Transcript Diagnostic and Laboratory Aspects of Japanese Encephalitis

Instructions for users
• This slide presentation provides an overview of the
diagnostic and laboratory testing aspects of JE.
• Below many of the slides, there are notes to explain
the information in the slide.
• You should adapt the presentation so it is relevant to
your audience in your country.
• This presentation is very long if used in total. Different
sections could be presented by different presenters or
in different sessions.
• Provide examples of the actual materials (e.g.,
specimen tubes) and include practical sessions when
possible. Suggestions are provided.
Diagnostic and Laboratory
Aspects of
Japanese Encephalitis
Learning Objectives
Participants will:
• Understand how diagnostics can enhance
surveillance for JE.
• Review different tests that can be done to
diagnose JE infection.
• Revise principles of MAC ELISA testing.
• Know types of specimens that can be tested.
• Be familiar with the collection, storage and
transport requirements for specimens.
Why do we need diagnostics?
• JE surveillance is based on surveillance for cases of
acute encephalitis syndrome (AES).
• AES has
—
Many potential agents (e.g., JE, herpes simplex, Ebstein
Barr viruses).
—
Several epidemic agents (e.g., JE, Enterovirus 71, Nipah
viruses).
• There are no specific signs or symptoms that
distinguish JE from other causes of AES, so
laboratory testing is needed to determine if JE is the
cause of illness.
How can you use diagnostic
information?
• Allows you to determine the proportion of all
encephalitis (AES) cases that are due to JE.
—
e.g., to estimate the specific disease burden from
JE
• Allows you to determine if any of the
encephalitis (AES) cases are due to JE.
—
e.g., is an outbreak of encephalitis due to JE or
another agent?
Different countries, different
stages, different purposes (1)
No previous JE control (e.g., Cambodia)
• Aim of surveillance
—
Determine disease burden from JE.
• Strategy
—
Monitor AES cases nationwide.
—
Use diagnostics in certain sentinel sites.
—
Estimate proportion of all AES cases due to JE.
Different countries, different
stages, different purposes (2)
Introduced control program for JE (e.g.,
China)
• Aim of surveillance
—
Monitor progress towards control of disease.
—
Identify at-risk areas and populations that require
further interventions.
• Strategy
—
Confirmatory test required for most cases.
Laboratory criteria for diagnosis:
WHO surveillance guidelines for JE
Laboratory confirmation of a JE virus infection includes:
1.
Presence of JE virus-specific IgM antibody in a single sample of cerebrospinal fluid (CSF)
or serum as detected by an IgM-capture ELISA specifically for JE virus;
OR
2.
Detection of JE virus antigens in tissue by immunohistochemistry;
OR
3.
Detection of JE virus genome in serum, plasma, blood, CSF, or tissue by reverse
transcriptase PCR or an equally sensitive and specific nucleic acid amplification test;
OR
4.
Isolation of JE virus in serum, plasma, blood, CSF, or tissue;
OR
5.
A four-fold or greater rise in JE virus-specific antibody as measured by hemagglutination
inhibition (HI) or plaque reduction neutralization assay (PRNT) in acute and convalescentphase serum samples. The samples should be collected 14 days apart.
Options for diagnostic tests in humans
• Isolation, amplification, or antigen detection?
–
Viraemia in JE is low and short so the likelihood of a
positive result is low.
–
High technology requirement and expensive.
• Antibody-based (serology) methods?
–
High sensitivity (if properly timed samples).
–
Options include PRNT, ELISA, and HI.
Antibody-based (serology) methods
• Plaque reduction neutralization assay (PRNT)
—
Least cross-reactivity; most specific.
—
Up to 14 days to complete test.
—
Costly
—
High biosafety level requirements.
—
Early acute phase specimen less likely to test positive with
PRNT than ELISA
—
Requires acute- & convalescent-phase serum.
• Haemagglutination Inhibition (HI)
—
High levels of cross-reactivity with other flaviviruses, low
specificity.
—
Anamnestic response with secondary flavivirus infection.
Antibody-based (serology) methods (2)
• JE-specific IgM capture ELISA
—
Advantages
– Simple.
– Sensitivity of IgM detection good as competition by IgG
molecules eliminated.
—
Disadvantages
– Some cross-reactivity with other flaviruses.
Summary: ELISA is most feasible testing methodology
Equipment for performing the
ELISA test
Pipettes
ELISA reader
Incubator
How does the JE IgM capture
enzyme linked immunosorbent
assay (ELISA) work?
Reporter molecule
Y
Anti-EJEV Mab
*
Japanese encephalitis E protein
Y
Sample containing human IgM
Y
Anti-human IgM
Y
Y
Y
*Y Y*
Y
*Y
Y
Y
*Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Type of specimens (1)
• CSF
—
Preferred sample whenever possible.
• SERUM
—
Most common sample for testing.
Type of specimens (2)
• Serum
—
Easy to collect.
—
A JE IgM positive result
from the serum of a patient
with encephalitis is a good
indicator of acute infection
(even though there is a
problem of some crossreactivity with other
flaviviruses such as dengue
virus).
Type of specimens (3)
• CSF
—
The finding of JE IgM in CSF confirms the
diagnosis of JE.
—
IgM to JE virus rises earlier in CSF than in
serum and rises to higher levels in CSF
than in serum (2 to 4 times)
—
Whenever possible when CSF is collected
for management purposes, an additional
tube should be collected for ELISA testing.
Additional tests
Further confirmatory tests (testing for other
flaviviruses that circulate in the area) should
be carried out if:
• There is circulation of dengue virus in
addition to JE.
• There is a suspected case in an area not
having data supportive of JE transmission.
• When JE vaccination coverage is very high.
Timing of specimen collection
• First specimens (blood and CSF) should be
collected on admission to hospital or when
patient first seen.
• A follow up specimen (blood) should be
collected at least 10 days after onset (or
before discharge or death).
Rationale for timing of specimen
collection
• IgM antibody levels rise steadily after onset of
encephalitis.
• The percentage of patients with IgM
detectable in serum increases with days after
onset.
Study: the kinetics of IgM and IgG
Percent of patients with JE IgM
120
Percentage
100
80
Serum
60
CSF
40
20
On admission,
only 53% of
serum specimens
and 68% CSF
specimens were
positive.
0
Day 1
Day 7
Day 30
Days after admission
Kinetics of IgM and IgG responses to JE virus in human serum and
cerebrospinal fluid. Burke DS, Nisalak A, Ussery MA et al
Journal of Infectious Diseases 1985. 151: 1093-1099
Therefore, it is important to collect a follow
up blood specimen at least 10 days after
onset (or approximately 7 days after
admission) or before discharge or death.
Typical course of acute
JE infection:
presence of virus and viral
markers
Encephalitis onset
Concentration
Concentration
Concentration
Concentration
JE virus in blood
Infection
Day 4- 6 illness
Encephalitis onset
Incubation: 5 - 15days
JEV - Blood
Concentration
Concentration
JEV - CNS & tissue
Infection
D4 – D6 illness
Encephalitis onset
Incubation: 5 - 15d
D14 – D21 illness
JEV - Blood
JEV - CNS & tissue
Concentration
Concentration
Serum & CSF IgM Ab
Infection
D4 – D6 illness
Encephalitis onset
Incubation: 5 - 15d
D14 – D21 illness
1Y after illness
JEV - Blood
JEV - CNS & tissue
Concentration
Concentration
Serum & CSF IgM Ab
IgG, HI & Neut Ab
Infection
D4 – D6 illness
Encephalitis onset
Incubation: 5 - 15d
D14 – D21 illness
1Y after illness
Collection and processing of specimens
Step 1: Complete forms and
specimen identification
• Fill in the information on tube (identifying
information, date of collection, and other
information as required).
• Fill in the laboratory form that will accompany
specimens.
Collection and processing of serum
• Collect blood in a tube that does not contain any
chemicals or anticoagulants.
• Collect 5mL of whole blood (for very small children
collect 1mL).
• Place tube upright for 30-60 minutes then when firm
clot has formed, centrifuge tube for 20 minutes at
2500rpm.
• Remove serum with a pipette and place in a plastic
storage tube (2-3mL microtube or cryovial).
• If 5mL of blood was collected it will result in about
2mL of serum.
Collection of CSF for JE ELISA
testing
• Collect 2mL of cerebrospinal fluid into a
sterile, plastic microtube or cryovial.
• Place in the refrigerator or, if ELISA testing
will not occur within 7 days, place
immediately in the freezer.
2.0 ml microtubes
OR
2.0 ml cryovials
Storage of specimens
Principles of storage of
serum and CSF (1)
• Both serum and CSF samples should be
stored in microtubes or cryovials.
• If a serum or CSF sample can be tested
within 7 days, it should be refrigerated until
tested (4°C).
• If it will be tested after more than 7 days, it
should be frozen (-20°C) (-70°C also
acceptable for serum)
Principles of storage of
serum and CSF (2)
• Periodically check the temperature of the refrigerator.
• There should be a constant electricity supply to the
refrigerator and freezer; if possible, have generator back
up.
• If electricity fails for longer than 2 to 3 hours during the
storage of samples, the date and length of time of the
power failure should be recorded.
• Do not use the refrigerator/freezer for storage of other
goods such as food or drink.
Transport of specimens
Principles of transport of
serum and CSF specimens
• The sample should be kept cold during transport
and arrive at the testing lab cold (or frozen if it
has been previously frozen).
• The transport time should be kept to a minimum.
• Ensure samples will arrive at the testing
laboratory when a staff member can receive
them and immediately put them into cold
storage.
• Ensure all forms with patient information are
complete and sent with the specimen.
Options for transport
• Cold box with dry ice.
• Container filled with liquid nitrogen.
• Cold box with wet ice.
Transport of serum and CSF
specimens
• Tubes (with lids tightly closed) should be packed with
absorbent material between tubes (e.g., cotton wool) in a
sealed container (e.g., 250-500mL screw top plastic
container or can)
• These containers and the dry ice (or wet ice) are then
packed in outer packaging.
• If ice is used, the outer package must be leak-proof.
• If dry ice is used, the outer package must permit the
release of carbon dioxide gas.
Rules to follow to avoid problems
with specimen quality
• If a serum or cerebrospinal fluid sample is
being stored frozen, keep it frozen until it is
thawed for immediate testing. Repeated
freezing and thawing will damage the
antibody.
• Do not keep serum and cerebrospinal fluid
samples for JE testing in warm conditions
(such as a warm room) for more than 2 to 3
hours.
Summary of requirements
Specimen Timing of
collection
Processing Storage
Transport
Serum
Admission
and 10
days after
onset
Collect
blood, allow
clot to form,
centrifuge,
collect
serum
Refrigerator Cold or
or freezer
frozen (dry
or wet ice)
(depending
on when
testing will
occur)
CSF
Admission
-
Same as
serum
(above)
Same as
serum
(above)
Commercial JE diagnostics:
Where are we now?
• There are several commercial ELISA diagnostic
kits that are being evaluated for sensitivity,
specificity and usability.
• The advantages of commercial ELISA kits include
—
No long blocking or incubation steps.
—
Shorter time to run tests (4 to 8 hours).
Acknowledgements
Please include the following acknowledgement
if you use this slide set:
This slide set was adapted from a slide set
prepared by PATH’s Japanese Encephalitis
Project.
For information: www.JEproject.org