Midterms 04. Laboratory Diagnosis of Viral Infections

target organ(s)

clinical symptoms of diseases often point to the ___ involved, which can help determine the most appropriate specimen/s to collect

• blood (buffy coat)

• CSF

• portals of entry (oral or respiratory tract)

• portal of exit (urine or stool)

for systemic, congenital, or generalized disease: specimen from multiple sites are appropriate

enteroviruses

• cause respiratory infections

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

• major cause of aseptic meningitis

• can also be isolated from urine specimens

early

best specimens are collected as ____ as possible

aseptically

specimens should be collected

aspirated

secretions preferred

dacron or rayon

swabs are easier to use and must be made of

• calcium alginate

• charcoal

• swabs w/ wooden shafts

do not use

moist

tissue samples must be kept moist

• viral transport medium

• saline

• trypticase soy broth

help keep tissue samples moist

• buffered isotonic solution w/ protein

• antibacterial & antifungal agents

several viral transport systems commercially available contains:

• respiratory

• swab

• tissue samples

samples that can be collected w/ viral transport media

• blood

• bone marrow

• CSF

• amniotic fluid

• urine

• pericardial and pleural fluids

samples that should be collected w/o viral transport media

12-24 hours

process viral specimens for culture immediately, at best within ____ of collection

4 deg C, <5 days

if there is delay, store specimens at

freeze @ -70 deg c, ≥6 days

for longer delays, store specimen at

ice crystals

• which will disrupt the host cells and result in loss of viral viability

• formed @ -20 deg C

-20 deg C

specimens should never be stored @ what temperature bc this is when ice crystals form

1. direct detection

2. nucleic acid-based detection

3. isolation of viruses

4. serologic assays

methods in diagnostic virology

• microscopy

• enzyme immunoassays

direct detection of the virus in clinical specimens

serologic assays

used to detect antibodies to virus

bright field light microscopy

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

electron microscopy

with greater magnification → can detect virions

electron microscopy

useful to detect non-culturable viruses

cytopathic effect

distinct and characteristic visual changes in infected cells

bright field microscopy

cytopathic effect is detected in cell scrapings from infected sites via

Herpes simplex virus

cytopathic effect: inclusion body—Cowdry Type A bodies

Cowdry Type A bodies

intranuclear eosinophilic droplet like bodies

Human papillomavirus

cytopathic effect: inclusion body—HPV-associated koilocytes

HPV-associated koilocytes

squamous cells with an enlarged nucleus surrounded by a nonstaining halo

Rabies virus

cytopathic effect: inclusion body—Negri bodies

Negri bodies

eosinophilic cytoplasmic inclusions in neurons

Cytomegalovirus

cytopathic effect: inclusion body—owl eye inclusions

owl eye inclusions

inclusion body seen in Cytomegalovirus

Measles virus

cytopathic effect: multinucleated giant cell (syncytia) and Warthin-Finkeldey cell

syncytia

multinucleated giant cell

• multinucleated giant cell (syncytia)

• Warthin-Finkeldey cell

cytopathic effect seen in Measles virus

Direct Fluorescent Antibody tests

• cells from a patient are fixed to a microscope slide

• fluorescence-labeled antibodies are added

• if viral antigens are present, the labeled antibody will bind → fluorescence will be seen microscopically

fluorescence

in DFA tests, if antigens are present → labeled antibody will bind → ?? will be seen microscopically

respiratory specimens

DFA from what specimen

• adenovirus

• influenza virus (A & B)

• measles virus

• parainfluenza viruses 1-4

• respiratory syncytial virus

cutaneous lesion material

DFA from what specimen

• Herpes simplex virus 1 & 2

• Varicella zoster virus

blood

DFA from what specimen

• cytomegalovirus

Enzyme immunoassays

detects:

• RSV & Influenza A

• Hepa B

• HIV-1

• Enteric Adenoviruses

• HSV

respiratory specimens

EIA from what specimen

• RSV & Influenza A

serum or plasma

EIA from what specimen

• HBV

• HIV-1

stool

EIA from what specimen

• enteric adenoviruses

cutaneous lesions & conjunctival swabs

EIA from what specimen

• HSV

Enzyme immunoassay

often less sensitive than cell cultures or IF tests

• cell culture

• IF

• nucleic acid-based tests

EIA negative results are confirmed with

Nucleic Acid-Based Detection

• Hybridization assay

• PCR assays

• Flow cytometry

• faster TAT

• more sensitive

• quantitative

• detect

  • non-culturable viruses

  • multiple viruses simultaneously

• characterizes virus genetically

viral isolation

gold standard against which all other methods in clinical virology

• cell culture (most common)

• animal inoculation (costly)

• embryonated eggs (rarely used)

3 methods for isolation of viruses

• primary

• low passage (finite)

• continuous

cell cultures can be divided into 3 categories

primary cell cultures

obtained from tissue removed from an animal

splitting or passaging

cell viability is maintained via

primary monkey kidney (PMK) cells

example of a commonly used primary cell culture

low passage (finite or diploid)

• limited to 50 generations

• must have at least 75% of cells with same karyotype as normal cells

• W1-38

• MRC-5

examples under low passage (finite or diploid)

continuous (heteroploid or immortal)

• infinite passage

• <75% normal cells, >25% of cells possess abnormal karyotype

• malignant tissues

• HeLa

• Hep-2

• KB

• A-549

• Vero

examples under continuous (heteroploid or immortal) cell culture

cytopathic effect

• inspection of cultures

• indicated by areas of dead or dying cells

inverted light or phase contrast microscope (LPO)

used to view cytopathic effect

• rounding

• fusion or syncytial formation

• destruction & lysis

• clumping

• giant multinucleated cells

• vacuolation

• granulation

cytopathic effect include

unstained monkey kidney cell culture

• shows cell monolayer

• advanced enteroviral cytopathic effect

  • cell rounding

  • cell detachment

  • necrosis

HSV-1-infected Vero cells

• rounded cells

• multinucleated cells

• loss of monolayer

• w/ syncytia

• hemadsorption

• hemagglutination

little or no detectable CPE

hemagglutination

viruses produce virus-specific hemagglutinins in the monolayer which combine w/ RBCs of certain animals

(+) plaques of RBC

seen in hemagglutination

Influenza A & B

possess hemadsorption and hemagglutination

• parainfluenza

• mumps

exhibit hemadsorption

hemadsorption

adherence of RBC to surface of cells

• ELISA

• Western Blot

• Complement fixation

• Indirect immunofluorescence

serological tests

serological tests (serology)

diagnosis of infections w/ nonculturable agents: HBV

serology

determination of immune status: rubella, measles, VZV, HAV, HBV

serology

monitoring of patients who are immunosuppressed or have had transplants

serology

used in epidemiologic or prevalence studies