Dried Blood Spots (DBS) for HIV-1 viral load and Drug Resistance

5èmes journées du Site ANRS-Cameroun
Yaoundé, 3-4 juin 2013
ANRS-12235
Le sang total séché sur papier filtre, DBS (Dried Blood Spot), est-il utilisable pour
le suivi virologique en routine des patients VIH+ traités et suivis dans les
programmes nationaux : étude multicentrique en Afrique et en Asie du Sud-Est
AC11/AC12 ANRS:
Groupe Résistance au Sud
Coordination: M. Peeters/ML. Chaix
ANRS-12235:
Coordination: A. Ayouba/A. Aghokeng
MEB: M. Monleau
Biostatistiques: S. Eymard-Duvernay
Context and Objectives
Context


Increasing number of patients on HAART in developing countries;
limited access to biological monitoring for those located far from
specialized laboratories;
Main objective
To evaluate the reliability of DBS to detect virological failure and drug resistance
mutations in HIV infected patients in real-life conditions and following routine processes
in resource-limited settings which are diverse in terms of climate characteristics with
different ambient temperature and humidity level, but are also different in terms of
available infrastructures and program functioning.
Specific Objectives
1.
Can DBS replace plasma for HIV viral load testing, regardless the viral load system used and the
setting?
2.
Are DRM profiles observed from plasma and DBS similar?
3.
How many failing patients with DRM will be missed if DBS were used for HIV-1 genotyping?
Methods (1): Study Sites
•
Burkina-Faso (Dr D. KANIA)
–
•
Cameroun (Dr A. AGHOKENG)
–
•
3 sites de recrutement: Biyemassi, Cité Verte
et Nkolndongo
Sénégal (Pr C. TOURE-KANE)
–
•
1 site de recrutement: hôpital de jour de
Sano-Soro de Bobo-Dioulasso
5 sites de recrutement (CTA, CRCF, CPS, HALD,
Roi Baudoin)
Thaïlande (Dr N.NGO-GIANG HUONG)
–
5 sites de recrutement (Mae Chan Hospital,
Chonburi Hospital, Hat Yai hospital, Maharaj
Nakornratchasrima Hospital, Ratchaburi Hospital)
•
Togo (Dr A. Yaoste DAGNRA)
–
5 sites de recrutement (CHU TOKOIN, Association
EVT, Association CRIPS, Association AMC, Asprofem)
•
Vietnam (Dr L. Truong XUAN)
–
3 sites de recrutement (OPC 1, OPC2, OPC4)
Methods (2): STUDY FLOWCHART
Blood samples from 1,621 adult patients receiving first line ART for 24 months were
used to prepare in parallel plasma aliquots and DBS
DBS
Plasma
-2-4 weeks at room temperature
-Transfer to -20°C until use
Nucleic acids extraction methods:
- Qiagen viral RNA in Labs A and F
- m2000rt, Abbott in Labs B, C, D
- MiniMag, Biomérieux in Lab E
Nucleic acids extraction methods:
- m2000rt, Abbott in Labs B, C,D
- MiniMag, Biomérieux in Labs A, E, F
HIV-1 VL quantification by:
-Generic Viral load (Biocentric) in Labs A & F
-m2000rt (Abbott) in Labs B, C, D
-EasyQ (Biomerieux) in Lab E
If VL ≥1,000 copies/mL in Plasma and ≥5,000 in DBS
amplification and sequencing of reverse transcriptase (RT) by nested RT-PCR. (ANRS protocol).
Results(1): FEASIBILITY
Parameter
Mean
[Interquartile Range]
Ambient T° during DBS
preparation (°C)
24
[22-26]
10
[4-18]
7
[0-9]
24
[-40-28]
1
[0-1]
DBS drying (hours)
DBS stockage on site (days)
T° During transfer to Ref lab.
(°C)
Transfer duration (days)
Results (2):
Sensitivity and specificity to detect virological failure
(>1,000 copies/ml) in DBS compared to plasma
Viral load
Assay
N
Abbott
173
Lab B
Lab C
Lab D
Generic VL
(Biocentric)
Lab A
Lab F
60
Plasma VL
>1,000 cp/ml
False
Negative*
Sensitivity
[IC95%]
False
Positive**
Specificity
[IC95%]
PL-DBS Pairs with
detectable VL
Nb diff < 0.5
log (%)
24
0
100
1
97.2
26
24
(40%)
53
13
[85.8-100]
0
(25%)
60
12
(20%)
100
7
[75.3-100]
3
75.0
(92%)
[85.5-99.9]
82.5
18
[67.2-92.7]
1
[42.8-94.5]
97.9
12
(67%)
10
8
(80%)
[88.9-99.9]
118
60
16
1
(27%)
58
23
93.8
17
[69.8-99.8]
2
(40%)
91.3
61.4
23
(70%)
[45.5-75.6]
14
[72.0-98.9]
60.0
16
37
18
(49%)
[42.1-76.1]
EasyQ
(Biomerieux
Lab F
91
67
(74%)
10
85.1
[74.3-92.6]
1
95.8
[78.9-99.9]
76
28
(37%)
*False Negative : VL >1,000 cp/ml in Plasma but <1,000 cp/ml in DBS. **False Positive : VL <1,000 cp/ml in Plasma but >1,000 cp/ml in DBS
DBS VL (Log10 copies/ml)
Results (3):
Correlation between PL and DBS VL.
Lab A Generic VL
8
7
6
5
4
3
2
8
7
6
5
4
3
2
(r=0.91)
Lab B m2000rt
8
7
6
5
4
3
2
(r=0.98)
Lab C m2000rt
8
7
6
5
4
3
2
(r=0.99)
Depends on:
2 3 4 5 6 7 8
2 3 4 5 6 7 8
2 3 4 5 6 7 8
Lab E EasyQ
Lab D m2000rt
Lab F Generic VL
(r=0.82)
2 3 4 5 6 7 8
8
7
6
5
4
3
2
8
(r=0.95)
7
(r=0.84)
6
5
4
3
2
2 3 4 5 6 7 8
2
3
4
5
6
7
8
Plasma VL (Log10 copies/ml)
VL assay used
Lab experience
Results (5):
Can DBS replace plasma for genotypic drug resistance testing: Are
nucleotides sequences observed from plasma and DBS identical?
45
number of samples
40
 Plasma vs DBS nucleotide sequences
35
30
<1% nt difference
1-1.99% nt difference
2-2.99% nt difference
3-3.99% difference
>4% nt difference
25
20
15
10
5
0
lab A
N=11
Lab B
N=20
Lab C
N=11
Lab D
N=3
Lab E
N=38
Lab F
N=15
Total
N=98
>80% of PL/DBS
sequences pairs
presented less
than 3%
nucleotides
differences
Results (6):
Can DBS replace plasma for genotypic drug resistance testing: Are
DRM profiles observed from plasma and DBS similar?
Genotyped
Any DRM
Identical DRM profile
Discordant DRM profile
DRM in Plasma NOT DBS
DRM in DBS NOT Plasma
Different DRM profiles
Plasma
98
90
DBS
98
81
75
23
10
3
10
Results (7):
Quality control of NT sequences of discordant samples:
Origin of discordant DRM profiles
E-2558DBS
E-2558PL
F-701DBS
F-701PL
D-2149DBS
D-2149PL
C-1179DBS
C-1179PL
F-493DBS
F-493PL
F-307DBS
F-307PL
F-451DBS
F-451PL
F-622DBS
F-622PL
F-600DBS
F-688DBS
F-600PL
F-704DBS
F-252DBS
F-252PL
F-704PL
F-688PL
B-3175DBS
B-3175PL
C-2423DBS
C-2423PL
A-1224DBS
A-1224PL
0.01
E-2292DBS
E-2292PL
B-1320DBS
B-1320PL
B-3161DBS
B-3161PL
C-4471DBS
C-4471PL
E-3120DBS
E-3120PL
B-2129DBS
B-2129PL
E-2055DBS
E-2055PL
E-3341DBS
E-3341PL
 Some sequences obtained from
Plasma and DBS of the same
samples are different
Results (8):
How many drug resistant samples were not detected on
DBS?
Plasma
study
VL
DRM in FN*
site
>1,000 DBS VL <1,000 DBS samples
Lab
Lab
Lab
Lab
Lab
Lab
A
B
C
D
E
F
16/60
24/60
13/53
12/60
67/91
23/58
1/16
0/24
0/13
3/12
10/67
2/23
0/1
2/3
10/10
2/2
Discordant DRM
Plasma/DBS
1/11
4/20
3/11 (1 no DRM at all)
1/3
5/38 (1 no DRM at all)
9/15 (8 no DRM at all)
14/16 samples with DBS VL<1,000 cp/ml harbored HIV-1 with DRM
8/23 samples with discordant results showed no DRM.
Conclusion
• The present study shows that DBS can be an
alternative to plasma for the follow-up of HIV-1
infected patients in routine settings;
• Performances depend on:
– VL assay characteristics ;
• Detection of proviral DNA or not;
– Redefine VL threshold for some assays?
• Viral diversity;
– Staff training
• Necessity of staff training for HIV-1 genotyping
using DBS and of external Quality Control;
Acknowledgements
The members of the AC11/AC12
“Résistance dans les pays du Sud” study group
The patients who participated to this study
- B. Bazin
- C. Rekacewicz
- G. Collin
- M. Peeters
- E. Delaporte