Pathogen Identification-Part 1-Stains, Biochemical, Antigen Detection, PCR

Question 1

Microbial Detection in the Clinical Laboratory

Answer 1

Staining; classic means of detection.
Fluorescent antibody
Antigen Detection
Nucleic acid detection (rapid diagnostics); not MALDI; particular genetic marker in an organism.
Culture; gold standard
Appropriate AB therapy early makes a big difference in patients; 50% reduction.

Question 2

Staining Techniques

Answer 2

Wet Mount
Potassium Hydroxide
Gram Stain
Acid-fast Stain
Lactphenol cotton blue
Calcofluor white stain
Trichrome stain
India Ink
Giemsa and Wright-Giemsa Stain
Spore stain (spores in clostridium and Bacillus).

Question 3

Gram Stain

Answer 3

Developed in 1884 by Hans Christian Gram
Used for bacteria only
Most commonly used in microbiology lab to test for organism ID
Purple die that stains cell wall in GP
Hits layer of membrane in GN, fix to wall, iodine stays in GP and doesn’t do anything in GN.
Decolorize with alcohol, GN is not fixed here, washes away=clear, GP stays purple.
Counter stain the safari, stays in GN, GP do not stain with safarin bc have purple.
-GP=purple/blue
-GN=pink/red
Guides initial AB choice.
Basic stain; what does it look like, what is it.
Shape and how they form together.
Don’t need complete culture, can take what is in a sample.
No GP, may not have to use Vancomycin.
Broad spectrum negative drug.
No GN=no augmentin.
Rod vs Cocci?
Formation of cells together in sample?
Size?
Can be performed on any body fluid or tissue as long as organism can be brought out of sample.
Information gained
-Cell wall form
-Shape
-Quantity; 10^5 is the breakpoint that distinguishes infection from contamination.
-Other cells in the sample: WBCs (inflammation), RBCs (blood contamination, difficult to assess white count because blood has WBC and protein in it-difficult to see meningitis), eosinophils, inclusion bodies (things related to infection that may help differentiate what type of pathogen dealing with).
Gram Stain Utilization
-Success based on quality of sample
Quality dependent upon…
1. Minimal contamination from site of collection; decontamination, clean collection.
2. Use of correct tube for collection; preparation and handling.
3. Interpretation of findings; growth of organisms, culture media, preparation of sample.

Question 4

Gram positive bacteria

Answer 4

Staphylococci spp
Streptococci spp
Enterococcus spp
Costridium spp
Corynebacterium spp
Bacillus spp
Lactabacillus spp
Listeria spp
Nocardia spp
Propionibacterium
Mycoplasma
Streptomyces

Question 5

Coagulase Test

Answer 5

Once in culture with gram positives…
Purpose: to differentiate staphylococcus spp
-Coagulase positive: S. aureus; pathogen causes significant morbidity and mortality and cause a lot of drug resistance.
-Coagulase negative: all other Staphylococcus app
Detect clumping factor using rabbit plasma reagent with EDTA.
-Slide test: observe for formation of white clumps
-Tube test: observe for clotting.
Simple and rapid, can do in same day as you get culture.

Question 6

Gram negative bacteria

Answer 6

Enterobacteriaceae
- E coli
-Klebsiella spp
-Proteus spp
-Salmonella spp
-Serratia spp
-Shigella spp
-Citrobacter spp
-Morganella spp
Non-enterobacteriaceae
-Pseudomonas spp
-Aceinetobacter spp
-Strenotrophomonas maltophilia
-Berkholderia spp
All of these gram stain

Question 7

Acid-fast Stain

Answer 7

For bacteria resistant to traditional Gram-staining
-Bacteria resistant to Gram stain due to thin PTG and large amounts of mycolic acid.
GP organisms and have a hard time taking up stain.
Slow growing so difficult to ID in culture.
Detects Mycobacterium spp (TB) and Nocardia spp (GP organism)
Acid-fast stain produces makes bacteria resistant to decolorization and retain carbol fochsin (red); all other cells will decolorize and counterstain with methylene blue.
Low sensitivity: requires > 5000 cells/ml; does not require a lot of cells to detect.
Stain as a ghost gram stain and non stainable organism.

Question 8

Lactophenol cotton blue

Answer 8

Basic mounting media for fungi consisting of phenol, lactic acid, glycerol, and aniline (cotton) blue dye.
Organisms are killed due to the presence of phenol.
High concentrations of the phenol deactivates lytic cellular enzymes so cells do not lyse.
Cotton blue is an acid dye that stains the chitin present in the cell walls of fungi.
Fungal or bacteria infection?
Quickly
Does not differentiate within fungal species.

Question 9

Calcofluor white stain

Answer 9

Non-specific fluorochromes that bind beta1,3 and beta 1,4 polysaccharides (cellulose and chitin) of cell walls in fungi.
Rapid detection of fungal specimens
Fungal elements appear blush white against a dark background under UV
Nonspecific.
More specifically for fungal disease.
Doesn’t differentiate fungal pathogen.
More rapid than cotton blue.
UV light background; clinical lab sees if potential pathogen in culture.
Differentiates between fungus and bacteria.

Question 10

India Ink (Colloidal Carbon)

Answer 10

Used to detect Cryptococcus neoformans (involved in patients with immunocompromised states, difficult to detect by traditional culturing, trouble growing) and other encapsulated fungi:
CSF commonly tested
Low sensitivity vs cryptococcal antigen
Background is stained that makes any capsule around the organisms visible as a halo.
Rapid test by stain.

Question 11

Potassium Hydroxide (KOH)

Answer 11

Stain used for fungal pathogens.
Purpose: differential diagnosis of infections produced by dermatophytes and candida albicans from other skin disorders.
KOH mixed with blue-black dye and added to tissue sample.
Spaghetti and meatball appearance=dermatophyte.
Any type of tissue sample; quite rapid.
Filamented picture along with organism; organism is spaghetti.

Question 12

Giemsa and Wright-Giemsa stains

Answer 12

Differential stain for detection of bacteria (Borrelia, Enrlichia, Anaplasma, Chlamydia), fungi (toxoplasma, pneumocystis), and parasites (microfilariae, babesia, plasmodium falciparum) in blood.
Detection of intracellular pathogens; into RBC, have to be stained inside RBC for detection.
Classic means of detecting malaria in patients.
Positive or negative; based also on presentation.
Tick born diseases; helps with patient background to say that this is what the patient might have.
Insensitive, but a positive stain is rapid confirmation of organisms presence.
Doesn’t give you a lot of level of detail.

Question 13

Fluorescent Antibody Techniques

Answer 13

General Procedure:
1. Take sample from patient tissue
2. Combine immunoglobulin (commercially available) and due with patient sample.
3. Incubate to allow dye to attach to immunoglobulin
4. Complex attaches to microbe and visualized under fluorescent light.
Is the patient producing an antibody to this particular pathogen or not?
Directed towards particular pathogens.

Question 14

Fluorescent stains

Answer 14

In theory, this should increase sensitivity and specificity.
You would think this would work very well and be very specific.
Due to technical and interpretation difficulties, few are used clinically.
-Pneumocystis jiroveci
-Legionella spp
-Cryptosporidium spp
-HSV, VZV (varicella), CMV
Fluorscent antibody stains are more commonly used for viruses rather than bacteria.
Bacterial identification has often been replaced with antigen tests in most labs.

Question 15

Antigen Detection

Answer 15

Antigens are organism products that can be detected when organisms are in insufficient quantity for culture: toxin or organism surface protein.
Antigen specific for that pathogen; number that are clinically available, detectable assays, only use a couple in clinical
Bacteria, virus, fungi, parasites.

Question 16

Antigen detection common uses in specific fluids

Answer 16

CSF antibody tests
-Streptococcus pneumoniae
-Neisseria meningitidis
-Haemophilus influenzae
-Streptococcus pyognes
-Cryptococcus neoformans (only fungus)
Blood
-Cryptococcus neoformans
-Malaria
-Viruses
Urine
-Histoplasma capsulatum (regional mold)
-Legionella pneumophilia
Cultures that have difficulty ID organism otherwise.

Question 17

Serology

Answer 17

Confirms infection by measuring antibody titers in patients that is consistent with the pathogen of interest.
Useful for identifying microbes that are difficult to detect otherwise.
Limitation: immunocompromised patients may not mount an immune response.
If can’t culture or reside in such low quantities in a specific site-cannot get to.
Bacteria can do this for:
-beta hemolytic strep
-Strep pneumoniae
-Neisseria meningitidis
-Haemophilus influenzae
-E coli; O157:H7 of interest in food born disease.
-Salmonella enterica
-Shigella spp
-Legionella pneumophila

Question 18

Galactomannan antigen detection

Answer 18

Galactomannan is a polysaccharide cell wall component that is released by Aspergillus (invasive mold that causes infection in immunocompromised patients, severe, advanced chemo/cancer, severe HIV) during growth.
ELISA approved for surveillance for invasive Aspergillosis.
Serum galactomannan can be detected 7-14 days before other diagnostic clues become apparent.
8-14% false positive rate; food, benign fungi, and mold, semi-synthetic beta lactate antibiotics; derived from fungus penicillium, derived somewhat from original mold leads to false positive rate.
Natural pencilin would not have as high of false positive rate because no derived from fungus penicillium, still a risk; would avoid, don’t get the test.
Specific for Aspergillus; a lot of resistant, candida not; good to differentiate between this and more of a mold infection.
Difficult to culture or detect; not enough organism clinically detectable until advanced; nice way to ID relatively early.

Question 19

Standard Culture

Answer 19

Traditional means of detecting organisms
Gold standard; sometimes have to do this to grow the organism, then take it out of culture and ID it, test for susceptibility testing.
Take a longer time.
Rely on as a truly identifiable test.
Automated techniques
Culture media and agar
-Non-selective media: supports growth of most bacteria.
-Selective media: contains inhibitory substances or antimicrobials which allow growth of some organisms and suppress growth of others.
-Differential media: contain substances which allow detection of organism characteristics; detection my be color change or growth patterns.
-Broth media: to enhance growth of difficult or small organism samples.

Question 20

Immunological and Molecule Methods for Pathogen Detection: THE FUTURE OF IDENTIFICATION IS HERE!

Answer 20

-Difficult to culture organism using traditional method of where rapid detection is necessary.
-Antibody/Antigen Detection: HIV, group A streptococci etc.
-Molecular techniques: RNA and DNA amplification techniques; specific DNA or RNA sequence (probe) which “hybridizes” to a complementary nucleotide of the organism,
Probe emits a signal (radiolabeled or chemoluminescent).
Direct PCR testing of virus, bacteria, fungi.
Particular for a pathogen of interest.
Faster
Difficult to culture or take a long time you use this for

Question 21

DNA and RNA Signal Amplification

Answer 21

Using a probe to attach to certain area of interest in pathogen
Attaches to area and us fluorescent microscope or light to see that it glows under fluorescence; like DNA.
Probe can be complicated

Question 22

Immunological and Molecule Methods for Pathogen ID

Answer 22

Nucleic Acid Amplification: PCR; produces copies of low levels of organism DNA or RNA without the use of probes.
DNA primers (excess) matching sequence areas of organism to be amplified are added to sample along with DNA polymerase.
-Cycle of heating and coiling separates DNA
-Heat stable DNA polymerase elongates strand allowing for copy to be made.
-Signals and is detected in large quantities; specific for organisms.
-Particular primers have to be developed.
-Rapid and can be done with pretty much any culture.

Question 23

FDA-cleared methods for bacteria and yeast

Answer 23

PCR- GP (including s. aureus/MRSA-susceptible or resistant to beta lactams, MecA element (beta lactam resistance) amplified) and GN.
Time to results: 1-2 h
PNA FISH; fluorescent probe-GP, GN, yeast; able to speciate down; aspergillum, candida and other organisms in culture.
Time to results: 1-2 h
Bacteriophage: S aureus, including MRSA; differentiates MRSA from MSSA.
Time to results: 5 h
For other organisms, MALDI replaces this; MALDI does not really recognize resistance; can do at same rate and see many other organisms.
Fast

Question 24

PCR

Answer 24

Amplification and detection of a target sequence using 2 flanking primers and a fluorescnetly labeled probe.

Question 25

PNA FISH

Answer 25

Hybridization of fluorescently labeled peptide nucleic acid DNA analogue probes to rRNA in intact bacteria and subsequent detection by fluorescent microscopy.

Question 26

Bacteriophage

Answer 26

Bacterial species-specific phage amplification as a surrogate marker for bacterial viability.

Question 27

Challenges with Rapid Microbial Identification Tests

Answer 27

• Costs for equipment and per test.
• Cost savings due to rapid identification of organisms is realized outside of he microbiology lab; reduced length of stay and pharmacy costs.
• Technical complexity; clinical lab improvement amendments (CLIA) certified complexity of the test and personnel skills; technician time.
• Lab space requirements
• Sensitivity and Specificity; work really well with particular pathogens of interest, but with new resistance mechanisms; hundreds of beta lactamases, not sensitive to detect infrequent or new ones.
• Ability to document impact on antibiotic selection and patient outcomes
• Antimicrobial stewardship programs can be the best advocate for justifying and implementing new diagnostic tests.

Question 28

Higher all-cause morality and length of stay with inappropriate empirical antibiotic treatment

Answer 28

Innappropriate (20%, appropriate therapy is 11%) empirical antibiotic treatment was associated with prolonged hospital stay (p=.002)
What can we do to do this faster?
-Mean duration of shoptalk stay was more than 2 days longer in patients who received inappropriate empirical antibiotic treatment.

Question 29

ID and Susceptibility Determination by rapid molecular ID allows earlier initiation of optimal therapy

Answer 29

Traditionally,
At hour 0, take sample, lab gram stain, then culture, ID what is pathogen specifically (1 day later, 18-48 hours, culture results), then do AB susceptibility testing require at least another day of growth (another 24-48 hours); 96 hours window.
All depends on how fast the organism grows in culture.
Rapid testing,
Skin sample and want to know if there is MRSA or MSSA in the culture, do quick test and within 1-5 hours you would know what the pathogen is.
Reality, not often 1-5 hours; often batched together for work flow.
Done within 12 hour period.
Knowing sensitivity within 96 hours to maybe within 6 hours.
Blood cultures are different.
Need to grow in vials (7-48 h), once this you can ID pathogen quickly; 18-48 h for culture results and then an additional 1-5 h for ID and susceptibility testing.
Not getting AB on board quickly is not good especially in s aureus; need a test and a pharmacist and apply to the patient.
60 hours for intervention (without clinical intervention)
With clinical intervention, took 39 hours, almost a day earlier.
Lab to patient.

Question 30

Identification of yeast from positive blood cultures with PNA FISH

Answer 30

Differentiate different candida species.
Other drugs work against different candidas.
Probe that binds to he yeast cell, if candida albicans and/or parapsilosis, they stain green; if tropical they stain yellow, if glabrata and/or krusei they stain red.
90 min
Treat different organisms with different anti fungals.
Rapid ID and also specific ID to direct therapy.

Question 31

Organism ID and the invitation of Targeted AB Therapy

Answer 31

Helps get AB on board, but allows to de escalate very quickly.
Usual, broad spectrum approach, should do bc need to get the right AB quickly.
BUT don’t want to do this for too long, if they don’t need a drug, narrow down to something more specific.
Rapid test can minimize window and narrow spectrum quickly.
Old way:
Up to 3 days of empiric and broad spectrum therapy and then having culture result.
New way:
Quick ID: tale off broad spectrum and narrow quickly, within first day of empiric treatment you narrow down; prevents long term resistance to AB.
COST OF MALDI IS GOING DOWN AS EFFICIENCY IMPROVES; HALF COST.
A lot of inappropriate use of AB in community setting.