Viruses 2022, 14, 2208. https://doi.org/10.3390/v14102208 www.mdpi.com/journal/viruses Article Towards Next‐Generation Sequencing for HIV‐1 Drug Resistance Testing in a Clinical Setting Calesta Hui Yi Teo 1,2,† , Nurul Hannah Binte Norhisham 2,3,† , Ogestelli Fabia Lee 4 , Siyu Png 5 , Chean Nee Chai 5 , Gabriel Yan 6 , Julian Wei‐Tze Tang 7 and Chun Kiat Lee 5, * 1 Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore 2 School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore 599490, Singapore 3 School of Social Sciences, Nanyang Technological University, Singapore 639818, Singapore 4 School of Life and Physical Sciences, PSB Academy, Singapore 039594, Singapore 5 Department of Laboratory Medicine, National University Health System, Singapore 119228, Singapore 6 Department of Medicine, National University Health System, Singapore 119228, Singapore 7 Respiratory Sciences, University of Leicester, Leicester LE1 7RH, UK * Correspondence: chun_kiat_lee@nuhs.edu.sg or chunkiatlee1983@gmail.com † Both authors contributed equally to the work. Abstract: The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high‐throughput or next‐ generation sequencing has caused a paradigm shift towards the detection and characterization of low‐abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug‐resistant mutations (DRMs). Here, we eval‐ uated the diagnostic performance of a laboratory‐developed, high‐throughput, sequencing‐based GRT using 103 archived clinical samples that were previously tested for drug resistance using pop‐ ulation sequencing. As expected, high‐throughput sequencing found all the DRMs that were de‐ tectable by population sequencing. Significantly, 78 additional DRMs were identified only by high‐ throughput sequencing, which is statistically significant based on McNemar’s test. Overall, our re‐ sults complement previous studies, supporting the notion that the two methods are well correlated, and the high‐throughput sequencing method appears to be an excellent alternative for drug re‐ sistance testing in a clinical setting. Keywords: HIV‐1; drug resistance; genotypic resistance testing; next‐generation sequencing; high‐throughput sequencing; Sanger; population sequencing 1. Introduction Human immunodeficiency virus type 1 (HIV‐1, family Retroviridae, genus Lentivirus) has been a global health problem for many decades. In 2020, the Joint United Nations Program on HIV/AIDS reported over 37.7 million cases of human HIV‐1 infections and 680,000 HIV/AIDS‐related deaths worldwide [1]. Despite the absence of a complete cure for the disease, research has made great advances in providing treatment regimens to mitigate the transmission of the virus. The development of antiretroviral therapies (ART), such as the highly active antiretroviral therapy (HAART), has proved to be effective in the suppression of HIV replication [2], and numerous studies have shown that the use of HIV drug resistance tests to guide therapy is associated with improved treatment out‐ comes [3–6]. The global scale‐up of HAART has also dramatically decreased the rates of AIDS‐related mortality and morbidity [7]. Citation: Teo, C.H.Y.; Norhisham, N.H.B.; Lee, O.F.; Png, S.; Chai, C.N.; Yan, G.; Tang, J.W.‐T.; Lee, C.K. Towards‐Next Generation Sequencing for HIV‐1 Drug Resistance Testing in a Clinical Setting. Viruses 2022, 14, 2208. https://doi.org/10.3390/v14102208 Academic Editor: Maria Cecilia Garibaldi Marcondes Received: 4 September 2022 Accepted: 5 October 2022 Published: 7 October 2022 Publisher’s Note: MDPI stays neu‐ tral with regard to jurisdictional claims in published maps and institu‐ tional affiliations. Copyright: © 2022 by the authors. Li‐ censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con‐ ditions of the Creative Commons At‐ tribution (CC BY) license (https://cre‐ ativecommons.org/licenses/by/4.0/).