New Diagnostic Tools for Rabies in Animals

Anthony R. Fooks

(1) AHVLA, Weybridge, UK

OIE Reference Laboratory for Rabies

WHO Collaborating Centre for Rabies

(2) University of Liverpool, Liverpool, UK

Overview

I. OIE resolutionsI. 1st International Conference on rabies in

Europe held in Kiev Ukraine - June 2005

II. Towards the Elimination of Rabies in Eurasia

held in Paris France - May 2007

II. Collection and storage of samples

III. OIE prescribed tests for rabies

IV. 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;– A definitive diagnosis can only be made by laboratory investigations;

• OIE reference laboratories and WHO collaborating centres work together;– International harmonisation of laboratory methods for diagnosis;

• The recommended primary diagnostic test for rabies is the fluorescent antibody test;

– 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 CNS-related diseases

– 14 (10.5%) – rabies

• Three of 26 (11.5%) clinically diagnosed cases of cerebral malaria were laboratory-confirmed as rabies

Mallawa et al., 2007

Objectives:

• Use of standardized diagnostic tests with regular inter-

laboratory 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 resolutionsI. 1st International Conference on rabies in

Europe held in Kiev Ukraine - June 2005

II. Towards the Elimination of Rabies in Eurasia

held in Paris France - May 2007

II. Collection and storage of samples

III. OIE prescribed tests for rabies

IV. Alternative tests for rabies

iii. (non-prescribed)

Collection of samples: occipital foramen

route for brain sampling from animals• 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;

– 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.

Barrat. WHO Laboratory Techniques in Rabies, 4th Edition

FTA cards: Advantages and benefits

• 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.

Whatman FTA devices format

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 resolutionsI. 1st International Conference on rabies in

Europe held in Kiev Ukraine - June 2005

II. Towards the Elimination of Rabies in Eurasia

held in Paris France - May 2007

II. Collection and storage of samples

III. OIE prescribed tests for rabies

IV. Alternative tests for rabies

iii. (non-prescribed)

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

Fluorescent Antibody Virus Neutralisation Test

Measuring Antibody levels

Moderate 3 days High High

RT-PCR Screening Cheap 2 days (inc RNA

extraction)

High High

Taqman Real-Time RT-PCR Sequencing/phylogenetics

Screening Cheap 1 day (inc RNA extraction)

High High

Commonly used OIE prescribed tests for rabies

AHVLA

OIE prescribed tests for rabies

Routine laboratory Tests

• Histological identification of characteristic cell lesions– Detection of „Negri bodies‟ [no longer recommended]

• Immunochemical identification of rabies virus antigen– Fluorescent antibody test (FAT)

• Detection of replication of un-inactivated rabies virus after inoculation

– Virus isolation• Rabies tissue-culture infection test (RTCIT)

• Mouse inoculation test (MIT)

• Current methods for rabies serology (antibody detection)– Rapid fluorescent focus inhibition test (RFFIT)

– Fluorescent antibody virus neutralisation test (FAVN)• 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

• 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

WHO Laboratory Techniques in Rabies

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 resolutionsI. 1st International Conference on rabies in

Europe held in Kiev Ukraine - June 2005

II. Towards the Elimination of Rabies in Eurasia

held in Paris France - May 2007

II. Collection and storage of samples

III. OIE prescribed tests for rabies

IV. 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

3 plasmid transient

transfection into 293T cells1

2Harvest virus and titrate on

target cells

Rabies G-protein

HIV Gag and Pol

Vector encoding GFP/Luc

3Measure the NAb titre

of serum using GFP

or luciferase readout

Pseudotype construction

100

102

104

106

108

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

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

Murine hippocampous (ICC)

- Mab staining of cortex

Vaculation of bovine Purkinje cells

In-situ hybridization (RNA detection)

Finnegan et al., 2004

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

Direct Immunohistochemistry test (dRIT)

• Detects antigen

• Sensitivity and specificity equivalent to the DFA / FAT (100%)

• Frozen and glycerol-preserved brain samples

• No specialised equipment or infrastructure required

• Ideal for use in developing countries, especially under field conditions

Lembo et al. 2006; Niezgoda et al., 2009

dRIT Analysis

rabies

negativerabies

positive

(A) dRIT

(B) FAT

Penside technology: Human pregnancy tests

• Proof-of-principle– Point-of care testing

• Gold standard for point of care testing

• Un-trained user

• Rapid – 1 to 3 minutes

• >99% accurate

• Informative

• Cheap (<US$3 / test)

Slide courtesy of Andrew Soldan (AHVLA, UK)

Rabies immunochromatographic diagnostic

test (RIDC)

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

Kang et al.. 2007; Markotter et al., 2009

α Flc

α Btn

Direction of flow

+ -

Molecular techniques

• Real-time PCRs

• Nucleic acid sequence-based amplification (NASBA)

• Loop-mediated isothermal amplification (LAMP)

• Requires a precision instrument for heating and cooling

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 10e810e7

10e6

10e5

10e4

10e8

10e7

10e6

10e5

10e4

10e3

NTC

RT-LAMPReal-time PCR

RT-LAMP was less sensitive (~ 1 log) than RT-PCR

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 RT-

LAMP

10/10 worked in RT-

PCR

Possible “in field” applicationPositive

FTA Card Samples

RT-LAMP

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

α Flc

α Btn

Direction of flow

+ -

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

• Fully portable instrument

• 12V battery

• 300mm (W) x 200mm (D) x 80mm (H); 1kg

• 2 Independently programmable heating blocks

• Excitation 475nm; Emission >510nm

CVS rRT-LAMP on Genie I

0

5000

10000

15000

20000

25000

30000

35000

40000

0 500 1000 1500 2000 2500 3000 3500 4000

Time (s)

Flu

ore

sc

en

ce

CVS1

CVS2

CVS3

NTC

12 min

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

National Reference Laboratories

Regional Laboratories

Point-of-care testing

OIE

OIE Reference Laboratories

Conclusions

• Technology is advancing rapidly

• New tests will appear

– Serology – rapid

– Molecular – no / little equipment costs

• 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

– 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

• AHVLA, Weybridge, UK– 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

• CDC, Atlanta, USA– Dr Charles Rupprecht

• FLI, Wusterhausen, GER– Dr Thomas Muller

• Institut Pasteur, Paris, FRA– Dr Noel Tordo

• U. of Westminster, UK– Dr Ed Wright

• U. of Glasgow, UK– Dr Sarah Cleaveland

• CSU, USA– Dr David Hayman

• Global Alliance for Rabies Control

– Dr Debbie Briggs

• ANSES, Nancy, FRA– Dr Florence Cliquet