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 studies
The 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 isolation
Clinical manifestations
etiological agents
and
common Source of specimen for virus isolation
clinical
1/ Upper respiratiory tract infections
Rhinovirus
Parainfluenzavirus
-Throat
swab
secretions
postmortem
or
nasal
Adenovirus
-Throat swab and feces
Lower respiratory tract infections
Influenzavirus
Parainfluenzavirus
SRV
Cutaneous and mucous membrane diseases
==vesicular
Small pox and vaccine
Herpes simplex
Varicella –Zoster
==exanthemous
Measles
Rubella
Enterovirus
-throat swab and sputum
-lung, bronchus, trachea
-Vesicle fluids and scrapings
Lung, liver, spleen
and brain
-throat swab
-throat swab
-feces and throat swab
Specimens for virus isolation
Central nervous system infections
Enterovirus
-Feces, and CSF
Herpes simplex
Lymphocytic chroriomeningitis
-brain, tissue, intestinal contents
-brain tissue
-brain biopsy and
CSF
-brain tissue
-blood and CSF
Arbovirus
Rabies
Parotidis
Mumps
Congenital anomalies
Cytomegalovirus
-blood and CSF
-saliva
-throat swab and urine
-Urine and throat swab
-Throat swab,m CSF and urine
Rubella
Hepatitis viruses
Enteritis
Rotavirus
Astrovirus
Adenovirus
Hemorrhagic fevers
Lassa
Ebola
Marburg
Machupo
Junin
Hantaan
-brain tissue
-brain tissue
-kidney, lung and other tissues
-lymph nodes, lung, spleen and other
tissues
Agents not recoverable
-feces
-blood, urine and throat swab
-liver
Specimen Collection
Specimen
Collection
7
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
Lower respiratory Swabs
Wrong position
Correct position
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 infection
We currently used two kinds of settings in
laboratory diagnostics:
– the direct diagnostics
– the indirect diagnostics or serological settings
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
 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
view of Calcivirus
picture of bright microcope of direct
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
Neg.
Pos.
1/1024
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
Patient
1/2
– Titer
– Prozone
Titer
1
2
3
64
8
512
4
5
<2
32
6
7
8
128
32
4
• Practical considerations
– Easy
– Semi-quantitative
1/512
1/256
1/128
1/64
1/32
1/16
1/8
1/4
• Definition
• Qualitative test
• Quantitative test
1/2
Agglutination/Hemagglutination
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
– Use predetermined
amounts of labeled Ag
and Ab and add a
sample containing
unlabeled Ag as a
competitor
Prior to Test
↔
+
Labeled
Ag
Test
+
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
Test
+
Solid Labeled
Ag
Phase
+
↔
Patient’s
sample
+
Solid
Phase
– Concentration determined from a standard curve
using known amounts of unlabeled Ag
• Quantitative
– Most sensitive test
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
Labeled
Anti-Ig
Ab in
Patient’s
sample
Immobilized
Ag
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
Labeled
Ab
Ag in
Patient’s
sample
Ag
Immobilized
Solid
Phase
Picture of ELISA results: colored are positive and colorless
negative
Pictures of apparatus
specimens for ELISA
of
distribution
of
Assays Based on Complement
Lattice formation not required
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
No Ag
Ag
Ag
Patient’s
serum
Ag