Replication capacity, biological phenotype, and drug resistance of HIV strains isolated from patients failing antiretroviral therapy

Nicastri, Emanuele; Sarmati, Loredana; d’Ettorre, Gabriella; Palmisano, Lucia; Parisi, Saverio Giuseppe; Uccella, Ilaria; Rianda, Alessia; Concia, Ercole; Vullo, Vincenzo; Vella, Stefano; Andreoni, Massimo

doi:10.1002/jmv.10269

Cited by 55 publications

(38 citation statements)

References 11 publications

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“…Clinical trials have shown that HIV-1 replication fitness may play an important role in the outcome of disease (4,17,19,20), and reliable assays are needed to determine relative viral fitness in a heterogeneous population of viruses. Several investigators have used a dye-labeled primer system with automated DNA sequencer to measure the replication fitness of viruses by comparing the peak heights of two viruses at a single locus in the RT or protease genes (7,8,12).…”

Section: Discussionmentioning

confidence: 99%

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

2003

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The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.

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Section: Discussionmentioning

confidence: 99%

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

2003

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“…Previous reports showed that subjects with primary HIV infections had a high prevalence of transmission of HIV-resistant strains (2, 9) and of NNRTIresistant isolates (6). A possible explanation of these findings is that HIV-1 strains with the primary mutations related to NNRTI resistance (6) show no significant difference in terms of replication capacity with respect to the wild-type virus. Therefore, a sustained viral fitness could explain the transmission of drug-resistant virus and the presence of the same mutational pattern in different compartments.…”

mentioning

confidence: 97%

“…DNA was extracted from 3 ϫ 10 6 PBMCs with a High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany).…”

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confidence: 99%

Drug-Associated Resistance Mutations in Plasma and Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus Type 1-Infected Patients for Whom Highly Active Antiretroviral Therapy Is Failing

et al. 2003

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In 32 patients for whom highly active antiretroviral therapy was failing, a good agreement between drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) was found (k ‫؍‬ 0.85). The mutations with the lowest agreement were 20R, 63P, and 84V in the protease gene and 184V in the reverse transcriptase gene. In eight patients, primary drug resistance mutations were detected only in PBMCs.The presence of resistant viral strains is routinely verified in plasma samples only, since circulating virus variants are considered representative of the viral population escaping the drug pressure. Nevertheless, archival human immunodeficiency virus (HIV) DNA present in PBMCs might represent the reservoir of additional drug-resistant viral variants (8).This study was designed to assess the level of agreement between the drug resistance-associated mutations in plasma and peripheral blood mononuclear cells (PBMCs) in 32 patients for whom highly active antiretroviral therapy (HAART) was failing.A commercial kit was used to identify mutations in the pol gene of HIV type 1 (HIV-1) (ViroSeq HIV-1 V2 genotyping system; PE Biosystems, Foster City, Calif.). DNA was extracted from 3 ϫ 10 6 PBMCs with a High Pure PCR template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany).The Cohen k test was used to determine the correlation between the presence of HIV-1 drug resistance in plasma and PBMCs. Cohen k agreement is defined as poor if k is Յ0.20, fair if k is Ն0.21 and Յ0.60, substantial if k is Ն0.61 and Յ0.80, and good if k is Ͼ0.80 (5). The association between determinant factors and discordance in plasma and PBMC genotypic analysis was assessed by crude and adjusted odds ratios (OR) and their 95% confidence intervals (CI) through univariate and multivariate models.Patients received a mean of 5.5 antiretroviral drugs (range, 3 to 8) during a mean of 57 months of treatment (range, 9 to 126). Twenty-eight (87%) and 16 (50%) of 32 subjects were exposed to protease inhibitors (PI) and nonnucleoside reverse transcriptase inhibitors (NNRTI), respectively.In 492 (21.9%) of 2,240 codons analyzed, there was evidence of drug resistance. In plasma and PBMC genotypic analysis, means of 7.4 (Ϯ4.7 [standard deviation]) and 7.9 (Ϯ5.5) drug resistance mutations were detected, respectively. When total numbers of mutations were calculated for each sample, they were higher in plasma than in PBMCs in 9 of 32 samples (28%), higher in PBMCs than plasma in 14 of 32 samples (43%), and the same in plasma and PBMCs in 9 of 32 samples (28%). A significant correlation between the time of the last ART and the number of protease (PR)-related drug resistance mutations detected in plasma (r ϭ 0.42; P ϭ 0.024) and in PBMCs (r ϭ 0.52; P ϭ 0.006) was found. No significant correlation between mutations in the reverse transcriptase (RT) gene in PBMCs and plasma and the time of drug exposure was detected.The agreement values (mean Cohen k) between drug resistance mutations in plasma and PBMCs are shown in Table 1. The mean of t...

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“…However, the V75I mutation, in combination with the Q151M complex, causes high level resistance to all NRTIs [16] while the K101Q has been identified to have a clinically significant impact on NNRTI activity [17].…”

Section: Discussionmentioning

confidence: 99%

Hiv-1 Drug Resistance in Treatment-Naive, Chronically Infected Patients in Jamaica

Barrow

Hylton-Kong²,

Rodríguez

et al. 2013

Antiviral Therapy

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Background: HIV-1 drug resistance in treatment-naive patients has a significant impact on the individual patient as well as implications for the wider population. These effects are amplified in the context of resource-limited settings, which are rapidly expanding access to antiretroviral therapy. Methods: This cross-sectional survey at a single treatment site in Kingston, Jamaica was designed to identify the prevalence of HIV-1 drug-resistant mutations in chronically infected, treatment-naive patients. Mutations were identified using the Stanford HIV database algorithm and the World Health Organization (WHO) HIV Drug Resistance (HIVDR) surveillance mutations. Results: The inclusion of 103 cases in the study resulted in 79 (76.6%) amplifiable samples. Genotype analysis revealed that 12.6% (95% CI 5.3, 19.9) were identified as having clinically significant mutations, while 10.1% (95% CI 3.5, 16.7) had WHO HIVDR surveillance mutations. Conclusions: According to the WHO standard, this study population has a moderate level of HIVDR in treatmentnaive patients and strongly implies the need to introduce HIVDR surveillance in Jamaica.

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Replication capacity, biological phenotype, and drug resistance of HIV strains isolated from patients failing antiretroviral therapy

Cited by 55 publications

References 11 publications

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

Drug-Associated Resistance Mutations in Plasma and Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus Type 1-Infected Patients for Whom Highly Active Antiretroviral Therapy Is Failing

Hiv-1 Drug Resistance in Treatment-Naive, Chronically Infected Patients in Jamaica

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