Improvement in highly sensitive plasma HIV-1 RNA assays requires sufficiently large volume plasma processing, viral concentration and efficient exclusion of PCR inhibitors and detection of inhibition. This has enabled the detection of low-level viral persistence at levels of less than 1 copy per milliliter of plasma . Similarly, for HIV-1 DNA detection, greater sampling through large blood volume draws or leukapheresis improves the chance of detecting rare HIV-infected cells, whereas the background human DNA signal can be reduced by purification of CD4+ cells or resting CD4+ memory cells [52▪▪].
High HIV diversity contributes to reduced sensitivity by delaying the threshold cycle when primers or probes mismatch a viral template [53▪▪]. To address this, the choice of conserved genome targets in integrase, gag or the long terminal repeat (LTR) region have improved assay performance and the inclusion of more than one genome target in a multiplex assay has reduced the risk of mismatches to all targets [54,55]. Digitalization of PCR reactions into individual nanoliter or picoliter reactions, followed by detection of the number of positive reactions, has been reported to be more robust to primer mismatches  but has limited throughput and is prone to background signal that could be reduced by touchdown PCR .
An innovative approach combining the principles of quantitative PCR with digitalization is the use of real-time PCR with multiple replicates at the highest dilution. This is less prone to nonspecific background and does not rely on the cycle threshold for the quantification of the highest dilutions .
When reporting cure assay results, it is important to report the denominator of cells actually assayed [52▪▪]. This could be achieved by parallel quantification of a human reference gene, which controls for all assay steps.
Advances in NGS, single cell and fractional expression assays, and assays of integrated provirus are providing new tools to characterize HIV reservoirs.
Improvements in PCR and NGS will result in higher throughput assays for intact provirus. DNA polymerases with improved processivity and proofreading (3′–5′ exonuclease activity) allows amplification of near–full-length amplicons, but when relying on multiple Sanger sequencing reactions, have limited throughput. Recent NGS platforms (e.g. Pacific Biosciences, Menlo Park, California, USA) have improved template read length that allows the sequencing of whole HIV genomes [58▪▪]. This high-throughput approach has the potential to characterize individual full-length viral genomes and determine which are intact. Viral templates must be diluted to one template per PCR reaction to avoid artifacts from recombination between multiple viral genomes, but this endpoint dilution limits throughput.
Recently, a limiting dilution assay has been developed to quantify cellular RNA levels expressed by individual cells, showing that the reduction of cellular HIV RNA during successful ART is not due to a smaller proportion of infected cells expressing HIV RNA but due to a smaller fraction of cells expressing high levels of RNA [59▪▪]. New developments in single cell assays will soon allow the simultaneous investigation of different characteristics of a single cell: the cellular phenotype, HIV-1 DNA and mRNA expression and virion production. Sequencing at a single cellular level could also investigate whether individual genomes are intact or defective [60▪].
Two assays to detect HIV integration sites have been developed. The one HIV integration site loop amplification assay makes use of primers that have a random 3′ decamer tail and an LTR U5-specific 5′ region, which through several steps generates a stem-loop structure with a known HIV-1 LTR sequence in the stem region and unknown human genome sequence in the loop region. Limiting dilution PCR and sequencing of the individual integration sites allow the design of integration-site specific primers, which together with envelope specific primers allow amplification and sequencing of integration sites and HIV 3′ LTR to envelope. Although elegant, this approach is very labor-intensive and requires multiple PCR and Sanger sequencing reactions . The other integration site assay (ISA) approach involves random ultrasonic shearing of HIV-1 DNA, blunt-end ligation of PCR linkers and amplification of the integration sites with and HIV-1 LTR-specific and linker-specific primer followed by a heminested PCR with another internal HIV-1-specific LTR-specific and linker-specific primer that enriches for HIV integration sites. Integration sites are then characterized by high-throughput Illumina sequencing. This provides an efficient approach but the very short HIV-1 sequence does not allow the linkage of the human genome integration site with specific proviral species . Current proviral ISAs are too insensitive to examine the effect of curative interventions on individual clones and therefore need further development. Future assays should also link full HIV genomes to their integration sites. This will likely be facilitated by long fragment PCR and newer NGS platforms that allow single read sequencing of whole HIV genomes [58▪▪] to investigate the survival and expansion of cells with specific integrated proviruses (Table 2). This capability will accelerate the understanding of whether experimental interventions affect most cells containing intact proviruses or only a subset as a consequence of variation in host cell and proviral biology.
Population-based Sanger sequencing of HIV provided essential, initial insights into HIVDR and has been used for patient monitoring in well resourced settings, but recent advances in sample collection, automated sample processing and sequencing technologies are poised to greatly expand availability of resistance monitoring. Because low-frequency mutations have recently been shown to affect treatment outcome, NGS platforms offer the additional advantage of greater sensitivity than population sequencing for detection of minor viral variants.
Higher-throughput quantitative assays of proviral competence are a high priority to assess the effects of interventions on the HIV reservoir size. Recent developments in full-length sequencing show promise and could identify intact proviruses, but have limited throughput. Current assays for HIV integration sites have increased our understanding of clonal expansion as a key mechanism of HIV persistence but further assay refinements are needed to assess the impact of curative interventions on individual clonal populations of infected cells. Such assay refinements and other advances will undoubtedly occur and provide much greater insight into HIV reservoirs and the impact of interventions designed to achieve an HIV cure.
Papers of particular interest, published within the annual period of review, have been highlighted as:
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4. Hütter G, Nowak D, Mossner M, et al. Long-term control of HIV by CCR5 delta32/delta32 stem-cell transplantation. N Engl J Med 2009; 360:692–698.
5. Inzaule SC, Ondoa P, Peter T, et al. Affordable HIV drug-resistance testing for monitoring of antiretroviral therapy in sub-Saharan Africa. Lancet Infect Dis 2016; 16:e267–e275.
6▪▪. Van Laethem K, Theys K, Vandamme AM. HIV-1 genotypic drug resistance testing: digging deep, reaching wide? Curr Opin Virol 2015; 14:16–23.
A thorough review of HIV drug resistance (HIVDR) strategies including the strengths and limitations of surveillance technologies with a focus on next-generation sequencing (NGS) and its application for ‘Deep’ versus ‘Wide’ sequencing analysis.
7. Grant RM, Kuritzkes DR, Johnson VA, et al. Accuracy of the TRUGENE HIV-1 genotyping kit. J Clin Microbiol 2003; 41:1586–1593.
8. Chew KK, Ng KY, Khong WX, et al. Clinical evaluation of an in-house human immunodeficiency virus (HIV) genotyping assay for the detection of drug resistance mutations in HIV-1 infected patients in Singapore. Ann Acad Med Singapore 2012; 41:553–558.
9. Lee CK, Lee HK, Loh TP, et al. An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1. J Med Virol 2012; 84:394–401.
10. Aitken SC, Bronze M, Wallis CL, et al. A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings by use of a novel genotyping assay targeting the reverse transcriptase-encoding region only. J Clin Microbiol 2013; 51:1757–1761.
11▪. Keys JR, Zhou S, Anderson JA, et al. Primer ID informs next-generation sequencing platforms and reveals preexisting drug resistance mutations in the HIV-1 reverse transcriptase coding domain. AIDS Res Hum Retroviruses 2015; 31:658–668.
This manuscript demonstrates the tagging of individual cDNAs with primer IDs to correct for PCR resampling and sequencing errors, facilitating accurate deep sequencing analysis to identify low-frequency mutations in mixed populations.
12. Ji H, Li Y, Graham M, et al. Next-generation sequencing of dried blood spot specimens: a novel approach to HIV drug-resistance surveillance. Antivir Ther 2011; 16:871–878.
13. Paredes R, Marconi VC, Campbell TB, Kuritzkes DR. Systematic evaluation of allele-specific real-time PCR for the detection of minor HIV-1 variants with pol and env resistance mutations. J Virol Methods 2007; 146:136–146.
14. Edelstein RE, Nickerson DA, Tobe VO, et al. Oligonucleotide ligation assay for detecting mutations in the human immunodeficiency virus type 1 pol gene that are associated with resistance to zidovudine, didanosine, and lamivudine. J Clin Microbiol 1998; 36:569–572.
15. Chung MH, Beck IA, Dross S, et al. Oligonucleotide ligation assay detects HIV drug resistance associated with virologic failure among antiretroviral-naive adults in Kenya. J Acquir Immune Defic Syndr 2014; 67:246–253.
16. Villahermosa ML, Beck I, Perez-Alvarez L, et al. Detection and quantification of multiple drug resistance mutations in HIV-1 reverse transcriptase by an oligonucleotide ligation assay. J Hum Virol 2001; 4:238–248.
17. Zhang L, Wang J, Coetzer M, et al. One-step ligation on RNA amplification for the detection of point mutations. J Mol Diagn 2015; 17:679–688.
18. Landegren U, Kaiser R, Sanders J, Hood L. A ligase-mediated gene detection technique. Science 1988; 241:1077–1080.
19. Kuritzkes DR, Grant RM, Feorino P, et al. Performance characteristics of the TRUGENE HIV-1 genotyping kit and the opengene DNA sequencing system. J Clin Microbiol 2003; 41:1594–1599.
20. Mouafo LC, Pere H, Ndjoyi-Mbiguino A, et al. Letter to the Editor performance of the ViroSeq(R) HIV-1 genotyping system v2.0 in Central Africa. Open AIDS J 2015; 9:9–13.
21. Thiam M, Diop-Ndiaye H, Kebe K, et al. Performance of the ViroSeq HIV-1 genotyping system v2.0 on HIV-1 strains circulating in Senegal. J Virol Methods 2013; 188:97–103.
22. Eshleman SH, Crutcher G, Petrauskene O, et al. Sensitivity and specificity of the ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for detection of HIV-1 drug resistance mutations by use of an ABI PRISM 3100 genetic analyzer. J Clin Microbiol 2005; 43:813–817.
23. Church JD, Towler WI, Hoover DR, et al. Comparison of LigAmp and an ASPCR assay for detection and quantification of K103N-containing HIV variants. AIDS Res Hum Retroviruses 2008; 24:595–605.
24. MacLeod IJ, Rowley CF, Essex M. Pan Degenerate Amplification and Adaptation for Highly Sensitive Detection of ARV Drug Resistance. Conference on Retroviruses and Opportunistic Infections, CROI 2014; 2014 March 3–6, 2014; Boston, MA, USA.
25. Rowley CF, Boutwell CL, Lockman S, Essex M. Improvement in allele-specific PCR assay with the use of polymorphism-specific primers for the analysis of minor variant drug resistance in HIV-1 subtype C. J Virol Methods 2008; 149:69–75.
26. Boltz VF, Maldarelli F, Martinson N, et al. Optimization of allele-specific PCR using patient-specific HIV consensus sequences for primer design. J Virol Methods 2010; 164:122–126.
27. Ellis GM, Vlaskin TA, Koth A, et al. Simultaneous and sensitive detection of human immunodeficiency virus type 1 (HIV) drug resistant genotypes by multiplex oligonucleotide ligation assay. J Virol Methods 2013; 192:39–43.
28▪▪. Clutter DS, Jordan MR, Bertagnolio S, Shafer RW. HIV-1 drug resistance and resistance testing. Infect Genet Evol 2016; 46:292–307.
Comprehensive overview of HIVDR monitoring detailing the genetic mechanisms, epidemiology and management of HIVDR as well as the implications of HIVDR for patient management at both individual and population levels across diverse economic and geographic settings.
29. Hauser A, Kuecherer C, Kunz A, et al. Comparison of 454 ultra-deep sequencing and allele-specific real-time PCR with regard to the detection of emerging drug-resistant minor HIV-1 variants after antiretroviral prophylaxis for vertical transmission. PLoS One 2015; 10:e0140809.
30. Casadella M, Paredes R. Deep sequencing for HIV-1 clinical management. Virus Res 2016; [Epub ahead of print].
31. Ekici H, Rao SD, Sonnerborg A, et al. Cost-efficient HIV-1 drug resistance surveillance using multiplexed high-throughput amplicon sequencing: implications for use in low- and middle-income countries. J Antimicrob Chemother 2014; 69:3349–3355.
32. Luo C, Tsementzi D, Kyrpides N, et al. Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample. PLoS One 2012; 7:e30087.
33. Zhou S, Jones C, Mieczkowski P, Swanstrom R. Primer ID validates template sampling depth and greatly reduces the error rate of next-generation sequencing of HIV-1 genomic RNA populations. J Virol 2015; 89:8540–8555.
34. Jabara CB, Jones CD, Roach J, et al. Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc Natl Acad Sci U S A 2011; 108:20166–20171.
35. Boltz VF, Rausch J, Shao W, et al. Analysis of resistance haplotypes using primer IDs and next gen sequencing of HIV RNA. Seattle, WA, USA: Conference on Retroviruses and Opportunistic Infections (CROI 2015); 2015.
36. Bertagnolio S, Parkin NT, Jordan M, et al. Dried blood spots for HIV-1 drug resistance and viral load testing: a review of current knowledge and WHO efforts for global HIV drug resistance surveillance. AIDS Rev 2010; 12:195–208.
37. Hearps AC, Ryan CE, Morris LM, et al. Stability of dried blood spots for HIV-1 drug resistance analysis. Curr HIV Res 2010; 8:134–140.
38▪. Salazar-Gonzalez JF, Salazar MG, Tully DC, et al. Use of dried blood spots to elucidate full-length transmitted/founder HIV-1 genomes. Pathog Immun 2016; 1:129–153.
Study showing that 454 pyrosequencing NGS can be used to genotype from dried blood spot with a high concordance to standard genotyping, from samples with a viral load as low as 1700 copies/ml and an estimated ∼50 viral copies per blood spot.
39. Li Z, Wu J, Deleo CJ. RNA damage and surveillance under oxidative stress. IUBMB Life 2006; 58:581–588.
40. Brooks K, DeLong A, Balamane M, et al. HemaSpot, a novel blood storage device for HIV-1 drug resistance testing. J Clin Microbiol 2016; 54:223–225.
41. Gous N, Scott L, Stevens W. Can dried blood spots or whole blood liquid transport media extend access to HIV viral load testing? Cape Town, South Africa: African Society for Laboratory Medicine; 2014; Abstract 663.
42. Ananworanich J, Puthanakit T, Suntarattiwong P, et al. Reduced markers of HIV persistence and restricted HIV-specific immune responses after early antiretroviral therapy in children. AIDS 2014; 28:1015–1020.
43. Strain MC, Little SJ, Daar ES, et al. Effect of treatment, during primary infection, on establishment and clearance of cellular reservoirs of HIV-1. J Infect Dis 2005; 191:1410–1418.
44. Josefsson L, von Stockenstrom S, Faria NR, et al. The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time. Proc Natl Acad Sci U S A 2013; 110:E4987–E4996.
45. Josefsson L, Palmer S, Faria NR, et al. Single cell analysis of lymph node tissue from HIV-1 infected patients reveals that the majority of CD4+ T-cells contain one HIV-1 DNA molecule. PLoS Pathog 2013; 9:e1003432.
46▪▪. Bruner KM, Murray AJ, Pollack RA, et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 2016; 22:1043–1049.
This study describes the proportion of HIV DNA that is intact after acute and chronic infection. It emphasizes the small proportion of intact HIV DNA genomes making up the ‘true’ reservoir.
47. Ho YC, Shan L, Hosmane NN, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 2013; 155:540–551.
48▪. Bruner KM, Hosmane NN, Siliciano RF. Towards an HIV-1 cure: measuring the latent reservoir. Trends Microbiol 2015; 23:192–203.
This article reviews current viral culture and PCR-based methods to characterize and quantify the latent reservoir.
49. Cillo A, Sobolewski M, Buckley T, Bui J, Cyktor J, Mellors J. Latent HIV is missed by assaying only resting CD4+ T cells. Conference on Retroviruses and Opportunistic Infections (CROI 2017); 2017 February 13-16, 2017; Seattle, WA, USA2017.
50▪. Procopio FA, Fromentin R, Kulpa DA, et al. A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine 2015; 2:874–883.
This manuscript describes the tat/rev induced limiting dilution assay, an inducible virus recovery assay that quantifies cells producing multiply spliced mRNA, after stimulation. It is more practicable than the quantitative virus recovery assay but is not specific for replication competent provirus.
51. Cillo AR, Vagratian D, Bedison MA, et al. Improved single-copy assays for quantification of persistent HIV-1 viremia in patients on suppressive antiretroviral therapy. J Clin Microbiol 2014; 52:3944–3951.
52▪▪. Hong F, Aga E, Cillo AR, et al. Novel assays for measurement of total cell-associated HIV-1 DNA and RNA. J Clin Microbiol 2016; 54:902–911.
This manuscript describes the development of accurate assays to measure total HIV-1 DNA and RNA; the use of a cellular gene quantified in parallel controls for cell input and extraction steps and provides an accurate denominator for the cells assayed.
53▪▪. Riddler SA, Aga E, Bosch RJ, et al. Continued slow decay of the residual plasma viremia level in HIV-1-infected adults receiving long-term antiretroviral therapy. J Infect Dis 2016; 213:556–560.
This study describes the decay rate of residual HIV-1 plasma RNA using an improved single copy assay.
54. Somsouk M, Dunham RM, Cohen M, et al. The immunologic effects of mesalamine in treated HIV-infected individuals with incomplete CD4+ T cell recovery: a randomized crossover trial. PLoS One 2014; 9:e116306.
55. Sizmann D, Glaubitz J, Simon CO, et al. Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 test, v2.0 using a novel dual-target approach. J Clin Virol 2010; 49:41–46.
56. Strain MC, Lada SM, Luong T, et al. Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 2013; 8:e55943.
57. De Spiegelaere W, Malatinkova E, Kiselinova M, et al. Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome. Anal Biochem 2013; 439:201–203.
58▪▪. Dilernia DA, Chien JT, Monaco DC, et al. Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing. Nucleic Acids Res 2015; 43:e129.
This manuscript describes continuous long read single genome sequencing of HIV genomes with Pacific Biosciences SMRT technology; a method that may allow an accurate study of near full genome HIV haplotypes.
59▪▪. Hong F, Spindler J, Musick A, et al. ART Reduces Cellular HIV RNA but Not the Fraction of Proviruses Transcribing RNA. Conference on Retroviruses and Opportunistic Infections (CROI 2016); 2016 February 22–25, 2016; Boston, MA, USA.
This abstract compares the proportion of cells transcribing HIV RNA and the level of HIV RNA expression of infected cells in viremic and successfully treated individuals. It found that the proportion of cells expressing HIV RNA is not significantly different on therapy, but fewer cells on therapy express high levels of HIV RNA.
60▪. Wiegand A, Spindler J, Shao W, et al. Analysis of HIV RNA in Single Cells Reveals Clonal Expansions and Defective Genomes. Conference on Retroviruses and Opportunistic Infections (CROI 2015); 2015 February 23–26, 2015; Seattle, WA, USA.
This investigation describes a method sequencing single HIV DNA genomes of cells and the mRNA that they express, which allowed the investigation of clonal expansion and the detection of defective genomes expressing mRNA.
61. Wagner TA, McLaughlin S, Garg K, et al. HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection. Science 2014; 345:570–573.
62. Maldarelli F, Wu X, Su L, et al. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science 2014; 345:179–183.