1. REAL-TIME RT-PCR BASED ASSAY ON BLOOD CLOT SPECIMENS FOR DIAGNOSIS OF HIV-1 INFECTION IN CHILDREN, MALAWI Hua Yang 1, Rita Helfand 2, Desiree Witte 3, Ashley C. LaMonte 2, Yashieka Blount 1, Robin Broadhead 3, Felicity Cutts 4, Richard Fudzulani 3, Chin-Yih Ou 1, Renu B. Lal 1, Chunfu Yang 1 1 Divisions of HIV/AIDS Prevention, NCHSTP and 2 Division of Viral and Rickettsial Diseases, NCID, CDC, Atlanta; 3 University of Malawi; 4 London School of Hygiene and Tropical Medicine Abstract Background: Mother to child HIV-1 transmission is the most common route of HIV-1 infection in children. HIV antibody testing is unreliable for children born to HIV-infected mothers under 18 months of age due to maternal antibodies. Therefore, in children younger than 18 months of age, HIV-1 infection is diagnosed by detecting HIV-1 viral sequences. We have developed a duplex, one-tube, one-step real-time RT-PCR- based assay for HIV-1 diagnosis. In this study, we evaluated this assay for its ability to detect HIV infection in blood clot specimens in children born to HIV-1-infected mothers under the age of 18 months in Malawi. Methods: We collected 1ml of blood by fingerstick at 6, 9, 12, 18-24, and 30-36 months of age during a study of measles vaccination of HIV-infected and -uninfected children outside of Blantyre, Malawi. We performed HIV rapid antibody testing on blood collected > 18 months of age from children born to HIV- infected mothers. Antibody positivity ≥ 18 months was taken as the indicator of HIV-1 infection in children. Total nucleic acids were extracted from blood clot specimens following digestion with streptokinase, and real-time RT-PCR was performed using the primers in the HIV-1 LTR region. We tested blood clots from 34 children with positive HIV antibody results and 24 of 146 children with negative HIV antibody tests. Results: To evaluate the sensitivity and specificity of the assay, we first tested 34 children who were consistently HIV antibody positive ≥ 18 months of age and all of them had positive RT-PCR results from samples taken on at least two time points. We then tested 24 HIV-1 antibody negative children. Of the 24 children, 23 were HIV-1 negative by RT-PCR while one had positive HIV-1 RT-PCR at 6, 12, 18 and 24 months and undetectable antibody at 18 and 24 months by two rapid HIV tests. HIV infection was confirmed in this child by amplification of the HIV-1 gag region and sequencing. Conclusion: The one tube and one step Real-time RT-PCR used here was both sensitive and specific in detecting true HIV-1 infection from blood clot specimens in children born to HIV-1-infected mothers under the age of 18 months. Introduction Mother to child HIV-1 transmission (MTCT) is the most common route of HIV-1 infection in children despite the advantage of ARV. Because of maternal antibodies, HIV-1 infection in children born to HIV-1 infected mothers under the age of 18 months is diagnosed by detecting HIV-1 viral sequences. Although HIV-1 DNA PCR assay for HIV-1 diagnosis is commercially available, it is expensive and labor intensive for resource-limited countries. Here we report an adaptation of a duplex, one-step real- time RT-PCR assay for detection of HIV-1 total nucleic acid using blood clot specimens for the diagnosis of HIV-1 infection in children born to HIV-1 infected mothers. Materials and Methods Participants are part of a study of Measles vaccination of HIV-1 infected and – uninfected children born to HIV-1 infected mothers from outside of Blantyre, Malawi. One milliliter of blood was collected by fingerstick at 6, 9, 12, 18-24 and 30-36 months of age. HIV rapid tests, Unigold and Determine were performed on blood collected ≥ 18 months. Sera were collected for anti-Measles antibody testing, the remaining blood clots were stored at -20°C freezer for HIV-1 viral sequence detection. GeneSequencesFunctions HIV-1 LTR5’tgcttaagcctcaataaagcttgccttgaForward Primer HIV-1 LTR5’:tctgagggatctctagttaccagReverse Primer HIV-1 LTR5’Fam-aagtagtgtgtgcccgtctgt-Qsy-7Probe RNaseP5’ agatttggacctgcgagcgInternal Control Farward Primer RNaseP5’gagcggctgtctccacaagtInternal Control Reverse Primer RNaseP5’Hex-ttctgacctgaaggctctgcgcg-Qsy-7Probe Digestion of blood clots with streptokinase and extraction total nucleic acid Blood clot specimens Add 300 μL of PBS Add 20 units/vial of Streptokinase Incubate at 37°C for O/N Extract total nucleic acid with QIAamp Blood DNA mini kit using 200 μL of the O/N digests Real Time RT-PCR For a 25 μL of real-Time RT-PCR Reaction 1.Add 12.5 μL of QuantiTect Probe RT-PCR Master Mix 2.0.25 μL of /Quantitect RT Mix 3.1 μL of HIV-1-specific primer and probe mix 4.1 μL of Human RNAse P primer and probe mix 5.0.25 μL of RNase-free water 15 μL Add 10 μL of total nucleic acid extract Real Time RT-PCR: 50°C, 30 minutes; 95°C, 15 minutes; 60 cycles at 95°C for 15 second, 52°C for 1 minutes Primers and Probes Results Sensitivity of the Assay RT-PCR positive (month) No. of specimens (34) 6912182024 + + 2+ 1+++ 1+++ 1++ 9+++ + 4++ + 3 2 HIV rapid tests positive ≥ 18 months 30 10++ 1 Distribution of HIV-1 RT-PCR positivity based on the first positive RT-PCR Specificity of the assay No. SpecimenNegativePositiveHIV-1 sequence amplified Rapid tests ≥ months 24 (100%) 0 RT-PCR2423 (95.8%) 1* (4.2%) Yes Summary  In this study, we used a duplex, one-tube, one step real-time RT-PCR assay to detect HIV-1 infection from blood clot specimens collected from children born to HIV-1 infected mothers from Blantyre, Malawi.  We tested 34 children who were HIV antibody positive the age of ≥18 months using two rapid tests, Unigold and Determine, and all of them had positive RT-PCR results, giving a 100% testing sensitivity.  We also tested 24 children with negative HIV antibody tests. 23 of them had negative HIV-1 RT-PCR results while one child gave positive RT-PCR results at 6, 12, 18 and 24 months. HIV status of the child was further confirmed by the amplification and sequencing of the gag gene.  Based on the excellent sensitivity and specificity of the assay, we then tested 144 children who didn’t have HIV antibody tests due to unavailable specimens ≥ 18 months. We found 32 had two positive RT-PCR, 28 had one positive RT-PCR; 61 had two negative RT-PCR and 23 had one negative RT-PCR. Conclusions  The duplex, one tube, one step real-time RT- PCR used here was both sensitive and specific in detecting true HIV-1 infection from blood clot specimens in children born to HIV- 1-infected mothers under the age of 18 months.  This study confirms that blood clot specimens can be used for the detection of HIV viral sequences, opening a new avenue for HIV detection in children in terms of specimen specification. CDC, Mail Stop D-12 1600 Clifton Road Atlanta, GA 30333 cyang1@cdc.gov 404-639-4975 No. 39 * This child was HIV-1 real-time RT-PCR positive at 6, 12, 18 and 24 months.