Viral diagnostics eac for finals

VIRAL
DIAGNOSTICS
February 2012

Friday, March 2, 2012

REMINDER:
REMAINING SCHEDULE
Modiﬁcations:

PH Viruses
merged with
Emerging and
Re-emerging
Viruses
(March 2)


PLENARY GROUPS:
Advances in Laboratory Diagnostics (Then and Now)

• INFLUENZA (AH1N1 • HIV by Lao to Manahan
and SEASONAL FLU) by Group
Advincula to Bundang
Group • HEPATITIS A/B/C by
Pedregosa to Santos, Dana
• INFLUENZA (AVIAN Group
FLU) by Cailao to
Emerciana Group • SARS by Santos, Fatima to
Velasco Group
• DENGUE by Faderog to
Jamias Group


LABORATORY
DIAGNOSIS OF
VIRAL INFECTIONS


STORAGE AND COLLECTION OF
BIOLOGICAL SPECIMENS FOR
VIRAL TESTING


VIRAL TESTING
• What types of specimens are
collected to diagnose?


VIRAL TESTING

• Respiratory tract infections: Nasal
and bronchial washings, throat
and nasal swabs, sputum


VIRAL TESTING


• Eye infections: throat and
conjunctival swab/scraping


VIRAL TESTING



• Gastrointestinal tract infections:
stool and rectal swabs


VIRAL TESTING




• Vesicular rash: vesicle ﬂuid, skin
scrapings

VIRAL TESTING
• What types of specimens are • Maculopapular rash: throat, stool,
collected to diagnose? and rectal swabs




scrapings

VIRAL TESTING

• Respiratory tract infections: Nasal • CNS (encephalitis and meningitis
and bronchial washings, throat cases): stool, tissue, saliva, brain
and nasal swabs, sputum biopsy, cerebrospinal ﬂuid



scrapings

VIRAL TESTING


• Eye infections: throat and • Genital infections: vesicle ﬂuid or
conjunctival swab/scraping swab


scrapings

VIRAL TESTING



• Gastrointestinal tract infections: • Urinary tract infections: urine

scrapings

VIRAL TESTING



• Gastrointestinal tract infections: • Urinary tract infections: urine
• Bloodborne infections: blood
scrapings

General Categories

• Direct Examination
• Indirect Examination
(Virus Isolation)
• Serology


DIRECT
EXAMINATION


DIRECT: clinical specimen is
examined directly for the presence of virus
particles, virus antigen or viral nucleic acids

• Electron Microscopy morphology / immunoelectron
microscopy
• Light microscopy histological appearance - e.g.
inclusion bodies
• Antigen detection immunoﬂuorescence, ELISA etc.
• Molecular techniques for the direct detection of
viral genomes

ELECTRON MICROSCOPY
• BASIS: morphology

• MAGNIFICATION: 50,000

• USE:

• diagnosis of viral gastroenteritis by detecting viruses in faeces
e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-
like viruses

• detection of viruses in vesicles and other skin lesions, such as
herpesviruses and papillomaviruses

• NOTE: With the availability of reliable antigen
detection and molecular methods for the detection of
viruses associated with viral gastroenteritis, EM is
becoming less and less widely used

ELECTRON MICROSCOPY
• SENSITIVITY & SPECIFICITY ISSUES:

• sensitivity and speciﬁcity of EM may be enhanced by immune
electron microscopy

• virus speciﬁc antibody is used to agglutinate virus
particles together and thus making them easier to
recognize, or to capture virus particles onto the EM grid

• DISADVANTAGE:
• expense involved in purchasing and maintaining the facility
• poor sensitivity (at least 10 to 10 virus particles per ml in
5 6

the sample required for visualization)

• observer must be highly skilled

Electronmicrographs of viruses commonly found in stool specimens from patients suffering from gastroenteritis. From
left to right: rotavirus, adenovirus, astroviruses, Norwalk-like viruses. (Courtesy of Linda M. Stannard, University of
Cape Town, http://www.uct.ac.za/depts/mmi/stannard/emimages.html)

Inﬂuenza Virus

Ebola Virus


IMMUNOELECTRON
MICROSCOPY
PARAMYXOVIRUSES
Int. J. Morphol., 28(2):627-636, 2010

COXSACKIE B4 VIRUS
(http://www.rightdiagnosis.com)

NEW CASTLE DISEASE
(Veits J et al. PNAS 2006;103:8197-8202)

LIGHT
MICROSCOPY
• ASSUMPTION: Replicating virus often produce
histological changes in infected cells.

• Viral inclusion bodies: collections of replicating virus
particles either in the nucleus or cytoplasm

• EXAMPLES: negri bodies (RABIES) and cytomegalic
inclusion bodies (CYTOMEGALOVIRUS / CMV)

• Not sensitive or speciﬁc; BUT useful adjunct in the
diagnosis of certain viral infections


LIGHT
MICROSCOPY

RABIES
http://infectionnet.org’;
http://virology-online.com

CYTOMEGALOVIRUS
http://www.meddean.luc.edu
http://en.citizendium.org

ANTIGEN DETECTION
Immunofluorescence (IF)
• specimen: nasopharyngeal aspirates for respiratory viruses (e.g.. RSV, flu
A, flu B, and adenoviruses)

• specimen: stool (rotavirus)

• specimen: skin scrapings (HSV)

• specimen: serum (HepB)*

• Advantage: rapid to perform; result being available within a few hours

• Disadvantage: technique is often tedious and time consuming; result
difficult to read and interpret; sensitivity and specificity poor

• NOTE: quality of the specimen obtained is of utmost
importance in order for the test to work properly
• * also categorized as serological assay


ANTIGEN DETECTION

CYTOMEGALOVIRUS
http://www.med.upenn.edu


ANTIGEN DETECTION

VARICELLA ZOSTER VIRUS
BMJ Case Reports 2009; doi:10.1136/bcr.07.2008.0461

AFRICAN SWINE FEVER
Journal of General Virology March 2005; 86 (3)


ANTIGEN DETECTION
Molecular Probes

• Dot-blot and Southern-blot: use of specific DNA/RNA
probes for hybridization

• Specificity: depends on the conditions used for
hybridization

• Allow for the quantification of DNA/RNA present in the
specimen

• Sensitivity: not better than conventional viral diagnostic
methods.


ANTIGEN DETECTION
Molecular Probes

CYTOMEGALOVIRUS EPSTEIN-BARR VIRUS
http://www.fgsc.net
Mol Path 2000;53:255-261 doi:10.1136/mp.53.5.255


ANTIGEN DETECTION
Molecular Probes (PCR)
• extremely sensitive
technique (1 DNA
molecule in a clinical
specimen)

• ISSUES:
contamination (danger
of false + result); +
result may not
necessarily indicate the INFLUENZA
presence of disease http://www.nanohelix.net


ANTIGEN DETECTION
Molecular Probes (PCR)


INDIRECT
EXAMINATION


INDIRECT: the specimen into cell
culture, eggs or animals in an attempt to grow the
virus (virus isolation)

• Cell Culture - cytopathic effect, haemadsorption,
confirmation by neutralization, interference,
immunofluorescence etc.
• Eggs pocks on CAM - haemagglutination, inclusion
bodies
• Animals disease or death confirmation by
neutralization


RECALL:
Koch’s Postulates


RECALL: 1. Organism present only in
Koch’s Postulates diseased individuals


RECALL: 1. Organism present only in
Koch’s Postulates diseased individuals
2. Organism cultivated in pure
culture from diseased
individual


3. Organism causes disease
RECALL: when injected into
Koch’s Postulates healthy individuals


3. Organism causes disease
RECALL: when injected into
Koch’s Postulates healthy individuals
4. Organism re-isolated from
infected individual from
point 3.


MODIFICATION TO THE
KOCH’S POSTULATE
(T.M. River, 1937)

• Isolate virus from diseased hosts
• Cultivation of virus in host cells
• Proof of ﬁlterability
• Production of a comparable disease when the
cultivated virus is used to infect experimental animals
• Re-isolation of the same virus from the infected
experimental animal
• Detection of a speciﬁc immune response to the
virus

TYPES OF CELL CULTURE
• Primary cells - e.g. Monkey Kidney

• essentially normal cells obtained from freshly killed adult
animals; can only be passaged once or twice

• Semi-continuous cells - e.g. Human embryonic kidney and
skin fibroblasts

• taken from embryonic tissue; may be passaged up to 50 times

• Continuous cells - e.g. HeLa,Vero, Hep2, LLC-MK2, BGM

• immortalized cells i.e. tumour cell lines; may be passaged
indefinitely
NOTE: Primary cell culture are widely acknowledged as the best cell culture systems
available since they support the widest range of viruses BUT are very expensive and it is
often difficult to obtain a reliable supply. Continuous cells are the most easy to handle
BUT range of viruses supported is often limited

PRESENCE OF GROWING
VIRUS IS USUALLY
DETECTED BY:
• Cytopathic Effect (CPE) - may be specific or
non-specific e.g. HSV and CMV produces a specific
CPE, whereas enteroviruses do not
• Haemadsorption - cells acquire the ability to stick
to mammalian red blood cells
• mainly used for the detection of influenza and
parainfluenzaviruses.
NOTE: Confirmation of the identity of the virus may be carried out using neutralization,
haemadsorption-inhibition, immunofluorescence, or molecular tests


Detection of Herpes Virus Simplex 1
using the shell vial technique and
immunoﬂuorescence

Tissue culture cells are grown on coverslips on
the bottom of shell vials


Quantitative
Assays

Quantitative Plaque Assays


CYTOPATHIC EFFECTS


CYTOPATHIC
EFFECTS
• Visible results of viral infection
• Cell death by
• Multiplying viruses
• Inhibition of DNA, RNA or protein synthesis
• Effects on permeability of membrane

CYTOPATHIC
EFFECTS
• Cytopathic effects (CPEs) of infected cells can be observed with
inverted light microscopes

• Rounding/detachment from plastic ﬂask

• Syncytia/fusion (Fusion of cells)

• Shrinkage

• Increased refractility

• Aggregation

• Loss of adherence Cytopathic effect of HSV, enterovirus 71, and RSV in cell culture. Note the
ballooning of cells in the cases of HSV and enterovirus 71. Note syncytia
formation in the case of RSV. (Linda Stannard. University of Cape Town,
• Cell lysis/death
Virology Laboratory,Yale-New Haven Hospital)

Quantitative Assays
 Tissue Culture Infectious Dose: TCID50
 Measure cytopathic effects other than lysis
 Concentration of virus it takes to produce
cytopathic effect (CPE) in 50% of the dishes of
cells infected with virus


Quantitative Assays

TCID50 Assays

PROBLEMS WITH
CELL CULTURE
• Long period (up to 4 weeks) required for a result to be
available
• Sensitivity is often poor and depends on many factors,
such as the condition of the specimen, and the
condition of the cell sheet
• Very susceptible to bacterial contamination and toxic
substances in the specimen
• Many viruses will not grow in cell culture at all e.g.
Hepatitis B and C, Diarrhoeal viruses, parvovirus etc.

SEROLOGY


SEROLOGICAL TESTS
• ASSUMPTION:

• Primary Exposure: first antibody to appear is IgM, which is followed by a
much higher titre of IgG

• Secondary exposure or Re-infection: level of specific IgM either remain the
same or rises slightly But IgG shoots up rapidly and far more earlier than in a
primary infection.

• ASSAYS AVAILABLE:

• EIA and RIA, one can look specifically for IgM or IgG (most sensitive)

• CFT and HAI, one can only detect total antibody, which comprises mainly
IgG (not so sensitive)

• The sensitivity and specificity of the assays depend greatly on the antigen used

• Assays that use recombinant protein or synthetic peptide antigens tend to be
more specific than those using whole or disrupted virus particles

SEROLOGY: detection of rising titres
of antibody between acute and convalescent stages of
infection


NOTE: CRITERIA FOR
PRIMARY INFECTION

• A significant rise in titre of IgG/total antibody
between acute and convalescent sera

• however, a significant rise is very difficult to define and
depends greatly on the assay used

• CFT and HAI: normally taken as a four-fold or greater
increase in titre

• The main problem is that diagnosis is usually
retrospective because by the time the convalescent
serum is taken, the patient had probably recovered


NOTE: CRITERIA FOR
PRIMARY INFECTION

• Presence of IgM
• EIA, RIA, and IF may be used for the detection of IgM
• offers a rapid means of diagnosis
• PROBLEMS: interference by rheumatoid factor, re-
infection by the virus, and unexplained persistence of
IgM years after the primary infection


NOTE: CRITERIA FOR
PRIMARY INFECTION
• Seroconversion
• changing from a previously antibody negative state
to a positive state e.g. seroconversion against HIV
following a needle-stick injury, or against rubella
following contact with a known case
• A single high titre of IgG (or total
antibody)
• very unreliable means of serological diagnosis since
the cut-off is very difﬁcult to deﬁne

SEROLOGY: detection of rising titres
of antibody between acute and convalescent stages of
infection

• Classical Techniques • Newer Techniques
• Complement Fixation Test • Radioimmunoassay (RIA)
• Hemagglutination Inhibition • Enzyme Immunoassay (EIA)
Test
• Particle Agglutination Tests
• Immunoﬂuorescence
Technique (IF • Western Blot (WB)
• Neutralization Tests • Recombinant Immunoblot
assay (RIBA)
• Single Radial Hemolysis


LIMITATIONS OF SEROLOGY
• For viruses such as rubella and hepatitis A, the onset of
clinical symptoms coincide with the development
of antibodies (detection of IgM or rising titres of IgG in
the serum of the patient would indicate active disease)

• Many viruses often produce clinical disease before
the appearance of antibodies such as respiratory and
diarrheal viruses (any serological diagnosis would be
retrospective and therefore will not be that useful)

• There are also viruses which produce clinical disease
months or years after seroconversion e.g. HIV and
rabies (mere presence of antibody is sufﬁcient to make a
deﬁnitive diagnosis)

DISADVANTAGES OF SEROLOGY


• Long length of time required for diagnosis for paired acute and
convalescent sera


convalescent sera

• Mild local infections may not produce a detectable humoral immune
response (e.g. HSV genitalis)


convalescent sera


• Extensive antigenic cross-reactivity between related viruses may lead to
false positive results (e.g. Japanese B encephalitis and Dengue)


convalescent sera



• Immunocompromised patients often give a reduced or absent humoral
immune response


convalescent sera



immune response

• Patients with infectious mononucleosis and those with connective tissue
diseases such as SLE may react non-speciﬁcally giving a false positive
result


convalescent sera



immune response

• Patients with infectious mononucleosis and those with connective tissue
diseases such as SLE may react non-speciﬁcally giving a false positive
result

• Patients given blood or blood products may give a false positive result
due to the transfer of antibody

HEMAGGLUTINATION/
HEMAGGLUTINATION-INHIBITION/
COMPLEMENT FIXATION TEST


• Some viruses
agglutinate RBCs
• Mumps, measles,
inﬂuenza
• Hemagglutination
• Clumps RBCs


HEMADSORPTION TEST

Hemadsorption of red blood cells onto the surface of a
cell sheet infected by mumps virus (Courtesy of Linda
Stannard, University of Cape Town).

ENZYME
-LINKED
IMMUNOSORBENT

ASSAY
(ELISA)


HIV & ELISA


WESTERN BLOT TO
CONFIRM HIV


(b) (c)
Image courtesy of Bio-Rad Laboratories

Figure 5.21c: The typical results of a Western blot
Figure 5.21b: The structure of HIV-1.
testing patient serum for HIV-1 antibodies.


SEROLOGY AND
HEPATITIS


USUALLY DIAGNOSED
BY SEROLOGY
• Hepatitis Viruses - hepatitis A, B and C infections
are usually diagnosed by serology as these viruses
cannot be routinely cultured
• including the test for HBsAg
• HIV - HIV infection is normally diagnosed by
serology
• The only instance when serology cannot be relied
on is in diagnosing HIV infection in the newborn

USUALLY DIAGNOSED
BY SEROLOGY
• Rubella and parvovirus - rubella and parvovirus
infections are usually diagnosed by serology

• difﬁcult to isolate and parvovirus cannot be isolated by
routine cell culture

• onset of clinical symptoms for these infections coincide
with the appearance of antibodies and thus there is little
need for other means of diagnosis

• EBV - although EBV serology is reliable, the heterophile
antibody test is usually used for diagnosing cases of
infectious mononucleosis

MAY BE DIAGNOSED BY SEROLOGY
BUT NOT METHOD OF CHOICE
• HSV - although CFT and other serological tests are available for
HSV, HSV infections are usually diagnosed by cell culture

• Electron microscopy, immunoﬂuorescence and PCR are available
as rapid diagnostic methods

• Serology is not that reliable in the case of HSV infections, in
particular reactivations

• CMV - although serology is available for diagnosing CMV infections,
it is not reliable as most cases of CMV infections are a result of
reactivation/reinfection

• Cell culture (including the DEAFF test) and rapid methods such as
the CMV antigenaemia test and PCR are preferred means of
diagnosis


• Respiratory viruses - diagnosis of respiratory virus
infections is more commonly made by cell culture or more
rapidly by immunoﬂuorescence of the clinical material

• CFT and HAI techniques are usually used for serology and
any diagnosis is going to be retrospective

• Enteroviruses - enterovirus infections are usually
diagnosed by cell culture

• Serology has a very limited role to play as available tests
such as neutralization, are cumbersome to perform and in
any case, the diagnosis would be retrospective



• Rabies - serology is used along with other direct
detection methods in diagnosing rabies and it may be used
to check for immunity after vaccination

• Arboviruses - arbovirus infections may be diagnosed by
serology or virus isolation

• Arboviruses will not usually grow in routine cell
cultures and may require mosquito cell lines or animal
inoculation.


NOT NORMALLY DIAGNOSED
BY SEROLOGY
• Diarrhoeal viruses - diagnosis is going to be
retrospective
• normally diagnosed by electron microscopy and
the detection of viral antigens by ELISA or particle
agglutination
• Papovavirus - serology is of virtually no value in
diagnosing papovavirus infections
• Poxviruses - serology is of little value in diagnosing
poxvirus infections

SAFETY FIRST!

Figure 5.25b: A CDC researcher working on
a BSL-4 infectious agent.

Figure 5.25c: A CDC scientist showers
in a protective suit before leaving a
BSL-4 laboratory.

QUIZ TIME!

• Draw the general serologic course of a viral
disease/infection and label properly (6 points)
• Give one direct examination to diagnose a
given viral pathogen and cite one virus that can
use this method for diagnosis (8 points)
• Give one indirect examination to diagnose a
given viral pathogen cite one virus that can use
this method for diagnosis (8 points)
• Give one serological examination to diagnose a
given viral pathogen cite one virus that can use
this method for diagnosis (8 points)

Viral diagnostics eac for finals

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