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:   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.

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

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

Specimen Collection

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

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 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 Y + ↔ 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 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

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

Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y + ↔ 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 Y + ↔ Prior to Test Y + ↔ 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 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

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

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

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

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 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