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Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

Cheung KW, Peng Q, He L, Cai K, Jiang Q, Zhou B, To SW, Yam WC, Liu L, Chen Z, Wang H - PLoS ONE (2016)

Bottom Line: The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains.Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections.In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

View Article: PubMed Central - PubMed

Affiliation: AIDS Institute and Department of Microbiology, Research Centre for Infection and Immunity, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

No MeSH data available.


Summary of call rate per sample for the 159 clinical specimensA summary of call rate per sample for the total 159 clinical specimens and specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC are demonstrated. i) The total call rate per sample of all 159 clinical specimens; ii) The call rate per sample of 109 specimens of CRF01_AE; iii) The call rate per sample of 12 specimens of subtype B and iv) The call rate per sample of 26 specimens of CRF07_BC and 2 specimens of CRF08_BC.
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pone.0153641.g002: Summary of call rate per sample for the 159 clinical specimensA summary of call rate per sample for the total 159 clinical specimens and specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC are demonstrated. i) The total call rate per sample of all 159 clinical specimens; ii) The call rate per sample of 109 specimens of CRF01_AE; iii) The call rate per sample of 12 specimens of subtype B and iv) The call rate per sample of 26 specimens of CRF07_BC and 2 specimens of CRF08_BC.

Mentions: The call rate per sample was subsequently evaluated in order to examine the effectiveness for clinical application. As shown in Fig 2I, 130 out of 159 (81.8%) specimens had a call rate greater than 90%, 149 out of 159 (93.7%) specimens had a call rate greater than 80%. We further evaluated the call rate per sample for specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC. For CRF01_AE, 90 out of 109 (82.6%) specimens had a call rate greater than 90%, and 104 out of 109 (95.4%) specimens had a call rate greater than 80%. For subtype B, 6 out of 12 (50%) specimens had a call rate greater than 90%, and 10 out of 12 (83.3%) specimens had a call rate greater than 80%. For CRF07_BC, 26 out of 26 (100%) specimens had a call rate greater than 90% and the call rate of the 2 CRF08_BC specimens were 70%. Results were demonstrated in Fig 2II, 2III and 2IV respectively. The discrepancies of the call rate per sample shown in Fig 2 were found to be mainly due to the genetic variations of HIV-1 among specimens after sequence alignment. Other possible factors for the discrepancies included the concentration of virus in the specimens and the quality of the PCR products. Although the call rate per sample was not completely ideal, the assay was able to detect major resistance mutations (M41L, K65R, M184V and G190A) in 150 out of 159 (94.3%) specimens simultaneously.


Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

Cheung KW, Peng Q, He L, Cai K, Jiang Q, Zhou B, To SW, Yam WC, Liu L, Chen Z, Wang H - PLoS ONE (2016)

Summary of call rate per sample for the 159 clinical specimensA summary of call rate per sample for the total 159 clinical specimens and specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC are demonstrated. i) The total call rate per sample of all 159 clinical specimens; ii) The call rate per sample of 109 specimens of CRF01_AE; iii) The call rate per sample of 12 specimens of subtype B and iv) The call rate per sample of 26 specimens of CRF07_BC and 2 specimens of CRF08_BC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836728&req=5

pone.0153641.g002: Summary of call rate per sample for the 159 clinical specimensA summary of call rate per sample for the total 159 clinical specimens and specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC are demonstrated. i) The total call rate per sample of all 159 clinical specimens; ii) The call rate per sample of 109 specimens of CRF01_AE; iii) The call rate per sample of 12 specimens of subtype B and iv) The call rate per sample of 26 specimens of CRF07_BC and 2 specimens of CRF08_BC.
Mentions: The call rate per sample was subsequently evaluated in order to examine the effectiveness for clinical application. As shown in Fig 2I, 130 out of 159 (81.8%) specimens had a call rate greater than 90%, 149 out of 159 (93.7%) specimens had a call rate greater than 80%. We further evaluated the call rate per sample for specimens of HIV-1 CRF01_AE, subtype B, CRF07_BC and CRF08_BC. For CRF01_AE, 90 out of 109 (82.6%) specimens had a call rate greater than 90%, and 104 out of 109 (95.4%) specimens had a call rate greater than 80%. For subtype B, 6 out of 12 (50%) specimens had a call rate greater than 90%, and 10 out of 12 (83.3%) specimens had a call rate greater than 80%. For CRF07_BC, 26 out of 26 (100%) specimens had a call rate greater than 90% and the call rate of the 2 CRF08_BC specimens were 70%. Results were demonstrated in Fig 2II, 2III and 2IV respectively. The discrepancies of the call rate per sample shown in Fig 2 were found to be mainly due to the genetic variations of HIV-1 among specimens after sequence alignment. Other possible factors for the discrepancies included the concentration of virus in the specimens and the quality of the PCR products. Although the call rate per sample was not completely ideal, the assay was able to detect major resistance mutations (M41L, K65R, M184V and G190A) in 150 out of 159 (94.3%) specimens simultaneously.

Bottom Line: The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains.Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections.In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

View Article: PubMed Central - PubMed

Affiliation: AIDS Institute and Department of Microbiology, Research Centre for Infection and Immunity, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, People's Republic of China.

ABSTRACT
The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

No MeSH data available.