Nan-Yu Chen1, Shu-Wei Kao1, Zhuo-Hao Liu2, Ting-Shu Wu1, Chia-Lung Tsai3, Hsi-Hsun Lin4, Wing-Wai Wong5, Yea-Yuan Chang6, Shu-Sheng Chen5, Stephane Wen-Wei Ku7. 1. Division of Infectious Diseases, Department of Internal Medicine, Chang Gung Memorial Hospital Linkou Branch, Chang Gung University College of Medicine, Taiwan. 2. Department of Neurosurgery, Chang Gung Memorial Hospital Linkou Branch, Taiwan. 3. Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taiwan. 4. General Clinical Research Centre, Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan; Institute of Public Health, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 5. Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan. 6. Division of Infectious Diseases, Department of Internal Medicine, National Yang-Ming University Hospital, Yilan, Taiwan; Institute of Clinical Medicine and AIDS Prevention and Research Centre, National Yang-Ming University, Taipei, Taiwan; Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Infectious Diseases, Department of Medicine, Taipei City Hospital Ren-Ai Branch, Taiwan. 7. Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; Division of Infectious Diseases, Department of Medicine, Taipei City Hospital Ren-Ai Branch, Taiwan. Electronic address: stephaneku@gmail.com.
Abstract
BACKGROUND: The clinical utilisation of deep sequencing in HIV treatment has been hindered due to its unknown correlation with standard Sanger genotyping and the undetermined value of minority drug resistance mutation (DRM) detection. OBJECTIVES: To compare deep sequencing performance to standard Sanger genotyping with clinical samples, in an effort to delineate the correlation between the results from the two methods and to find the optimal deep sequencing threshold for clinical utilisation. METHODS: We conducted a retrospective study using stored plasma collected from August 2014 to March 2018 for HIV genotyping with the commercial Sanger genotyping kit. Samples with available Sanger genotyping reports were further deep sequenced. Drug resistance was interpreted according to the Stanford HIV drug resistance database algorithm. RESULTS: At 15-25% minority detection thresholds, 9-15% cases had underestimated DRMs by Sanger sequencing. The concordance between the Sanger and deep sequencing reports was 68-82% in protease-reverse transcriptase region and 88-97% in integrase region at 5-25% thresholds. The undetected drug resistant minority variants by Sanger sequencing contributed to the lower negative predictive value of Sanger genotyping in cases harbouring DRMs. CONCLUSIONS: Use of deep sequencing improved detection of antiretroviral resistance mutations especially in cases with virological failure or previous treatment interruption. Deep sequencing with 10-15% detection thresholds may be considered a suitable substitute for Sanger sequencing on antiretroviral DRM detection.
BACKGROUND: The clinical utilisation of deep sequencing in HIV treatment has been hindered due to its unknown correlation with standard Sanger genotyping and the undetermined value of minority drug resistance mutation (DRM) detection. OBJECTIVES: To compare deep sequencing performance to standard Sanger genotyping with clinical samples, in an effort to delineate the correlation between the results from the two methods and to find the optimal deep sequencing threshold for clinical utilisation. METHODS: We conducted a retrospective study using stored plasma collected from August 2014 to March 2018 for HIV genotyping with the commercial Sanger genotyping kit. Samples with available Sanger genotyping reports were further deep sequenced. Drug resistance was interpreted according to the Stanford HIV drug resistance database algorithm. RESULTS: At 15-25% minority detection thresholds, 9-15% cases had underestimated DRMs by Sanger sequencing. The concordance between the Sanger and deep sequencing reports was 68-82% in protease-reverse transcriptase region and 88-97% in integrase region at 5-25% thresholds. The undetected drug resistant minority variants by Sanger sequencing contributed to the lower negative predictive value of Sanger genotyping in cases harbouring DRMs. CONCLUSIONS: Use of deep sequencing improved detection of antiretroviral resistance mutations especially in cases with virological failure or previous treatment interruption. Deep sequencing with 10-15% detection thresholds may be considered a suitable substitute for Sanger sequencing on antiretroviral DRM detection.
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