Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®
Abstract
:1. Introduction
2. Materials and Methods
2.1. RNA Extraction, cDNA Generation and HIV Env V3 Amplification
2.2. MiSeq™ Data Analysis
2.3. HIV Tropism Determined by PSSM, Geno2pheno and Codon Analysis
2.4. Cost of Assay
3. Results
3.1. Clinical Plasma HIV RNA and HIV Template Recovery
3.2. Comparison of V3-UMI Sequences Interpreted by Genotypic Analysis to Trofile®
3.3. Analysis of Classifications Discordant by Trofile® vs. Genotypic Algorithms
3.4. Cost Analysis
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Purpose | Sequence |
---|---|---|
cDNA | cDNA synthesis | 5′-CCCGCGTGGCCTCCTGAATTAT[ill.1]-CCGCTCCGTCCGACGACTCACTATA[ill.2]-SSMVSBYNNNNN[UMI]-CAGTAGAAAAATTCCCCTCCACAATT[HIV7377-7353] -3′ |
env6880F | 1st round, forward | 5′-CCCCGGCTGGTTTTGCGATTCTAAAGTGTA[HIV6880-6909] -3′ |
ill.1 | 1st round, reverse | 5′-CCCGCGTGGCCTCCTGAATTAT -3′ |
envC2F6 | 2nd round, forward | 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[MiSeq adapter]-GCACAGTACAATGTACACATGGAATTA[HIV6952-6978] -3′ |
ill.2 | 2nd round, reverse | 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[MiSeq adapter]-CCGCTCCGTCCGACGACTCACTATA[ill.2] -3′ |
cDNA oligo, 5′-3′ (HXB2 7353-7377) | ||||||||||||||
99% | 99% | 90% | 95% | 99% | 95% | 99% | 99% | 90% | 99% | 99% | 90% | 99% | ||
5′-C | A | G | T | A | G | A | A | A | A | A | T | T- | ||
99% | 90% | 99% | 99% | 95% | 95% | 90% | 95% | 99% | 99% | 95% | 95% | 99% | ||
-C | C | C | C | T | C | C | A | C | A | A | T | T-3′ | ||
env6880, 5′-3′ (HXB2 6880-6909) | ||||||||||||||
99% | 99% | 99% | 90% | 75% | 99% | 99% | 99% | 99% | 99% | 95% | 99% | 95% | 99% | 99% |
5′-C | C | C | C | G | G | C | T | G | G | T | T | T | T | G- |
99% | 90% | 90% | 99% | 95% | 90% | 99% | 95% | 95% | 95% | 70% | 99% | 99% | 95% | 99% |
-C | G | A | T | T | C | T | A | A | A | G | T | G | T | A-3′ |
Model. (n = 52) | Sensitivity (%) | False Positives | Specificity (%) | False Negatives | Positive Predictive Value (PPV) | Negative Predictive Value (NPV) |
---|---|---|---|---|---|---|
g2p FPR 2 | 76% | 0 | 100% | 5 | 100% | 86% |
g2p FPR 3 | 81% | 2 | 93.5% | 4 | 89.5% | 88% |
g2p FPR 3.9 | 81% | 5 | 84% | 4 | 77% | 87% |
g2p FPR 4.8 | 81% | 7 | 77% | 4 | 71% | 86% |
g2p FPR 5.75 | 81% | 10 | 68% | 4 | 63% | 84% |
PSSM | 81% | 5 | 84% | 4 | 77% | 87% |
codon analysis | 76% | 3 | 90% | 5 | 84% | 85% |
Model (n = 31) | Sensitivity (%) | False Positives | Specificity (%) | False Negatives | Positive Predictive Value (PPV) | Negative Predictive Value (NPV) |
---|---|---|---|---|---|---|
g2p FPR 2 | 85% | 0 | 100% | 2 | 100% | 90% |
g2p FPR 3 | 92% | 1 | 94% | 1 | 92% | 94% |
g2p FPR 3.9 | 92% | 4 | 78% | 1 | 75% | 93% |
g2p FPR 4.8 | 92% | 6 | 67% | 1 | 67% | 92% |
g2p FPR 5.75 | 92% | 8 | 56% | 1 | 60% | 91% |
PSSM | 92% | 5 | 72% | 1 | 71% | 93% |
codon analysis | 92% | 2 | 89% | 1 | 86% | 94% |
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Ko, D.; McLaughlin, S.; Deng, W.; Mullins, J.I.; Dragavon, J.; Harb, S.; Coombs, R.W.; Frenkel, L.M. Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®. Viruses 2024, 16, 510. https://doi.org/10.3390/v16040510
Ko D, McLaughlin S, Deng W, Mullins JI, Dragavon J, Harb S, Coombs RW, Frenkel LM. Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®. Viruses. 2024; 16(4):510. https://doi.org/10.3390/v16040510
Chicago/Turabian StyleKo, Daisy, Sherry McLaughlin, Wenjie Deng, James I. Mullins, Joan Dragavon, Socorro Harb, Robert W. Coombs, and Lisa M. Frenkel. 2024. "Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®" Viruses 16, no. 4: 510. https://doi.org/10.3390/v16040510