1. MEANS OF viral infection DIAGNOSIS
Claude MUVUNYI M.D., Ph.D.

2. 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

3. 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.

4. Specimens for virus isolation
Clinical manifestations and common etiological agents
Source of specimen for virus isolation
clinical
postmortem
1/ Upper respiratiory tract infections
Rhinovirus
Parainfluenzavirus
Adenovirus
-Throat swab or nasal secretions
-Throat swab and feces
Lower respiratory tract infections
Influenzavirus
SRV
-throat swab and sputum
-lung, bronchus, trachea
Cutaneous and mucous membrane diseases
==vesicular
Small pox and vaccine
Herpes simplex
Varicella –Zoster
==exanthemous
Measles
Rubella
Enterovirus
-Vesicle fluids and scrapings
-throat swab
-feces and throat swab
Lung, liver, spleen
and brain

5. Specimens for virus isolation
Central nervous system infections
Enterovirus
Herpes simplex
Lymphocytic chroriomeningitis
-Feces, and CSF
-brain biopsy and
CSF
-blood and CSF
-brain, tissue, intestinal contents
-brain tissue
Arbovirus
Rabies
-saliva
Parotidis
Mumps
-throat swab and urine
Congenital anomalies
Cytomegalovirus
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
Enteritis
Rotavirus
Astrovirus
Adenovirus
-feces
Hemorrhagic fevers
Lassa
Ebola
Marburg
Machupo
Junin
Hantaan
-blood, urine and throat swab
-liver

6. Specimen Collection

7. Specimen Collection

8. Cerebral Spinal Fluid

9. Throat and nasal Swabs

10. Ear and eye Swabs-

11. Wound Swabs

12. Poor sample quality from Young child

13. Urine specimen from young child

14. Genital Swabs

15. Lower respiratory Swabs
Wrong position
Correct position

16. 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

17. Laboratory diagnosis of viral infection
We currently used two kinds of settings in laboratory diagnostics:
the direct diagnostics
the indirect diagnostics or serological settings

18. 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

19. 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

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

21. 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

22. Fig : PCR or Polymerase Chain Reaction

23. 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.

24. Agglutination Tests
Lattice Formation

25. Agglutination/Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen
Agglutinin/hemagglutinin Y + ↔
Qualitative agglutination test
Ag or Ab

26. Agglutination/Hemagglutination
Quantitative agglutination test
Titer
Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7

27. Agglutination/Hemagglutination
Definition
Qualitative test
Quantitative test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
Practical considerations
Easy
Semi-quantitative

28. Passive Agglutination/Hemagglutination
Definition - agglutination test done with a soluble antigen coated onto a particle Y + ↔
Applications
Measurement of antibodies to soluble antigens

29. Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y + ↔
Prior to Test Y + ↔
Test
Patient’s sample

30. Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA)
Lattice formation not required

31. Competitive RIA/ELISA for Ag
Method
Determine amount of Ab needed to bind to a known amount of labeled Ag Y + ↔
Prior to Test
Labeled Ag
Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor Y + ↔
Test
Patient’s
sample
Labeled Ag

32. Competitive RIA/ELISA for Ag
Method cont.
Determine amount of labeled Ag bound to Ab
↓ NH4SO4
↓ anti-Ig
Immobilize the Ab Y + ↔
Test
Patient’s
sample
Labeled Ag
Solid
Phase
Concentration determined from a standard curve using known amounts of unlabeled Ag
Quantitative
Most sensitive test

33. 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
Solid
Phase Y Ag
Immobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig
Quantitative

34. 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
Solid
Phase Y Ag
Immobilized
Ag in
Patient’s
sample
Labeled Ab
Quantitative

35. Picture of ELISA results: colored are positive and colorless negative

36. Pictures of apparatus of distribution of specimens for ELISA

37. Assays Based on Complement
Lattice formation not required

38. Complement Fixation Y Ag No Ag
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
No Ag Ag Y
Patient’s
serum Ag Y