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MEANS OF viral infection DIAGNOSIS. Claude MUVUNYI M.D., Ph.D. Clinical diagnosis. The patient’s history and symptoms provide the first clues to the diagnosis of a viral infection, but the diagnosis also includes the exclusion of other types of infection (e.g., bacterial, fungal )

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means of viral infection diagnosis

MEANS OF viral infectionDIAGNOSIS

Claude MUVUNYI M.D., Ph.D.

clinical diagnosis
Clinical diagnosis
  • The patient’s history and symptoms provide the first clues to the diagnosis of a viral infection,
  • but the diagnosis also includes the exclusion of other types of infection (e.g., bacterial, fungal)
  • Results from viral laboratory studies can confirm the clinical diagnosis by identifying the viral agent of the infection or detecting specific antigen antibodies
viral laboratory studies
Viral laboratory studies

The laboratory diagnosis of viral infection is based upon three general approaches:

  • Direct detection of viral antigens or structures, either in cells derived from infected tissues or free in fluid specimens;
  • Isolation and identification of viruses, usely accomplished in cell cultures;
  • Demonstration of a significant increase in serum antibodies to a etiological possible virus during the course of a illness; that’s by serological testing assays.
slide15

Lower respiratory Swabs

Correct position

Wrong position

laboratory diagnosis of viral infection
Laboratory diagnosis of viral infection
  • A clinical diagnosis of a viral infection can be confirmed in laboratory through the observation of:
    • Virus-induced cytopathogenic effets (CPE) on inoculated permissive cells
    • Electron microscope detection of viral particles
    • Isolation and growth of the virus
    • Detection of viral components or antigens ( e.g., proteins, enzymes, nucleic acid)
    • Evaluation of the patient’s immune response to the virus that may be by serology detecting specifics antibodies against viral antigens
laboratory diagnosis of viral infection1
Laboratory diagnosis of viral infection
  • We currently used two kinds of settings in laboratory diagnostics:
    • the direct diagnostics
    • the indirect diagnostics or serological settings
d irect diagnostics
Direct diagnostics
  • Direct diagnosis procedures concern :
    • Cytological examinations of CPE
    • Electron microscopy
    • Virus isolation and growth onto permissive cell’s culture
    • Detection of viral antigens: proteins, enzymes
    • Detection of viral genetic elememts , mainly genomic nucleic acid
direct diagnostics
Direct diagnostics
  • The “gold standard” for providing a viral etiology of a syndrome, infection or disease is the recovery and growth of infecting agent.
  • Isolation and growth studiesare very fastidious and mainly available only in referral laboratories.
  • CPEs can be detected by means of cytological examination.
  • Use of electronmicroscopyisn’t a standard clinical laboratory technique, but it can be used to detect some viruses if sufficient viral particles are presents, mainly in serum or feces such as new increminateRotavirus causative agents of children’s gastroenteritis
slide20

Picture of electron microscope picture of bright microcope of direct view of Calcivirusfluorescence

molecular biology techniques
Molecular biology techniques
  • Viral genome detection often after been applified in PCR. We also can quantify and detect DNA or RNA sequences
  • PCR or polymerase chain reaction and reverse transcriptae PCR (RT-PCR) are more used and becoming very important for viral detection
  • Used of the appropriate primers can promote a million fold amplicaficationof a target genomic sequence in few hours.
  • Then we can qualify and/or quantify the genome structures
i ndirect diagnostics
Indirect diagnostics
  • Indirect diagnostic procedures mean:
    • Serological different testing of hemagglutination
    • Inhibitionof hemagglutination,
    • Neutralizing of cytopathologiceffect,
    • Indirect immunofluorescence,
    • ELISA,
    • Immunobotting,
    • Western blots.
agglutination tests

Agglutination Tests

Lattice Formation

slide25

Qualitative agglutination test

    • Ag or Ab

Y

+

Y

Y

Agglutination/Hemagglutination

  • Definition - tests that have as their endpoint the agglutination of a particulate antigen
    • Agglutinin/hemagglutinin
agglutination hemagglutination

1/1024

1/256

1/512

1/128

1/16

1/64

1/32

Pos.

1/8

Neg.

1/4

1/2

Titer

Patient

64

1

8

2

512

3

<2

4

32

5

128

6

32

7

4

8

Agglutination/Hemagglutination
  • Quantitative agglutination test
    • Titer
    • Prozone
agglutination hemagglutination1

1/256

1/512

1/128

1/16

1/64

1/32

1/8

1/4

1/2

Agglutination/Hemagglutination
  • Definition
  • Qualitative test
  • Quantitative test
  • Practical considerations
    • Easy
    • Semi-quantitative
passive agglutination hemagglutination

Y

Y

+

Y

Passive Agglutination/Hemagglutination
  • Definition - agglutination test done with a soluble antigen coated onto a particle
  • Applications
    • Measurement of antibodies to soluble antigens
agglutination hemagglutination inhibition

Prior to Test

Y

Y

+

Y

Y

Test

Y

+

+

Y

Patient’s sample

Agglutination/Hemagglutination Inhibition
  • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag
radioimmuoassays ria enzyme linked immunosorbent assays elisa

Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA)

Lattice formation not required

competitive ria elisa for ag

Prior to Test

Y

Y

+

Labeled

Ag

Test

Y

Y

+

+

+

Labeled

Ag

Patient’s

sample

Competitive RIA/ELISA for Ag
  • Method
    • Determine amount of Ab needed to bind to a known amount of labeled Ag
  • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor
competitive ria elisa for ag1

Solid

Phase

Solid

Phase

Test

Y

Y

+

+

+

Labeled

Ag

Patient’s

sample

Competitive RIA/ELISA for Ag
  • Method cont.
    • Determine amount of labeled Ag bound to Ab
      • ↓NH4SO4
      • ↓ anti-Ig
      • Immobilize the Ab
  • Concentration determined from a standard curve using known amounts of unlabeled Ag
  • Quantitative
    • Most sensitive test
solid phase non competitive ria elisa

Labeled

Anti-Ig

Ab in

Patient’s

sample

Y

Y

Ag

Immobilized

Solid

Phase

Solid Phase Non-Competitive RIA/ELISA
  • Ab detection
    • Immobilize Ag
    • Incubate with sample
    • Add labeled anti-Ig
    • Amount of labeled Ab bound is proportional to amount of Ab in the sample
  • Quantitative
solid phase non competitive ria elisa1

Labeled

Ab

Ag in

Patient’s

sample

Y

Ag

Y

Immobilized

Solid

Phase

Solid Phase Non-Competitive RIA/ELISA
  • Ag detection
    • Immobilize Ab
    • Incubate with sample
    • Add labeled antibody
    • Amount of labeled Ab bound is proportional to the amount of Ag in the sample
  • Quantitative
assays based on complement

Assays Based on Complement

Lattice formation not required

complement fixation

No Ag

Ag

Patient’s

serum

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Complement Fixation
  • Methodology
  • Ag mixed with test serum to be assayed for Ab
  • Standard amount of complement is added
  • Erythrocytes coated with Abs is added
  • Amount of erythrocyte lysis is determined

Ag

Ag