CONSTRAINTS WITH THE AVALIABLE IMMUNO-SERO-VIROLOGICAL

DIAGNOSTIC TESTS IN RESSOURCE LIMITED COUNTRIES

François SimonCHU saint Louis – Lariboisière, Paris

• 1- Can we improve the HIV diagnosis strategy in POC ?

• 2- Can we move the viral load/resistance monitoring from Regional to District ?

HIV in RESSOURCE LIMITED COUNTRIES

HOW TO DO MORE WITH LESS !!

• HIV screening in UE

1- Test-and-Treat Strategy for HIV in Resource-Limited Settings ?

• in P O C

4th generation EIAAg & Ab detection

High sensitivity, similar to NAT

RDT on serumon capillary or whole blood

FDA approved 2010

- Large number of studies, including in P O C

- But no comparative studies between the different RDT assays

- Real time evaluation with oral fluid and capillary whole blood are difficult

- No primary infection with whole blood or OF , no reference panel for diversity

ARE THE RDT RELIABLE IN POC ?

> 80 RDT in 2010agressive marketing

HIV onto urineFDA labelled

in 2010

= number of RDT are commercialized by non- healthca re manufacturer with no visibility in quality assuranc e

TRANSACTION VOLUMES OF RAPID HIV TESTS (WHO 2009)

the overall RDT diagnostics sector has had sluggishgrowth but few major manufacturers involved

HIV RAPID DIAGNOSTIC TESTSARE THE RDT RELIABLE ?

Nucleic acid testing (NAT) in routine HIV in testing programs versus RDT , San Diego, California

• RDT : Oraquick

• 3151 persons tested

RDT missing 15 about 79 (19%) of the positive samples

Morris et al Ann Intern Med. 2010 Jun 15;152(12):I30

RDT IN A HEALTHCARE SETTING : A COMPARATIVE STUDY OF RDT SENSITIVITY

- Hôpital Saint Louis, Paris 2009

- 200 adults : HIV-1 (n=194) or HIV-2 infection (n=6)

- 5 HIV rapid tests using either oral fluid or finger-stick whole blood

Sensitivity ranged from 86.5% to 99%

Rapid HIV tests were -less sensitive on oral fluid than on whole blood- less sensitive on finger-stick whole blood than on serum

Pavie et al, PlosOne 2010

SENSITIVITY OF FIVE RAPID HIV TESTS IN 200 HIV-INFECTED PATIENTS

Oraquik OF Oraquick FSB

VikiaFSB

DetermineFSB

INSTIFSB

Determine ComboAg +Ab

FSB

Invalid - - - 4 2 33

Negative 27 11 3 10 2 7

Positive and weakly positive

173 189 197 186 196 160

Overall sensitivity % of valid tests

86.5%

[81-90.5]

94.5%

[90.4-96.9]

98.5%

[95.6-99.5]

94.9%

[90.8-97.2]

99%

[96.3-99.7]

95.8%

[91.6-97.9]

Pavie et al, Plosone 2010

Genotype

Oraquick sang

Vikia Determine INSTi Determine 4th GENAg +Ab

50(Chronic stage)

(HIV- 0) N + N + N

67 (primary infection) * B N N N N N

73 (primo infection) F N N N +/- N

184 (primary infection

4 months ago)

B + + N + N

* Ag P24 = 380 pg/mL

Pavie et al, Plosone 2010

HIV PRIMARY INFECTIONS AND VARIANTS TDR RESULTS ONTO WHOLE BLOOD

Hôpital Saint Louis, Paris 2009

REVISED RECOMMANDATIONS FOR THE SELECTION AND USE OF HIV ANTIBODY TESTS

* WHO – Wkly Epidemiol Rec 1997, 72: 81-87

1°°°° screening assay Result2°°°° screening assay

Screening assay ResultConfirmatory assay

Conventional strategy

Alternative strategy

1892/09 780/09 133/08 130/08 1723/07 1946/09 1937/09 Determine 3G HIV1/2 + + + + - + +

Immuno HIV 1/2 - + - + + + +

Vikia HIV 1/2 + - - -

Retro check - - +

SD bioline + - -

Ag P24 - - - - -

CV Taqman V2.0 - -

EIA Architect Abbott - - - - - - -

Conclusion OMS P P P P P P P Conclusion SLS N N N N N N N

FAILURE IN WHO ALGORITHM : A ROUTINE SCREENING IN CAMEROON IN 2009

Tokombéré, JM Huraux, 2009

Failure in WHO strategy II leading to a false positivity reportFailure in western blot identification

WHO conclusion

Specificities and PPVs of diagnostic HIV tests (% ; 95% CI)

Test specificities* PPVs**

Bioline

DetermineDoubleCheck

ImmunoFlow

OraQuick

Stat-PakUni-Gold

Vikia

323/348 (90-95)

136/348 (33-44)319/348 (88-94)314/348 (87-93)

341/348 (96-99)

342/348 (96-100)336/348 (94-98)

297/348 (81-89)

11/36 (15 - 45)

11/223 (2 - 7)

11/40 (13 - 41)11/45 (11 - 37)

11/18 (37 - 84)

11/17 (41 - 88)11/23 (26 - 68)

11/62 (8 - 27)

*Number of persons with negative test result/total number of persons with HIV-negative status

**Number of persons with HIV-positive status/number of persons with reactive test result.

RDT AND CONFIRMATORY STRATEGIESWHO RECOMMENDATIONS

The assumption that each test perform independently is the corner stoneof WHO 2 - tests strategy

11 % 89.6% 5 % 77.8% 2 % 57.6 %

Prevalence WHO 2-tests strategyPositive predictive value*

*

• Difficulties of conducting independent evaluations

• Lack of sensitivity and of specificity

• RDT and EIA serological assays are not independent , all being based onot gp41 reactivity

• WB is costly with an high rate undetermined samples

• A new algorithm is badly needed , based on the association of with a sensitive serological screening test and a specific molecularindependent assay

RDT : TO REDUCE THE COST BUT NOT AT ALL PRICE

HIV DIAGNOSIS IN AFRICA : PROPOSAL FOR A SERO-MOLECULAR ALGORITHM

RDTPOC

Whole bloodPlasma

HIV-1RNA NAT

VL on DBS

+

HIV-2 RDT

HIV-1 reportHIV-2 report

-

primaryinfection or variant

suspected

-

+

-New sample

andretest

RDT and Viral loadare both independent assays

Collect a new sample onto DBS

Refer tocentral lab

+

Persistance :Refer to Central for Blot

(LTNP) , peptides (Variant)

HIV MONITORING IN AFRICA

• Viral load alerts to non-adherence, treatment failure and HIV resistance

• HIV-1 diversity is now over-passed by the new assays

• Is Cost effective to Guide Switching Antiretroviral Therapy in Resource-limited Settings

Laboratory Monitoring to Guide Switching Antiretrov iral Therapy in Resource-limited Settings: Clinical Benefits and Co st-effectiveness

April D. Kimmel et al for the CEPAC-International Investigators

Is a • Simple • Cheap • Reliable • Sensitive* • Individual • Disposable• No power• No cold chain, • no freezer, • no maintenance• Rapid

HIV VIRAL LOAD MONITORING IN RESOURCE-POOR SETTINGS LOCATIONS

* At least equivalent to 3 to 4 log equivalent RNA copies/mL

available in a near** future ?

•Cepheid, SAMBA, Inverness, CIGHT, Iquum Liat, Micronics DxBox, Wave 80 ESCAPE-20•Most are still in proof of concept•No field evaluations

Parallel plasma & DBS stored at RT for 7 daysMiniMAG – HIV-1 Easy Q 82 patients :31 (37.8 % ) undetectable in both plasma and DBS

DBS+ Plasma VL

33% 50-400 copies67% 400-3000100 % > 3000

28 presentations in Vienna 2010 :

Plasma VL(Log

cop/ml)

DBS storage ( °°°°C) WO W2 W4 W8

< 1.6-20°°°°C

RT37

< 1.6 Not done< 1.6< 1.6< 1.6

< 1.6< 1.6< 1.6

1.8-20°°°°C

RT37

<1.6 Not done

< 1.6 < 1.6< 1.6

< 1.63.3

< 1.6

3.1-20°°°°C

RT37

3.63.63.73.6

3.63.53.7

3.53.73.8

3.5-20°°°°C

RT37

3.84.24.34.3

3.94.23.9

4.14.03.8

4.3-20°°°°C

RT37

4.6 Not done4.54.54.5

4.54.44.5

4.4-20°°°°C

RT37

4.64.95.35.2

4.64.94.9

4.54.94.7

4.5-20°°°°C

RT37

4.7 Not done4.64.84.8

4.14.44.5

Evaluation of VL and genotyping on DBS under different storage conditionsM Arredondo et Al

Dried Blood Spot HIV-1 RNA Quantification Using Open Real-Time Systems in South Africa and Burkina Faso

- 892 DBS HIV-1-positive pregnant women and their neonates

- For early infant diagnosis, the sensitivity and specificity were 100%

- DBS viral load kinetics were similar to those in plasma in treated patients

J Acquir Immune Defic Syndr. 2010 Aug 10. [Epub ahead of print]

Vilijoen et al, WHO-ANRS 1289 Kesho ora study group

IS ULTRA SENSITIVE P24 AG AN ALTERNATIVETO THE NAT IN POC ?

• The p 24 antigen or core antigen forms the conical core of the viral particle

• Interacts with Cyclophilin A, leading to its incorporation into the virion

• Complex crystal structure

• 13 potential antigenic sites with 2 major epitopes in the CypA binding loop and adjacent helices and at the end of the C-terminal domain.

HIV P24 : A HIGH GENETIC DIVERSITY

The mean SDR values for the group M

• p24 0.47 (±0.004), • p32 0.50 (±0.003) • Gp160 0.49 (±0.002

D Robertson AIDS 2007

Anti-p24 antibodies (subtype B) are broadly cross-reactive but p24 has a high genetic diversity

HIV P24 : A HIGH GENETIC DIVERSITY SAINT LOUIS 2010

0,01

0,1

1

10

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Série1

Série2

0,01

0,1

1

10

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Série1

Série2

14 samples Subtype B 14 samples non-subtype B

Viral load Log

Viral load Log

Agp24 reactivity ratio Agp24 reactivity ratio

28 HIV- patients naives of HAART, chronically infectedR Nabias 2010

VL

Ag p24 Ag p24

VL

ULTRASENSITIVE HIV-1 P24 ANTIGEN & DBS ?

• 38 Tanzanian children

• DBS-p24, 32 (84%) of 38 (subtype D FN)

• DBS DNA, 30 (79%) of 38 ; – plasma-p24, 23 (85%) of 27 ;– plasma RNA, 30 (100%) of 30

– But is the p24 EIA more simple to perform than VL assay ??

SchSchüüpbach Jpbach J.. J Acquir Immune Defic Syndr. 2007 Mar 1;44(3):247-53

Highly sensitive assay , could detect < 0.5 to 0.1 pg/mL

NP based Biobarcode amplification

THE FIRST COMMERCIAL AG P24 RAPID TEST LACKS OF SENSITIVIY

AgP24(pg/ml)

> 400 400-100 100-50 50-5

Référence

EIA P24

VIDAS

6 4 5 17

Determine 4eme G

Ag POSITIF

4 3 1 0

JC Tardy , Lyon 2009

32 HIV-1 p24 positive sera by EIA

Competitive field and technical improvement will come soonWith and without decomplexation

development of a low-cost dipstick p24 antigen assay

Heat shock methods to disrupt immune complex

Analytical sensitivityof 50 pg/mL

Equivalent to4.6 log or RNA

Ag only Ab + Ag

A NEW ACADEMIC SIMPLE AND CHEAP DIPSTICK FOR AG P24 AND AB+AG DETECTION

Ab only

c

D. Glencross, MOPE0080 Wien 2010

CD38 on CD8 = correlate with CD4 & VLReliable marker of declining VL

73 pts, regular follow up 180 weeksin 62% successfully used : VL decreasing in 12% rises in CD38 : increased VLin 26% fluctuations with no matching

? Elevated CD38 -> VL monitoring ?

CD38 ACTIVATION AS ALTERNATIVE TO VIRAL LOAD FOR MONITORING

CONCLUSIONS :

• In a POC setting

– - RDT : new sensitive and specific RDT badly needed – DBS and new confirmatory strategy for diagnosis– - US p24 unitary RTD sensitive and cheap : in progress

• At the district level– new immunological follow up like CD38 – DBS and viral load– new VL technologies

PROJET PASCALPROJET PASCAL

PAPArtnership for SCSCaling up AAccess to viral LLoad testing in resource-constrainedsettings

CHU Saint Louis Paris

Laboratoire associéau Centre National de Référence du VIH

Faculté de Médecine-Pharmacie

Université de Rouen

J. Christophe PlantierMarie Gueudin

François SimonConstance Delaugerre

J. Michel Molina

PATIENTS, NURSES, TECHNICIANS AND ALL THOSE MAKING THESE STUDIES POSSIBLE

Eric Laforgerie

AKNOWLEDGMENTS

HIV IN LOW-RESOURCE SETTINGS: HOW TO DO MORE WITH LESS