Microbio Final: Week 14

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

1. isolate bacterium from patient

2. pure culture - cell and colony morphology

3. gram stain

4. biochemical pathways/properties

5. 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:

1. wick coated with antibodies against hCG labeled with colored compound

2. test and control zones contain immobilized Abs against hCG or a control Ab

3. urine is soaked into the wick (capillary action)

4. if hCG is present, it and bound antibodies migrate to the test zone filled with unlabeled antibodies and concentrate there making a visible strip

5. 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