Pediatric Diagnosis of HIV-1
Infection Using Dried Blood Spots
Chin-Yih Ou, PhD
NCHSTP/DHAP
Centers for Disease Control and
Prevention
Objectives

To develop and validate a nucleic acid
based-assay for the diagnosis of HIV-1
infection in infants and young children in
resource-poor countries using dried blood
spots
Mother-to-Child Transmission: Crisis

Without antiretroviral intervention, 20-45% of HIV-infected
women transmit HIV to infants.

In 2004, between 650,000 to 750,000 children were newly
infected. About half a million of children died of AIDS.

Because antiretroviral drugs are becoming more
affordable, many developing countries are expanding
programs on prevention of mother-to-child transmission. It
is thus important to identify infected infants early to initiate
antiretroviral therapy.
Problems of Laboratory Tests for the
diagnosis of HIV-1 vertical transmission

Because of the presence of maternal antibodies in children under
the age of 18 months, serologic tests are not useful.

Enhanced HIV p24 assay is potentially useful, but remains to be
validated.

Nucleic Acid Technology (NAT) based Assays could be useful in
pediatric diagnosis:
Standard PCR testing on whole blood, cell pellets, and dried blood
spots (DBS) has been used; but each approach has its own
limitations related to cost, suitability and sustainability in
resource-limited sites.
Why is DBS important for pediatric
diagnosis?

Easier to get blood samples by heel stick than
venipuncture.

Ease of sample collection, storage and shipping.
Testing can be performed in well-qualified
central laboratories.
Problems with DBS
Small volume: about 50 - 100 ul per DBS spot
Extraction of nucleic acid from the blood card is
labor-intensive and automation of the process is
technically challenging
Presence of PCR Inhibitors
Current PCR based methods
DeVange Panteleeff et al; Rapid method for screening dried blood
samples on filter paper for HIV-1 DNA. J.Clin.Microbiol., 37:350,
1999
Fisher et al; Simple DNA extraction method for DBS and comparison
of two PCR assays for diagnosis of vertical HIV-1 transmission.
J. Clin. Microbiol. 42:16, 2004
Problems: Detection sensitivity is low and thus requires
nested amplification. These methods are
not suitable for clinical settings
Use HIV total nucleic acid as the targets to
increase the detection sensitivity


When stored properly in humidity-free
conditions, HIV RNA can be detected after
several months.
Storage conditions:
humidity-tight bag
desiccant packs and humidity indicator
room temperature to -70C freezer
Relative amount of HIV measured as
compared with that of 23C dry
conditions
Storage of DBS
1.0
12 days
0.8
20 days
0.6
0.4
0.2
0.0
23C
dry
-20C
wet
4C
wet
23C
wet
Storage conditions
37C
wet
Total Nucleic Acid (TNA) Extraction


6 mm punch (about 1/5 of a DBS circle, 15ul
whole blood)
Magnetic beads (Cortex)
TNA is eluted in 50ul water and
10 ul (about 3ul whole blood TNA) is
used for RT PCR assay to detect HIV
Real-time RT PCR assay to detect
HIV-1 Total Nucleic Acid
Duplex assay:
HIV primers and fluorescent probe are
derived from a conserved region of
HIV Long Terminal Repeat:
Subtype-independent
Internal Control: Human RNaseP gene
Positive
HIV signal
Internal Control
Negative
Lack of HIV signal
Internal Control
Invalid Findings:
Internal Control:
1
1
0.8
0.8
0.6
0.6
Failed
0.4
0.4
0.2
0.2
0
0
-0.2 0
20
40
60
Improper TNA isolation or amplification
-0.2 0
Late
20
40
60
Determination of Real-Time Assay Results
DBS
Nucleic Acids
RT PCR Assay
HIV Signal
Positive
Negative
HIV-1 detected
Internal control signal
Positive
Negative or weak
HIV-1 not detected
Invalid result
Repeat extraction
Performance of total nucleic acid (TNA)
assay using DBS from Uganda: specificity


Samples: DBS from 52 un-infected infants and young
children (with negative plasma viral load)
Age: 12 to 60 weeks (mean =23.3 weeks)
N=52
TNA Positive
TNA Negative
Plasma VL
negative
1*
51
*: also positive for HIV gag and integrase sequence
Total nucleic acid (TNA) vs DNA alone


Samples: DBS from 76 infected infants and young
children (with positive plasma viral load)
Age: 8 to 80 weeks (mean = 40 weeks)
DNA Positive
DNA Negative
TNA
Positive
74
2
TNA
Negative
0
0
Although concordance was 97%, the signals from the TNA assay were
stronger than those from the DNA assay
Total Nucleic Acid is a better target
than proviral DNA alone
16
Normalized DNA Ct
14
12
10
8
6
4
2
0
0
2
4
6
8
10
12
14
16
Normalized total nucleic acids Ct
On average, the TNA signal appeared 2 cycles earlier
than the DNA signal.
Evaluation Of The Real-Time TNA Assay
Using Field Specimens - Cameroon
Heel-stick DBS
______________________________________________________
Real-time TNA Results
-------------------------------------------------------Amplicor DNA
Positive
Negative
______________________________________________________
Positive (50)
50
0
Negative (265)
2*
263
______________________________________________________
Total
(315)
52
263
______________________________________________________
*: Concordance = 99.4%
These two samples were found to be positive by another run of realtime assay based on LTR sequence and were also positive by gag
and integrase.
Issues to consider




Performed in a centralized facility with well-trained lab
staff.
Well-calibrated duplex assay reagents (HIV and internal
control primers and fluorescent probes)
DBS control panel
DBS proficiency panel
Performance of TNA assay: KiBS
Study, Kenya
Positive
Negative
Invalid
Roche
(n=85)
4
77
4*
TaqMan
(n=85)
5
80
0
*Presence of inhibitor. Upon re-extraction, these 4 samples were free of
inhibitors. Three of them were found to be Taqman negative and one of them
positive.
Performance of DBS TNA assay on
PMTCT plus program, Kenya
Positive
Negative
Invalid
Roche DNA
(n=120)
23
90
7*
TaqMan
(n=120)
23
97
0
Upon re-extraction, 5 of these 7 samples were free of inhibitors
and were all negative by Roche 1.5 DNA tests.
Summary
Do we have the right tool to diagnose HIV infection
in children under the age of 18 months?

We have developed a protocol using DBS to determine HIV
infection status in infants and children less than 1.5 years of age.
This approach has been validated using DBS specimens from
Uganda, Cameroon and Kenya.

The detection assay is a real-time duplex reaction and with
internal control built-in.

Cost of the entire test including nucleic acid isolation is 5 USD,
which is significantly cheaper than a commercial DNA-targeted
assay.

Technology transfer to Uganda, Kenya , Thailand, and South
Africa is in progress.