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)

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 studiesThe laboratory diagnosis of viral infection is based upon three general approaches: 1. Direct detection of viral antigens or structures, either in

cells derived from infected tissues or free in fluid specimens;

2. Isolation and identification of viruses, usely accomplished in cell cultures;

3. 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.

Specimens for virus isolationClinical manifestations and common etiological agents

Source of specimen for virus isolation

  clinical postmortem1/ Upper respiratiory tract infectionsRhinovirusParainfluenzavirus Adenovirus

  -Throat swab or nasal secretions -Throat swab and feces 

 

Lower respiratory tract infectionsInfluenzavirusParainfluenzavirus SRV 

  -throat swab and sputum

  -lung, bronchus, trachea

Cutaneous and mucous membrane diseases==vesicularSmall pox and vaccineHerpes simplexVaricella –Zoster ==exanthemousMeaslesRubellaEnterovirus 

   -Vesicle fluids and scrapings   -throat swab-throat swab-feces and throat swab

   Lung, liver, spleen and brain     

Specimens for virus isolationCentral nervous system infectionsEnterovirus  Herpes simplex Lymphocytic chroriomeningitis

  -Feces, and CSF  -brain biopsy and CSF -blood and CSF 

  -brain, tissue, intestinal contents -brain tissue  -brain tissue

ArbovirusRabies

-blood and CSF-saliva

-brain tissue-brain tissue

ParotidisMumps 

 -throat swab and urine

 

Congenital anomaliesCytomegalovirus Rubella

 -Urine and throat swab-Throat swab,m CSF and urine

 -kidney, lung and other tissues-lymph nodes, lung, spleen and other tissues

Hepatitis viruses Agents not recoverable  

EnteritisRotavirusAstrovirus Adenovirus

 -feces

 

Hemorrhagic feversLassaEbolaMarburgMachupoJuninHantaan

 -blood, urine and throat swab

 -liver

Specimen Collection

7

Specimen Collection

Cerebral Spinal Fluid

Throat and nasal Swabs

Ear and eye Swabs-

Wound Swabs

Poor sample quality from Young child

Urine specimen from young child

Genital Swabs

Wrong position Correct position

Lower respiratory Swabs

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 infectionWe currently used two kinds of settings in

laboratory diagnostics:– the direct diagnostics– the indirect diagnostics or serological settings

Direct diagnosticsDirect 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 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 studies are very fastidious and mainly available only in referral laboratories.

CPEs can be detected by means of cytological examination.

Use of electron microscopy isn’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 increminate Rotavirus causative agents of children’s gastroenteritis

Picture of electron microscope picture of bright microcope of direct view of Calcivirus fluorescence

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 amplicafication of a target genomic sequence in few hours.

Then we can qualify and/or quantify the genome structures

Fig : PCR or Polymerase Chain Reaction

Indirect diagnostics Indirect diagnostic procedures mean:

– Serological different testing of hemagglutination

– Inhibition of hemagglutination, – Neutralizing of cytopathologic effect, – Indirect immunofluorescence,

– ELISA,

– Immuno botting,

– Western blots.

Agglutination Tests

Lattice Formation

Agglutination/Hemagglutination• Definition - tests that have as their endpoint the agglutination of a particulate

antigen

– Agglutinin/hemagglutinin

+ ↔

• Qualitative agglutination test

– Ag or Ab

Agglutination/Hemagglutination• Quantitative agglutination test– Titer– Prozone

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

24

Pos

.

Neg

.

Titer

64

8

512

<2

32

128

32

4

Patient

1

2

3

4

5

6

7

8

Agglutination/Hemagglutination

• Definition • Qualitative test• Quantitative test

• Practical considerations– Easy– Semi-quantitative

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

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

• Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

+ ↔

Prior to Test

+ ↔+

Test

Patient’s sample

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

Lattice formation not required

Competitive RIA/ELISA for Ag

• Method– Determine amount of

Ab needed to bind to a known amount of labeled Ag

+ ↔

Prior to Test

Labeled Ag

+ ↔

Test

+Patient’ssample

LabeledAg

+

– Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

Competitive RIA/ELISA for Ag

• Method cont.– Determine amount

of labeled Ag bound to Ab• ↓ NH4SO4

• ↓ anti-Ig • Immobilize the Ab

• Quantitative– Most sensitive test

+ ↔

Test

+Patient’ssample

LabeledAg

+

– Concentration determined from a standard curve using known amounts of unlabeled Ag

SolidPhase

SolidPhase

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

SolidPhase

AgImmobilized

Ab in Patient’ssample

LabeledAnti-Ig

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

SolidPhase

Ag

Immobilized

Ag in Patient’ssample

LabeledAb

Picture of ELISA results: colored are positive and colorless negative

Pictures of apparatus of distribution of specimens for ELISA

Assays Based on Complement

Lattice formation not required

Complement Fixation

– 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

Patient’sserum

Ag No Ag

Ag

• Methodology