Micro Exam 8 Review

Ziehl-Neelsen (ZN) Acid Fast Stain

• Basic stain: Carbolfuchsin

• Decolorizer: 3% Acid-Alcohol

• Counterstain: Methylene blue

• Procedure: Heating required (Mycobacteria are stained RED and the background LIGHT BLUE)

Kinyoun Acid Fast Stain

• Basic stain: Carbolfuchsin

• Decolorizer: 3% Acid-Alcohol

• Counterstain: Methylene blue

• Procedure: Cold procedure; no heating required (Mycobacteria are stained RED and the background LIGHT BLUE)

Auramine Fluorochrome Acid Fast Stain

• Basic stain: Phenolic Auramine

• Decolorizer: 0.5% Acid-Alcohol

• Counterstain: Potassium permanganate

• Procedure: No heating required (Mycobacteria are stained YELLOWISH-ORANGE against a dark background)

How are the ZN and Kinyoun stains similar?

Both are carbolfuchsin stains

What dye is taken up in the Kinyoun stain?

Carbolfuchsin

What color are the mycobacteria on a carbolfuchsin stain?

Red against a blue or green background

What is the decolorizer in the acid-fast stain?

Acid alcohol

What is the advantage of the fluorescent stain (auramine flurochrome)?

Faster to read

When around mycobacteria, what should concern you as a lab worker?

* Risk of aerosols
* Work in a BSC, BSL3

What has to be added to gastric aspirates if the specimen will not be processed quickly?

Sodium carbonate to neutralize the sample since gastric acid kills AFB

When is the best time to collect sputum samples?

Shortly after the patient awakens in the morning; when mycobacteria are in the highest concentration to maximize the chances of mycobacteria recovery

What type of urine specimen should be collected?

First morning, midstream collected on 3 consecutive days to maximize the chances of AFB recovery

What specimens do not require digestion/decontamination?

* Sterile specimen – no normal flora present
* CSF
* Bone Marrow
* Blood
* Biopsied Tissue

What is the function of NALC in the digestion/decontamination process?

* It liquefies mucus
* splits disulfide bonds

What is the function of the NaOH in the digestion/decontamination process?

It is a decontaminant that kills any normal flora that may be present in the specimen

A first morning sputum specimen is received for AFB cx. The specimen is centrifuged and the sediment is inoculated on 2 LJ slants, which are incubated at 35 degrees C in 5-10% CO2. After 1 week, the slants show abundant growth over the entire surface. Stains reveal Gram negative bacilli. To avoid this problem:

Decontaminate the specimen with sodium hydroxide

Recovery of M. avium-intracellulare from stool specimens

• First prepare a direct smear and stain for acid-fast bacilli

• If negative for AFB = specimen is not processed further

• If positive for AFB = proceed with inoculation into appropriate media after due digestion-decontamination

What is the significance of detecting AFB in fecal samples?

The recovery of M. avium-intracellulare from stool specimens is predictive of disseminated disease.

Mycobacteria Media & Incubation

• Selective & non-selective Mycobacteria media available

  • Malachite green

• Recommended to have a solid media and liquid media for AFB recovery in culture

  • AFB grows quicker in liquid media than in solid media

What automated methods are there for AFB cx’s?

• MGIT Tubes

• BacT/ALERT

• VersaTREK

What rapid methods are available for identifying mycobacteria?

• High-Performance Liquid Chromatography (HPLC)

• Nucleic acid probes

• Nucleic acid amplification test

• Mass spectrometry

M. tuberculosis

* ==Grows best at 37°C==
* Poorly or not at all at 30°C or 42°C

M. ulcerans, M. marinum, M. haemophilum

• Infect the skin

• Grow best at the temperature of the skin (30-32°C)

  • Incubator set at 30°C be used for all specimens from suspected skin or subcutaneous mycobacterial infections

M. xenopi

* ==Grows best at 42°C==
* An incubator set at 42°C can be helpful in recovering M. xenopi
* Hot water taps, including water storage tanks and hot water generators of hospitals , are potential sources for nosocomial infections

Mycobacteria isolated from the hot-water system of a down-town hospital grew at  42°C. Colonies on Lowenstein-Jensen medium were not pigmented after exposure to light and negative for niacin accumulation and nitrate reduction. The most likely identification is:

M. xenopi

M. xenopi

* Often recovered from hot-water taps and contaminated water systems and is a possible source of nosocomial infection

M. marinum, M. ulcerans, M. haemophilum

• Cause skin infections

• Grow on media below 42°C.

Niacin

• Most mycobacteria possess the enzyme that converts free niacin  to niacin ribonucleotide

• 95% of M. tuberculosis produce free niacin (nicotinic acid) but lack the enzyme

  • Niacin accumulates in the medium and is detected as nicotinic acid (YELLOW color)

• Most commonly used biochemical test for M. tuberculosis.

All of the following Mycobacterium spp. are capable of producing the enzyme required to convert niacin to niacin ribonucleotide except:

M. tuberculosis

All of the following Mycobacterium spp. are capable of producing the enzyme required to convert niacin to niacin ribonucleotide except:

• M. tuberculosis

• All mycobacteria are capable of producing niacin. However, not all of them possess the enzyme necessary for converting niacin into niacin ribonucleotide. M. tuberculosis lacks this enzyme and so niacin accumulates in the culture medium to give a +ve test.  M. simiae and some strains of M. marinum, M. cholenae and M. bovis also lack this enzyme.

Nitrate reduction test

• M. tuberculosis: Nitrate POSITIVE (RED color)

• To confirm a true negative, zinc is added to detect nitrate which results in a PINK color change.

Heat-stable Catalase test

• Not all mycobacteria have heat-stable catalase

  • Heat to 68°C or 20 minutes and look for bubbles

• M. tuberculosis and some other members of its complex are incapable of producing heat-stable catalase.

  • Why this test is done? To confirm it is not M. tuberculosis

Photochromogens

• YELLOW pigment when exposed to light after being grown in the dark

• M. kansasii, M. marinum

Scotochromogens

• YELLOW pigment when grown in light or dark.

• M. gordonae, M. scrofulaceum

Nonphotochromogens

• NO pigment produced in light or dark.

• M. ulcerans, M. avium/intracellulare complex

Rapid Growers

• Colonies appear on solid media in less than 7 days

• M. Fortuitum, M. chelonae

Mycobacteria that produce pigment only after exposure to light are classified as:

Photochromogens

Which of the following mycobacteria produces pigmented colonies in the dark (i.e. is a scotochromogen)?

M. szulgai

M. szulgai

A scotochromogen

M. kansasii

A photochromogen

M. tuberculosis

does NOT produce pigmentation in the dark or after exposure to light

M. scrofulaceum and X. xenopi

Pathogenic scotochromogens

M. gordonae

A common tap water scotochromogen

The growth inhibition by thiophene-2-carboxylic hydrazide (T2H) is used to differentiate M. tuberculosis from which other Mycobacterium spp.?

M. bovis

M. tuberculosis

Not inhibited by T2H

M. bovis

Inhibited by T2H

Which bacteria are included in the M. tuberculosis complex?

• M. tuberculosis

• M. bovis

• M. africanum

M. gordonae

Common contaminant of tap water

M. marinum

Contracted in swimming pools

M. tuberculosis colonies on solid media

• Colonies resemble bread crumbs: buff, rough, and dry

• Growth is described as eugenic (luxurious).

What are the main identifying characteristics of M. tuberculosis?

• Slow growing (14-28 days)

• Nonpigmented

• Niacin POSITIVE

• Nitrate POSITIVE

• 68°C catalase NEGATIVE

Which is the single best for the biochemical identification of M. tuberculosis?

Niacin test

PPD Skin test

• This is a skin test (Mantoux test) used to determine if an individual has developed some immune response to tuberculosis

  • Purified protein derivative (PPD) injected into the top layers of the forearm and read 48-72 h post injection

  • Appearance or absence of an induration is noted

• A positive skin test = recent or past exposure to TB

• Those who have received a BCG vaccine in the past may also have a positive skin test. A false positive can occur in this situation

  • made from attenuated strains of Mycobacterium bovis

QuantiFERON-TB Gold (QFT) test

Used for diagnosing latent tuberculosis infection as well as active TB. Test has a much higher sensitivity than the tuberculin skin test

QuantiFERON-TB Gold (QFT) test

* How does it work?
* Based on the measurement of the amount of interferon-gamma (IFN-λ) secreted by those T-cells that had been previously exposed to Mycobacterium tuberculosis. It uses a triad of M. tuberculosis specific antigens (ESAT-6, CFP-10 & TB 7.7.6). These antigens are lacking in all BCG vaccine strains and absent from most environmental mycobacteria(except in M. marinum, M. kansisii and M. szulgai). When these T cells are stimulated by these antigens, they secrete IFN-λ.

M. tuberculosis

Human tuberculosis

M. bovis

Bovine & human tuberculosis

M. ulcerans and M. marinum

associated with skin infections

M. africanum

Between M. tuberculosis and M. bovis

M. szulgai

Chronic pulmonary and extra pulmonary disease

M. avium/intracellulare complex

Associated with AIDS patients

M. haemophilum

associated with skin lesions in immuno-suppressed patients

All of the following Mycobacteria are associated with skin infections except:

M. kansasii

M. kansasii

* a photochromogen that causes chronic pulmonary disease (classic tuberculosis)

M. marinum, M. haemophilum, M. ulcerans

associated with skin infections

M. fortuitum, M. chelonae, M. abscessus, M. mucogenicum  

• Rapid Growers

• Belong to Runyon group IV

• Growth in less than 7 days (an unknown acid-fast isolate can be suspected of belonging to this group if growth is observed after 2-4 days incubation)

• Associated with skin and lung infection. May cause nosocomial infection

• M. fortuitum (Nitrate +ve) – distinguishing it from the other rapid growers

• M. fortuitum, M. abscessus and M. chelonae = Can grow at 28°C on special MacConkey agar(devoid of crystal violet).

Which of the following Mycobacterium spp. are used as controls for rapid growers and slow growers?

M. fortuitum and M. tuberculosis

M. fortuitum

• Grows within 3-5 days at 37°C

• Used as the control for rapid growers

M. tuberculosis

• Grows in 12-25 days at 37°C

• Control organism for slow growers

Mycobacterium leprae

Causative agent of leprosy

To which Runyon’s group does M. leprae belong?  

None

How is leprosy diagnosed?  

• AFB smears of skin ulcers & skin biopsy

• Cannot be grown on artificial culture media

A primary drug used for the treatment of M. tuberculosis is:

Rifampin

What is MDR-TB?

• Multidrug-resistant TB

• Resistant to INH, rifampin

What is XDR-TB?

• Extensively drug-resistant TB

• Resistant to INH, rifampin, any fluoroquinolone, and at least one of 3 injectable second-line drugs (amikacin, kanamycin, capreomycin).

Virus specimen transport - Temperature

• Kept at 4 degrees, freezer only at -70C

• Never at 20C

Viral transport media - what is in it?

VTM – buffered isotonic solution with proteins and antimicrobials

What is the optimum time to collect specimens for viral studies?

Within the first 2-3 days of illness

Coxsackieviruses A

* Hand, foot, and mouth disease
* Billae on hands and feet
* Maculopapular rash on hands, feet and buttocks

Rubeola

* Causes measles
* Koplik’s spots and rash on head and trunk

Patient X has unexplained meningitis and encephalitis. Patient X recently went on a hike and was bitten by a mosquito. What flavivirus is most likely the cause of patient X’s symptoms?

West Nile Virus

Naked or Non-enveloped viruses

• Much more virulent & can cause host cell lysis. E.g. Parvovirus

• The capsid is the outermost covering and is made of proteins

• Resistant to drying, acids & heat; can retain its infectivity even after drying

• Capable of surviving inside the gastrointestinal tract (GIT)

• Transmitted via dust, fecal/oral matter and formites.

Enveloped viruses

• Less virulent & seldom cause host cell lysis. E.g. Herpes simplex virus

• The envelope consists of phospholipids, proteins or glycoprotein

• Sensitive to drying, acids and heat; often lose their infectivity on drying.

• Generally cannot survive inside the GIT

• Transmitted via blood, secretions, lesions or organ transplants.

Sequence of viral replication

Attachment or Adsorption > Penetration > Uncoating > Assembly or Maturation > Release

Viral Replication - Attachment

Specific for cell receptors

Viral Replication - Penetration

• Direct penetration of the membrane (naked viruses)

• Envelope-membrane fusion (enveloped viruses)

• Endocytosis: enter cell in a cytoplasmic vacoule

Viral Replication - Uncoating

* Release the genome from capsid
* RNA in cytoplasm
* DNA in the nucleus

Viral Replication - Replication

Viral genome directs host cell to make viral proteins and replicate viral genome

Viral Replication - Assembly or maturation

Viruses associate to form virions

Viral Replication - Release

• Lysis (naked viruses)

• Budding (enveloped viruses)

What is a virion?

The virus itself

Naked virions

Can penetrate the host cell membrane directly

Uncoating

During the process of viral replication, the stage in which the virus loses its protein coat once inside the cell

During the release stage:

the new virions are released by lysis if they are naked viruses or by budding if they are enveloped viruses.  

HSV

Known to grow rapidly on many different cell lines and to frequently produce CPE within 24 hours.

RSV

Produce syncytia or giant multinucleated cells in human laryngeal carcinoma cell lines

RNA viruses

• Influenza A

• Parainfluenza

• Mumps

• Measles

• Respiratory syncytial virus

• Rotavirus

• Rabies

• HIV

How are retroviruses unique?

They possess a reverse transcriptase that allows the viral RNA genome to be replicated into DNA, rather than RNA

How are influenza viruses unique?

• They alter their genetic composition so new vaccines must be formulated each year

• Antigenic drift vs antigenic shift

RSV

CPE: Syncytia