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Pyrosequencing dried blood spots reveals differences in HIV drug resistance between treatment naive and experienced patients.

ABSTRACT: Dried blood spots (DBS) are an alternative specimen collection format for HIV-1 genotyping. DBS produce HIV genotyping results that are robust and equivalent to plasma when using conventional sequencing methods. However, using tagged, pooled pyrosequencing, we demonstrate that concordance between plasma and DBS is not absolute and varies according to viral load (VL), duration of HIV infection and antiretroviral therapy (ART) status. The plasma/DBS concordance is the highest when VL is ?5,000 copies/ml and/or the patient has no ART exposure and/or when the duration of HIV infection is ?2 years. Stepwise regression analysis revealed that VL is most important independent predictor for concordance of DBS with plasma genotypes. This is the first study to use next generation sequencing to identify discordance between DBS and plasma genotypes. Consideration should be given to VL, duration of infection, and ART exposure when interpreting DBS genotypes produced using next generation sequencing. These findings are of particular significance when DBS are to be used for clinical monitoring purposes.

SUBMITTER: Ji H

PROVIDER: S-EPMC3567018 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Pyrosequencing dried blood spots reveals differences in HIV drug resistance between treatment naïve and experienced patients.

Ji Hezhao H Li Yang Y Liang Binhua B Pilon Richard R MacPherson Paul P Bergeron Michèle M Kim John J Graham Morag M Van Domselaar Gary G Sandstrom Paul P Brooks James J

PloS one 20130207 2

Dried blood spots (DBS) are an alternative specimen collection format for HIV-1 genotyping. DBS produce HIV genotyping results that are robust and equivalent to plasma when using conventional sequencing methods. However, using tagged, pooled pyrosequencing, we demonstrate that concordance between plasma and DBS is not absolute and varies according to viral load (VL), duration of HIV infection and antiretroviral therapy (ART) status. The plasma/DBS concordance is the highest when VL is ≥5,000 cop ...[more]

PMID: 23409150

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Project description:BACKGROUND: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. METHODOLOGY/PRINCIPAL FINDINGS: The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies >/=5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods. CONCLUSIONS/SIGNIFICANCE: We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required.

Project description:BACKGROUND:The presence of drug resistance mutations (DRMs) against antiretroviral agents is one of the main concerns in the clinical management of individuals with human immunodeficiency virus-1 (HIV-1) infection, especially in regions of the world where treatment options are limited. The current study aimed at assessing the prevalence of HIV-1 DRMs among naïve and treatment-experienced HIV-1-infected patients in Iran. METHODS:From April 2013 to September 2018, the HIV-1 protease and reverse transcriptase genes were amplified and sequenced in plasma specimens of 60 newly diagnosed antiretroviral-naive individuals and 46 participants receiving antiretroviral therapies (ARTs) for at least six months with an HIV viral load of more than 1000 IU/mL to determine the HIV-1 DRMs and subtypes. RESULTS:Among the 60 treatment-naïve HIV-1-infected participants, 8.3% were infected with HIV-1 variants with surveillance DRMs (SDRMs). The SDRMs, D67N and D67E, belonged to the NRTIs class in two patients and K103N and V106A belonged to the NNRTIs class in three patients. The phylogenetic analysis showed that 91.7% of the subjects were infected with subtype CRF35_AD, followed by subtype B (5.0%) and CRF01_AE (3.3%). Among the 46 ART-experienced participants, 33 (71.7%) carried HIV-1 variants with SDRMs (9.1% against PIs, 78.8% against NRTIs, and 100% against NNRTIs). M46I and I47V were the most common mutations for PIs, M184V was the most common mutation for the NRTIs, and K103N/S was the most common mutation for NNRTIs. Phylogenetic analysis of the polymerase region showed that all of the 46 HIV-1-infected patients who failed on ART carried CRF35_AD. CONCLUSIONS:The moderate prevalence of SDRMs (8.3%) in treatment-naïve and ART-failed (77.1%) Iranian patients with HIV-1-infection emphasizes the need for systematic viral load monitoring, expanding drug resistance testing, carefully surveilling individuals on ART regimens, and facilitating access to new antiretrovirals by health authorities.

Dataset Information

Pyrosequencing dried blood spots reveals differences in HIV drug resistance between treatment naive and experienced patients.

Publications

Pyrosequencing dried blood spots reveals differences in HIV drug resistance between treatment naïve and experienced patients.

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