HIV

VIDAS Training

VIDAS HIV

Objectives
Objectives

•• know
knowthe
thebasis
basisof
ofHIV
HIVinfections
infections
•• know
know the main technicalfeatures
the main technical featuresof
ofthe
theVIDAS
VIDASreagents
reagents
•• Know
Knowthe
themain
mainsales
salesarguments
argumentsand
andtools
tools

GCS - TM 0009 – 04/01/2007 – part 1 - p.1

Hepatitis profile

What do you know

about HIV epidemiology ?

GCS - TM 0009 – 04/01/2007 – part 1 - p.2

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 1

 HIV

HIV/AIDS: The Global Pandemic Continues

Every 6 seconds
someone is newly HIV infected

Every 10 seconds
someone dies from AIDS

33 millions :
the number of people living with HIV in the world today (2007 )

35 millions :
the number of people who have died of AIDS – related diseases

GCS - TM 0009 – 04/01/2007 – part 1 - p.3

Epidemic

http://data.unaids.org/pub/GlobalReport/2008/JC1511_GR08_ExecutiveSummary_en.pdf
GCS - TM 0009 – 04/01/2007 – part 1 - p.4

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 2

 HIV

HIV / AIDS adult prevalence

Western Europe 3 per 1000

North America 6 per 1000

Latin America 6 per 1000

South / South East Asia 5 per 1000

Sub Saharian Africa 88 per 1000

(Adults of 15-49 years old)

WHO - ONUSIDA dec 2005

GCS - TM 0009 – 04/01/2007 – part 1 - p.5

HIV patients in industrialized countries

30 % of people
do not know to be infected

GCS - TM 0009 – 04/01/2007 – part 1 - p.6

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 3

 HIV

New cases of AIDS

depending on the status

1400 800 800

Homosexuals
Homosexuels Drug
Usagersaddicts
de drogues Heterosexuals
Hétérosexuels
1200 700 700

600 600
1000

500 500
800
400 400
600
300 300

400
200 200

200 100
100

0 0 - 36% 0
94-1 96-1 98-1 '00-1 '02-1 94-1 96-1 98-1 '00-1 '02-1 94-1 96-1 98-1 '00-1 '02-1

not aware of HIV status

InVS – nov 2002 awareness of HIV status but no treatment
FRANCE anti-retroviral treatment before AIDS
GCS - TM 0009 – 04/01/2007 – part 1 - p.7

Hepatitis profile

Why is it important
to detect the infection early?

GCS - TM 0009 – 04/01/2007 – part 1 - p.8

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 4

 HIV

The main risk of contamination

is during the primary infection

Risk of
contamination by 30 %
contact

Primary
infection

Asymptomatic
10 %
stage
AIDS
0.1 % 0.1 %
0.01 %
0
0
Weeks Years
GCS - TM 0009 – 04/01/2007 – part 1 - p.9

Advantages of an early detection

of the HIV infection

Up to 90% of new HIV infections

are caused by people at the
primary stage of infection

Prevention of transmission

Early diagnosis
*Berrey et al., J.Infect Dis 2001
GCS - TM 0009 – 04/01/2007 – part 1 - p.10

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 5

 HIV

VIDAS Training

GCS - TM 0009 – 04/01/2007 – part 1 - p.11

HIV

What else do you know

about HIV infection?

2 - How to diagnose an HIV infection ?

3 – bioMérieux position in HIV
4 – How to confirm results of VIDAS HIV Duo Quick/Ultra

5 – VIDAS HIV Duo Ultra and Quick in front of competition

6 – VIDAS HIV Quick/Ultra sales tools

7–
GCS - TM 0009 – 04/01/2007 – part 1 - p.12

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 6

 HIV

HIV

Acquired
Immuno
Deficiency
Syndrome
GCS - TM 0009 – 04/01/2007 – part 1 - p.13

HIV : a deadly virus

direct attack to the immune system

target cell

Lymphocyte T

GCS - TM 0009 – 04/01/2007 – part 1 - p.14

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 7

 HIV

Risks to be contaminated depending on

the mean of transmission

sexual
intercourses

blood
(transfusions,
contaminated needles)

mother to child
breast milk

GCS - TM 0009 – 04/01/2007 – part 1 - p.15

The maximum contamination risk

is during primary-infection

Risk of contamination
per contact 30 %
Primary
infection

Asymptomatic
10 %
stage
AIDS
0.1 % 0.1 %
0.01 %
0
0
Weeks Years
GCS - TM 0009 – 04/01/2007 – part 1 - p.16

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 8

 HIV

50 to 70 % of primary infections
DO HAVE clinical symptoms

non-specific : mononucleosis type

fever 96%
adenopathy 75%
common pharyngitis 70%
rash 70%
arthralgia 50%
diarrhea 30%

confirmation with HIV detection test

“IN EMERGENCY"
GCS - TM 0009 – 04/01/2007 – part 1 - p.17

Several clinical phases

(without treatment )

AIDS
clinical symptoms
primary
primary
infection asymptomatic
period

2 to 15 years time

AIDS
Opportunistic infections
Kaposi sarcoma
CMV
toxoplasmosis
mycobacteria infections
herpes
aspergillosis ...

GCS - TM 0009 – 04/01/2007 – part 1 - p.18

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 9

 HIV

An efficient therapy that helps but not cures

no vaccine yet

antiviral therapy :
to be given as early as possible

mutant virus -> resistance

GCS - TM 0009 – 04/01/2007 – part 1 - p.19

HIV 1 is the most widespread virus

The predominant virus is HIV1. More than 90% of

HIV1 infection belong to HIV1 group M

HIV 2 is mainly concentrated in endemic zones

like as West Africa
M : Major
O : Outlier
N : New
CRF : circulating
recombinant forms

GCS - TM 0009 – 04/01/2007 – part 1 - p.20

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 10

 HIV

HIV virus

Viral enveloppe:
Two layers of lipids
Spikes made of 3 gp120
and gp41 subunits

HIV matrix:
Made of p17
a lie between enveloppe and core

Viral Core :
Made of p24
p7nucleocapsid protein

2 single strands of RNA

3 enzymes: reverse transcriptase,
integrase and protease.

GCS - TM 0009 – 04/01/2007 – part 1 - p.21

Hepatitis profile

video Link

GCS - TM 0009 – 04/01/2007 – part 1 - p.22

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 11

 HIV

HIV life cycle

REVERSE
TRANSCRIPTASE
conversion of the viral RNA into
double-stranded viral DNA

ENZYME INTEGRASE
Integration of the viral DNA
Into the host DNA

VIRAL PROTEASE
Cleavage of longer proteins into
smaller proteins

GCS - TM 0009 – 04/01/2007 – part 1 - p.23

HIV

1 - What else do you know about HIV infection ?

2 - How to diagnose an HIV

infection ?

3 – bioMérieux position in HIV

4 – How to confirm results of VIDAS HIV Duo Quick/Ultra
5 – VIDAS HIV Duo Ultra and Quick in front of competition
6 – VIDAS HIV Quick/Ultra sales tools
7–
GCS - TM 0009 – 04/01/2007 – part 1 - p.24

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 12

 HIV

How to diagnose a primary infection ?

Fourth
Generation

Third
HIV
Generation
Reactivity

RNA

Second
Generation-

p24 First
Ag
Generation
IgM
Anti- IgG
HIV Anti-HIV

0 1 2 3 4 5 6 7 8
Weeks post-infection
GCS - TM 0009 – 04/01/2007 – part 1 - p.26

Characteristics of anti-HIV Elisa

tests generations

1st gen 2nd gen 3rd gen 4th gen

recombinant recombinant recombinant
proteins proteins proteins
Antigen used Viral lysate
and peptides and peptides and peptides

Elisa indirect indirect sandwich indirect

anti-HIV Ig IgG IgG IgG Antibodies

detection IgM Ag P24

Sensitivity + ++ +++ ++++

Specificity + ++ + +++ +++

Year appeared 1985 1987 1989 1997

GCS - TM 0009 – 04/01/2007 – part 1 - p.27

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 13

 HIV

HIV

1 - What else do you know about HIV infection ?

2 - How to diagnose an HIV infection ?

3 – bioMérieux position in HIV

4 – How to confirm results of VIDAS HIV Duo Quick/Ultra

5 – VIDAS HIV Duo Ultra and Quick in front of competition
6 – VIDAS HIV Quick/Ultra sales tools
7–
GCS - TM 0009 – 04/01/2007 – part 1 - p.28

1995:
HIV : Nasba HIV1 QT
History of the disease,
our story 1993: 1st HIV viral load 2004:
VIDAS DuoUltra
VIDAS P24 Ag
1991: 2 signals
1st automated The most
VIDAS HIV 1+2 test available sensisitve
1985: 1st automated 2002:
Vironostika anti test available
Real time Easy Q
HTLV III on the market Quantitative viral load
1st test
1998:
available for
screening 4th generation
1st tests
Microtiter plates
and automated

GCS - TM 0009 – 04/01/2007 – part 1 - p.29

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 14

 HIV

What types of tools for HIV infections ?

Antibodies detection Virus detection

ELISA method
ARN = viral load
NucliSens EasyQ HIV

VIDAS HIV Duo Ultra

VIRONOSTIKA HIV
Ag HIV (p24)ELISA
Uniform II Ag/Ab (rutina)

VIDAS HIV P24 /

Vironostika HIV 1 Ag

Western Blot
(confirmatory)

bioMerieux’ offer is large !

GCS - TM 0009 – 04/01/2007 – part 1 - p.30

HIV bioMérieux :
The experience from screening to follow up

VIDAS HIV Duo – ULTRA - QUICK : Precocity of detection

Vironostika HIV Uniform II Ag/Ab: Microplates for high volume throughput

VIDAS HIV P24 : For the specific quantification of the HIV Ag in blood

Vironostika HIV 1 Ag: microplate tools for Ag detection with high throughput

NucliSens EasyQ HIV : Viral Load : quantitative detection of HIV-1 RNA with
REAL TIME NASBA: based amplification

VIKIA HIV: rapid test

VIDIA (full automated instrument)

mini Vidas Vidas DaVinci

Quattro NucliSens EasyQ VIKIA
GCS - TM 0009 – 04/01/2007 – part 1 - p.31

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 15

 HIV

VIDAS : an early diagnosis of HIV infections

2 tests to satisfy customers needs

VIDAS HIV Duo QUICK

VIDAS HIV Duo ULTRA

GCS - TM 0009 – 04/01/2007 – part 1 - p.32

VIDAS : an early diagnosis of HIV infections

2 tests to satisfy customers needs

VIDAS HIV Duo QUICK

VIDAS HIV Duo ULTRA

GCS - TM 0009 – 04/01/2007 – part 1 - p.34

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 16

 HIV

VIDAS HIV DUO QUICK

Simoultaneous detection of Ag/Ab

2 - antigen
detection

1 - antibody
detection

Detection of
HIV 1 IgG and IgM, Groupe O, HIV 2,

HIV 1 Ag

GCS - TM 0009 – 04/01/2007 – part 1 - p.35

VIDAS HIV DUO QUICK - Clinical sensitivity

All the subtypes tested

were found positive

VIDAS HIV Duo Quick

sensitivity in Ag
is similar to
a HIV p24 Ag test :

4.5 pg/ml
pg/ml in p24 Ag

GCS - TM 0009 – 04/01/2007 – part 1 - p.36

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 17

 HIV

VIDAS HIV DUO QUICK - Specificity

Results from testing 5027 blood donor samples

from 3 blood banks :

Specificity = 99.88 % (99.74-99.93)

GCS - TM 0009 – 04/01/2007 – part 1 - p.37

VIDAS : an early diagnosis of HIV infections

2 tests to satisfy customers needs

VIDAS HIV Duo QUICK

VIDAS HIV Duo ULTRA

GCS - TM 0009 – 04/01/2007 – part 1 - p.38

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 18

 HIV

VIDAS HIV DUO ULTRA

Separate signals for Ag and Ab

2 - antigen
detection

1 - antibody
detection

Detection of
HIV 1 IgG and IgM, Groupe O, HIV 2,

HIV 1 Ag
Ultra about 2 hours
GCS - TM 0009 – 04/01/2007 – part 1 - p.39

VIDAS HIV DUO ULTRA - sensitivity

very high sensitivity

in P24 Ag
compared to other 4th gen. tests

Earlier detection on panels,

compared to other
4thgeneration assays
and HIV Ag assays.

3.15 pg/ml P24 Ag

GCS - TM 0009 – 04/01/2007 – part 1 - p.40

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 19

 HIV

VIDAS HIV DUO ULTRA - Specificity

Results from testing 5008 blood donor samples

from 3 blood banks :

Specificity = 99.88 % (99.73-99.95)

GCS - TM 0009 – 04/01/2007 – part 1 - p.41

Seroconversion panels

A seroconversion panel consists of a series of samples

obtained from an individual during the early phases of
infection. The time intervals between the serial samples of a
seroconversion panel may vary.

The earliest is the detection of the virus, the best is the

sensitivity of the kit.

GCS - TM 0009 – 04/01/2007 – part 1 - p.43

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 20

 HIV

VIDAS HIV DUO ULTRA VIDAS HIV DUO QUICK

P24 Ag of HIV 1 –
Detection
IgG and IgM of HIV 1 and 2
of Real sandwich for Ab phase

Calibration S1 (std Ab) S2 (std Ag) S1

C1 (pos Ab) C2 (neg Ab) C3 (pos Ag) C1 (pos Ab) C2 (neg Ab) C3 (pos Ag)
All lypoh except C2 (liquid) All lypoh except C2 (liquid°

Duration 2h Short protocol: 1 h 20 min.

Samples Serum or plasma

on lithium heparinate or EDTA
type
Samples < 2 days :2-8 °C
>2 days : -25 +6°C
storage "Avoid successive freezing and thawing“
Written in package insert :
“If necessary , clarify samples by centrifugation”
centrifugation”
Sample 200 µl exactly
volume
SPR storage Clip seal provided in the kit
Results Separated results for Ag and Ab Global results
Interpretation < 0.25 : neg < 0.25 : neg in Ab AND Ag
> 0.25 : pos > 0.25 : pos in Ab OR Ag

Sensitivity 11.5 pg / ml HIV Ag 16.5 pg / ml HIV Ag

in Ag ie : 3.1 pg / ml P24 Ag ie : 4.5 pg / ml P24 Ag.

GCS - TM 0009 – 04/01/2007 – part 1 - p.44

VIDAS HIV kits :

Technical recommendations

 Centrifuge properly the tubes : at least 10 minutes.

 Samples can be stored at 2-8 °C for up to 2 days. If
longer storage is required, freeze at -25 ± 6°C.
 Only remove the required reagents from the refrigerator and allow
them to come to room temperature for at least 30 minutes

 Pipette precisely 200 µl of sample

 VIDAS Instruments have to be maintained by bioMérieux’s service
engineer periodically and we advise you to perform in priority the
preventive maintenance on all VIDAS using HIV.

GCS - TM 0009 – 04/01/2007 – part 1 - p.45

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 21

 HIV

Clinical impact

Early HIV Detection with

ADVANCED 4th generation assays

means :

Preventing infection

Preventing transmission

Improving patient care/ life quality

GCS - TM 0009 – 04/01/2007 – part 1 - p.46

Anti HIV tests generations

Depends Fourth
VIDAS HIV Duo on the kit Generation
Quick & Ultra
Third
HIV
Generation
Reactivity

RNA

Second
Generation-

p24 First
Ag
Generation
IgM
Anti- IgG
HIV Anti-HIV

0 1 2 3 4 5 6 7 8
Weeks post-infection
GCS - TM 0009 – 04/01/2007 – part 1 - p.47

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 22

 HIV

HIV

1 - What else do you know about HIV infection ?

2 - How to diagnose an HIV infection ?
3 – bioMérieux position in HIV

4 – How to confirm results of

VIDAS HIV Duo Quick/Ultra ?

5 – VIDAS HIV Duo Ultra and Quick in front of competition

6 – VIDAS HIV Quick/Ultra sales tools
7–
GCS - TM 0009 – 04/01/2007 – part 1 - p.48

Screening tests have to be confirmed

Screening
Confirmation

Ag + Ab Antibodies only

P24 Ag Western Blot / Western Blot /

Immunoblot Immunoblot

GCS - TM 0009 – 04/01/2007 – part 1 - p.49

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 23

 HIV

What is a Western Blot ?

1 2 3
Electrophoresis Transfert Sera incubation
Revelation
+ + + +
ECHANTILLONS
SAMPLES

marked Ac

- - - - Visualisation of proteins
transfert on nitrocellulose filter specifically known by
Separation of nitrocellulose + corresponding Ab
proteins transferred proteins
filter

Application to HIV

Qualitative detection of anti HIV IgG

GCS - TM 0009 – 04/01/2007 – part 1 - p.50

To be known concerning Western Blot

IgG only

1 WB for HIV 1 / 1 WB for HIV 2

used for specificity

limited in sensitivity : less than ELISA techniques

variations between - suppliers
- batchs from a same supplier
 the lab has to «validate» each lot before use

Poor reagents stability

Interferences : blood, bilirubin, auto-immune diseases...

Necessity to use controls for interpretation

Subjective interpretation : visual
the interpretation depends on the local registration

GCS - TM 0009 – 04/01/2007 – part 1 - p.51

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 24

 HIV

P24 Ag - a marker of HIV infection

marker of viral replication

appears approximately one week before Ab

No detectable after
during asymptomatic period, P24 is complexed with anti P24

GCS - TM 0009 – 04/01/2007 – part 1 - p.53

VIDAS HIV P24 II – ref 30117

Principle : direct Sandwich

• 30 tests Anti-
Anti-P24
• 90 min
Anti LH
•
•
200 µl exactly
test code : P24
α E labeled
P24
serum

Quantitative detection of HIV1 p24 antigen (all sub-

sub-types)

Limit of sensitivity : 3 - 5 pg/ml of p24 Ag

30 tests

confirmation with VIDAS ref 30444

2 units – pg/ml P24 Ag (recombinant) (customer's choice)
– pg/ml HIV Ag (whole virus)

1 pg/ml P24 Ag = 3.65 pg/ml HIV Ag

GCS - TM 0009 – 04/01/2007 – part 1 - p.54

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 25

 HIV

POSITIVE VIDAS HIV P24 II

should be confirmed by neutralization

60 tests

90 mins

same protocol as VIDAS HIV P24 II

test code : P24

any P24 Ag positive result should be confirmed

should be used with the kit HIV P24 II
dilute the samples > 400 pg/ml Ag P24
it is mandatory to test the neg Control of HIV P24 II

GCS - TM 0009 – 04/01/2007 – part 1 - p.55

VIDAS HIV P24 II confirmation - Principle

VIDAS reaction
conjugate

anti P24
Ag P24 E +
monoclonal *

Confirmation reaction :
1 - neutralization 2 - VIDAS reaction

Ag P24
4

-
P2

neutralizing anti P24 Antibody

Ag

GCS - TM 0009 – 04/01/2007 – part 1 - p.56

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 26

 HIV

VIDAS HIV P24 II confirmation - Procedure

250 µl sample

10 µl neg serum 10 µl neutralizing Ab

Procedure
30 min
37 °C

200 µl treated
sample

P24 II Ag strip

N-P N = RFV sample containing Negative serum.

Interpretation R= P = RFV sample containing Neutralizing antibody.
N-C C = RFV obtained for the negative control
If R is > 60 %, the presence of p24 antigen is confirmed.
If R is < 60 %, the presence of p24 antigen is not confirmed.
If N or P are negative, the presence of p24 antigen is not confirmed

GCS - TM 0009 – 04/01/2007 – part 1 - p.57

Interpretation algorithm for

VIDAS HIV Duo Quick and Ultra

GCS - TM 0009 – 04/01/2007 – part 1 - p.58

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 27

 HIV

Interpretation algorithm for

VIDAS HIV Duo Quick and Ultra

Negative result Positive result

Retested twice using the

same reagent on the same sample

Results in Duplicate Results in Duplicate

2 neg results 2 pos results or
1 pos / 1 neg

Perform a complementary analysis

No HIV No HIV using the same sample and a « fresh »
sample collection to confirm HIV
Infection Infection Infection See specific algorithm

This algorithm is proposed as an indication and is valid for the majority of cases. Please take into consideration the recommendations in force in your country.
(1) If clinical signs or risk factors require a new bleeding two weeks after, it is also possible to ask for a viral load on a 2nd immediate bleeding to provide the
patient with a more rapid diagnosis orientation.
GCS - TM 0009 – 04/01/2007 – part 1 - p.59

GCS - TM 0009 – 04/01/2007 – part 1 - p.64

VIDAS HIV – GCS - TM 0009 - Revision 04/01/2007 part 1 - p 28