Detection of Pediatric HIV Infection Using Dried Blood Spots
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: Subtypeindependent 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 0.4 Failed 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 Plasma VL negative TNA Positive TNA 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 ______________________________________________________ time TNA Results Real- -------------------------------------------------------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 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
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.
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