Viral Diagnostics

Question 1

Steps in collection & handling of clinical specimens

Answer 1

1. Develop diagnostic hypothesis
2. Collect clinical specimens from affected area
   
   • Rectal swabs
   • Throat swabs
   • Nasopharyngeal washings
   • Scrapings from skin/mouth lesions
   • Lumbar puncture/CSF
3. Samples collected in sterile containers, kept cool, transported to lab
4. Used to detect presence of virus, virus antigen, viral nucleic acids, or virus-specific antibody

Question 2

Growth of virus in traditional tube culture

Answer 2

• 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)

Question 3

Growth of viruses in shell vial cultures

Answer 3

• 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

Question 4

Advantages & disadvantages of virus cultivation

Answer 4

• 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

Question 5

Nucleic acid tests: PCR

Answer 5

• 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)
• Nucleotide sequence can also be determined from PCR amplicons, compare with known viruses

Question 6

Nucleic acid tests: multiplex PCR assays

Answer 6

• 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

Question 7

Advantages & disadvantages of PCR detection

Answer 7

• 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

Question 8

Rapid immunoassays

Answer 8

• 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

Question 9

Advantages & disadvantages of rapid antigen detection

Answer 9

• 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

Question 10

Western blot

Answer 10

• 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)

Question 11

Direct immunofluorescence of infected cells from clinical specimens

Answer 11

• 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

Question 12

Serology

Answer 12

• 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

Question 13

ELISAs

Answer 13

• 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

Question 14

Western blot

Answer 14

• 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

Question 15

Classic methods in diagnostic virology

Answer 15

• 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

Question 16

Tests to determine size

Answer 16

• Tissue culture medium filtered through sterilized membranes of different pore sizes
• Then inoculated into tissue culture and observed for virus replication

Question 17

Tests to determine type of nucleic acid of unknown virus

Answer 17

• Virus inoculated into two tubes of tissue culture cells, one containing BrDU
  
  • Inhibits replication of DNA-containing viruses but not RNA-containing viruses

Question 18

Tests to determine presence of lipid-containing viral envelope

Answer 18

• Virus treated with chloroform or ether
  
  • Solubilizes envelope
  • Inactivates most enveloped viruses
• Does not affect infectivity of non-enveloped viruses

Question 19

Virus neutralization test

Answer 19

• Reference antisera against different strains of virus mixed with patient’s virus then inoculated into new tissue culture
• Evidence of virus replication indicates that virus was not neutralized by antiserum & is not same as reference virus
• OR neutralization indicates that it was the same as the reference virus or is closely related to standard reference virus

Question 20

Hemagglutination assay

Answer 20

• Virus dilutions mixed with RBCs in 96-well plate
• Unabsorbed RBCs form sharp dot in bottom of well
• Agglutinated RBCs form lattice that coats entire well

Question 21

Hemagglutination inhibition (HAI)

Answer 21

• If virus is identified by hemagglutination, tx of virus with antiviral Ab can prevent hemagglutination
• Dilutions of serum incubated with virus and RBCs

Question 22

Evaluation of drug resistance of virus isolates

Answer 22

• Mutants that arise spontaneously are able to replicate in presence of drug have strong in vivo selective advantage
• Evaluated by growing virus isolate in cells in presence of different concentrations of drug
• PCR or RT-PCR can be used to determined deduced AA sequence of viral drug-resistance mutations
• Tx with cocktails of several anti-viral drugs b/c it is less likely that drugs will develop resistance to > 1 drug at a time

Question 24

Identification of vaccine vs. virulent viruses

Answer 24

• Live attenuated virus vaccines - contain marker mutations that distinguish them from virulent wild-type virus
• Viruses isolated from patients inoculated with LAVV sometimes contain mutations that revert vaccine strain to virulence
• To determine if virus found in patient is virulent or from vaccine, labs use PCR + DNA sequencing to look for known nucleotide markers of vaccine strain

Question 26

Diagnostic virology assays performed at UCH

Answer 26

1. Virus cultivation/identification with traditional tube cultures and shell vial cultures
2. Singleplex and some multiplex qualitative PCR and quantitative PCR assays
3. Rapid virus antigen detection
4. Direct immunofluoresnce of infected cels
5. Serology