med microbio--MODULE 14. ACID-FAST BACTERIA

What is meant by “acid-fast” and what cell wall component(s) are unique to the AFB.

• what is necessary to stain (2)

• sig property is due to what

• rez to removal by___

• When most bacteria are stained with dyes, they stain easily but color is quickly removed when treated with acid alcohol

  • Ex:  methylene blue, Crystal violet, safranin (both used in gram stains)

•  acid-fast back bacteria are difficult to stain with dyes→ heat and extended drying times are necessary

•  resistant to gram stains

•  once acid fast bacteria stain, they retain the dye even if treated with acid alcohol (thus the name acid fast)

  •  this property is due to composition of the cell wall

  •  acid fast means organisms, once stained, are resistant to stain removal with acid alcohol

Cell wall composition of mycobacterium:

• high ___ component

• what do TB specific drugs target as mech of action

• 3 rez

• Free mycolic acids in CW are unique to this genus

•  acid-fast staining properties:

  •  due to unique lipid surfaces predominantly composed of long chain fatty acids ( mycolic acids)

  •  mycolic acids are common to genus mycobacterium, thus providing a unique signature for it

  •  TB-specific drugs (isoniazid, ethionamide) target mycolic acid synthesis as their mechanism of action

•  mycolic acids:

  •  find basic dyes such as carbolfushin

  •  are high and lipid content– up to 60%

  •  resist staining with many dyes like Crystal Violet and saffron

  •  are resistant to acids, alkali, drying

  •  have hydrophobic surface:

    • dyes are not absorbed immediately by mycobacterium 

    • dyes are bound upon use of heat and increased staining time 

Associate reagents, procedures/protocols and names and results of stains/staining procedures used for the identification of AFB.

• main stain and procedure

  • primary stain, decolorizer, counterstain

• interpretation if red vs blue

• how many organisms present does it take to ID TB

Describe acceptable specimens and general protocol for growth and identification of mycobacteria.

Describe laboratory and equipment necessities for performing Mycobacterial work.

Acid fast stain (Ziehl-Neelsen stain) procedure:

• Fix smear with heat

•  Apply carbolfuchsin (primary stain)-->redish pink

•  heat to steaming (or 5 minutes at room temp)

•  rinse with H2O

•  decolorize with acid alcohol (decolorizer)

•  staying with methylene blue (counter stain to see background)

•  blot slide dry

•  observe microscopically

•  Interpretation: red= acid fast bacteria, blue=background

  • It only takes one organism to ID as TB, and will look at whole slide, not just one field of view

evolution of acid fast stain 

• 3 ppl

• Koch in 1882 described TB

  •  original staining method with basic dies for 20 to 24 hours at room temperature or 30 to 60 Minutes at 40° C

• Ehrlich in 1882 improved Koch's method

• Ziehl in 1882 described a new method using acidic rather than basic dyes:

  •  this stain was less damaging to tissue preparations of tubercles, yet permitted visualization of causative bacteria

  •  this stain is routinely used today

•  these stains were enormously important in convincing doctors and scientists worldwide the real cause of TB was mycobacterium tuberculosis (acid-fast bacilli)

Acid fast bacteria of 3 genuses mycobacterium of importance:

• M. leprae

  •  causative agent of , ____ which presents as ___

• M. tuberculosis

  •   causative agent of , ____ which presents as ___

• M. avium-intracellulare complex (MAC)

  •  causative agent of , ____ which presents as ___

• M. leprae

  •  causative agent of leprosy ( Hansen's Disease)

  •  a chronic disease of skin, mucous membranes, and nerve tissue– stops conduction of nerve impulse and thus lose sense of touch

• M. tuberculosis

  •  causative agent of TB in humans

  •  chronic pulmonary infection– infects lungs most commonly but can affect other locations

• M. avium-intracellulare complex (MAC)

  •  causative agent of disseminated disease in AIDS patients

  •  causative agent of pulmonary infection in non-AIDS patients

State the disease caused by M. leprae. Be familiar with both names the disease is called.

• where are most cases globally (3)

leprosy (Hansen's Disease): Chronic communicable disease that targets skin, mucous membranes, peripheral nerves

•  most cases globally are in india, brazil, Indonesia 

source, transmission, clinical infect of M. leprae

• how contageous

• incubation period

• sub clinical infect?

• 2 major forms of disease

source= humans are most common

Transmission:

• Direct contact via inhalation of aerosol droplets of bacteria

•  humans have a high resistance to development of disease– disease usually follows prolonged exposure because not extremely contagious

  •  prolonged, close contact with someone with untreated leprosy over many months is needed to catch the disease

  •  disease is not transmitted by casual contact, such as shaking hands, hugging, sitting next to someone

Clinical infect:

•  Incubation period is 2 to 7 years (long)

•  sub clinical infection exists: no symptoms, but positive skin test ( symptoms may take up to 20 years to appear)

•  two major forms of disease exist: tuberculoid and lepromatous

treatment and prev (2) of M. leprae

• curable?

• name of vacc

Treatment:

• Three drug regimen of dapstone, rifampin, clofazimine

•  multi drug therapy (MDT) does not cure but does control disease

Prev:

•  Isolation

  •  leper colonies: now called Hansen's Disease centers

  •  centers in the US are in Carville Louisiana and Molokai Hawaii

    • Fr. Damien tour was to understand leprosy and how it affects people around the world

•  Vaccine

  • BCG: bacillus and calmette and Guerin ( historical vaccination)

  • Attenuated TB strain used for vacc

Associate “lepra cells” with M. leprae.

• uses and appearence

Acid fast bacteria are present inside leper cells, which are modified mononuclear cells

•  cells with lots of acid fast bacteria inside and thus become larger in comparison to other cells in the area

• Can be used to ID in lab

Describe the morphology of M. leprae.

Rods inside leper cells

Differentiate “tuberculoid form” and “lepromatous form” of leprosy.

• which is localized

• which is more serious

• which has nodules of tumor like lesions

• which is a feather test done

• which is bc lack if cell mediated response

• which do lesions open up

• which is non-pus forming

Tuberculoid leprosy:

• Localized mild form of disease

•  erythematous macules or papules appear on involved regions of body

  •  shallow, reddish, painless papules (inflammatory skin elevations that are not superlative– pus-forming)

•  peripheral nerve involvement in damage produce areas of neuropathy exemplified by lack of feeling or touch Sensations

•  host response is to wall off organism from healthy neighboring tissues

•  Lepra cells are well confined within small nodules that do not spread because of strong cell mediated immune response

•  feather test:  feather is tickled over face and trunk to determine loss of fine sensitivity to touch– assessment of peripheral nerve damage and can provide evidence of early infection

Lepromatous leprosy:

•  nodule form

•  more serious

•  disseminated form of disease– all over body

•  usually occurs due to deficient cell-mediated immunity ( primary host response to leprosy)

•  nodules of tumor like lesions are evident on skin and in mucosal membranes

•  lesions open up in→ can lead to secondary infections that enter the open wound

•  facial deformation is typical of lepromas,  clawing and wasting are due to nerve damage that interferes with musculoskeletal activity, individuals in a later phase can lose their fingers 

Tuberculoid vs lepromatous:

T: milder, few bacilli in lesions, few shallow skin lesions in many areas, loss of pain sensation and lesions, no skin nodules and occasional mutilation of extremities, lymph nodes are not infiltrated by bacilli, well developed cell mediated/ T-cell response, localized 

L: severe, many bacilliant lesions, numerous deep lesions concentrated in cooler areas of the body, sensory loss is more generalized and occurs late in disease, skin nodules and mutilation of extremities is common, lymph nodes massively infiltrated by bacilli, poorly developed T-cell response, disseminated

Discuss and describe the laboratory diagnosis of leprosy.

• found inside what

• grow on media?

• main way to culture

• Acid fast bacteria are present inside leper cells, which are modified mononuclear cells

  •  cells with lots of acid fast bacteria inside and thus become larger in comparison to other cells in the area

•  do not grow on traditional culture media→ we have never been able to culture and thus we can't ID this way and must use molecular tools instead

•  only cultures possible are growth of M. leprae in foot pads of armadillos, since these animals are the only natural hosts of leprosy aside from humans

  •  older way of identification

  •  large portion of armadillo body is covered with armor which makes skin lesions not highly visible– hence reason for foot pads used in testing 

State the disease caused by M. tuberculosis and the major determinant for its pathogenicity

• what forms when immune sys walls off and why

Tuberculosis (more common disease in genus than others)--TB

• Chronic granulomatous infection

  • Lung disease 

  • Granulomas form when the immune system attempts to wall off substances perceived as foreign: immune system walls off since it is unable to eliminate infection

    •  “walling off” is when host defence surrounds bacteria like a capsule to slow down infection since it can't spread

    •  mechanism of lungs to fight off infection– also used to fight off fungal lung infection 

• Incidence: worldwide (1 bil infect, 3 mil die–in US 10 mil infect, 20,000 new cases/yr)

• Any tissue or organ can dev M. tuberculosis infect, however has a predilection for the lungs (preference)

  • Pulm–infection in the lungs

  • Extrapulm–infection at other locations other than lungs

2 MTB Determinants of patho:

• Cord factor

  • Trehalose 6,6’-dimycolate, a cell wall glycolipid

  • Contributes to virulence of MTB

  • Causes granulomatous lesions

• Survives intracellularly

  • Protected from functional processes of phagocyte due to cell wall lipids

  • Once inside phagocytes, MTB inhib a critical step of phagocytosis (phagosome-lysosome fusion)

 Describe the morphology of M. tuberculosis. and culture char

• what stain most commonly used

Red rods on blue background that bunch together in cord visible on Ziehl-Neelsen stain (most commonly used)

granular, waxy pattern of growth on culture plate

State the definitive biochemical characteristics of M. tuberculosis. (3)

• Pos for niacin accum (pos=yellow)

• Pos for nitrate red (pos=pink)

• Pos for catalase (pos=bubble form)

lab diag MTB: what 2 things must be done prior

• how fast does MTB grow

• 3 steps to grow MTB

• 3 possible staining tech

Lab diag:

• Sample digestion and decontam is necessary:

  • Sputum is most common samp received for TB testing bc most commonly goes to lungs and sputum coughed up as part of symp

• Sputum and mouth have mucus and bac (normal flora) that contam the culture 

• TB cultures req incubation (after digestion and decontam) for weeks to be pos: slow grower

  • At ~2 wks will begin to show growth, but will wait 4 wks before rule out as neg

• Steps include:

  • Digestion with alkaline (gets rid of mucus), liquifying, mucolytic agent (N-acetyl-L-cystine or NALC)

  • Decontam with NaOH

  • Conc of sample put in culture or media

• Mycobac will survive all these steps bc very resilient 

• AFB stain (acid fast bacillus stain)

  • Serpentine cording is an indicator of virulence

    • Due to cord factor:

      • Inhib phagocytosis

      • Inhib neutrophil (PMN) migration–which are actively recruited via bloodstream

      • Is toxic to mammalian cells

  • Look for red rods that bunch together in cord visible on Ziehl-Neelsen stain (most commonly used)

• Culture: aerobic, inc CO2, 35-37 deg C

  • Slow grower: 2-8 wks

  • Typical granular, waxy pattern of growth

• Other stains:

  • Auramine rhodamine florochrome stain for mycobac

  • Kinyoun positive stain for mycobac

Define PPD and relate it to the principle of tests such as the Tine Test and Mantoux Test.

TB clinical testing:

• Tuberculin: Mantoux test

  • Is purified protein derivative (PPD) of M. TB–not whole organism injected

  • Is used to test if a person has been exposed to tuberculin protein from TB bac, vacc, or exposure

  • Is injected under the skin on the forearm and read 48-72 hrs later to test if a person has anti-TB antibodies

  • Pos skin rxn: hard, dense wheal (red, itchy, raised welt that is 10-33 mm induration at 48-72 hrs) indicating prior exposure either through prev vacc with BCG, env exposure, or current infect

    • Pos is when site is pressed, very firm w/ no indention of where finger was–redness and raised bump does not necessarily mean pos, just inflam rxn

    • Induration= hardened mass due to inc amt of fibrous elements in tissue, marked by loss of elasticity and pliability

  • Neg skin rxn: no hard, dense, raised mass

tine test MTB

• diag or screening

  • what follow up needs to happen

• Tine test (screening test)

  • Not freq used in US

  • A multiple puncture tuberculin skin test used in med diag of TB

  • Tine test uses a small button that has 4-6 needles (tines) coated with tuberculin (TB antigens)

  • Tines are pressed into the skin, usually forearm, forcing antigens into the skin

  • Test is read 48-72 hrs later by measuring size of papule induration (papule)--a neg result is presence of no papules

  • Bc it is not possible to precisely control the amt of tuberculin used in the tine test, a pos test should be verified with Mantoux test

Diag of active pulm TB:

• Chest x-ray reveals infiltrates and cavities in lung lobe(s)

• Acid-fast bac in specimen (resp, intestinal, meningeal)

• More bac mean more progressed stage

• Can see calcified lesions (walled off organisms)

Describe the characteristic symptoms associated with chronic pulmonary tuberculosis and identify extrapulmonary sites for tuberculosis.

• 3 in adults, 4 in kids

• inherent host rez?

• primary TB

• active TB

  • lacks what immune response

  • what happens to lung appearance

• typical host response (humoral or cell med)

Infection sites: (most common sites listed first)

• Adults: lungs, intestines, kidneys

• Children: lungs, meninges, bone, lymph nodes

Clinical infection:

• People are sus to infect but tend to have a level of inherent rez to disease

  • Whether or not a person dev disease is det by his/her cellular (cell-mediated) immun, amt of exposure (microbial dose), and virulence of the strain

• Disease progression: inhal of infected droplets, MTB multiplication, primary pulm infect

  • Bacteremia may follow primary infection w dissemination of MTB to multiple organs

  • Tuberculin skin test turns pos w/in 6 wks post infect

• Primary TB: when in lungs

  • Alveolar macrophages in the lungs phagocytose the MTB and form granulomatous tubercules

    • Walled off areas of lungs containing MTB organisms

    • Walling off is result of immune response

  • Tubercules disintegrate to form a cheese-like mass (caseation–tubercules act on lungs themselves to create holes and masses) in the lungs which then becomes a cavity

  • W adequate cell-mediated immunity (CMI), T-lymphocytes become active w/in 4-6 wks, lesions heal, injured tissue calcifies 

    • w/ inadequate CMI, active TB follows

  • Potential for TB reactivation exists, usually due to breakdown of cellular immune sys as occurs in patients w HIV/AIDS, carcinoma, diabetes

    • Compounding concern for these indivs

• Active TB:

  • Lack of localized containment of infect

  • Liquefaction of the caseous mass (necrotic process leading to gross appearance of the lungs to be cheesy)

  • Microbe number inc and organisms spread (definition of infect)

  • Lesions result

Common symps of each type:

• Active pulm TB:

• Intestinal TB:

• Meningeal TB:—is this transmissible

• Active pulm TB:

  • Fever, chills, night sweats, weight loss, cough, hemoptysis (bloody sputum–highly sugg of TB and of greatest concern)

• Intestinal TB:

  • Lesions in intestinal tract, ulcerations and bleeding, pain and diarrhea

• Meningeal TB:

  • Acute lesions of meninges (when in, not transmissible); usually occurs in children

List three drugs commonly used in the treatment of tuberculosis and discuss multi-drug resistant Mycobacterium tuberculosis.

• sus testing? probs w it?

Treatment:

• Multi-drug strategies are now norm (due to multi-drug rez MTB: MDR TB that has emerged in the last 10 yrs)

• Isoniazid (INH) and rifampin for 6-9 mo

  • Used to be used by itself

• Pyrazinamide (PZA) is added to the treatment regimen during the first 2 mo–usually bc patient has low cell med immune response

• Ethambutol is used in regimen in areas where MTB strains are re to INH or rifampin (4% of TB patients)

• Sus testing rec bc of dev of resistant organisms, however, it takes 3 wks for results to be reported due to slow growth of organism

• Rest, balanced diet, vit supp (esp vit C)

prev MTB

• prophylactic treatment and vacc

• how effective is vacc

• Isoniazid

  • A change in skin test result from neg to pos with no corresponding symptom strongly sugg that indiv has been infected w/ MTB–prophylactic treatment w isoniazid (INH) is rec

    • 1 yr treatment to help patient primarily if in early stage of disease

• BCG vacc: bacillus Calmette-Guerin

  • Vacc against TB prepared from a strain of attenuated bovine tuberculosis bacillus, Mycobacterium bovis, that has lost it virulence (lost virulence factors either genetically or naturally) in humans by being specially subcultured

    • Attenuated strains: live bac with dramatically red virulence

  • BCG vacc is used in some geographic locations; however, is protective effect varies greatly (0-80%)

    • Not reg used in US, but is often given to infants and small children in other countries where TB is common–BCG does not always protect ppl from getting TB

State the mode of M. tuberculosis transmission, source, natural reservoir, frequency of disease and elicited host response.

• host response, source, tranmis (3—one is most common), host defense and 2 tests to test for this

• what type of response is detected in PPD tests

Host response: cell mediated (CMI)--not Ab related (humoral)

Sus pops: very young, old, immunocomp due to lowered CMI response

• Other sus groups: IV drug abusers, those living in overcrowded and/or poor living cond, indv with poor nutrition

source= infected humans

transmission=human to human

• Inhalation of droplets from infected indiv 

  • Sneeze or cough

  • Most common way of infect

• Ingestion

  • Occurs when primary lesions are in the mouth or intestines

• Contact of broken skin with organism

Host defense:

• Hypersensitivity response occurs 6 wks post exposure

• Hypersensitivity is detectable by:

  • Tine test–multiple punctures of PPD

  • Mantoux test–intradermal injection of PPD

• Host defense is cell-mediated immunity indicating requirement for T-lymphocytes (recruitment is part of pos rxn for tests)

Define animals for which M. bovis is virulent.

• how transmis

Causes TB in cows

• Highly virulent to man

• Transmissible to humans via ingestion of milk (rare in US bc of pasteurization)

Clinical infection:

• Associated with TB of the lungs, bone, viscera and GI tract

Distinguish M. bovis from M. tuberculosis from a laboratory diagnostic perspective.

Lab diag M. bovis:

• Distinguish from MTB bc M. bovis is niacin neg (doesn’t turn yellow)

Identify the major source of M. avium-intracellulare.

Mainly contam water via ingestion

Identify and state a high-risk population for developing disease from M. avium-intracellulare.

• treatment

Clinical infect: similar to TB

• Cough, fever, weight loss, night sweats

• Disseminated disease (whole body, not just lungs)

• Most common systemic bac infect in HIV/AIDS patients

Treatment:

• Multidrug regimen–ethambutol, rifamycin, clofazimine, aminoglycoside

• Not always effective, esp w/ AIDS patients

what is primary stain in Ziehl-Neelsen, what is decolorizer and counterstain

carbolfuchsin= prim

acid alc= decolor

methylene blue= counter

AIDS vs non AIDS MAC (Mycobacterium avium complex) disease

A—dissem

NA—pulm infect

which disease incubates 2-7 yrs and has subclinical infects

M. leprae

which form of leprosy is characterized by inc CMI response, nonsuperlative papules, neuorpathy, and loc

tuberculoid

which form of leprosy is nodular, dissem, dec CMI, lesions that open and drain

lepromatous

is leprosy chronic or curable

chronic

how do granulomatous form and in what disease

MTB bc immune sys attempt to wall off

most common TB sample

sputum from lungs (cough)

how rel long does it take MTB to be cultured

wks (2-8)

which bac is cording on an AFB stain a sign of virulence

MTB

why is the tine test a screen only

unprecise ctrl amt TB Ag thus must verify pos tests

what virulence factor causes MTB gramatulous lesions

cord factor

where do MTB survive in body and how do they make worse

phagocytes—>prev phagosome-lysosome fusion

what is case-ation

primary TB tubercles that disintegrate to form cheese like masses

prognosis of MTB primary infect

adequate CMI=heal, calcification of injured tissue

inad CMI= (HIV/AIDS, carcinoma, diabetes) active TB infect and reactivation of disease

what is sign of active MTB

lesions and cheese like lobes appears

who does meningeal TB usually involve

children

what bac does BCG vacc get its attenuated strain from

M. bovis

what is systemic TB in HIV/AIDS patients from (ca)

M. avium-intracellulare complex