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New molecular-based tools for the detection, diagnosis and follow-up of CE

INSTITUTO DE RECURSOS NATURALES Y AGROBIOLOGIA DE SALAMANCA

1.

IMAGE (ULTRASONOGRAPHY)

DIAGNOSIS AND FOLLOW-UP OF HUMAN CE

INACTIVE TRANSITIONAL ACTIVE STAGE

IMAGE

CE5 CE4 CE3b CE3a CE2 CE1 CL IWGE-WHO

2003

Type V Type IV Type III Type II Type III Type I GHARBI 1981

IMAGE SHORTCOMINGS

- Interpretation (specificity)

- Small-hidden lesions (sensitivity)

- Follow-up (drug treatment, PAIR). “Reversible” stages

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DIAGNOSIS AND FOLLOW-UP OF HUMAN CE

2.

SEROLOGY (ANTIBODY DETECTION)

AGAINST HYDATID FLUID

“screening”

ELISA

“confirmation”

IMMUNOBLOT

SEROLOGY (WITH HYDATID

FLUID) SHORTCOMINGS

- Where to collect the hydatid fluid? (variability)

- Low specificity, low sensitivity

- Follow-up not posible (antibody persistence after cure)

WHAT ABOUT RECOMBINANT ANTIGENS?

VERY VARIABLE RESULTS IN SPECIFICITY AND SENSITIVITY (FOR THE SAME ANTIGEN!)

VERY FEW DEFINED FOR (POTENTIAL) FOLLOW-UP

Antigen B

A

ntigen 5

Carmena et al., 2006

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RECOMBINANT ANTIGENS SHORTCOMINGS

FOR DIAGNOSIS AND FOLLOW-UP OF CE

NO SINGLE HIGH-QUALITY COMPARATIVE

TRIAL OF SERODIAGNOSIS/FOLLOW-UP

OPTIONS WITH RECOMBINANT ANTIGENS

IS AVAILABLE

Antigen

Technique

Immunoglobulin isotype

Statistics

Sera panel

Clinical data

WHICH VARIABLES COULD INFLUENCE THE

PERFORMANCE OF A DEFINED CE

SERODIAGNOSTIC TEST?

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SOME ANTIGENS WITH HIGH

SENSITIVITY AND SPECIFICITY

In spite of contradictory results, these could be good candidates for CE serodiagnosis,

but need to be properly defined.

If not, results cannot be assessed and compared.

Antigen CE Other Donors Technique Sp Se Authors

rAgB8/1 129 65 203 IgG ELISA 84 91 Virginio et al., 2003

rAgB8/2 129 65 203 IgG ELISA 93 99 Virginio et al., 2003

rEgcMDH 129 65 203 IgG ELISA 90 95 Virginio et al., 2003

rEgCaBP2 129 65 203 IgG ELISA 84 97 Virginio et al., 2003

rEgAFFP 129 65 203 IgG ELISA 59 96 Virginio et al., 2003

rEgAg5 34 36 18 IgG ELISA 65 89 Lorenzo et al., 2006

ONLY PRELIMINARY DATA ARE AVAILABLE TO DATE

TOOLS MUST BE VALIDATED THROUGH A MULTICENTER STUDY,

INCLUDING:

- ELLABORATION OF CLINICAL PROTOCOLS

- ELLABORATION OF DATABASES FOR CLINICAL DATA

- COLLECTION OF SERA WITH A COMMON PROTOCOL (SERA BANK)

- PRODUCTION AND TESTING OF RECOMBINANT ANTIGENS

- STANDARDIZATION OF DIAGNOSTIC/FOLLOW-UP TOOLS

- OBTAINMENT OF A MARKETABLE (POINT-OF-CARE) KIT

ARE RECOMBINANT ANTIGENS (IS SEROLOGY) USEFUL FOR THE DIAGNOSIS

AND FOLLOW-UP OF CYSTIC ECHINOCOCCOSIS PATIENTS?

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A MULTICENTER STUDY IS PLANNED IN THE FRAME OF THE EUROPEAN PROJECT HEALTH-F3-2013-602051-2

“Human cystic Echinococcosis ReseArch in CentraL and Eastern Societies” (HERACLES)

TASK 2.1: Sample repository (Months 1-42)

- Mode of collection, storage and treatment of prospective and retrospective

samples (blood/serum and cyst material from patients and animals).

(+WP3).

DRAFT PROTOCOL READY DEC. 2013

- Ellaboration of clinical protocols for the definition of cohorts and samples,

and for the study design.

DRAFT PROTOCOL SHOULD BE READY FOR FEB-MARCH 2014. WE

HAVE A PREVIOUS DRAFT PROTOCOL ELLABORATED IN

COLLABORATION WITH Profs. JUNGHANSS AND BRUNETTI

DEFINITION OF SAMPLE NUMBER FOR EACH CLINICAL GROUP,

DEFINITION OF DATA EXTRACTION AND STATISTICS TO BE APPLIED

FOR DIAGNOSIS AND FOLLOW-UP — STATISTICIAN

(Preliminarily defined in Task 2.3: prospective: at least 500 human serum

samples (5 samples per person) and 200 animal serum samples).

TASKS WP2

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TASK 2.2: Database development (Months 1-12)

Patients database, including clinical and epidemiological data needed for

diagnostic tools assesment. Designed to allow for a direct numeric extraction of

the dataset to Excel and SPSS programs to facilitate statistical analyses.

Human data should be linked with CE registry developed in Italy, including:

http://161.111.251.249/

mar.irnasa@hoplite.es; test.mar

Animal data, included in collection protocol, where are they hosted?

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TASK 2.3: Production and testing of recombinant antigens (Months 1-42)

- The recombinant antigens 2B2t and P29 wich have shown to be a potential

tool for a sensitive and specific detection of CE and for the follow-up of CE,

will be produced.

Sequences of 2B2t, B1t, MDH, AFFP, Ag5 and CBP have been obtained and

cloned in sequencing/expression vectors (psCA, pGEX). Expression trials

have been done for all but Ag5, showing al to be soluble proteins. P29?

The MTA from CSIC to Vircell has been prepared.

READY

- A shared ELISA protocol will be elaborated. IgG, IgG2, IgG4

SHUOLD BE READY IN FEB 2014

- Antigens will be preliminarily tested in ELISA by CSIC and by Vircell with a

selected retrospective panel of sera (CE, cross-reactive; around 2,500 sera

already available at CSIC).

SHOULD BE READY IN JUNE-JULY 2014

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TASK 2.3 (cont.):

- The selected antigens will be produced in a 35-litre automated fermentor

under ISO 9001 requirements. Production will be optimized to be cost-

effective.

SHOULD BE READY IN DEC. 2015

- The antigens will be preliminarily tested in POC and LOC kits.

SHOULD BE READY IN FEB. 2016

- Plates (or others) with selected antigens will be tested by partner 3 with the

serum repository, and distributed to other Partners (on demand) to test

serum samples independently in several laboratories.

SHOULD BE READY IN FEB 2016. RESULTS READY IN JUNE-JULY 2016

- Serology results will be loaded in the database developed in Task 2.2.

STARTING IN DEC. 2015

TASK 2.4: Development of POC-LOC devices (Months 12-48)

- Refinement/adjustment of POC-LOC devices and testing, resulting

in a pre-commercial product.

VIRCELL

MICROGUNE (SPAIN)

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ADDITIONALLY….

-Antibodies against 2B2t (polyclonal, hyperimmune, purified IgG) are

ready and could be used in human and animal cyst samples (fluid). The

antibodies have already been tested in an immunocromatography format,

showing high specificity (no cross-reactivity with fluid from E.

multilocularis, Taenia solium, T. saginata and T. hydatigena)

COULD BE USED FOR RAPID IDENTIFICATION OF THE PARASITE IN

CYST MATERIAL EXTRACTED FROM PATIENTS OR ANIMALS

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Rapid tests Main features of the market and products

About us

Vircell is a biotechnology company established from private capital in 1991.

Founded by clinical microbiologists with previous experience working in hospitals, our activity is

focused on the production of ready-to-use kits for the diagnosis of esoteric infectious diseases

by means of different techniques.

Our international projection translates into worldwide export of our products. Vircell is well

introduced in laboratories of more than 60 countries in Europe, Latin-America, Asia and Middle East.

QUALITY

Clear customer orientation from the

product conception to after-sale service.

Focused on the production of

reagents of the highest quality

CUSTOMER

ORIENTATION

INNOVATION Development of innovative products

bringing significant advances

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VIRCELL is a microbiology-biotechnology company which develops, manufactures, markets, sales

and distributes ready-to-use kits for the diagnosis of esoteric infectious diseases. Our business

culture is based upon our people, our high quality products, science, our know how and support

through R&D.

Vision:

We pursue to become a gold standard for the microbiology and biotechnology sector by

developing innovate products and techniques for the diagnosis of esoteric infectious diseases.

In a two years time frame, we aim for a qualitative and quantitative major turn around to become an

international company by consolidating our global presence.

Since its foundation VIRCELL has three main objectives:

1. To produce reagents of the highest quality

2. To maintain along the whole process a clear customer orientation that can be seen all the way from

the product concept to the after-sales service.

3. To develop innovative products for obtaining significant advances.

ISO 9001: 2000. ISO 13485: 2003. CE mark. IQNet.

SFDA (China). GMP / FDA (on process)

Mission:

Mission & Vision

The team 35% Lab technicians 44%

Bachelor Science

21% PhD

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Distribution network + 70 countries; 130 partners; one shared vision

Product pipeline:

Chlamydia trachomatis IgG

Mycoplasma pneumoniae IgM

Parvovirus IgG/IgM

Cysticercosis DUO (Ag+Ab)

Dengue DUO (Ag+Ab)

Ab detection

Ag detection

Ab detection

Ab detection

VIRapid® analysis

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The main benefits of immunochromatographic tests are:

- Rapid detection and visual reading

- Easy performance

- No need of skilled personnel or expensive automated systems (field test)

- Appropriate pack size for diagnosis of rare disease or urgent analysis, etc.

Nowadays the tendency for the diagnosis lies in the use of more flexible techniques that are easy to perform and interpret, in order to achieve a more rapid diagnosis for the development of effective treatments.

Immunochromatographic test

- This product line has 4 references:

- VIRapid® MONO M&G

- VIRapid® Legionella Culture

- VIRapid® Hydatidosis

- VIRapid® Tularemia

- New market opportunities, a good chance to grow and diversify sales

VIRapid®

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VIRapid® HYDATIDOSIS

The first rapid test for hydatidosis diagnosis

VIRapid® Hydatidosis

• Rapid detection- Visual reading in just 30 minutes

• User friendly protocol with little manipulation

• All the necessary reagents included in the kit

• Product control line on each device

• Based on specific and highly purified antigen (conjugate and line)

• Excellent performance with 94.7% sensitivity and 99.5% specificity

• CE marked (IVD)

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• Cyst hydatidosis is normally diagnosed by image tests. However, serological.

confirmation is always necessary as it can be confused with other pathologies like tumors

• ELISA and Indirect hemagglutination (IHA) are the most popular techniques: More than

20 ELISA kits available in the market and Fumouze IHA is the market leader.

• VIRapid® Hydatidosis is unique in the market and could be a great opportunity.

• Immunochromatographic tests are user friendly and more convenient for the customer.

•A HPLC-purified E. granulosus 5/B enriched antigen is used in VIRAPID® HYDATIDOSIS.

VIRapid® Hydatidosis

• The test is not able to distinguish between patients with active or inactive cysts.

• Cross-reactions with Echinococus alveolaris have been reported.

• Sensitivity and specificity should be improved with recombinant antigens, instead of

native antigens.

VIRapid® Hydatidosis

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Strip Automatic Reading System

The new version of the automatic reading system includes scanner, VIRapid®

kit bar-coded cassette base, updated software and license for its use

The automatic reading of the test and will reduce the subjectivity of the technician

creating PDF reports for documenting the results of the analyzed samples.

Rapid tests Main features of the market and products

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Over 20 years developing diagnostic solutions for

HUMAN INFECTIOUS DISEASES