S2-1 NewDiagnosticTools_ProfFooks

New Diagnostic Tools for Rabies in Animals
Anthony R. Fooks
(1)
(2)
AHVLA, Weybridge, UK
OIE Reference Laboratory for Rabies
WHO Collaborating Centre for Rabies
University of Liverpool, Liverpool, UK
Overview
I.
OIE resolutions
I.
II.
II.
III.
IV.
1st International Conference on rabies in
Europe held in Kiev Ukraine - June 2005
Towards the Elimination of Rabies in Eurasia
held in Paris France - May 2007
Collection and storage of samples
OIE prescribed tests for rabies
Alternative tests for rabies
iii.
(non-prescribed)
1st International Conference on rabies in Europe (Kiev Ukraine - 2005)
Towards the Elimination of Rabies in Eurasia (Paris France - 2007)
Resolutions – rabies diagnosis:
• Clinical diagnosis of rabies is not reliable;
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•
OIE reference laboratories and WHO collaborating centres work together;
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•
International harmonisation of laboratory methods for diagnosis;
The recommended primary diagnostic test for rabies is the fluorescent
antibody test;
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A definitive diagnosis can only be made by laboratory investigations;
Confirmatory diagnosis, where required, should be undertaken using the rabies
tissue-culture infection test;
The mouse inoculation test should only be used if tissue culture is not available;
The use of the polymerase chain reaction and other amplification techniques is not
currently recommended for the routine diagnosis of rabies;
Serological methods should not be used for routine rabies diagnosis;
–
There is a requirement for rapid and accurate serological methods (i.e. ELISA
tests) to replace currently used virus neutralisation tests.
First International Conference on Rabies in Europe (2006) B. Dodet, A. Schudel, P-P. Pastoret, M. Lombard (eds).
Developments in Biologicals 125.
OIE Terrestrial Manual; WHO Laboratory Techniques in Rabies.
Rabies is an under-reported disease
• In a cohort of 133
children with CNSrelated diseases
– 14 (10.5%) – rabies
• Three of 26 (11.5%)
clinically diagnosed
cases of cerebral
malaria were laboratoryconfirmed as rabies
Mallawa et al., 2007
Objectives:
• Use of standardized diagnostic tests with regular interlaboratory tests in compliance with OIE standards
– Robust / Accurate
• High specificity / sensitivity
– Cheap
• Affordable
– Tests that do not involve the use of expensive
equipment / specialised laboratories
– Tests that are easy to use
• Tests that must be available and cost-effective for use
in rabies-endemic countries
Diagnostic testing
SPEED
QUALITY
COST
Overview
I.
OIE resolutions
I.
II.
II.
III.
IV.
1st International Conference on rabies in
Europe held in Kiev Ukraine - June 2005
Towards the Elimination of Rabies in Eurasia
held in Paris France - May 2007
Collection and storage of samples
OIE prescribed tests for rabies
Alternative tests for rabies
iii.
(non-prescribed)
Collection of samples: occipital foramen
route for brain sampling from animals
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•
•
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Ideal for use in the field;
Useful in an outbreak situation;
Less invasive procedure than the
traditional method of brain
removal;
More testing to be done in the
laboratory
Specifically targets 3 regions
confirmed to show initial rabies
infection -Medulla, Cerebellum,
Hippocampus;
Once the sample is in glycerol, it
will not require refrigeration during
transit to the laboratory;
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•
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•
Barrat. WHO Laboratory Techniques in Rabies, 4th Edition
RNA still degrades unless at 4oC
Requires less equipment that the
standard method of brain removal;
Multiple samples dealt with much
faster;
Much safer procedure for
laboratory staff.
FTA cards: Advantages and benefits
Whatman FTA devices format
•
•
•
Capture nucleic acid in one step;
Captured nucleic acid is ready in
less than 30 minutes;
FTA Cards are stored at room
temperature;
– DNA collected on FTA Cards
is preserved for years at room
temperature;
•
FTA Cards change colour upon
sample application to facilitate
handling of colourless samples.
Electron micrograph showing DNA
entrapped within the FTA matrix
(Magnification x 10,000)
http://www.whatman.com
Picard-Meyer et al., 2007
Overview
I.
OIE resolutions
I.
II.
II.
III.
IV.
1st International Conference on rabies in
Europe held in Kiev Ukraine - June 2005
Towards the Elimination of Rabies in Eurasia
held in Paris France - May 2007
Collection and storage of samples
OIE prescribed tests for rabies
Alternative tests for rabies
iii.
(non-prescribed)
Commonly used OIE prescribed tests for rabies
Test
Abbrev.
Use
Unit Cost
Turn-around
time
Sensitivity %
Specificity %
FAT for
Rabies
Confirmatory test
Cheap
2-4 hours
Medium
High
RTCIT virus
isolation
Confirmatory test
Moderate
5 days
High
Medium
Mouse
inoculation
test
‘Gold Standard’
Confirmatory test
Expensive
28 days
High
Medium
Measuring
Antibody levels
Moderate
3 days
High
High
RT-PCR
Screening
Cheap
2 days (inc
RNA
extraction)
High
High
Taqman RealTime RT-PCR
Sequencing/
phylogenetics
Screening
Cheap
1 day (inc RNA
extraction)
High
High
Fluorescent
Antibody
Virus
Neutralisation
Test
AHVLA
OIE prescribed tests for rabies
Routine laboratory Tests
•
Histological identification of characteristic cell lesions
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Immunochemical identification of rabies virus antigen
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Detection of „Negri bodies‟ [no longer recommended]
Fluorescent antibody test (FAT)
Detection of replication of un-inactivated rabies virus after
inoculation
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Virus isolation
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Rabies tissue-culture infection test (RTCIT)
Mouse inoculation test (MIT)
Current methods for rabies serology (antibody detection)
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Rapid fluorescent focus inhibition test (RFFIT)
Fluorescent antibody virus neutralisation test (FAVN)
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Gold standard test
Determination of rabies virus-specific antibodies
Evaluation of ORV programmes
International trade in companion animals
Seroconversion following rabies pre-vaccination
FAVN: Smith et al., 1973; Zalan et al., 1979; Perrin et al., 1985; Cliquet et al., 1998Cliquet et al., 2004;
Servat et al., 2007;
OIE Terrestrial Manual
Direct Microscopic Examination Detection of ‘Negri bodies’
•
Negri bodies (H&E)
– Acidophilic staining reaction
– Pink / purplish / magenta
colour
•
Negri body
– Identified within the
cytoplasm of the neuron
•
•
•
•
•
•
WHO Laboratory Techniques in Rabies
Poor specificity
Low sensitivity, especially in
autolysed samples
Screening tool
Relatively cheap
No need for expensive
equipment or reagents
No longer recommended if
other OIE-prescribed tests are
available
Fluorescent Antibody test (FAT)
• „Gold-standard‟ test
• Antigen detection
• Brain smear
– Hippocampus, medulla,
cerebellum
• Apple green staining
• <2 hrs
• Fresh tissue should be
examined, whenever
possible
• 99% agreement between
FAT and MIT
– Sensitivity 90 – 100%
– Sensitivity reduced in
autolysed samples
Goldwasser and Kissling, 1959; Dean and Abelseth, 1973; Bourhy et al., 1989;
OIE Terrestrial Manual; WHO Laboratory Techniques in Rabies.
Rabies tissue-culture infection test (RTCIT)
• Neuroblastoma or Baby Hamster kidney cells
inoculated with brain homogenate
• Incubated for 24hrs, supernatant removed and
incubated for further 72hours
• Fixed in acetone and stained with fluorescent
antibody
Mouse inoculation Test (MIT)
• Only recommended where tissue culture is not
available
• Inoculation of brain homogenate into
anaesthetised 3-4 wk old mice
• Observe for at least 28-days
• Any deaths 5-28 days confirm by FAT
Evaluation of FAT, MIT and DME
• 337 dog brains analysed by RITM Philippines
• Using MIT as reference test:
– DME
• 9 (9.5%) false-negative
• 3 (3.4%) false-positive
– FAT
• 1 (1.1%) false-negative
• 1 (1.1%) false-positive
• FAT more sensitive than DME
– (99.9% versus 90.5%)
• FAT more specific than DME
– (99.6% versus 98.8%)
Robles and Miranda, 1992
Overview
I.
OIE resolutions
I.
II.
II.
III.
IV.
1st International Conference on rabies in
Europe held in Kiev Ukraine - June 2005
Towards the Elimination of Rabies in Eurasia
held in Paris France - May 2007
Collection and storage of samples
OIE prescribed tests for rabies
Alternative tests for rabies
iii.
(non-prescribed)
Rabies serology
• Recent developments - not
prescribed
(antibody detection)
– Enzyme linked
immunoadsorbent assay
(ELISA)
• Lack of correlation with Gold
Standard serological assays
• Sensitivity poor near cut-off
– Pseudotype assay
– Rapid serological
immunochromatographic test
(ICTS)
Wright et al., 2008, 2009, 2010; Wang et al., 2010
OIE Terrestrial Manual; WHO Laboratory Techniques in Rabies
Pseudotype construction
2
1
Harvest virus and titrate on
target cells
3 plasmid transient
transfection into 293T cells
3
Rabies G-protein
Measure the NAb titre
of serum using GFP
or luciferase readout
108
106
HIV Gag and Pol
Vector encoding GFP/Luc
104
102
100
Summary
• Developed a virus neutralisation assay capable of
measuring rabies virus-specific antibodies
– Principle based on knockdown of GFP expression
• No need for expensive conjugate
– Use of reporter genes such as GFP or β-galactosidase will
allow the assay to be undertaken at low cost
• No need for the use of live rabies virus
• Each assay requires <10μl of serum
• High Spe / Sen
•
CVS-11 pseudotype assay had 100% concordance
with FAVN
• Test not yet validated – for research purposes only
– Ring trial in progress
Wright et al., 2008; 2009; 2010
Rapid serological immunochromatographic
test (ICTS)
 Similar to a pregnancy test
 Quick detection of rabies virus antibody
 No specialized equipment or infrastructure
required
 Potential for ready-to-use field test
Wang et al., 2010
Alternative diagnostic tests for rabies
• Histological identification of characteristic cell
lesions
– Immunocytochemisty (ICC)
– General pathology
• Intracellular spaces - vacuolation of Pukinje cells
• In-situ hybridization (ISH)
• Antigen detection based assays
– Direct immuno-histochemical test (dRIT)
– Immunochromatographic tests
• Rabies immunochromatographic diagnostic test (RIDC)
• Nucleic-acid based assays
– NASBA
– RT-PCR TaqMan
– RT-LAMP
Histological tools for rabies
Murine hippocampous (ICC)
- Mab staining of cortex
ICC
• Screening tool
• Relatively cheap
• No need for expensive
equipment or reagents
• Detection of antigen
• Poor specificity
• Sensitivity reduced in
autolysed samples
Vaculation
• Non-specific
• Poor sensitivity
Vaculation of bovine Purkinje cells
In-situ hybridization (RNA detection)
ISH DIG labelled genomic RNA staining of
periform cortex and accessory basal amygdaloid
nucleus
ISH DIG labelled messenger RNA staining of periform
cortex and accessory basal amygdaloid nucleus
Finnegan et al., 2004
Direct Immunohistochemistry test (dRIT)
dRIT Analysis
rabies
negative
rabies
positive
• Detects antigen
• Sensitivity and specificity
equivalent to the DFA /
FAT (100%)
• Frozen and glycerolpreserved brain samples
• No specialised equipment
or infrastructure required
• Ideal for use in
developing countries,
especially under field
conditions
(A) dRIT
(B) FAT
Lembo et al. 2006; Niezgoda et al., 2009
Penside technology: Human pregnancy tests
•
Proof-of-principle
– Point-of care testing
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Slide courtesy of Andrew Soldan (AHVLA, UK)
Gold standard for point of
care testing
Un-trained user
Rapid – 1 to 3 minutes
>99% accurate
Informative
Cheap (<US$3 / test)
Rabies immunochromatographic diagnostic
test (RIDC)
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Lateral flow device (LFD) - penside
Similar to a pregnancy test
Quick detection of rabies virus antigen
No specialized equipment or infrastructure required
Potential for ready-to-use field test
+
Direction of flow
α Flc
α Btn
Kang et al.. 2007; Markotter et al., 2009
Molecular techniques
• Real-time PCRs
• Nucleic acid sequence-based amplification (NASBA)
• Loop-mediated isothermal amplification (LAMP)
• Requires a precision instrument for heating and cooling
Real-time PCR
RT-LAMP
10e8
10e3
10e8
10e7
10e6
NTC
10e5
10e4
RT-LAMP was less sensitive (~ 1 log) than RT-PCR
TaqMan RT-PCR: Black et al., 2002; Hughes et al., 2004; Wakeley et al., 2005;
Wacharapluesadee et al., 2008; Hoffmann et al., 2010; Hayman et al., 2011a
NASBA: Wacharapluesadee et al., 2010
LAMP RT-PCR: Hayman et al., 2011b
10e8
10e7
10e6
10e5
10e4
Simplified Methodologies
10 Positive Turkish samples
sent on FTA cards
• Eluted the viral RNA off the
card
• Added directly to RT-LAMP
and RT-PCR
7/10 worked in RTLAMP
10/10 worked in RTPCR
Possible “in field” application
RT-LAMP
Positive
FTA Card Samples
LAMP LFD
• In order to facilitate detection lateral flow devices have
been used to capture the LAMP products
• LAMP products incorporates Btn and Flc labelled
primers
• LAMP products can be detected on an LFD based on
concentration of streptavidin coated blue nanospheres that
appear as a blue line in a positive reaction
+
Direction of flow
α Flc
α Btn
LFD detection of LAMP products
• 10 Ghanaian positive dog samples
• Tested using 12 primers in RT- LAMP penside format
Hayman et al., 2011
OptiGene Instrument
CVS rRT-LAMP on Genie I
40000
35000
Fluorescence
30000
25000
CVS1
CVS2
CVS3
20000
NTC
15000
10000
12 min
5000
0
0
500
1000
1500
2000
Time (s)
• Fully portable instrument
• 12V battery
• 300mm (W) x 200mm (D) x 80mm (H); 1kg
• 2 Independently programmable heating blocks
• Excitation 475nm; Emission >510nm
2500
3000
3500
4000
Rabies rRT-LAMP Performance
• RT-LAMP assay is almost as sensitive as RT-PCR using PicoGreen to
monitor reaction in both cases
• Rab1 RT-LAMP works well with samples from Africa (Morocco, Nigeria,
Kenya, Botswana, South Africa) and from various species
• Not possible to use degenerate primers so ended up with 12 primers
• Rab1/Rab4 RT-LAMP Detects both Cosmopolitan and Arctic-like strains
• Specificity of the assay may lead to some positives not being identified
• RT and LAMP occur concurrently – FAST!!
• Requires further optimisation
Microarray technologies
• Identification of rabies virus RNA
• Differential diagnosis for CNS-related diseases
Fooks et al., 2009
Issues with penside tests!
• Which is the best test to use?
– Needs knowledge of the tests
– Sometimes easier to send to laboratory!
• Fit into work flows
– How long does it take to get a result?
• Sample handling
– Labelling samples is a big enough challenge – following ID
through to result is a real issue
– How many tests to be done – 1 or 200?
What do we gain, what do we lose?
• What do we gain?
– Faster, cheaper tests;
– Reduced transport costs and time;
– Faster slaughter of animals infected with notifiable
diseases;
– More accurate and rapid treatment of animals infected
with endemic diseases.
• What do we lose?
– Central control over what tests are being used;
– Central control over who is running the tests;
– Central disease surveillance systems.
Reporting and Laboratory structure
OIE Reference Laboratories
OIE
National Reference Laboratories
Regional Laboratories
Point-of-care testing
Conclusions
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Technology is advancing rapidly
New tests will appear
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All tests must be fit-for-purpose
Tests must be validated and accepted by OIE
These tests will require little training to use
Tests will be cheaper and easier to use
There will be a move from central laboratories to local laboratories
Need for standardized reagents from OIE Standards Commission
Huge advantages for the developing world
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Serology – rapid
Molecular – no / little equipment costs
Highlights the importance of participation in projects that link laboratories from
the developed and the developing countries (OIE Twinning)
Recommend that OIE consider a universal molecular diagnostic test
New technological initiatives that combine advances in biology with other disciplines
will support the development of microchip, biosensor and robotics-based techniques
capable of high throughput testing with a low turnaround time for rabies diagnosis
Fooks et al., 2009
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AHVLA, Weybridge, UK
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Dan Horton
Nick Johnson
Ash Banyard
Lorraine McElhinney
Denise Marston
Karen Mansfield
Trudy Goddard
Dave Selden
Katja Voller
Hooman Goharriz
Graeme Harkess
Stacey Leech
Emma Wise
Philip Wakeley
Andrew Soldan
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CDC, Atlanta, USA
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FLI, Wusterhausen, GER
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Dr David Hayman
Global Alliance for Rabies
Control
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Dr Sarah Cleaveland
CSU, USA
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Dr Ed Wright
U. of Glasgow, UK
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Dr Noel Tordo
U. of Westminster, UK
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Dr Thomas Muller
Institut Pasteur, Paris, FRA
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Dr Charles Rupprecht
Dr Debbie Briggs
ANSES, Nancy, FRA
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Dr Florence Cliquet