Infectious Disease Microscopy And Diagnostics Flashcards
What is a pathogen?
microorganism that causes infectious disease
What is normal flora?
Bacteria that reside in a part of the body normally and it doesn’t cause infection
What is contamination?
an unintended introduction of potentially infectious material into a sample of body fluid
What is colonization?
Colonies of an organism that takes up residence in a part of the body where it would normally live but does not cause pathologic infection
What is an infection?
the invasion and multiplication of microorganisms into a part of the body resulting in illness
What is a sterile site?
Body part that is meant to be free of organisms
What are examples of sterile fluid?
Urine
CSF
What are non-sterile sites?
Parts of the body that contain normal flora
What are examples of non-sterile sites?
mouth
colon
What are the 5 categories of medical microbiology Lab testing for infectious diseases?
1) Antigen detection
2) Direct stains/examination
3) Cultures (aerobic and anaerobic) = ID from positive cultures and susceptibility testing
4) Nucleic acid detection
5) Serology
What is a direct specimen?
Surgical or needle aspiration of site
What is an example of a direct specimen?
Skin abscess
What is an indirect specimen?
Must pass through site with normal flora
What is an example of an indirect specimen?
Expectorated sputum
What is a sample from site with normal flora?
Both pathogen and normal flora are in the same site
Stool is an example of this
What are the two types of antigen detection tests?
Direct Fluorescent Antibody (DFA)
Indirect Fluorescent Antibody (IFA)
What is DFA?
Fluorescent labeled antibody binds to antigen of interest on a glass slide or other surface
What is IFA?
Looking for patients own native antibody
Antibody with is NOT fluorescent labeled binds to antigen of interest on a glass slide or other surface; fluorescent labeled antibody to IgG is then added and binds to antibody previously bound to antigen
Why do we need to have an indirect antigen detection test?
The antibodies used in DFA are synthetically made and because antigens often change, there can be problems with those antibodies that were made, not binding to the antigen
What tests are used as antimicrobial sensitivity testing?
Dilution method
Disk diffusion method
What is the dilution method?
Organism is put into tubes and a serial concentration amount is put into different tubes
Tubes are allowed to incubate and then looked at to see which concentration of antimicrobial agent inhibited the most of the organism
Turbidity of organism
What is the disk diffusion method?
Organism is spread over agar plate
Disks with differing concentrations and differing antimicrobial agents are put onto agar plate
zone of clearance is then measured after incubation to see how sensitive the organism is to the antimicrobial agent
What is the minimum inhibitory concentration (MIC)?
lowest concentration of an antimicrobial agent that inhibits growth
What does sensitive mean?
Antimicrobial is effective at retarding the growth of that pathogen
What does intermediate mean?
organism has some resistance to the antimicrobial agent
What does resistant mean?
Avoid this because pathogen isn’t susceptible to antimicrobial agent
What is Nucleic acid amplification (NAAT) testing?
Highly sensitive and specific
Valuable for difficult to detect or grow organisms
ONLY DETECTS ORGNIAMS THE ASSAY DETECTS
Ex. = HIV viral load
What is imperative that you know before using NAAT?
You must know what organism you are looking for in order to use this test
If the pathogen is no longer present = no results
What are serological tests?
Detect host antibodies
Not detectable early in disease
Can represent previous infection
What anitbodies are found in early infection?
IgM
What antibodies are found in late infection?
IgG
What diseases can be evaluated with serological assays?
Lyme disease Hep A Measles Mumps Varicella virus (chickenpox)
What is a strep throat test?
Detects Streptococcus pyogenes (Group A beta-hemolytic streptococcus)
What are the two types of strep throat tests?
1) Rapid antigen test
2) Throat culture
Which strep test is used more often?
Rapid antigen test
What is a rapid antigen detection test?
Detects Group A strep antigens using standard immunologic techniques
Results are available in 10-20 min. so patient can wait in office
90-95% Sensitivity
95-98% Specificity
This is only if sample is good
What should be done if a rapid antigen detection test comes back negative for strep throat?
A throat culture should be performed; although this does depend on the hospitals protocols
What is a throat culture?
Double swab of throat for strep
Culture is spread out on agar plate, incubated at a certain temp., and they wait for growth
What are blood cultures used for?
To detect bacteremia or fungemia
What diseases are blood cultures used to detect?
Sepsis Meningitis Osteomyelitis Arthritis Endocarditis Peritonitis Pneumonia Fever of unknown origin
What are the disadvantages of a blood culture?
Even in patients with bacteremia, they are likely to have a low number of bacteria in their blood and numbers often fluctuate
Therefore, multiple sets must be taken with sufficient volume of blood
Contamination from ubiquitous normal flora is possible and careful interpretation must be employed when a positive results returns
What should you remember about blood cultures?
If you order a blood culture, you are committed to act on those results
When should blood cultures absolutely be obtained?
prior to antibiotic therapy
What are the characteristics of Gram + bacteria?
Thick murein layer
Stain dark purple
What are the characteristics of Gram - bacteria?
Outer membrane, thin murein layer, and inner membrane
Stain pink
What are examples of Gram + bacteria?
Staph epidermis
Strep pyogenes
Clostridium tetani
What are examples of Gram - bacteria?
E. coli
Salmonella typhi
Vibrio cholerae
Bordetella pertussis
What are diplococci?
Cocci in pairs
Neisseria Gonorrhoaea
Gonorrhea
What are streptococci?
Cocci in chains
Streptococcus pyogenes
Strep throat
What are staphylococci?
Cocci in clusters
Staph aureus
Boils
What are tetrads?
A packet of 4 cocci
Micrococcus luteus
Rarely pathogenic
What are octads?
A packet of 8 cocci
Sarcina ventriculi
Rarely pathogenic
What are the different bacterial shapes?
Spheres
Rods
Spirals
What are Vibrio spp. and Campylobacter spp.?
Gram - curved rods
What is E. coli?
Gram - thin rods
What is Acinetobacter spp.?
Gram variable coccobaccili
What is Haemophilus?
Tiny gram -, pleomorphic diplococci
What is strep pneumoniae?
Gram +, kidney shaped diplococci
What is N. Gonorrhea?
Gram - intracellular diplococci
What is staph?
Gram + cocci in clusters
What is strep?
Gram + cocci in chains
What are acute phase proteins/reactants?
Those proteins/entities whose serum concentrations increase or decrease by at least 25% during inflammatory states
Acute phase response accompanies both acute and chronic inflammatory states associated with infection , trauma, infarcation, inflammatory arthrides, and systemic autoimmune diseases
What are the two Acute phase reactants?
1) Erythrocyte Sedimentation Rate (ESR)
2) C-reactive protein (CRP)
What is the ESR?
The rate in mm/hr at which erythrocytes suspended in plasma settle when placed in a vertical tube
It is an indirect measure of the Acute phase response and acute phase proteins (fibrinogen), in patients with acute or chronic inflammation
What is a function of CRP?
It binds phosphocholine which permits the recognition of both foreign pathogens that display this moiety and phospholipid constituents of damaged cells
What conditions can cause an elevated ESR that are not related to acute or chronic inflammation?
Increased age and female gender Anemia Renal disease Obesity Tilting of the ESR tube or high room temp.
What conditions can cause a decrease in ESR that are not related to acute or chronic inflammation?
Abnormalities of erythrocytes Extreme leukocytosis Extremely high serum bile salt levels Heart failure Hypofibrinogenemia Cachexia Clotting of sample
What inflammatory conditions cause an increase in ESR?
Tissue injury
Ischemia
Trauma
When do elevations of CRP typically occur?
In acute and chronic inflammation
In what type of infections are CRP levels markedly increased and why?
Bacterial infections because bacteria have more phosphocholine than viruses
What conditions can cause minor CRP elevations?
Obesity Cigarette smoking Diabetes Mellitus Uremia Hypertension Low levels of physical activity
What are nontreponemal tests?
Tests for reagin antibodies
Based on the reactivity of serum from patients with syphillis to a cardiolipin cholesterol lecithin anitgen
Semi-quantitative = amount of antibody present reflects activity of infection
Non-specific
Low cost
Used for initial syphillis testing
What tests are nontreponemal?
Venereal Disease Research Laboratory (VDRL)
Rapid Plasma Reagin (RPR)
Toludine Red Unheated Serum Test (TRUST)
What is a negative Rapid plasma reagin test?
Carbon particles remain unclumped
What is a positive Rapid plasma reagin test?
Clumped carbon from flocculation of the cardiolipin-based antigen by antibodies in the test serum
What are Treponemal tests?
Detect antibodies directed against specific treponemal antigens and thus are more specific than non-treponemal tests
Qualitative only
Reported as reactive or nonreactive
More complex and more expensive
Only used as a confirmatory test for syphillis when non-treponemal tests are positive
What are examples of treponemal tests?
Fluroescent treponemal antobody absorption (FTA-ABS)
Microhemagglutination test for anitbodies to T. pallidium (MHA-TP)
T. pallidium particle agglutination assay (TP-PA)
T. Palladium enzyme immunoassay (TP-EIA)
What does the CDC now recommend for HIV testing?
Lab based 4th generation assays that detect HIV p24 antigen and HIV antibodies
If test is positive, a confirmatory HIV-1/HIV-2 antibody differentiation immunoassay must be performed
What was the old way of testing for HIV?
ELISA and then Western Blot
When should an HIV RNA test be done?
If the results of serologic studies are indeterminate of ir acute HIV infection is suspected
What is the rapid HIV antibody test?
> 99% sensitive and specific
Perform on patient when healthcare worker is stuck with a needle they used on that patient
This test is only a preliminary test though and if it comes back positive it must be confirmed with a lab based combo antibody and antigen assay before a diagnosis of HIV can be made
What does a urinary dipstick provide?
Rapid semi-quantitative assessment of urinary characteristics on a series of test pads embedded on a reagent strip
What do dipsticks analyze urine for?
Heme Leukocyte esterase (WBCs) Nitrite Albumin (protein) Ketones pH Specific gravity (concentration) Glucose
What should occur if heme is detected in urine?
A confirmation of blood via microscopy
What may cause a false positive result for Leukocyte esterases in the urine?
Excessively dilute urine
What may cause a false negative result for leukocyte esterases in the urine?
Concentrated urine
Proteinuria
Glucosuria
What does nitrite presence in urine detect?
Infection
What are the test options for Influenza testing?
1) rapid antigen tests
2) Immunofluorescence assays
3) Reverse transcriptase Polymerase chain reaction (RT-PCR)
All are relatively rapid
What is the rapid antigen test for flu?
Sensitivity is awful
Immunoassays that detect influenza A and B nucleoprotein antigen in respiratory specimens
Qualitative results
15 minute results
FLu vaccine can cause a false positive
What is the more reliable flu test?
Immunofluorescence antibody tests
What is a direct or indirect immunofluorescence anitbody test?
Screening tests that yield result within hours
Can determine what type of flu it is: A vs. B
What is a viral culture for flu testing?
Flu virus is cultured from nasal washes, throat swabs, sputum, or bronchoalveolar lavage specimens
Gold standard for lab diagnosis
Takes 48-72 hours
Used for confirming screening tests
What do serologic tests for Lyme disease test for?
Antibodies to B. Burgdorferi
What is the most commonly used initial serologic test to detect Lyme disease?
ELISA tests
When can a false positive ELISA test be seen?
Patients with: Relapsing Fever Syphillis Leptospirosis Pinta Yaws Infective Endocarditis Viral illnesses Autoimmune diseases = Systemic lupus erythematosus, or rheumatoid arthritis EBV Malaria
What does the acid fast bacilli staining test/Ziehl-Neelsen Stain used for?
Detects Mycobacterium Tuberculosis
Monitors disease state
Positive test retains stain and red boxcars of macrophages seen
What is the technique for acid fast bacilli staining?
Fix sample in question
Stain with Fuchsin dye
Wash with an acid alcohol
What cannot be made from a positive acid fast bacilli stain test?
A diagnosis of TB; positive stain needs to be followed up with confirmatory test
What is the more specific and sensitive test for TB than a PPD?
Interferon Gamma Release Assay (IGRA)
What are the advantages of IGRA?
Single patient visit
Results in 24 hours
Does not cause boosting effect from subsequent test
No false positives from prior BCG vaccine
What are the disadvantages of IGRA?
Must be run within 8-30 hours of collection and sensitive to transport/testing conditions
Unknown if testing will have predictive power for TB disease
Limited data on children <5 y/o, recently exposed, immunocompromised, serial testing
Super expensive
What do we use to detect malaria?
Thick and thin blood smears which are drawn on febrile patients
What does a thick blood smear detect?
Thick, irregular amount of blood is dried and stained without fixing which increases the sensitivity of detection
Allows us to detect malaria
What does a thin blood smear detect?
Thin smear is fixed to slide and stained
Parasitemia and speciation of malaria can be determined
What does a lactate test detect?
Lactic acidosis which means patient is septic
What concentration of lactate in the blood means that the patient has lactic acidosis?
> 4 meq/L
What is responsible for the rise in lactate production in patients with sepsis?
Anaerobic metabolism is occuring
What are the types of stool sample tests?
1) Fecal Leukocytes (WBCs) = suggestive of infammation
2) stool culture
3) Stool for Ova and parasites
What is the stool culture positivity rate?
1.5-5.6%
What does a routine stool culture detect and why?
Salmonella
Campylobacter
Shigella
Because these organisms are shed continuously so a negative result is typically truly negative
What should you notify lab of testing stool for?
Yersinia
Aeromonas
Legionella
What types of organisms are shed intermittently and require a stool O and P?
Giardia
Cryptosporidium
Cyclospora
Entamoeba histolytica
What are possible indications that might require you to order a stool O and P?
Persistent diarrhea
Persistent diarrhea in the setting of foreign travel, particularly to mountainious areas
Persistent diarrhea with exposure to infants in daycare centers
Diarrhea in man who has had sex with men or a patient with AIDS
Community waterborne outbreak
Bloody diarrhea with few or no leukocytes
What tests are used for C. diff testing?
Enzyme Immunoassay (EIA)
Polymerase Chain reaction (PCR)
Selective Anaerobic culture
Cell culture cytotoxicity assay
What is the gold standard for C. diff detection?
Cell culture cytotoxicity assay
What is the cell culture cytotoxicity assay?
Culture liquid stool on a layer of cultured fibroblasts and detection of morphologic rounding of the fibroblasts
Confirmation obtained by addition of antiserum with concomitant revesral of the effects
Takes 2 days to complete
What is the more common test used for C. diff detection?
Initial EIA in acute setting
Tests for glutamate dehydrogenase antigen and toxins A/B
Results in 1 hour
What is PCR used for in C. diff testing?
Initial test of confirmation of an EIA test
Results in 1 hour
Does not test for active production of toxin A/B
Sensitive
Does not distinguish between symptomatic and asymptomatic patients
ONLY use to test symptomatic patients
What is the most sensitive test for C. diff?
Anaerobic cell culture
However, it can not distinguish between toxigenic and nontoxigenic strains
What is important to remember with C. diff testing?
C. diff toxins degrade at room temp. and may be undetectable within 2 hours = FRESH IS BEST and keep it refrigerated
What are is NAAT used to test for?
Chlamydia
Gonorrhea
What specific NAAT is used to test for Chlamydia and Gonorrhea?
XPert CT/NG assay
results in 90 minutes
What are culture methods limited use?
Research and reference labs only for chlamydia
What can be considered a positive result for men with gonorrhea?
Gram stain demonstrating polymorphonuclear leukocytes with intracellular gram - diplococci
