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

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Without antiretroviral intervention, 20-45% of HIV-infected women transmit HIV to infants.

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In 2004, between 650,000 to 750,000 children were newly infected. About half a million of children died of AIDS.

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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

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Because of the presence of maternal antibodies in children under the age of 18 months, serologic tests are not useful.

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Enhanced HIV p24 assay is potentially useful, but remains to be validated.

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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

1.0

0.8

0.6

0.4

0.2

0.0

12 days 20 days 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 Negative

HIV signal Internal Control Lack of HIV signal Internal Control

Invalid Findings:

Internal Control: 1 0.8

0.6

0.4

0.2

0 -0.2

0 20 Failed 40 60 Improper TNA isolation or amplification 1 0.8

0.6

0.4

0.2

0 -0.2

0 Late 20 40 60

Determination of Real-Time Assay Results

DBS Nucleic Acids RT PCR Assay Positive HIV-1 detected HIV Signal Negative Internal control signal Positive HIV-1 not detected Negative or weak 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 Plasma VL negative TNA Positive 1* TNA Negative *: also positive for HIV

gag

and

integrase

sequence 51

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 14 12 10 4 2 8 6 0 0 2 4 6 8 10 12

Normalized total nucleic acids Ct

14 16 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 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) TaqMan (n=85) 4 5 77 80 4* 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

Roche DNA (n=120) TaqMan (n=120) Positive 23 23 Negative 90 97 Invalid 7* 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?

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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.

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The detection assay is a real-time duplex reaction and with internal control built-in.

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Cost of the entire test including nucleic acid isolation is 5 USD, which is significantly cheaper than a commercial DNA-targeted assay.

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Technology transfer to Uganda, Kenya , Thailand, and South Africa is in progress.