A single variant sequencing method for sensitive and quantitative detection of HIV-1 minority variants

Abstract Objectives HIV-1 integrase inhibitors are recommended as first-line therapy by WHO, though efficacy and resistance data for non-B subtypes are limited. Two recent trials have identified the integrase L74I mutation to be associated with integrase inhibitor treatment failure in HIV-1 non-B subtypes. We sought to define the prevalence of integrase resistance mutations, including L74I, in West Africa. Methods We studied a Nigerian cohort of recipients prior to and during receipt of second-line PI-based therapy, who were integrase inhibitor-naive. Illumina next-generation sequencing with target enrichment was used on stored plasma samples. Drug resistance was interpreted using the Stanford Resistance Database and the IAS-USA 2019 mutation lists. Results Of 115 individuals, 59.1% harboured CRF02_AG HIV-1 and 40.9% harboured subtype G HIV-1. Four participants had major IAS-USA integrase resistance-associated mutations detected at low levels (2%–5% frequency). Two had Q148K minority variants and two had R263K (one of whom also had L74I). L74I was detected in plasma samples at over 2% frequency in 40% (46/115). Twelve (26.1%) had low-level minority variants of between 2% and 20% of the viral population sampled. The remaining 34 (73.9%) had L74I present at >20% frequency. L74I was more common among those with subtype G infection (55.3%, 26/47) than those with CRF02_AG infection (29.4%, 20/68) (P = 0.005). Conclusions HIV-1 subtypes circulating in West Africa appear to have very low prevalence of major integrase mutations, but significant prevalence of L74I. A combination of in vitro and clinical studies is warranted to understand the potential implications.

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Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing

Nature ◽

10.1038/365671a0 ◽

1993 ◽

Vol 365 (6447) ◽

pp. 671-673 ◽

Cited By ~ 139

Author(s):

B. A. Larder ◽

A. Kohli ◽

P. Kellam ◽

S. D. Kemp ◽

M. Kronick ◽

...

Keyword(s):

Drug Resistance ◽

Dna Sequencing ◽

Quantitative Detection ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Automated Dna Sequencing ◽

Hiv 1

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Whole blood as an alternative to peripheral blood mononuclear cell for detection of total HIV-1 DNA

BMC Infectious Diseases ◽

10.1186/s12879-020-05675-3 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Ling Lin ◽

Yong-Song Yue ◽

Ni-Dan Wang ◽

Lei-Yan Wei ◽

Yang Han ◽

...

Keyword(s):

Peripheral Blood Mononuclear Cell ◽

Mononuclear Cell ◽

Whole Blood ◽

Peripheral Blood ◽

Antiviral Treatment ◽

Quantitative Detection ◽

Peripheral Blood Mononuclear ◽

Blood Samples ◽

Whole Blood Samples ◽

Hiv 1

Abstract Background A more time saving, convenient, reproducible, and scalable method is needed to assess total HIV-1 DNA levels. Methods Frozen whole blood and peripheral blood mononuclear cell (PBMC) samples both 200 μl at the same point were used to detect total HIV-1 DNA. Automatic extraction of total HIV-1 DNA was used to ensure the consistency of sample extraction efficiency. The detection reagent was HIV-1 DNA quantitative detection kit and real-time quantitative PCR was utilized. Results Of the 44 included patients, 42 were male and 2 were female, with a median age of 33 years. Thirty-three cases were collected after receiving antiviral treatment, with a median duration of treatment of 3 months, and the other 11 cases were collected before antiviral treatment. The median viral load was 1.83 log10 copies/mL, the median CD4 and CD8 count were 94 and 680 cells/μL, and the median CD4/CD8 ratio was 0.18. The results of the two samples were 3.02 ± 0.39 log10 copies/106 PBMCs in PBMC samples and 3.05 ± 0.40 log10 copies/106 PBMCs in whole blood samples. The detection results of the two methods were highly correlated and consistent by using paired t test (P = 0.370), pearson correlation (r = 0.887, P < 0.0001) and intra-group correlation coefficient (ICC = 0.887, P < 0.0001) and bland-altman [4.55% points were outside the 95% limits of agreement (− 0.340 ~ 0.390)]. Conclusions The results of the whole blood sample test for total HIV-1 DNA are consistent with those of PBMC samples. In a clinical setting it is recommended to use whole blood samples directly for the evaluation of the HIV reservoir.

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A4 An amplicon-based approach for universal amplification, sequencing, and assembly of full-length HIV-1 samples from the DRC

Virus Evolution ◽

10.1093/ve/vez002.003 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

M Bletsa ◽

N Vidal ◽

B Vrancken ◽

S Lequime ◽

M Peeters ◽

...

Keyword(s):

Complete Genome ◽

Democratic Republic Of Congo ◽

Central Africa ◽

Human Populations ◽

Genome Data ◽

Sequencing Method ◽

History Of ◽

Key Aspects ◽

Traditional Approaches ◽

Hiv 1

Abstract Phylogenetic studies have contributed to our understanding of the early epidemic onset of HIV-1 in the Democratic Republic of Congo (DRC); however, the factors driving its early emergence and establishment in human populations still remain unresolved. In order to determine the key aspects of its successful epidemic spread, complete genome data are required from samples representative of the viral diversity in the DRC. In this study, we have established a universal PCR-assay that uses seven different panels of primers to produce overlapping amplicons covering the complete HIV genome. To circumvent the limitations of purifying these fragments and sequencing them with traditional approaches, we have developed a massive parallel sequencing method and a protocol for efficiently assembling HIV-1 genomes. A total of thirty-six samples, collected between 1997 and 2001 from different locations across the DRC, have been obtained, and, at this stage, we are focusing on complementing our dataset with more archival samples that can be used as HIV ‘molecular fossils’. By generating complete genome phylogeographic data from the DRC, we aim to create a genomic window into the past evolutionary and epidemiological dynamics of HIV-1 in Central Africa and understand the natural history of this devastating pandemic.

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