Viral Diagnostics Flashcards
1
Q
Steps in collection & handling of clinical specimens
A
- Develop diagnostic hypothesis
- Collect clinical specimens from affected area
- Rectal swabs
- Throat swabs
- Nasopharyngeal washings
- Scrapings from skin/mouth lesions
- Lumbar puncture/CSF
- Samples collected in sterile containers, kept cool, transported to lab
- Used to detect presence of virus, virus antigen, viral nucleic acids, or virus-specific antibody
2
Q
Growth of virus in traditional tube culture
A
- Cell monolayer adheres from midpoint to bottom of one side of tube
- Incubate for days –> weeks
- Examined by light microscopy for development of cytopathic effects (e.g. cell vacuolization, rounding up of cells, cell fusion, cell lysis)
- Alternative: **hemadsorption **test
- Cell culture medium removed from culture tubes, incubated and examined by light microscopy
- If virus is hemadsorbing - erythrocytes adhere in clumps to cell monolayer
- ID usually requires confirmation by second assay (PCR or RT-PCR)
3
Q
Growth of viruses in shell vial cultures
A
- Cell monolayers grown on round glass coverslip
- Cell growth medium removed and processed clinical specimen placed directly on cells to inoculate cells with clinical specimen
- Vial centrifuged at low speed to enhance virus attachment to cells and viral penetration of cells
- Fresh culture medium added, vials incubated
- Viral infection detected by staining cells on coverslips with labeled virus-specific monoclonal Abs
- Stained coverslips evaluated by fluorescent microscopy
4
Q
Advantages & disadvantages of virus cultivation
A
- Advantages
- Capacity to isolate wide variety of viruses
- Provides virus isolate that can used for further studies like antiviral susceptibility testing, serotyping, epidemiology
- Shorter turnaround times
- Do not require development of CPE
- Disadvantages
- Long incubation times required for some viruses
- Inability of some viruses to proliferate in cell cultures
- Need for technical expertise to evaluate cell cultures for various forms of CPE
5
Q
Nucleic acid tests: PCR
A
- Most commonly used method for rapid viral diagnosis
- Quick, sensitive, can be automated
- DNA/RNA isolated from patient’s clinical specimen or from cultured cells
- **Reverse transcriptase **used to transcribe RNA to make cDNA copy
- Patient’s DNA then amplified by PCR using oligonucleotide primers specific for suspected virus pathogen
- Can be qualitative or quantitative
- Qualitative: size of amplified DNA identified in agarose gels
- Quantitative: amount of nucleic acid amplified determined from standard curve (used when viral load is clinically significant - HIV, HCV, CMV)
- Can be qualitative or quantitative
- Nucleotide sequence can also be determined from PCR amplicons, compare with known viruses
6
Q
Nucleic acid tests: multiplex PCR assays
A
- Recently approved for use in clinical lab by FDA
- Designed to allow simultaneous detection of multiple different viruses that might be found in particular anatomical site
- Ex. multiplex PCR assay to simultaneously detect HSV-1 and HSV-2
7
Q
Advantages & disadvantages of PCR detection
A
- Advantages
- High sensitivity and specificity
- Short turnaround time
- Ability to detect evidence of virus infection for viruses that cannot be cultured in traditional cell cultures
- Disadvantages
- Lack of standardized protocols or FDA-approved kits
- Technical expertise required in-house for assay development
- Cost of equipment and reagents
8
Q
Rapid immunoassays
A
- Available for rapid detection of influenza A, influenza B, and RSV
- Very rapid (10-15 minutes for results)
- Sensitivity: 50-90%
- Specificity: 90-95%
- Most can be done with variety of clinical specimen types
- Accuracy of tests varies based on type of specimen collected
- Clinical specimen migrates via capillary action, viral proteins react with labeled antibodies as complex migrates down strip –> colored line on test strip indicates protein is present in sample
9
Q
Advantages & disadvantages of rapid antigen detection
A
- Advantages
- High specificity for RSV and influenza A/B viruses
- Very rapid (15 minutes or less)
- Minimal technical expertise required to perform tests
- Disadvantages
- Comparatively poor sensitivity (false negatives common)
- Some distinguish influenza A from B - others do not
- Do not provide information on influenza A subtypes
- Many factors influence accuracy: time from illness onset, collection of specimen for testing, type of specimen tested, handling of specimen
10
Q
Western blot
A
- Uses antibodies to recognize **viral proteins **separated in SDS polyacrylamide gel
- Samples subjected to gel electrophoresis
- Separates proteins based on sizes
- Proteins transferred onto nitrocellulose membranes
- Virus-specific antibodies recognize bands containing antigenic viral proteins
- Can be used to detect if patient sera contain specific antibodies against known virus proteins (e.g. in HIV testing)
11
Q
Direct immunofluorescence of infected cells from clinical specimens
A
- Ex. nasopharyngeal washes - contain virus-infected cells
- Samples centrifuged onto glass slides
- Monoclonal Abs against each of viruses that commonly infect that anatomic site used to detect viral antigens in cells
- Fast and definitive - but labor intensive
12
Q
Serology
A
- Necessary in order to prove that virus present in a person caused the disease
- Obtain serum specimen from patient on first visit of acute infection, and second specimen several weeks later during convalescence
- Positive test requires at least four-fold increase in circulating Ab titer against isolated virus
13
Q
ELISAs
A
- Use specific viral proteins most frequently targeted by Ab response
- Proteins purified from virus-infected cells or produced by recombinant DNA technology, attached to solid phase
- Proteins incubated with serum, washed to eliminate nonspecific Abs, allowed to react with enzyme-linked reagent to detect IgG or IgM specifically adhering to viral antigen
- Amount of Ab then can be quantitated by intensity of a color reaction mediated by linked enzyme
- Can be sensitive and automated
14
Q
Western blot
A
- Used to confirm presence of Ab to multiple viral proteins
- Virus proteins separated by size and transferred to inert membrane, incubated with serum Abs
- Have internal specificity control - level of reactivity for viral proteins can be compared with that for cellular proteins in same sample
- Require individual evaluation, inherently difficult to quantitate or automate
15
Q
Classic methods in diagnostic virology
A
- Used for virus isolation and characterization before molecular techniques were available
- Tests to determine size
- Tests to determine type of nucleic acid in unknown virus
- Tests to determine presence of lipid-containing viral envelope
- Virus neutralization test
- Hemagglutination assay
- Hemagglutination inhibition
