A sensitive genotyping assay for detection of drug resistance mutations in reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried blood spots or frozen RNA extracts

Ziemniak, Carrie; George-Agwu, Allison; Moss, William J.; Ray, Stuart C.; Persaud, Deborah

doi:10.1016/j.jviromet.2006.05.030

Cited by 39 publications

(52 citation statements)

References 21 publications

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“…The results of the study presented confirmed the feasibility of extracting and subsequently sequencing HIV-1 DNA from DBS as shown by others (24,43). The sensitivity of our inhouse genotyping assay for sequencing DNA from DBS was only slightly lower than the sensitivity for sequencing DNA extracted from whole blood cells.…”

Section: Continued On Facing Pagesupporting

confidence: 85%

“…Few studies examined the possibilities of performing drug resistance testing on dried blood, plasma, or serum on filter paper (24,33,43). Because of the ease to collect, store, and transport DBS, use of DBS is the ideal method for blood sampling in resource-poor rural settings.…”

Section: Discussionmentioning

confidence: 99%

“…The fact that a reverse transcription step is no longer needed for the amplification of the genes of interest also reduces the overall cost of the procedure. Blood spots collected on filter paper have been used for DNA amplification purposes, but reports on the feasibility of performing genotypic analysis for HIV-1 drug resistance remain scarce (24,33,43). In resource-poor settings, dried blood spots (DBS) are the preferred method of sample collection in the…”

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confidence: 99%

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Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

et al. 2007

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Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study.Today, more than one million people in low-and middleincome countries have access to antiretroviral treatment (ART). Even though this is only 23% of the estimated 4.6 million human immunodeficiency virus type 1 (HIV-1)-infected individuals who are in need of highly active ART (HAART), it proves that the delivery of ART in resource-poor settings is feasible (41). Experiences from Europe and the United States, however, indicate that the emergence of HIV drug resistance remains an important obstacle to the long-term success of therapy. An adequate follow-up of patients on treatment, in order to identify cases in which therapy has failed, is essential to avoiding accumulation of drug resistance. Efforts to lower the prices for CD4 and viral load testing are being made, and alternative methods for measuring these parameters in resource-poor settings are being developed and evaluated (8,12,17). Unfortunately, genotypic resistance testing remains almost inaccessible due to its high cost, the need for specialized laboratories, and the logistical challenges, such as the requirements for a cold chain to transp...

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confidence: 85%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

et al. 2007

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“…A major advantage of sampling blood or plasma as dried spots on absorbent paper is that samples can be shipped easily and safely and that no cold chain is required for preservation. Dried blood, plasma, and serum spots (DBS, DPS, and DSS, respectively) have been tested for HIV serology (4,8,22), molecular diagnosis (11), CD4 ϩ lymphocyte enumeration (18), and more recently, viral RNA quantification (1-3, 10, 11, 15, 16, 23) and genotypic drug resistance (6,17,19,26). However, the use of dried spots can be recommended only as long as the results are comparable to those obtained with fresh or frozen plasma.…”

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confidence: 99%

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

et al. 2009

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The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays-a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)-were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n ‫؍‬ 47; median VL, 4.13 log 10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log 10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log 10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log 10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.Due to the efforts of national programs and the support of a wide range of international partners, the number of people receiving antiretroviral therapy (ART) in resource-limited countries has increased significantly over recent years, most notably in sub-Saharan Africa (24). In order to limit the emergence of resistance to antiretroviral drugs, human immunodeficiency virus (HIV) treatment should ideally be accompanied by periodic virological monitoring, such as viral load and resistance testing. However, in resource-limited countries, viral load monitoring and drug resistance tests are not yet available at an affordable cost, and infrastructure requirements limit the scale-up in access to these tests. Treatment initiation and modifications are thus guided mainly by clinical disease progression and by CD4 cell counts, when possible (13, 24). As a consequence, many people with virological failure stay on inadequate drug regimens for long periods. Rapid or uncontrolled emergence of HIV drug resistance is thus feared as a potential consequence of ART scale-up in resource-limited countries. Therefore, ART resistance monitoring is not recommended at the individual patient level in low-income countries, and the WHO has established a sys...

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“…Briefly, viral RNA was extracted from virions pelleted at 23,500 ϫ g for 2 h at 4°C from four 1-ml aliquots of thawed plasma, using a QIAamp viral RNA isolation kit (Qiagen, Valencia, CA). Following DNase treatment to ensure removal of cell-associated DNA (Invitrogen, Carlsbad, CA), eight independent one-step RT-PCRs were set up for the DNase-treated RNA, followed by a nested PCR with previously published primers (49). The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and directly sequenced bidirectionally using gene-specific primers.…”

Section: Methodsmentioning

confidence: 99%

Identification of Ongoing Human Immunodeficiency Virus Type 1 (HIV-1) Replication in Residual Viremia during Recombinant HIV-1 Poxvirus Immunizations in Patients with Clinically Undetectable Viral Loads on Durable Suppressive Highly Active Antiretroviral Therapy

Shiu

Cunningham

Greenough

et al. 2009

J Virol

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In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P ‫؍‬ 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8؉ T-cell selective pressures.Highly active antiretroviral therapy (HAART) inhibits human immunodeficiency virus type 1 (HIV-1) replication, decreasing plasma viremia below the detection limit (Ͻ50 copies/ ml) of clinically used viral load assays (32). Most patients receiving standard HAART, however, have low levels of viremia (median, 3.1 copies/ml) detectable by more sensitive assays (11,17,22,28,31,34), the source of which remains unclear and controversial. Low-level viremia may arise from viral expression from cellular reservoirs established before HAART (2, 21-23, 25, 29, 31, 34, 46) or from low levels of complete cycles of virus replication (7, 46). Evidence supporting the former includes the stable persistence of low-level viremia during long-term HAART (2,11,23,25,28,31,34), intermingling of HIV-1 sequences from free plasma virus with persistent replication-competent viral sequences in resting memory CD4 ϩ T cells or proviral DNA in peripheral blood mononuclear cells (PBMC) (2,23,34,46), and a lack of decay in low-level viremia with treatment intensification of standard three-drug HAART regimens (10, 24). The contribution of ongoing complete cycles of virus replication during long-term suppressive HAART is supported by findings of sequence evolution with new drugresistant variants in a subset of patients during seemingly effective HAART (7, 46), decreases in plasma viremia with treatment intensification (22), and transient increases in episomal cDNA following treatment intensification with an integrase ...

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A sensitive genotyping assay for detection of drug resistance mutations in reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried blood spots or frozen RNA extracts

Cited by 39 publications

References 21 publications

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

Identification of Ongoing Human Immunodeficiency Virus Type 1 (HIV-1) Replication in Residual Viremia during Recombinant HIV-1 Poxvirus Immunizations in Patients with Clinically Undetectable Viral Loads on Durable Suppressive Highly Active Antiretroviral Therapy

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