TOPIC 4 VIRO MIDTERM

target organs involved

The clinical symptoms of diseases often point to _____ which can help determine the most appropriate specimens to collect

true

Patients symptoms and history, including recent travel, the season of the year and presumptive diagnosis, help determine the appropriate procedures to be used to identify a viral agent

true or false?

the affected site

For optical recovery, specimens for viral isolation should be collected from?

RESPIRATORY TRACT

Antigen Detection

Adenovirus, Herpes Simplex Virus (HSV)

Cytomegalovirus (CMV)

Influenza virus type A/B

Parainfluenza virus

Respiratory syncytial virus (RSV)

RESPIRATORY TRACT

Serology

Adenovirus

Coxsackie A and B virus

Echovirus

HSV

CMV

Influenza A and B

Para influenza virus

RSV

Nasal aspirate

Nasopharynx (NP) or Throat swabs

Bronchoalveolar lavage

Lung biopsy

Culture specimen for respiratory tract

GASTROINTESTINAL

Antigen Detection

Adenovirus 40 and 41 rotavirus

GASTROINTESTINAL

Virus Isolation

Adenovirus 40 and 41

Coxsackie A

Reovirus

GASTROINTESTINAL

Serology

Adenovirus 40 and 41

Coxsackie A

Stool and swab

Culture specimen for Gastrointestinal tract

Liver

Antigen detection

Virus Isolation

None

Liver

Serology

Hepatitis A, B, C, D, E

Epstein barr virus

none

Culture specimen for Liver

Cutaneous

Antigen Detection

HSV

Adenovirus

Varcicella-zoster virus (VZV)

Cutaneous

Virus Isolation

HSV

Adenovirus

Coxsackie group A and B virus

Echovirus

Enterovirus

Measles virus

VZV

Reovirus

Rubella virus

Vaccinia virus

Cutaneous

Serology

HSV

Adenovirus

Coxsackie group A and B virus

Echovirus

Human herpesvirus 6 (HHV-6)

Measles virus

VZV

Parvovirus B19

Rubella virus

Vaccinia virus

Vesicle aspirate

NP aspirate

Stool

Lesion swab

Culture specimen for Cutaneous

Central nervous system

Antigen detection

HSV

Murrrips virus

Central nervous system

Virus isolation

Coxsackie group A and B virus

Echovirus

Enterovirus

Poliovirus

HSV

Murrips virus

Central nervous system

Serology

Coxsackie group A and B virus

Echovirus

Poliovirus

HSV

HHV-6

Murrips virus

CSF

Brain biopsy

NP Swabs

Stool

CNS Culture specimen

Ocular

Antigen detection

Adenovirus

HSV

Ocular Virus Isolation

Adenovirus

HSV

Coxsackie Group A

Enterovirus

Ocular Serology

HSV

Coxsackie group A virus

Ocular Culture specimens

Cornmeal swabs

Conjunctival scrapings

Genital

Antigen detection and virus isolation

HSV

Culture specimen for Genital

Vesicle-Aspirate

Vesicle swab

true

For systemic, congenital or generalized disease:

Specimens from multiple sites including the blood (buffy coat), CSF and the other portals of entry (oral or respiratory tract) exit (urine stool) are appropriate

true or false?

respiratory infections

Enteroviruses can cause?

Enteroviruses

may be recovered from the stool after the respiratory shedding has ceased

Enteroviruses

major cause of aseptic meningitis and can also be isolated from urine specimens

As soon as possible

how fast Best specimen are collected?

aseptically

Specimen should be collected

Aspirated secretions

preferred specimen

Dacron or wooden shafts

Swab are easier to use must made of?

Calcium alginate, charcoal

Don't use______ in collecting specimens

swabs with wooden shafts

Don't use______ in collecting specimens

True

Tissue samples must kept moist (to prevent drying out and degradation)

true or false

Types of Liquid medium

1.Viral Transport Medium (VTM)

2.Saline

3.Trypticase soy broth (TSB)

buffered isotonic solution with protein

Several viral transport systems are commercially available

Contains?

antibacterial and antifungal agents

Several viral transport systems are commercially available

Contains? (2)

respiratory, swab and tissue samples

Samples that can be collected with viral transport media (VTM) are ?

Samples that should be collected without using viral transport media

Blood

Bone marrow

CSF

Amniotic fluid

Urine

Pericardial and pleural fluids

No need for VTM

does blood need VTM?

No need for VTM

does CSF need VTM?

No need for VTM

does Amniotic fluid need VTM?

No need for VTM

does Urine need VTM?

No need for VTM

does Pericardial and pleural fluid need VTM?

yes it is needed to use VTM

does Respiratory swab and tissue sample need VTM?

12 to 24 hrs

best hours to process viral specimens

store 4C no more than 5 days

If theres a delay store it on ? how many day?

Freeze st -70C 6 or more days

For 6 or more than 6 days delayed store it on?

-20C

Don't store viral specimens at ____ can form ice crystal that can disrupt host cell and result in loss of viral viability

Ice crystal

Don't store viral specimens at -20C because it can form ____ that can disrupt host cell and result in loss of viral viability

METHODS IN DIAGNOSTIC VIROLOGY

1.Direct detection

2.Nucleic acid based detection

3.Isolation or viruses

4.Serologic assays

DIRECT DETECTION METHODS MICROSCOPY

MICROSCOPY

Cytopathic Effect (CPE)

Direct Fluorescent Antibody (DFA) test

Enzyme Immunoassay (EIA)

Bright field light microscopy

Best for poxviruses; all other virus particles are too small to be seen

Electron microscopy

Greater magnification; can detect virions

Electron microscopy

Expensive, Labor intensive, not very sensitive

Electron microscopy

Rarely used in clinical laboratories

Electron microscopy

Suited for large teaching or research institutions

Electron microscopy

Useful to detect non culturable viruses

Cytopathic Effect (CPE)

Distinct and characteristics visual changes in infected cells

Cytopathic Effect (CPE):

Detected in cell scrapings from infected sites via bright field microscopy

Herpes simplex virus (HSV)

Cytopathic effect:Inclusion body

Cowdry Type A bodies

Intranuclear eosinophilic droplet like bodies

Cowdry Type A bodies

Intranuclear eosinophilic droplet like bodies

Intranuclear eosinophilic droplet like bodies

What does this mean?🔹 "Intranuclear" = Found inside the nucleus.🔹 "Eosinophilic" = Stains pink/red with certain dyes.🔹 "Droplet-like" = Appears as small, round droplets inside the nucleus.

Cowdry Type A bodies

What are they?🔹 Large, round inclusion bodies inside the nucleus of infected cells.🔹 Surrounded by a clear halo (empty space around them).

Human papillomavirus

Cytopathic effect: Inclusion body

HPV-associated koilocytes

Squamous cells with an enlarged nucleus surrounded by a non staining halo

HPV-associated koilocytes

Squamous epithelial cells (flat skin/mucosal cells) that are altered due to HPV infection.

HPV-associated koilocytes

Squamous cells with an enlarged nucleus surrounded by a non staining halo

Rabies virus

Cytopathic effect:Inclusion body

Negri bodies

Eosinophilic cytoplasmic inclusions in neurons

Negri bodies

What are they?🔹 Eosinophilic (pink/red-staining) cytoplasmic inclusions found inside neurons (nerve cells).🔹 Seen in the brain tissue of rabies-infected animals or humans.

Negri bodies

Eosinophilic cytoplasmic inclusions in neurons

Cytomegalovirus (CMV)

Cytopathic effect:Inclusion body

Owl eye inclusions

Owl eye inclusions

Owl eye inclusions

Direct Fluorescent Antibody (DFA) test

cells from a patient are fixed to a microscope slide and fluorescence labeled antibodies are added.

Direct Fluorescent Antibody (DFA) test

If viral antigens are present in the sample, the labeled antibody will bind and fluorescence will be seen microscopically.

Direct Fluorescent Antibody (DFA) test

DFA identities:

Adenovirus

Influenza viruses A and B

Measles virus

Parainfluenza viruses (PIV's) 1 through 4

Direct Fluorescent Antibody (DFA) test

test that detects

RSV from respiratory specimens

HSV-1 and 2

VZV from cutaneous lesion materials

Cytomegalovirus or CMV

Enzyme Immunoassay (EIA)

EIA test kits are available commercially

Enzyme Immunoassay (EIA)

Test that detects

RSV and Influenza A (Respiratory Specimens)

Hepatitis B virus and HIV-1 (serum or plasma)

Enteric Adenoviruses (stool)

HSV (Cutaneous lesions and conjuctivals swabs)

Enzyme Immunoassay (EIA)

test are often less sensitive than cell cultures or IF test so negative results are confirmed with cell culture or IF or nucleic acid based test

Enzyme Immunoassay (EIA)

Nucleic Acid Based Detection

Hybridization assay

PCR assays

Flow cytometry

Enzyme Immunoassay (EIA)

Faster TAT

More sensitive than cell culture and DFA

Enzyme Immunoassay (EIA)

Can be quantitative

Detect non culturable viruses

Enzyme Immunoassay (EIA)

Detect multiple viruses simultaneously

Characterize virus genetically

Enzyme Immunoassay (EIA)

Expensive

Need for specialized training

Enzyme Immunoassay (EIA)

More complex facilities

Lack FDA cleared assays

isolating viruses

In clinical virology,_____ is still the gold standard against which all other methods are compared.

3 methods for isolation of viruses:

Cell culture (most common)

Animal inoculation (extremely costly)

Embryonated eggs (rarely used)

Primary

Low passage

Continuous

CELLCULTURES divided into 3 categories

Primary viral isolation

A isolation that Obtained from tissue removed from animal

Primary viral isolation

A isolation that animal

Only minimal cell division occurs

Primary viral isolation

A isolation Cell viability is maintained via splitting or passaging

Primary viral isolation

cell lines can only be passaged a few times before new cells must be obtained

Primary cell lines

can only be passaged a few times before new cells must be obtained

Primary viral isolation

Primary monkey kidney (PMK) is exa,ple of this isolation

Finite or Diploid

Low passage is also called

Low passage or Finite or Diploid

Isolation that is Limited to 50 generations