Pediatric Diagnosis of HIV-1 Infection Using Dried Blood Spots

Chin-Yih Ou, PhDNCHSTP/DHAPCenters 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

Storage of DBS

0.0

0.2

0.4

0.6

0.8

1.0

23C dry

-20Cwet

4C wet

23C wet

37C wet

Storage conditions

Rel

ativ

e am

ou

nt

of

HIV

mea

sure

d a

s co

mp

ared

wit

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hat

of

23C

dry

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12 days

20 days

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 Negative

HIV signal

Internal Control Internal Control

Lack of HIV signal

Invalid Findings:

Internal Control:

-0.2

0

0.2

0.4

0.6

0.8

1

0 20 40 60-0.2

0

0.2

0.4

0.6

0.8

1

0 20 40 60

Failed Late

Improper TNA isolation or amplification

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

Positive74 2

TNA

Negative0 0

Although concordance was 97%, the signals from the TNA assay werestronger than those from the DNA assay

Total Nucleic Acid is a better target than proviral DNA alone

0

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4

6

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16

0 2 4 6 8 10 12 14 16

Normalized total nucleic acids Ct

No

rmal

ized

DN

A C

t

On average, the TNA signal appeared 2 cycles earlierthan 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 real-time 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 ofinhibitors. 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 inhibitorsand were all negative by Roche 1.5 DNA tests.

SummaryDo 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.