a really thick peptidoglycan layer, and that’s what causes them to be gram
positive. They retain gram stain. They also have cytoplasmic membrane that looks the same for essentially
the different types of organisms.
b. Gram-negative have a smaller peptidoglycan layer, but have an outer membrane. Have lipopolysaccharides,
endotoxins. Less peptidoglycans causes them to decolorize and be gram-negative.
c. The mycobacterium have peptidoglycan layer and are technically gram-positive, but don’t stain gram positive
because the stain won’t go into them. They also have many other constituents (arabinogalactan – involved in
immunologic response). Have very thick cell wall and membrane, and are very different from others.
V. Obligate Pathogenic Mycobacteria [S5]
a. Mycobacteria tuberculosis was the first bacteria shown that causes disease. Koch’s postulates based on this.
(can remove the organism, grow it in culture, then reintroduce it to an animal and it will cause disease)
b. M. leprae causes leprosy. Is an important disease a lot of places. Not so much here.
VI. Prehistory of Tuberculosis [S6]
a. Just to say has been around a long time. Looking in mummies in both the new world and old world and can
find traces of tuberculosis there.
VII. Graph [S7]
a. This is England and Wales, starting in 1860 to 1950.
b. Even before Tubercle bacillus was identified, the epidemic was already decreasing in number of deaths.
c. Could be due to nutrition, sunlight, and social systems. Also genetics (people who are very susceptible to
tuberculosis die off, leaving people more resistant to TB)
d. Skin test developed – didn’t make a difference in the rate of decline.
e. World War 1 and World War 2 had effects.
f. BCG vaccination not used here, because it causes positive skin tests. Is used other places with higher rates
of infection. Doesn’t prevent infection or spreading, but can prevent the disease.
VIII. Mycobacterium tuberculosis [S8]
a. Humans are the only natural host. Other animals can be infected (often zoo animals can become infected)
b. 1/3rd of world is infected. Less than 5% of people in U.S. are skin test positive, however. Other parts of the
world much higher.
c. Most people that are infected never actually develop the disease. However still 1.7 million deaths per year.
IX. Tuberculosis cases and deaths US [S9]
a. Can see that in the 80’s, we had 25000 or so, continuing to go down.
b. Then, stalled out and increased (largely driven by the HIV population)
c. Some large cities were putting their AIDS patients together, but not watching TB closely, so resulted in large
number of TB deaths.
d. Budget for TB control increased, and cases have since decreased. Still .5% of those cases die.
CLASS: Hour 1
DATE: 10-20-10
PROFESSOR: Benjamin
Mycobacteriology
e. Any time TB is not actively controlled, it can easily reemerge.
Scribe:Spencer Terry
Proof:Ashley Tate
Page 2 of 6
X. Number of TB cases in U.S-born vs Foreign born persons [S10]
a. This shows the number of foreign born cases of disease of TB in the U.S. has stayed constant for a number
of years.
b. Native born people, as older people who have been infected for decades are dying off, the rate of TB is
decreasing.
c. More than half the people now that have TB now were likely infected outside the U.S.
d. However, to transmit TB, must be pretty close to someone for quite a while, so shouldn’t be too concerned
with being infected by these people.
e. The strains of bacteria that foreign born have, are not very often transmitted to native born.
XI. Alabama Tuberculosis Cases [S11]
a. Alabama has a very good TB control system and anybody that is infected and has disease will have the
public health service come give them their drugs and watch them take the drugs for 6 months.
b. Therefore, don’t have much transmission here. Initially had higher than national average.
c. Even have a prison cell where they can hold people if necessary. The cell has negative air flow.
XII. Examples of high prevalence countries[S12]
a. Some other places of the world. Zambia is a fairly large country. Much higher numbers of tuberculosis per
100,000 people.
b. However, these countries don’t do a very good job of diagnosing.
XIII. Transmission of Tuberculosis [S13]
a. As I mentioned before, transmission is by infectious droplet. Droplets are very small.
b. When someone coughs, bacteria will come out dry and float in the air, so the droplets can really only contain
one to three bacteria. Droplets settle very slow in the air (9 mm/minute). Takes 90 minutes or so to land from
breathing space. So area is infectious for a while.
c. A person really only puts out 1.25 infectious droplet per hour, so it is rare to make these nicely infection
droplets. It takes 5-200 droplets to cause an infection.
d. Few cases produce more droplets than this.
e. Was determined by running air from TB patient’s room to a guinea pig’s room and then determine how much
air is breathed and how many infectious droplets put out/taken in.
XIV.
Exposure  No infection [S14]
a. 50% of cases, even with a lot of exposure, don’t get infected as measured by the skin test. The skin test is
how we tell if someone is infected.
b. 50% do get infected. 5% of those go ahead and develop disease. A lot of these people are
immunosuppressed (HIV patients, young kids, older people). A few have a defect that isn’t understand and
therefore develop the disease.
c. 95% of people infected will have latent TB, and 90% of those will never reactivate.
d. 5% will develop TB sometime in their lifetime.
e. Anything that causes you to become immunosuppressed really increases chances, so these people will likely
develop the disease as their CD4 cell count continues to decrease.
XV. Mantoux Skin test
a. On left – Canadian, native American, Eskimos. Can see x axis is size of reaction, so when PPD is injected
into the skin, should see a white whelp in the skin. This is measured after 48 to 72 hours by taking a ballpoint
pen and drawing a line until you reach an edge. Measure distance between pen marks.
b. 33% of these Eskimos were skin test negative. Most of them had 20 millimeters or more, showing they were
really infected with tuberculosis.
c. Atlanta on the right. 90% of the people were skin test negative. But, there were many in the middle which we
can’t tell. This is because there are many other mycobacteria in our environment that aren’t found in the
northern areas.
d. Many of these other species cause a positive skin test. Often results in false positives.
XVI.
Signification induration on Mantoux
a. 15mm probably always indications TB infection.
b. 10 mm is positive for populations at risk. (read slide)
CLASS: Hour 1
Scribe:Spencer Terry
DATE: 10-20-10
Proof:Ashley Tate
PROFESSOR: Benjamin
Mycobacteriology
Page 3 of 6
c. 5 mm called positive in people that are immunosuppressed, or if it is someone that has very recently been
exposed. Chest film used to verify.
XVII. Quantiferon-Gold
a. This test is a blood test, so you don’t have to return to have a skin test read. The major advantage is that this
doesn’t have the antigens that are present in BCG, so if someone has been vaccinated by BCG, they won’t
respond in this test, while they will to the skin test.
b. A small amount of blood in 3 different tubes. 1 has no antigens. 1 has TB antigens, and the other has a
mitogen. – causes T cells to release interferon gamma. (names aren’t important).
c. Read the different amount of interferon gamma released in these tubes and from that can determine whether
infected with TB.
XVIII.
T-Spot TB
a. Won’t discuss this. Works about the same way.
XIX.
M. Tuberculosis infection without disease
a. What happens if you are infected with TB and don’t have disease?
b. You inhale one of these infectious droplets and it gets down into the alveoli and down into the lungs. If it is a
larger droplet and gets stuck in an upper airway, you will cough it up and swallow it. Rarely are people
infected if this happens.
c. When the droplet gets all the way down into the alveoli, it is taken up by alveolar macrophage, where it can
survive and grow in the lymph nodes. This causes caseous necrosis in that area. Hilar and peribronchiolar
nodes are infected in this way.
d. Then the organism spreads all over body, called lymphohematogenous dissemination. Takes 6-8 weeks (not
really sick during this time).
e. Then, the immune system walls it off, forming tubercles. The organism can then sit there for decades, still
alive but not causing problems. As long as the person has an immune system, it will keep it contained.
f. Dystrophic calcification – this is how we tell if someone is infected. They will have a calcium deposit where
the first lesion or granuloma was, down in the center part of the lung where most of the air goes. Will also
have a spot where first lymph node was infected. These two calcified regions are called Ghon complex, and
suggests TB infection.
XX. Tuberculosis Risk Factors
a. People with AIDS have a CD4 count that is very low. This is really the only infectious disease that AIDS
people are at risk for spreading to the rest of the population.
b. TNF-alpha inhibitors are used to inhibit the immune system in people who have auto-immune disease. These
are good at activating latent TB. People being placed on these drugs should be screen for TB first.
c. Age- young and old people are really at risk for developing disease from TB.
d. Alcoholism, malnourishment, diabetes and genetics are all things that predispose people.
e. There are people in the Amazon basin that have not really been exposed to TB. Around 80% of these people
who are exposed then die within 6 months. Almost no one goes on to develop latent TB because they haven’t
been exposed to the organism before.
XXI.
Types of Tuberculous disease
a. Childhood tuberculosis is the primary disease. This is when someone gets infected and develops the disease
right away. More common in children.
b. Adult or reactivation disease is when someone has been infected a long time and the organism comes out
other places in the body where the organism is spreading to. The most common place is the apices of the
lung. Cavities in upper area of lungs suggests TB. This area because it is the highest oxygen concentration,
and TB likes oxygen.
c. Other places it can reactivate are the bones, spinal column, joints.
d. Acute tuberculous pneumonia is found in people with suppressed immune response. These people have a
much different presentation of TB. Most of the pathology in adult reactivation disease is due to their own
immune response trying to get rid of TB.
i. So, TB in immunosuppressed people looks much different.
e. Someone kind of in the middle will have miliary tuberculosis. Name comes from millet seeds. Results in lots
of little tubercles, and the disease keeps spreading around causing more. Very serious disease.
f. Cold abscess means you can have a lesion that grows very slowly and doesn’t have a lot of erythema or
induration.
g. Addison’s disease – when TB infects adrenal glands and causes adrenal insufficiency.
XXII.
Tuberculosis prevention
a. Environmental – want to decrease exposure. If someone is coughing, try to get away from them.
CLASS: Hour 1
Scribe:Spencer Terry
DATE: 10-20-10
Proof:Ashley Tate
PROFESSOR: Benjamin
Mycobacteriology
Page 4 of 6
b. UV irradiation is efficient at killing TB. Take the coughing person outside….
c. Air exchanges – in the TB lab, have 12 air exchanges an hour. Is almost windy. Lots of buildings such as
hospitals will have negative pressure to prevent transmission.
d. Chemoprophylaxis – if someone has positive skin test, would then think about giving them one drug to keep
them from reactivating. Take this for 9 months.
e. Want to rule out disease first. People with the disease have so many organisms that in that population there
will be resistant organisms, so a single drug wouldn’t be enough. In this country, would give at least 4 drugs
to someone that has the disease. Latent carriers get the 1 drug for 9 months because they have far fewer
numbers of organisms.
f. BCG vaccine causes positive skin test. Used in nearly all the rest of the world. Really prevents childhood
disease.
XXIII. Types of Disease caused by M. tuberculosis
a. Pathologists name pathologies after food, so caseous necrosis means cheese. Look like cheesy spots in the
lung (middle). Happens when parts of the lung liquefy and then are coughed out, leaving cavities.
b. On the right, miliary TB. Can see little granulomas everywhere. Large numbers, obvious a lot of lung has
been destroyed.
XXIV. Environmental resistance of M. tuberculosis
a. TB really survives drying. So dry sputum could be lying around and have live organisms. If a roach eats the
sputum, the feces would have live organisms as well.
b. Susceptible to UV irradiation. A couple of hours in sunlight will kill it. Any naked droplet would be killed
rapidly.
c. Resistant to many disinfectants, so be sure to read that it is tuberculocidal. Chlorine and phenols are best.
d. Pasteuriation was developed to kill M. bovis in cow’s milk. Lots of people were being infected.
e. HEPA = high efficiency particulate air filters. Used in labs, etc.
XXV. Germicidal Ultraviolet light
a. In the hood, we have a UV light. Can also be used in air ducts. UV lights can be shined in the upper room.
Many homeless shelters have UV lights to try and control TB.
XXVI. UV Exposed Culture Plate
a. Demonstration I did. Took TB and spread it all over the plate. On the left, was not exposed to UV, and can
see organisms all over the plate. On the right, the lid was moved. Created a shadow of the lid and prevented
UV light from going through. Where the UV light went in for 1 minute, all the organisms were killed. Very
susceptible.
b. But in liquid, it only penetrates a few mm.
XXVII. Diagnosis of Tuberculosis
a. Acid fast bacillus smear done if you think someone has TB.
b. Skin test we discussed (mantoux)
c. X-ray and the culture are all ways of diagnosing TB.
XXVIII. M. tuberculosis direct smear
a. This is what we would see. Can see one organism in the middle. This is an acid fast smear. Around 90% of
specimens are negative.
XXIX. AFB Cultures at UAB
a. At UAB, we do nearly 5000 cultures a year. Around 5% of those grow some mycobacteria. Only around 1% of
the cultures are positive for TB.
b. Most of these are in immunosuppressed patients.
XXX. Diagnostic M. tuberculosis Cultures
a. How do we diagnose it from that?
b. These are the number of patients that had TB at UAB. In 2009, only had 13 patients.
c. Days to grow is how long it took to tell something was growing. Should be around 14 days.
d. Use a molecular method to identify the growth once it occurs. Takes a few more days.
e. Have to report the smear within 24 hours after specimen is collected. Worldwide, around 60-70% are AFB
smear positive. So, in addition to the smear, we also do culture. Another thing to look at is what percent of
smear positives are actually TB. Tends to be only around 33%
f. A lot of AIDS patients especially will have mycobacteria avium that tests smear positive as well.
XXXI. Number of Patients each species of mycobacteria
a. This is a list of the other organisms we have isolated. Can see that M. avium, which is really common in HIV
patients, is still the most common organism.
XXXII. Microbiological Diagnosis of Tuberculosis
a. So how do you culture TB?
CLASS: Hour 1
Scribe:Spencer Terry
DATE: 10-20-10
Proof:Ashley Tate
PROFESSOR: Benjamin
Mycobacteriology
Page 5 of 6
b. Use mucolytic agents to digest the sputum. Sodium hydroxide will do this, but also kills TB. So we decrease
the amount of it and add some other things. Use about 1% sodium hydroxide to kill other bacteria, but not the
TB.
c. Then concentrate it to decrease the volume.
d. Then we do an acid fast stain of that and culture it. Culture it in both solid media and liquid media. Liquid
culture is faster, but if anything is growing besides mycobacteria, then it can get overgrown.
e. Anti-mycobacterial susceptibilities is required to be done for TB. The state has a law that you must send it to
them to do the susceptibilities, so they can monitor the amount of drug resistant TB in the state. UAB doesn’t
get enough cases to be able to do it themselves.
XXXIII. Picture of hood
a. This is the hood where the technician is working. This is done at night here.
XXXIV. Decontamination and Concentration of AFB cultures
a. The mucolytic agent we use is N-acetyl-L-cysteine
b. NaOH could be important. It really digests the sputum, and also kills other things.
c. Centrifuge it at 3000g for 30 minutes. Have to centrifuge them hard to get them to settle to be able to stain.
XXXV. M. tuberculosis concentrated smear
a. This is what a concentrated smear looks like. Huge number of organisms. This is what someone coughed up.
b. It turns out that even on an airplane, it is only about 3 feet around someone that you are really at risk. For the
most part, there is not much transmission on planes.
XXXVI. Mycobacteria Culture media
a. The solid media takes 3 to 4 weeks to see colonies. There are couple of different kinds. Not important
b. Liquid media – should be able to diagnose most respiratory disease within 2 weeks. Several different
methods are used.
c. One we use now is MGIT. Has a fluorescent media at the bottom of a tube. As any organism in that tube
uses oxygen, it decreases the oxygen, and that oxygen is quenching the fluorescence. So when oxygen is
gone, the medium at the bottom will fluoresce.
XXXVII. Colony Morphology of M. gordonae and M. tuberculosis
a. The colonies look kinda like bacteria.
b. On left is M. gordonae – is not a pathogen. Is very crusty and solid. Lots of electrostatic properties.
XXXVIII.
Liquid culture systems
a. This is the liquid system.
XXXIX. Growth in Liquid Medium:
a. This is what it looks like in the tube.
b. On left is M. tuberculosis growing in a bottle. Is all in big clumps.
c. Other organisms really can be individual organisms. (on right) You can see they are all acid fast because they
stain red. The blue is yeast, which aren’t acid fast.
XL. Identification of Mycobacterium
a. Biochemical tests take too long. Not really done anymore.
b. GenProbe takes a couple of hours. We do this once a week.
c. High performance liquid chromatography is what the state uses to identify things we don’t identify.
XLI.
Hybridization Protection Assay
a. Skipped
XLII.
Mycobacterium avium complex
a. Mycobacterium avium complex. This is the most common thing we’ve found.
b. Is really found in soil and tap water. Interesting that hospital showers have M. avium in them.
c. M. avium causes bird tuberculosis. It likes hotter temperatures, so lives in the hot water in showers.
d. For immunosuppressed patients, this can be a problem.
e. Can cause a pulmonary disease like tuberculosis.
f. 50% of early AIDS autopsies had M. avium
XLIII. Other important MOTT
a. M. kansasii can cause tuberculosis like disease, but it is not transmitted from person to person.
b. M. marinum is in water. Also produces pigment.
c. Scrofulaceum, fortuitum, chelonei really grow quickly and usually cause skin infections, but can cause
pulmonary disease.
XLIV. M. marinum
a. These are really non-healing lesions. They go up the lymph system of the infected area.
b. Some patients have had fingers amputated before knowing it was a M. marinum infection.
XLV. M. marinum
a. Grows in the dark without pigment. Expose it to the light and the colonies that are there turn yellow.
CLASS: Hour 1
Scribe:Spencer Terry
DATE: 10-20-10
Proof:Ashley Tate
PROFESSOR: Benjamin
Mycobacteriology
Page 6 of 6
b. This causes a positive skin test.
XLVI. Mycobacterium leprae
a. Read slide
b. Fewer cases than TB.
c. Lepromatous patients don’t have a good immune response to the bacilli and are very infectious.
XLVII. Leprosy
a. Do not know how to grow it on media.
b. Lepromin test – grind up a leprous person’s skin and inject it into someone as a skin test.
c. Treatment is efficient – dapsone and rifampicin for at least a year.
d. Skin scraping – scraping the skin in the knees and elbows and stain it to test if organisms are still present
after treatment. The organisms can hang around a long time.
e. Prevention is basically just isolation.
XLVIII. M. leprae epidemiology
a. Worldwide epidemic of leprosy is going down fairly well.
b. Ratio of tuberculoid to lepromatous leprosies. The lepromatous ones are the ones that are infectious, have
real high dose because they don’t make an immune response to the organisms.
c. Tuberculoid have low number of organisms so aren’t very infectious, but these have a high immune response
to the organisms.
XLIX. US Leprosy
a. Found in the western gulf.
b. In 1975, found a wild armadillo infected. Also found tissue of armadillos infected before the experiments, so it
shows armadillos are naturally infected, probably before humans were infected here.
c. It grows internally in armadillos because of the lower body temperature. In humans, it typically only infects the
skin due to higher body temperature.
L. Clinical Types of Leprosy
a. Read slide
LI. Lepromatous leprosy
a. Read slide. CMI = cell mediated immune response
b. Testes especially involved due to colder temperature.
c. Bacteremia and localized infections in nerves and skin; leonine facies
LII. Lepromatous leprosy
a. Can see the organisms have causes a lot of disfiguration and can see when you stain it, there are huge
numbers of organisms in the lesions on the skin.
LIII. Tuberculoid leprosy
a. Tuberculoid is non-progressive, so what you have is what you get when diagnosed.
b. Grow in nerves in cooler parts of the body; cutaneous loss of sensation and nerve damage due to cell
mediated immunity
c. Read slide.
LIV.
Tuberculous leprosy
a. The immune response causes a hypopigmentation. Also, is anesthetic, so can poke it and not feel it. Middle
picture shows swollen nerve.
b. Losing fingers is not from the disease, but from loss of sensation. They can’t feel it, so they will reach into
fires and such. Also, the story about the missionary in India that found out rats were eating fingers of leprous
kids while they slept.
[End 54:00 mins]