JAC Advance Access published online on October 16, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn412
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Original research |
Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance
1 Virus Reference Department, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK 2 Camden Primary Care Trust, London, UK 3 Division of Infection and Immunity, University College, London, UK
* Corresponding author. Tel: +44-20-8327-6470; Fax: +44-20-8200-1569; E-mail: andrew.buckton{at}hpa.org.uk
Received 22 May 2008; returned 4 August 2008; revised 11 August 2008; accepted 6 September 2008
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Abstract |
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Objectives: We present the evaluation of a methodology for the genotypic assessment of human immunodeficiency virus type-1 (HIV-1) drug resistance, optimized for use with dried blood spots (DBS).
Methods: The ability to generate HIV-1 protease (PR) and reverse transcriptase (RT) contiguous amplicons and nucleotide sequences from DBS was evaluated. Different collection matrices and extraction methodologies were compared. The relative subtype sensitivity of the amplification strategy was assessed using a comprehensive panel of plasmids representing A–H subtypes. A panel of DBS and plasma specimens was subjected to HIV genotyping. Sequences generated from each sample type were compared.
Results: Extensive replicate testing revealed most sensitivity with the use of 903 filter paper and silica/guanidine extraction, which had an estimated 95% inclusivity endpoint of 1542 proviral copies/mL, as compared with 21 573 proviral copies/mL for the FTA system. All HIV-1 group M subtypes analysed—with the exception of subtypes A2, AE, AG, F and H—had a relative sensitivity of 
10 plasmid copies/PCR reaction. The PCR was multiplexed to include amplification of a human housekeeping gene to monitor the integrity of the human genomic DNA. Using a panel of clinical samples, we demonstrated the ability to amplify and sequence from 83% (n = 10) in the PR region and 100% (n = 12) in the RT region, of samples with detectable viral load. All specimens with an HIV-1 RNA load 
1000 copies/mL were successfully amplified and sequenced. Twelve specimens had pol genotyping from both plasma and DBS samples. Sequence analysis and drug resistance interpretation revealed that 10 (83%) provided concordant drug resistance interpretation.
Conclusions: Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.
Key Words: HIV-1 , filter paper , antiretrovirals , scale-up
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Introduction |
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Filter papers have been used to store blood specimens for many years and are widely employed in the UK for the diagnostic testing of metabolic and inherited disorders in newborn babies.1–4 Dried blood spots (DBS) were first successfully applied to detect markers of human immunodeficiency virus type-1 (HIV-1) infection during post-natal sero-surveillance studies in the late 1980s.5 More recently, the application of HIV DBS testing has been broadened to include the following laboratory assays: nucleic acid detection,6–8 viral load determination by quantitative real-time PCR techniques,9–11 env V3 loop serotyping,12 anti-HIV antibody detection by gelatin particle agglutination,13 p24 antigen detection14 and, recently, drug resistance genotyping.15–19
Blood spot technology offers a number of advantages over standard diagnostic testing procedures. First, it circumvents the requirement for trained phlebotomists, given the simplicity of sample collection in the field. Secondly, DBS can be safely transported from the place of collection to the laboratory at ambient temperature, via standard postal systems. This permits remote testing without the requirement of specialist low temperature shipping, as is required for plasma RNA testing. Taken together, DBS offer a simple, safe and cost-effective method for monitoring HIV-1 drug resistance in resource-poor settings. However, this approach has some potential disadvantages compared with standard genotyping methodologies: DBS testing approaches may lack sensitivity when compared with plasma RNA-based methods and the contribution of HIV proviral DNA may also misrepresent historically archived sequences,15 and this may skew interpretation of the drug resistance genotype of the viraemic strain.
The recent innovation of several DBS-based genotyping assays shows global interest in the development and implementation of such tests;15,18 however, no consensus as to optimal collection matrices, storage conditions, nucleic acid extraction and amplification strategies has yet been determined.
The studies presented here provide information to guide the most appropriate testing procedures for HIV-1 DBS drug resistance genotyping. We highlight the importance of filter paper and extraction method selection for the success of DBS genotyping methodologies. In addition, we report the effect of storage temperature on DBS stability and show that the integrity of DBS nucleic acid may be monitored by the incorporation of human housekeeping gene primers into the amplification strategy. Finally, using a small patient study, we compared HIV pol consensus sequences derived from plasma and DBS nucleic acid extracts.
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Materials and methods |
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Optimization and validation reagents
The human lymphocyte cell line 8E5/LAV20 was cultured in vitro. This contains a single HIV DNA proviral genome per cell. Appropriate dilutions were made, using normal human whole blood collected on sodium citrate, from pellets containing 1 x 106 8E5 lymphocytes. The use of this cell line for optimization studies provides the best reagent to simulate infected lymphocytes, which may be co-amplified along with viral RNA in plasma using DBS technology, containing both integrated provirus and transcript RNA. A log10 diminishing dilution series, with a lower dilution of 100 8E5/LAV lymphocytes/mL, was prepared. These dilutions were used to make three sets of blood spots on 903 filter paper. These DBS sets were stored for 3 months, but at different temperatures: room temperature, 4°C and –20°C. DBS were stored in zip-locked plastic bags along with a silica gel desiccant sachet, as described below. A panel of HIV-1 group M subtype clones was obtained from NIBSC, Potters Bar, UK. This panel contained a diverse range of group M subtypes (Table 1); subtype information and country of origin were provided on the accompanying datasheet. The concentration of each was determined using Quant-IT PicoGreen dsDNA quantification (Invitrogen, Paisley, UK), and each plasmid clone was diluted into working concentrations comprising 100 000, 31 600, 3160 and 1000 HIV copies/mL. Panels were titrated using TE buffer supplemented with a background of human genomic DNA, extracted from normal human whole blood and diluted to a concentration that was approximately equal to the cellular equivalents per reaction. These dilutions were used to assess the relative sensitivity of the amplification strategy to function across a range of group M subtypes.
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Human specimens
EDTA-containing whole blood specimens from 33 adult HIV-infected clinic attendees were collected (The Mortimer Market Centre, University College Hospital, London, UK). Specimens were blinded with regard to clinical and patient information, and informed consent was sought at enrolment.
DBS were prepared as follows: 4x 50 µL were spotted onto 903 filter paper (Whatman, Maidstone, UK) for each sample in a Class II biosafety cabinet. These were air-dried for 1 h at room temperature and then placed into a zip-locked bag along with a silica gel desiccant sachet. Humidity is known to adversely affect amplification of HIV nucleic acids from DBS.21 Blood spots were stored at –20°C. Plasma from each sample was collected, and each viral load was quantified using the COBAS AmpliPrep HIV Taqman Assay (Roche, Lewes, UK). Remaining plasma specimens were then stored at –80°C. Both DBS and plasma specimens were submitted for HIV drug resistance genotyping. Nucleic acid was extracted from DBS using the silica/guanidine method (discussed subsequently) and from plasma using an Ultra Sens Viral RNA Kit (Qiagen, Hilden, Germany). Extracted specimens were PCR-amplified and sequenced, as detailed below.
Two 6 mm diameter filter paper spots were punched, using a standard office hole-punch, from a 903 filter paper placed into a sterile 2 mL microtube, containing 150 µL of PBS and 600 µL of L6 guanidine lysis buffer (Severn Biotech, Kidderminster, UK). We estimate that this is equivalent to 100 µL of whole blood per extract. DBS were resuspended by gentle rotation for 2 h at room temperature. The lysate was separated from the filter paper debris by brief centrifugation, transferred to a fresh microtube, along with 10 µL of silica suspension (Severn Biotech), and rotated gently for 10 min at room temperature. The silica was then pelleted by brief centrifugation and washed twice with excess buffer L2 (Severn Biotech), twice with excess 70% ethanol and once with excess 100% acetone. The silica pellets were dried for 10 min at 56°C. Specimens were resuspended in 60 µL of nuclease-free water containing 40 U of recombinant ribonuclease inhibitor (Promega, Madison, WI, USA) and incubated at 56°C for 10 min, followed by centrifugation at 13 000 g for 5 min. The eluate was transferred to a fresh microtube for further processing. Extracted material (10 µL) was subjected to PCR amplification.
cDNA synthesis and PCR amplification
Nucleic acid extracts were subjected to RT–PCR using the OneStep RT–PCR Kit (Qiagen), according to the manufacturer's guidelines. The addition of this step ensures that the total HIV nucleic acids present in DBS extracts are available for amplification. Briefly, two separate reactions with contiguous amplicons spanning positions 2057–2979 and 2813–3618 of the HIV genome (numbering according to the HXB2 reference strain K03455 [GenBank] 22) were prepared for each specimen. All HIV-specific oligonucleotide sequences are based on existing WHO/HIVResNet Global HIV Drug Resistance primers; 15 pmol of each (at a concentration higher than that of β-globin primers to take account of the relative proportions of both template materials) of the protease (PR) primers, P1 (5'-TGA ARG AIT GYA CTG ARA GRC AGG CTA AT) and P2 (5'-AYC TIA TYC CTG GTG TYT CAT TRT T), or of the reverse transcriptase (RT) primers, pR5 (5'-GGA AGT TCA ATT AGG AAT ACC ACD) and R2 (5'-CCT CIT TYT TGC ATA YTT YCC TGT T), were added. In order to monitor DBS integrity, 5 pmol of each of the primers GH20 (5'-GAA GAG CCA AGG ACA GGT AC) and PCO4 (5'-CAA CTT CAT CCA CGT TCA CC) were added to amplify a portion of the human β-globin gene. Reactions were cycled using a Dyad Thermal Cycler (Bio-Rad, Hercules, CA, USA) with an initial incubation of 40 min at 50°C and denaturation at 95°C for 15 min, followed by 35 cycles of 30 s at 94°C, 30 s at 53°C and 1 min at 72°C, with a final extension of 4 min at 72°C.
For nested round amplification, a 1 µL portion of each RT–PCR reaction product was added to the following mixture: 20 mM Tris (pH 8.4), 72 mM KCl, 0.2 mM deoxynucleotide triphosphate mixture (Invitrogen) and 2.5 U of Platinum Taq DNA polymerase (Invitrogen). To this mixture, for the PR region, 1 mM MgCl2 and 15 pmol of each of the PR primers, P7-R (5'-CTT TAR CTT CCC TCA RAT CAC TCT) and P8 (5'-TCC TGA AGT CTT YAT CTA AGG GAA C), were added. For the RT fragment, 2 mM MgCl2 and 15 pmol of each of the RT primers, R7 (5'-AAT CAG TAA CAG TAC TGG ATG TGG GT) and R8 (5'-GGC TCT TGA TAA ATT TGA TAT GTC CAT), were added. Reactions were adjusted to 50 µL with nuclease-free water and amplified for 5 min at 95°C, followed by 35 cycles of 30 s at 90°C, 30 s at 50.3°C and 30 s at 65°C, with a final extension of 2 min at 72°C.
Amplification of both pol and β-globin fragments was assessed by agarose gel electrophoresis.
Purified amplicons generated from the clinically derived specimens were sequenced using an ABI capillary 3130xl sequencer (Applied Biosystems, Foster City, CA, USA). DNA sequencing reactions were performed using the second-round PCR primers, as required, with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). Sequence data were edited using Sequencher 4.7 software (Genecodes, Ann Arbor, MI, USA). Subtype determination and drug resistance interpretation for codons 5–99 of PR and 1–320 of RT were performed using the Genotypic Drug Resistance Interpretation Algorithm, University of Stanford (http://hivdb.stanford.edu/pages/algs/HIVdb.html).
The nucleotide sequences derived in this study have been placed in the GenBank sequence repository (EU401678 [GenBank] –EU401701 [GenBank] ).
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Optimization of filter paper collection matrices and extraction procedures
As a part of assay optimization studies, two commonly used filter media were evaluated to determine which had the greatest HIV nucleic acid recovery. Each filter paper type required a different extraction procedure: 903 paper with silica/guanidine and FTA paper with the FTA wash procedure. The FTA matrix is specially designed to lyse cells, denature proteins and protect nucleic acids from nucleases, oxidation and UV damage. This matrix is reported to inactivate blood-borne pathogens and to prevent the growth of bacteria and other microorganisms. As this paper is designed to be used downstream in molecular tests, it is supplied with its own extraction reagent. Five half-log10 dilution series of 8E5/LAV cells were prepared in anti-coagulated normal human whole blood. The dilution series ranged between 100 and 50 000 cells/mL. For each dilution, DBS were prepared on 903 and FTA filter papers in duplicate. DBS were extracted as described for the 903 paper and according to the manufacturer's instructions for the FTA wash procedure. Extracts were amplified using the PR-containing PCR amplification; the experimental procedure omitted the initial RT step, such that only proviral DNA was amplified. Amplification was assessed by agarose gel electrophoresis. Probit regression analysis (STATA version 8.2, Statacorp, TX, USA) was used to estimate the lower limits of PCR detection for the methods. This was calculated using the proportion of PCR positive samples at each point in a log10 dilution series (Table 2). The lowest number of HIV copies required to generate a positive signal, either 95% or 50% of the times the PCR is performed. The 95% inclusivity endpoint of the developed methodology, using 903 filter paper and silica/guanidine extraction, was 1542 proviral copies/mL, compared with 21 573 proviral copies/mL for the FTA system. The use of 903 filter paper and silica/guanidine extraction provided the best recovery of HIV-1 DNA. We also performed a small evaluation of DBS on 903 filter paper extracted using the external lysis protocol of the MagNA pure LC robot (Roche). We found the overall sensitivity cut-off to be 
45 750 proviral copies/mL (data not shown). These small, initial studies indicated to us that this system may not be applicable to automated extraction, so a larger evaluation was not performed.
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Sensitivity of the amplification strategy across group M plasmid clones
To determine the sensitivity of the PR- and RT-containing PCR amplification strategy to function across a broad range of group M isolates, plasmid clones were titrated and used as PCR template. Twenty-one group M clones (Table 1) were used to generate plasmid preparations from which each dilution series was prepared. Plasmid stocks were diluted such that panels were generated containing 1000, 316, 31.6 and 10 plasmid copies/reaction, in duplicate. Both independently prepared panels were then assayed by two technicians in duplicate, such that four data points per subtype/dilution were generated, for both the PR- and RT-containing amplification reactions. Probit analysis was used to calculate the relative sensitivity cut-off for each subtype for each genome (Table 2). These analyses reveal that our amplification strategy is broadly effective for many globally occurring HIV subtypes.
Monitoring DBS human genomic DNA integrity and storage temperature stability studies
In order to investigate the significance of DBS storage temperature, a 10-fold dilution series of 8E5/LAV lymphocytes was prepared and spotted onto 903 filter paper. DBS were stored at room temperature (20°C), 4°C and –20°C, for 3 months. DNA PCR amplification (for the PR region only) revealed no reduction in HIV-1 or human genomic (β-globin gene) DNA with increasing temperature, at any dilution. The nested PCR endpoint was 100 proviral copies, at room temperature, 4°C and –20°C. Agarose gel analysis of the first-round PCR products revealed an endpoint of 100–1000 HIV DNA genomes/mL at all three storage temperatures. The human β-globin gene was amplified (268 bp) in all dilutions and irrespective of the storage temperatures tested. In an attempt to observe DBS DNA degradation, DNA extracts were further tested using an in-house alternative method to amplify a larger pol fragment (not described), of 1.3 kb, comprising both PR and RT. As with the smaller PR product, no detectable degradation occurred with increasing temperature (data not shown).
Comparison of DBS and plasma-derived consensus pol sequences from patient specimens
Venous blood was taken from 33 HIV-infected outpatient clinic attendees to compare pol sequences generated from both plasma- and DBS-derived template material. Plasma samples were submitted for viral load testing, of which 12 (36%) had a viral load of >50 copies/mL, which ranged between 80 and 115 380 copies/mL (median 22 005 copies/mL) (Table 3). Those with detectable HIV-1 RNA load were submitted for pol genotyping. DBS from all samples were extracted and amplified across the pol region. The human β-globin product was amplified in each case. Of those with detectable plasma RNA, 10 (83%) for the PR fragment and 12/12 (100%) for the RT fragment were amplified. Of those with no detectable plasma RNA (<50 copies/mL), 12 (57%) for the PR fragment and 7/21 (33%) for the RT fragment were amplified. Table 3 shows the results from these studies. Sequence polymorphisms and subtype designation were determined using the Genotypic Drug Resistance Interpretation Algorithm, University of Stanford. Discrepant codons between the two sample types are shown in Table 4. These data show that of the 12 specimens, for which sequence data were generated from both plasma and DBS, 10 (83%) gave concordant drug resistance profiles across the amplicons analysed. In order to demonstrate that both sequences were generated from the same source material, phylogenetic analysis was performed using a 620 bp fragment of the RT gene (nucleotide positions 2889–3509 of the HXB2 HIV genome), which represented the region for which sequence data were obtained for all samples. Reference strains representing the subtypes present and an outlaying sequence (A, CRF02_AG, B, C and group O) were added to the tree generated using MegAlign (DNASTAR Inc., WI, USA) (Figure 1). It revealed the close sequence homology of the paired specimens, indicating that both sequences were indeed derived from the same patient.
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Discussion |
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In this article, we report the development, optimization and performance of a PCR-based assay using DBS for the purpose of HIV-1 drug resistance assessment and subtyping using the pol gene. Our studies confirm and extend the previous observations15–18 that DBS may provide a suitable sample for HIV-1 drug resistance genotyping. The approach described here confers a number of advantages over previously reported approaches. First, the use of a human gene internal amplification control enables evaluation of the integrity of human genomic DNA in DBS samples. Secondly, our amplification primers have been developed and rigorously tested against a broad panel of group M subtypes. This will enable the testing of DBS specimens from a wide range of geographical locations. Thirdly, the approach is sensitive. Our studies demonstrated an estimated 95% inclusivity cut-off of
1542 proviral copies/mL. Indeed, during the patient study reported here, all specimens with an HIV-1 RNA load 1000 copies/mL were successfully amplified and sequenced across the pol region. Fourth, the assay has been developed to minimize costs incurred to the testing laboratory, and this has been achieved in two ways: (i) the extraction method uses low-cost reagents, namely a silica matrix, a guanidine extraction buffer and ethanol-based wash buffers;23 and (ii) only four sequencing reactions are used to generate bi-directional, contiguous sequence data spanning 1245 nucleotides of the PR–RT-encoding region. One technical caveat of the assay should be noted. Here, this amplification strategy performed less efficiently against subtypes A2, AE, AG, F and H for the RT-containing amplicon. Amplification of the PR-containing product was also poorer performing on subtypes AG, F and H. Indeed, investigation of the primer and template sequence from subtype A failed to reveal any sequence mismatches between the sequences, which may have explained our observations. Thus, future versions of this test may require incorporation of primers that target subtype A variants with increased efficiency. In contrast, subtype A and CRF02_AG samples were successfully amplified and sequenced as part of the patient study reported here.
Previously published studies describing HIV genotyping from dried fluid spots have used Whatman 903 filter paper,15–18,24 although with apparently little justification. In this study, we directly compared two filter paper matrices and extraction procedures to determine which offered the greatest HIV nucleic acid recovery. Here, we compared 903 filter paper, coupled to a silica/guanidine extraction, and the FTA DBS system. The evaluation of the two systems demonstrated that the 903 filter paper and silica/guanidine extraction recovered most of the HIV nucleic acids, having a 95% inclusivity endpoint of 1542 proviral copies/mL, when compared with the FTA system at 21 573 proviral copies/mL. Our systematic appraisal of filter paper and extraction methods confirms the empirical observations of others that 903 paper provides the best capture and storage matrix for genotyping from DBS. Nevertheless, the use of the 8E5/LAV cell line for this evaluation may be flawed because a significant majority of viral RNA is cell-associated.
The incorporation of primers to amplify a fragment of the human β-globin gene provides this assay system with a means of identifying DBS that have undergone degradation. Its inclusion in the assay parameters is of value, given that DBS specimens may be stored and shipped in conditions damaging to their integrity. Of the 33 specimens tested as part of the patient study, all had a detectable human gene product and thus provided reassurance that DBS had been properly stored, even when HIV nucleic acids remained undetectable. The evaluation of our assay also involved stability testing of DBS proviral DNA dilution series at three different temperatures. The procedure was robust enough to detect the lowest dilution performed, 100 8E5 proviral DNA copies/mL, even when stored at 20°C for 3 months.
In order to assess the effectiveness of our DBS genotyping procedure, we tested 33 HIV clinic attendees. We observed that 13 (62%) of those specimens with plasma RNA of <50 copies/mL were detectable in either or both of the PCR reactions. Those specimens with a detectable RNA load were amplified, and consensus DNA sequencing was performed. These studies sought to evaluate the degree to which proviral DNA contributes to DBS and whether this can give rise to a drug resistance genotype discordant to that derived from the plasma compartment. We had hoped to include an estimation of CD4 cell count for study specimens. This would have enabled a correlation between CD4 and success of DBS resistance testing. The study design was such that these specimens were unlinked as to the outcome of other laboratory tests. The data presented in Table 4 show discrepant amino acids between the two samples. Only two specimens (432 and 437) had any observed drug resistance; in both cases, the DBS and plasma genotypes were discordant. For specimen 432, an RT mutation, of leucine to isoleucine at residue 100 (existing as a mixture with wild-type and valine at this position), which causes intermediate resistance to non-nucleotide RT inhibitors, was observed in the plasma only. This observation may be explained by the recent emergence of this strain, yet to be established in the proviral archive, as has been previously observed.25
For specimen 437, two key nucleotide RT inhibitor mutations, occurring at residues 184 and 215, were observed in the DBS sequence only. This may therefore represent failure to previous drug treatments not presently selected for in the circulating plasma virions. The sequence derived from the DBS in this case contained many discrepant amino acids, including three stop codons. The comparison of the nucleotide sequences derived for 437 showed the accumulation of 17 guanine to adenine mutations across the RT sequence alone, which gave rise to the observed codon changes. This observation in the proviral sequence is consistent with an APOBEC hypermutation effect.26 Gifford et al.27 recently postulated that artefactual mutations, such as M184I, might be observed in some DBS-derived sequences. Our studies are supportive of their suggestion. Specimens 432 and 437 had relatively low plasma RNA load. The discrepancy between plasma and DBS sequences may thus have arisen due to the preferential amplification of HIV DNA in these specimens.
These studies contribute to the general understanding of the most appropriate molecular testing procedure for DBS-derived HIV genotyping. Our studies support the suggestion that DBS may not always reflect the drug resistance genotype of virus isolated from the plasma compartment. Nevertheless, this system provides a robust, sensitive, internally controlled DBS-based alternative to plasma HIV genotyping assays.
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These studies were funded by the Health Protection Agency, England.
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None to declare.
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Acknowledgements |
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We would like to thank Jim Waite (University College) for performing the HIV-1 load testing on study samples. HIV-1 plasmid clones were sourced from the National Institute of Biological Standards and Controls (NIBSC), UK.
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