Microbiology & Immunology munson content

Midstream (clean-catch) steps

1. clean outer urethra with antiseptic, females clean labial area

2. first portion voided into toilet (get rid of flora of urethra)

3. midway through urination, collect urine (sampling the bladder)

when do you want to collect a specimen for sampling?

collect during acute phase (while infection is happening), before antimicrobial treatment

3 main methods for identification

1. direct detection

2. routine culture

3. pre-emptive screening

direct detection- what is it, what are 4 main examples of it?

detecting organism straight from a sample, without growing it!

1. antigen detection of the invader (ex. strep or cryptococcus)

2. serology- detecting antibodies from the patient's own immune system (ex. viruses and vector-borne illness)

3. primary stains (ex. fluorescence microscopy and gram stains

4. molecular diagnostics (ex. DNA or RNA)

what is antigen testing? what is the main problem with it?

detecting the invader's antigens (proteins)

- problem: some have LOW SENSITIVITY- higher number of false negatives!

real-time PCR

newly made DNA is tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle

(machine "watches" the DNA being copied)

- used to ID invaders or to ID certain genes/ mutations in patients

advantages of real time PCR

1. fast turnaround time

2. sensitive- finds very small amounts

3. specific- correctly identifies organism

3. can be quantitative- qPCR detects how much of invader DNA is present

4. less prone to contamination (everything is in the same tube)

5. more compatible with automation- the machine does it for you (less human error, etc)

what are some clinical applications of real-time PCR?

1. healthcare-associated infections (c. diff, MRSA screening)

2. women's health (strep, HPV, bacterial vaginosis)

3. STIs (gonorrhea, chlamydia, etc.)

4. Virology and pertussis (respiratory viruses, immunocompromised)

Lumbar puncture, how many tubes do you collect? where do these go after collection?

collecting CSF from between lumbar vertebrae, most commonly used when meningitis is suspected

- aseptic procedure

- three to four tubes collected (tube 1 might have skin contaminants)

- always STAT and very valuable (straight to lab)!!

- same remnants for esoteric testing

bacterial vs viral meningitis: why is it important to determine, and what are the main indicators you use

bacterial will be treated with antibiotics, viral won't

look at leukocytes, glucose, protein, direct smear and culture

bacterial vs viral meningitis: which is more common?

viral: higher prevalence in US (30,000-50,000 cases vs 4,000 for bacterial)

bacterial meningitis:

primary leukocytes? glucose? protein? direct smear and culture?

- neutrophils dominate!! (innate immune response)

- low glucose!! (bacteria eats that shii)

- high proteins

- usually positive direct smear and culture

viral meningitis: primary leukocytes? glucose? protein? direct smear and culture?

- lymphocytes dominate!! (adaptive immune response)

- normal glucose!! (viruses do NOT eat that shii)

- slightly elevated proteins

- negative direct smear and culture

for a primary sputum screening (mucous coughed up), would you want high or low epithelial cell count, and high or low specimen count?

- want low epithelial cell count (high would mean more contamination from mouth)

- want high specimens

why would a sputum be rejected?

predominance of squamous epithelial cells and low inflammatory cells likely means contamination from oropharynx

Kirby-Bauer Test

The test to determine the efficacy of antibiotics or the antibiotic resistance of bacteria (SUSCEPTIBILITY TESTING)

- also called disc diffusion test

- used when there is a clinically-significant infection, failure of empiric therapy (treating without knowing the organism), or desire to convert to directed therapy (knowing the organism)

when would you NOT use susceptibility testing?

- normal flora

- colonization (organism is present but not causing problems/ symptoms)

- polymicrobial infections (can treat empirically)

- known organism/ drug combos

- self-limiting infections (ex. salmonella will go away by itself)

bigger zone of inhibition means...

bacteria is more susceptible to antibiotic

smaller/ no zone of inhibition means...

bacteria is resistant to antibiotic

empirical therapy

The administration of antibiotics according to a patient's symptoms and one's understanding of infectious disease before the pathogen has been identified; used in life-threatening situations, such as meningitis.

WANT TO TREAT FAST

D-test

Detect a specific type of antibiotic resistance called inducible clindamycin resistance.

- Most commonly used for: Staphylococcus aureus, especially MRSA

1. put erythromycin and clindamycin on plate

2. if bacteria looks susceptible to clindamycin (has big zone of inhibition), repeat with erth. and clinda. closer together

3. if clindamycin has a zone of inhibition with a flat edge near erth. (the zone looks like a D), then INDUCIBLE CLINDAMYCIN RESISTANCE IS PRESENT, DO NOT TREAT WITH CLINDAMYCIN

(actually have to report staph. inducible resistance)

what does cefoxitin resistance mean for staphylococcus aureus?

NEED TO TEST FOR MRSA

what does it mean if there's mixed gram positive and gram negative flora in a culture?

probably contamination

For testing for pathogens in stool cultures, what do we do nowadays? "new school?" (vs what was "old school"?)

nowadays, we use PCR for pathogens, C. diff, etc

- old school- culture or EIA (enzyme immunoassay)

how do you collect blood cultures? do you take before, during, or after fever spikes?

- collect 2-3 bottles

- one aerobic, one anaerobic

- collect from 2 different locations (ex. one from R arm one from L) to increase chances of finding bacteria

- IF YOU KNOW WHEN FEVER HITS (ex. every 6 hours or so), TAKE SAMPLES 30-45 MINUTES BEFORE NEXT SPIKE-> this is when bacteria are highest, before your body has an immune response (fever)!

what is an antibiogram? how would you use it as a PA?

a hospital's/ community's "map" of which antibiotics usually work against which bacteria

- GUIDES EMPIRIC THERAPY SO YOU CAN TREAT BEFORE WAITING FOR CULTURE/ SUSCEPTIBILITY RESULTS

urine reflex

testing strategy where the lab first performs a urinalysis (UA) and only performs a urine culture if the UA suggests infection

signs from a UA that prompt a urine culture

might have a UTI/ need a urine culture with:

- high nitrites (bacteria in urine convert nitrates to nitrites)

- positive for leukocytes (pyuria) (WBCs in urine indicates infection, inflammation, contamination, or UTI)

- AND SYMPTOMS OF UTI/ PROBLEM

most common cause of UTI?

E. coli

asymptomatic bacteriuria

Presence of bacteria in urine without symptoms.

- do not order culture

where do you collect specimens for anaerobes from?

ALL DEEP SOURCES: abscess, body fluids, drainage, deep wounds (less O2 down there)

why would you use respiratory acid-fast bacilli test? what is it?

test for TB bacteria in lungs

1. acid-fast smear- look for acid-fast bacilli on smear, if positive, go to PCR

2. culture and report negative at 8 weeks

3. positive cultures get sent to Wisconsin State Lab of Hygiene (WSLH)

what happens when you inhale a droplet nuclei of TB? what step is latent TB?

1. engulfed by macrophages, which release cytokine interferon gamma

2. formation of granuloma (epithelial and other cells surround it) LATENT TB- 1/3 of people have this! 10% risk of it becoming active by:

3. caseation, invasion of bronchus

4. clinical symptoms, transmission

ways to test for TB

- acid-fast bacilli test

- PPD (skin test)- low specificity

- interferon-gamma release assays (IGRA)- high specificity (blood)

Ova & Parasites test

a stool test used to look for parasite eggs (ova), larvae, and adult parasites that may be causing gastrointestinal infection

most common parasites found in stool in the US

Giardia

Cryptosporidium (highest case ever in MKE!)

*if travel to other countries, need to consider international parasites!

how do you test for viruses? (sample type, conditions to grow)

- aspirates (fluid taken with needle) or tissue preferred sample, blood and urine also used

- keep them COLD!

- inoculate into host cells

- convert to molecular diagnostics

high prevalence means ___(high or low)___ PPV

high positive predictive value (out of the positive tests, more of them are actually positive)

low prevalence means ___(high or low)___ PPV

low PPV (out of the positive tests, less of them are actually positive)

innate immunity, what are the 2 types?

defenses against any pathogen; rapid, present at birth

- external defense

- internal defense

external defense

- physical barriers (skin, mucous membranes)

- mechanical barriers (coughing, urination)

- chemical barriers (lactic acid, HCl)

- competition for binding sites

internal defense

- cellular

- pathogen recognition receptors

- negative acute phase reactants

- positive acute phase reactants

myeloid cells

involved in innate immunity.

neutrophils, basophils, eosinophils, monocytes

neutrophils

Most abundant white blood cell

- phagocytosis

- antimicrobial and oxidative granules

eosinophils

kill larger parasites that cannot be phagocytized by neutrophils

basophils

granulocyte (has granules), makes histamine and present in allergic reactions

monocyte

phagocytosis

- can act as antigen presenting cell-> activates adaptive immune response (brings T cells over!)

macrophage

large phagocyte

- monocyte but in tissues

- ex. microglial in brain, osteoclasts in bone

- phagocytosis and antigen presenting cell!

mast cell

like big basophils but bigger and with more granules

- plays role in allergies

- adaptive immune response

- in skin, CT, mucosal epithelium

dendritic cell

"best eater, best giver"

- MOST EFFECTIVE PHAGOCYTIC CELL, MOST EFFECTIVE APC

- liver, lung, brain, bone, CT

ANCA- associated vasculitides, what main disease is associated with this?

ANCA = antibodies that mistakenly target neutrophils → neutrophils damage small blood vessels → vasculitis

- Wegener's granulomatosis is a type (aka GPA)

Wegener's granulomatosis/ Granulomatosis with polyangiitis (GPA)

- type of ANCA- associated vasculitides

- affects noses, sinuses, then lungs, then kidneys, hard to distinguish with TB

- The body makes an abnormal antibody called c-ANCA.

This antibody targets PR3 on neutrophils.

The neutrophils become activated and damage nearby blood vessels.

-90% die in a year if untreated!

negative acute phase reactants

antithrombin, albumin, transferrin

substances in the blood that decrease during inflammation/ innate immune response

positive acute phase reactants

C-reactive protein, procalcitonin, complement

- increase rapidly with infection/ injury/ trauma to tissue

C-Reactive Protein I (CRP)

most widely used marker for acute inflammation

- increases dramatically, peaks in first 48 hours

- declines rapidly with cessation of stimulus

CRP II

can detect very low levels of CRP, USED TO PREDICT CV RISK with infection/ inflammation

- chronic inflammation can cause serum CRP has now reached endothelial cells lining vessels, which can cause:

- vasoconstriction, platelet activation, thrombosis

high CRP means...

Active inflammation in the body

low CRP means...

no inflammation in body/ little inflammation

procalcitonin

used to detect BACTERIAL INFECTION

- ex. used to determine origin of pneumonia- could be bacterial or viral

high procalcitonin means...

bacterial infection

low procalcitonin means...

little evidence of bacterial infection

complement

synthesized in liver (mostly), adipose, macrophages

- DESTROYS AND CLEARS FOREIGN ANTIGEN

- pro-inflammatory function

- linked to coagulation system, anaphylaxis

- IN GENERAL, INCREASES WITH IMMUNE RESPONSE

- HOWEVER, CAN BE USED UP AND LOW WITH immune system activation/consumption (ex. with SLE (lupus)!)

low complement means...

autoimmune- lupus

high complement means...

acute inflammation/ infection (immune system is making more complement)

t-lymphocyte

- precursors in bone marrow

- made in thymus

- found in blood and lymph nodes

- makes cytokines- helps tell system there's an infection

- include clusters of determination- "ID badges": (all t cells are CD2 & CD3; differ by either CD4 or CD8)

CD4 Absolute Count, what is it and what is it used to monitor?

CD4= found on helper T cells

Job: coordinate the immune response

- USED TO MONITOR HIV

low CD4 means...

HIV/ AIDS

(HIV invades and destroys CD4 cells)

high CD4 means...

infection/ inflammation

B-lymphocyte

- precursors in bone marrow, develop there too (B cells= BONE, T cells= THYMUS)

- found in bone marrow, spleen, lymph nodes

- makes ANTIBODIES (immunoglobins/ Ig) that recognize a specific antigen (virus, bacteria, toxin, etc.), bind to it, and

help the immune system destroy it

- C19, CD20, C21, CD40

IgG

immunoglobulin G, 70-75% of serum Ig

- monomer

- made AFTER IgM

- suggests past infection or long term immunity

- can cross the PLACENTA to protect the fetus

- does Opsonization = "tag the foreigner so it gets eaten."

IgM

immunoglobulin M, 5-10% of serum Ig

- pentamer

- made FIRST with infection, but low affinity

- makes complement!

- also does Opsonization = "tag the foreigner so it gets eaten."

high IgM means...

ACUTE or RECENT infection

high IgG means...

PAST infection or IMMUNITY

what happens with Lupus (SLE)? what is the main way to test for it?

autoimmune disorder where the body makes IgG autoantibodies (antibodies against itself)

- autoantibodies attach to pt's own cells, and complement gets activated to come kill them

- over 100 autoantibodies associated with SLE, typical pt has 3

- some autoantibodies are associated with other autoimmune diseases

- main autoantibodies to test for= ANA. can also use anti-dsDNA and anti-Sm

requirements for diagnosing lupus

need 4 of 17 criteria, including at least 1 clinical and 1 immunologic

ex. clinical- hemolytic anemia, cutaneous lupus, etc.

ex. immunologic- ANA, anti-dsDNA, etc

signs of lupus (SLE)

- ↑ autoantibodies (especially ANA, anti-dsDNA)

- ↓ C3/C4 (complement consumption) during active disease

- Inflammation in multiple organs

- butterfly rash on face

what does high anti-nuclear antibodies (ANA) mean?

Lupus

what does high anti-dsDNA mean? what do they target?

possibly Lupus

- autoantibodies targeting double stranded DNA

what does high anti-Sm mean?

possibly Lupus

- autoantibodies targeting protein with uridine-rich RNA component

how to determine if autoantibodies are from lupus or another disease?

use physical to narrow down options/ make differential

how does ANA screening work? what do you do if the screen is positive?

indirect fluorescence assay- patient antibody (ANA autoantibody) binds to Antigen in the cell nucleus, which is detected by Fluorescent marker

ANA IS A TITER (measure how much ANA is present), AND A STAIN (look for pattern)

ANA stain: homogeneous/ diffuse pattern

anti-histone, anti-DNP, anti-dsDNA

- could suggest SLE, drug-induced lupus, RA

ANA stain: speckled pattern

anti-Sm, anti-RNP, anti-SS-A, anti-SS-B, anti-Jo-1, anti-scl-70

- could suggest SLE, scleroderma, CREST Sjogren's, mixed CT disease, polymyositis

ANA stain: Nucleolar Pattern

anti-nucleolar, anti-Scl-70

- could suggest SLE, scleroderma

ANA stain: centromere pattern

anti-centromere

- could suggest CREST

ANA stain: peripheral/ rim pattern

anti-dsDNA

- could suggest SLE

ANA stain: nucleus, kinetoplast pattern

anti-dsDNA

- could suggest SLE

Rheumatoid Arthritis, what is commonly formed? what test is used?

chronic, symmetric erosive arthritis of peripheral joints that can also affect hearts and lungs

- rheumatoid factor (RF) is frequently an IgM autoantibody made by B cells that target antigens in joint spaces and causes complements to "fix" (kill) them

- test with LATEX AGGLUTINATION FOR IgM RF AND anti-CC test to confirm

what population is more affected by RA and SLE?

women

what does latex agglutination for IgM RF test for ? what's the problem with it?

rheumatoid factor

- low sensitivity! (many false negatives). specificity is also not great (some false positives)

how can you confirm if someone has RA? (after latex test)

anti-ccp test

- increases specificity of RA testing (less false positives) (91-93% specificity with both tests)

- can predict RA before it happens

positive anti-ccp test means...

person might develop RA

traditional algorithm for syphilis

- used before 2010

1. initial nontreponemal test, if reactive/ positive, get:

2. treponemal test, if reactive/ positive:

they have syphilis

reverse algorithm for syphilis

- used nowadays

1. treponemal test, if positive, get:

2. nontreponemal test, if positive: they have syphilis. if negative, get:

3. TP-PA test. Positive = syphilis

positive treponemal and nontreponemal tests means...

they have syphilis (no matter if it's the traditional algorithm or reverse algorithm)