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Clinical Microbiology
a subdiscipline of microbiology whose focus is diagnosing infectious diseases by identifying pathogenic microbes and advising medical providers on treatment
these labs must identify pathogens safely, efficiently, and reliably
standard lab practices have been established to minimize pathogen risk of infections
Biosafety Level (BSL)
a way to classify clinical laboratories on their containment potential
designated as BSL-1 through BSL-4
Specimen Collection Sites
human body sites
blood
cerebrospinal fluid : lumbar puncture, direct microscopy and culture
pleural fluid
synovial fluid
peritoneal fluid
any internal organ tissue
specimen should be obtained from site of infection aseptically
Lab Identification of Pathogens
isolate bacterium from patient
pure culture - cell and colony morphology
gram stain
biochemical pathways/properties
metabolism, cell properties, and behavior study
Selective Media
allows some organisms to grow while inhibiting others
Differential Media
allows identification of organisms based on their growth and appearance on the medium
Biochemical Algorithm to Identify Bacteria
identifying gram positive pyogenic cocci
What is the Reliability of a Test Based on?
specificity and sensitivity
Specificity
the ability to recognize the target pathogen, minimizing false positives
Sensitivity
the minimum amount of a pathogen needed for the test to detect it, minimizing false negatives
Immunoassays
use antibiotics specific for pathogens and their products for in vitro tests designed to detect specific infectious agents
clinicians can often identify an infection by measuring the patients antibody levels
Serology
the study of an antigen - antibody reactions in vitro
if an individual is infected antibodies to that pathogen will become elevated
Antigen (Ag)
foreign agents to the body that provoke an immune response
ex: viruses, parasites, bacteria, fungi, chemicals, etc
Antibody (Ab)
immune responders produced by B cells
bind antigens for which they are specific to
Enzyme Immunoassay (EIA)
very sensitive immunological assay
widely used in clinical diagnostic and research applications
employ covalently bonded enzymes attached to antibody molecules
rapid tests are similar to these - both allow detection of an antigen-antibody complex
Direct ELISA
a type of enzyme immunoassay (EIA)
detects antigens
sample containing antigens is mixed with antibody
enzyme linked antibodies react with the antigen
detected by adding a substrate for the linked enzyme, a color is produced
Indirect ELISA
a type of enzyme immunoassay (EIA)
detects antibodies
Sandwich EIA
a type of enzyme immunoassay (EIA)
detection of antibody
uses immobilized pathogen antigen and enzyme labeled pathogen antigens to detect pathogen specific antibodies in patient samples such as blood
more sensitive than the direct or indirect methods
Point of Care Lab Tests
a test used directly at the site of patient care
Rapid Test
similar to EIAs except the results can often be reported within minutes instead of hours
provide point of care diagnostics but are generally not as specific or sensitive
reagents are absorbed to support material
body fluid is applied to the support matrix
matrix contains a soluble antigen conjugated to a colored molecule = chromophore
contains a line of fixed antigen which antibodies bind to, detect color change
ex: strep tests, influenza tests, HIV tests, pregnancy tests
Chromophore
in rapid tests - in matrix
a soluble antigen conjugated to a colored molecule
Pregnancy Tests
example of a rapid test
detects levels of human chorionic gonadotropic (hCG) horome (antigen)
test procedure:
wick coated with antibodies against hCG labeled with colored compound
test and control zones contain immobilized Abs against hCG or a control Ab
urine is soaked into the wick (capillary action)
if hCG is present, it and bound antibodies migrate to the test zone filled with unlabeled antibodies and concentrate there making a visible strip
any unbound Abs will bind in the control zone giving a line showing that the control worked
Nucleic Acid Based Clinical Assays
quantitative and reverse transcription PCR
presence of amplified gene segment confirms presence of pathogen
Reverse Transcription PCR (RT-PCR)
uses pathogen specific RNA to make cDNA
Quantitative Real Time PCR (qPCR)
uses fluorescently labeled PCR products
almost immediate results
Polymerase Chain Reaction
process of increasing (amplifying) small quantities of DNA for analysis
can make billions of copies of a target DNA within a few hours
used for diagnostic tests for genetic diseases and detecting pathogens
most widely used molecular method in the clinical laboratory
DNA primers can be made for specific pathogens
reverse-transcription PCR used mRNA as a template
Quantitative Reverse Transcription PCR (RT-qPCR)
involves the detection and quantification of RNA
detects pathogens such as viruses for the diagnosis of infectious diseases
combines the effects of reverse transcription and quantitative PCR or real time PCR to amplify and detect specific targets
at each cycle during this method, the quantity of DNA is measured in real time by a variety of fluorescent signals = hydrolysis probes
Influenza (Flu)
single stranded, negative sense, segmented RNA genome surrounded by an envelope composed of protein, a lipid bilayer, and external glycoproteins
three different types: A, B, and C
each strain can be identified by a unique set of surface glycoproteins
avian, swine, and mammalian strains
Hemagglutinin (HA) Spikes
recognize and attach to host cells
one type of its viral capsid and is named for the antigens it contains
Neuraminidase (NA) Spikes
help the virus separate from the infected cell
one type of its viral capsid and is named for the antigens it contains
Influenza A
alphafluenzavirus
can cause seasonal epidemics every winter
identified by variation in the HA and NA spikes
16 subtypes of HA, 9 subtypes of NA
human adapted: H1N1, H2N2, H3N2
Influenza B
betainfluenzavirus
Influenza C
gammainfluenzavirus
Why do Influenza Outbreaks Occur Annually?
the genome has lots of plasticity
Antigenic Drift
minor antigenic changes in HA and NA
allow the virus to elude some of the host immunity
Antigenic Shifts
changes great enough to evade most immunity
leads to pandemics
involves the reassortment of the eight RNA segments
Influenza Diagnosis
hard to diagnose from clinical symptoms
can be diagnosed with rapid antigen tests or PCrR
Influenza Prevention
immunization
careful worldwide surveillance
use of various drugs like tamiflu
multivalent vaccine for the most important strains
composition of the vaccine determined annually by the identification of circulating viruses = doesn’t provide long term immunity
1918-1919 Pandemic
20-50 million deaths worldwide
unusually lethal for young adults
rapid onset and death, likely caused by cytokine storm
invaded lungs and caused lethal hemorrhaging
pigs are more readily infected with influenza, so they were suggested as the original recipients of the virus that passed to humans
Influenza Pandemics
occur periodically
ex: 1957 asian flu outbreak
influenza A outbreak nicknamed swine flu spread rapidly
H1N1 was a classic case of influenza virus genome reassortment in swine and is the major virus health officials are monitoring today
H5N1 has been detected in birds in many countries, if it jumps to humans it could become deadly