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. 2013 Jun;19(6):E263-70.
doi: 10.1111/1469-0691.12167. Epub 2013 Mar 11.

Restriction Fragment Mass Polymorphism (RFMP) Analysis Based on MALDI-TOF Mass Spectrometry for Detecting Antiretroviral Resistance in HIV-1 Infected Patients

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Restriction Fragment Mass Polymorphism (RFMP) Analysis Based on MALDI-TOF Mass Spectrometry for Detecting Antiretroviral Resistance in HIV-1 Infected Patients

J-H Lee et al. Clin Microbiol Infect. .
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Abstract

Viral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance.

Figures

FIG. 1.
FIG. 1.
Evaluation of the sensitivity of the RFMP assay for detection of minor amounts of virus with a defined mixture of K103N. The MALDI-TOF MS spectra and direct sequencing chromatograms shown are representative of experiments repeated three times using mixed populations of wild-type (K103) and NNRTI mutant (N103). The wild-type plasmids were mixed with mutant type at various ratios as follows: (a) 100%, (b) 50%, (c) 20%, (d) 10%, (e) 5% and (f) 1%. Molecular masses of 3133.0 and 3157.0 correspond to N103 and K103, respectively. AI is absolute intensity; m/z is mass-to-charge ratio.
FIG. 2.
FIG. 2.
Comparison of the RFMP and direct sequencing assays for detection of mixed genotypes. Sera were taken from patients infected with HIV-1 carrying ARV drug-resistant mutations and examined by the RFMP and sequencing assays. (a) For codon 103, molecular masses of 3088.0/3157.0 and 3113.0/3133.0 represent Lys (AAA) and Asn (AAC), respectively. (b) For codon 46, molecular masses of 2988.0/3164.0 and 3003.0/3148.0 represent Met (ATG) and Ile (ATA), respectively. (c) For codon 215, molecular masses of 3740.4/3796.4 and 3724.4/3811.4 represent Thr (ACC) and Tyr (TAC), respectively. Each codon was indicated by a red box in the sequencing chromatogram. AI is absolute intensity; m/z is mass-to-charge ratio.

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