means of viral infection diagnosis claude muvunyi m.d., ph.d
Post on 22-Dec-2015
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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
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
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/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
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
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