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\begin{document}

\title{Prevalence and trends in human immunodeficiency virus-1 subtypes and
drug resistance in South Korea: Analysis of 5 years' data (2017-2022)}



\author[1]{Jinho Heo}%
\author[1]{Seung-Tae Lee}%
\author[1]{Sinyoung Kim}%
\author[1]{Borahm Kim}%
\author[1]{Younhee Park}%
\affil[1]{Yonsei University Department of Laboratory Medicine}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The clinical guidelines for acquired immunodeficiency syndrome (AIDS) in
HIV-infected Koreans recommend routine HIV drug resistance testing for
patients undergoing their first highly active antiretroviral therapy or
considering a change in medication after 2018. Herein, the trends in
HIV-1 subtypes and drug resistance were assessed from 2017 to 2022 by
retrospectively analyzing 2,107 HIV-1-infected patients' data. The
Stanford HIV Drug Resistance Database was used to analyze each patient's
HIV-1 polymerase ( \emph{pol}) gene sequences. Subtype B infections were
predominant in the study population (75.7\%). Meanwhile, CRF01\_AE was
the most prevalent non-B subtype and increased from 58.4\% to 73.7\%
during the study period. Overall, all types of drug resistance mutations
(DRM) were detected in 34.7\% of the HIV-1 pol sequences. The prevalence
of DRMs and high-level resistance mutations decreased from 39.4\% to
31.6\% and 16.7\% to 7.7\%, respectively. The prevalence of DRM was
higher in patients treated with antiretroviral therapy (ART). These
findings indicate that the prevalence of non-B subtype HIV infection has
increased rapidly in South Korea, and the overall prevalence of DRMs
decreased between 2017 and 2022. Additionally, since DRM is high in
ART-treated patients, routine standard genotypic resistance testing
surveillance is important before and after the treatment.%
\end{abstract}%



\sloppy


\textbf{Prevalence and trends in human immunodeficiency virus-1 subtypes
and drug resistance in South Korea: Analysis of 5 years' data
(2017-2022)}

\textbf{Running title: HIV-1 subtypes and resistance in South Korea}

\par\null

Jinho Heo\textsuperscript{1,2}, Seung-Tae Lee\textsuperscript{1},
Sinyoung Kim\textsuperscript{1}, Borahm Kim\textsuperscript{1}, Younhee
Park\textsuperscript{1+}

\textsuperscript{1}Department of Laboratory Medicine, Severance
Hospital, Yonsei University College of Medicine, Seoul, Republic of
Korea

\textsuperscript{2}Present Address: Department of Laboratory Medicine,
National Health Insurance Service Ilsan Hospital, Goyang, Republic of
Korea

\textsuperscript{+}\textbf{Corresponding author} : Dr. Younhee Park,

Department of Laboratory Medicine, Severance Hospital, Yonsei University
College of Medicine,

50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea

Tel: +82-2-2228-2449

Fax: +82-2-2227-8353

E-mail: younheep@yuhs.ac

\textbf{ABSTRACT}

The clinical guidelines for acquired immunodeficiency syndrome (AIDS) in
HIV-infected Koreans recommend routine HIV drug resistance testing for
patients undergoing their first highly active antiretroviral therapy or
considering a change in medication after 2018. Herein, the trends in
HIV-1 subtypes and drug resistance were assessed from 2017 to 2022 by
retrospectively analyzing 2,107 HIV-1-infected patients' data. The
Stanford HIV Drug Resistance Database was used to analyze each patient's
HIV-1 polymerase (\emph{pol} ) gene sequences. Subtype B infections were
predominant in the study population (75.7\%). Meanwhile, CRF01\_AE was
the most prevalent non-B subtype and increased from 58.4\% to 73.7\%
during the study period. Overall, all types of drug resistance mutations
(DRM) were detected in 34.7\% of the HIV-1 pol sequences. The prevalence
of DRMs and high-level resistance mutations decreased from 39.4\% to
31.6\% and 16.7\% to 7.7\%, respectively. The prevalence of DRM was
higher in patients treated with antiretroviral therapy (ART). These
findings indicate that the prevalence of non-B subtype HIV infection has
increased rapidly in South Korea, and the overall prevalence of DRMs
decreased between 2017 and 2022. Additionally, since DRM is high in
ART-treated patients, routine standard genotypic resistance testing
surveillance is important before and after the treatment.

\textbf{Key words:} HIV-1 subtypes, drug resistance mutation, highly
active antiretroviral therapy, genotypic antiretroviral resistance test,
standard genotypic resistance testing



\textbf{INTRODUCTION}

Global efforts to prevent the spread of human immunodeficiency virus
(HIV) have reduced the incidence of new infections \textsuperscript{1}.
The importance of wide coverage and effective suppression of HIV viremia
is reflected by the 90-90-90 goals established by the Joint United
Nations Program on HIV/AIDS. In addition, Korea's national health
insurance fully covers HIV-related medical expenses for those subscribed
to the program to ensure that no patient is untreated for economic
reasons.

Highly active antiretroviral therapy (HAART) has significantly reduced
HIV-related morbidity and mortality \textsuperscript{2}. Recent
guidelines emphasize that antiretroviral therapy (ART) should be
initiated immediately after diagnosis to reduce the risk of HIV
transmission and improve the rate of virologic
suppression\textsuperscript{3,4}. However, the increase in ART use is
expected to increase the prevalence of acquired drug resistance among
patients infected with HIV undergoing treatment and transmitted drug
resistance (TDR) in newly infected patients \textsuperscript{5}.
Moreover, these drug-resistant mutations (DRM) can be transferred to
treatment-na\selectlanguage{ngerman}ïve patients, thereby negatively affecting the success of
primary care and individual prognosis \textsuperscript{6}.

Current treatment guidelines for using antiretroviral agents against
HIV-1 suggest that therapy for a treatment-naïve patient comprises two
nucleoside reverse transcriptase inhibitors (NRTIs) in combination with
a third active drug from one of three drug classes: integrase strand
transfer inhibitor (INSTI), nonnucleoside reverse transcriptase
inhibitor (NNRTI), or protease inhibitor (PI) \textsuperscript{3,4}. The
introduction of INSTIs is expected to change TDR demographics in
treatment-naïve patients. Furthermore, the emergence of INSTI resistance
mutations has been reported \textsuperscript{7}, including a major INSTI
resistance mutation, E92Q, in treatment-naive patients in South
Korea\textsuperscript{8}. These results suggest the need for routine
monitoring of TDR, including INSTI resistance mutations.

Since 2011, new antiretroviral drugs, such as etravirine (NNRTI) and
darunavir (PI), have been frequently prescribed in South Korea. HIV drug
resistance testing has been performed to assist in selecting active
drugs when altering ART regimens, especially in patients with virologic
failure. The clinical guidelines for HIV/AIDS in HIV-infected
Koreans\textsuperscript{9} after 2018 recommend routine HIV drug
resistance testing of patients undergoing their first HAART or those
considering a change in medication due to virologic failure. The
prevalence of HIV TDR in South Korea was 2.5--12.0\% between 1999 and
2019, showing an increasing tendency over time \textsuperscript{10-13}.

In South Korea, the most common subtype of HIV-1 is subtype B. In fact,
a nationwide study involving sample collection between 1999 and 2012
revealed that 93.1\% of the samples were subtype B \textsuperscript{14}.
In South Korea, subtype B primarily comprises Korean clade B
(B\textsuperscript{k}), a distinct clade from subtype B in other
countries, likely due to isolation from other geographic
locations\textsuperscript{15}. However, a recent study reported an
increase in non-B subtypes from 13.4\% to 27.4\% between 2015 and 2019,
with CRF01\_AE being the most prevalent non-B subtype
\textsuperscript{16}.

As of December 2021, the cumulative number of patients with HIV
infection in Korea, including non-ethnic Koreans, was 20,715.
Approximately 1,000 new HIV cases have been reported annually since
2013, with 90\% of the patients being Korean and 10\% non-ethnic
Koreans\textsuperscript{17}. The centralized laboratory conducted more
than 600 genotypic antiretroviral resistance test (GART) annually from
hospitals across the country since 2017; hence, more than half of
patients with HIV in Korea have undergone HIV-drug resistance (DR)
testing at the study institution. Therefore, the analysis conducted by
this institution might represent the current HIV-1 drug resistance
status and subtype statistics throughout South Korea. Accordingly, this
study assesses the trends in HIV-DR and HIV-1 subtypes based on this
large sample population.

\textbf{MATERIALS AND METHODS}

\textbf{Study design and patient population}

Genotypic antiretroviral resistance test (GART) data for 2,107 patients
infected with HIV-1 between August 2017 and August 2022 at Severance
Hospital, Seoul, South Korea, were retrospectively analyzed. The
clinical data included the patients' medical records. Treatment
information, including antiviral agents used, was only collected from
the patients' medical records at Severance Hospital. The study protocol
was reviewed and approved by the institutional review board of Severance
Hospital in Seoul, Korea (IRB No. 4-2019-1076). The requirement for
written informed consent was waived due to the study's nature (review of
medical records)---the study involved no more than minimal risk to the
patients, and their privacy was thoroughly protected.

\textbf{Sequencing of HIV-1 polymerase (\emph{pol}) (protease and
reverse transcriptase genes)}

HIV RNA was extracted from plasma using the QIAmp Viral RNA Mini Kit
(QIAGEN, Hilden, Germany) and reverse-transcribed into complementary DNA
(cDNA) using the RETROscript kit (Applied Biosystems, Foster City, CA,
USA), according to the manufacturer's instructions. A nested polymerase
chain reaction (PCR) was performed using 10 \selectlanguage{greek}μ\selectlanguage{english}L of diluted cDNA or
cellular DNA template added to 40 \selectlanguage{greek}μ\selectlanguage{english}L of the reaction mixture for the
first round. The reaction mixture comprised 5.0 \selectlanguage{greek}μ\selectlanguage{english}L of 10\selectlanguage{ngerman}× PCR buffer
containing magnesium chloride and 1.0 \selectlanguage{greek}μ\selectlanguage{english}L of 10 nM deoxynucleotide
triphosphate Mix (GeneAmp, Applied Biosystems, Foster City, CA, USA),
0.25 \selectlanguage{greek}μ\selectlanguage{english}L of Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN,
USA), 31.75 \selectlanguage{greek}μ\selectlanguage{english}L of molecular grade water, and 1 \selectlanguage{greek}μ\selectlanguage{english}L of each of the 20 \selectlanguage{greek}μ\selectlanguage{english}M
primers. The 50-\selectlanguage{greek}μ\selectlanguage{english}L samples were heated to 94 \selectlanguage{ngerman}°C for 2 min and subjected
to 35 cycles of 30 s at 94 °C, followed by 30 s at 52 °C and 60 s at 68
°C. Subsequently, the samples were heated to 68 °C for 10 min and stored
at 4 °C until use. The second round of PCR was performed using 5 \selectlanguage{greek}μ\selectlanguage{english}L of
the product from the first round as the template, which was added to 45
\selectlanguage{greek}μ\selectlanguage{english}L of the reaction mixture to obtain a final volume of 50 \selectlanguage{greek}μ\selectlanguage{english}L. This
reaction mixture comprised the same reagents, with the volume of
molecular-grade water increased to 36.75 \selectlanguage{greek}μ\selectlanguage{english}L. The thermal cycling
parameters were: 35 cycles of 30 s at 94 \selectlanguage{ngerman}°C, followed by 30 s at 55 °C
and 60 s at 72 °C. The PCR products from each sample were sequenced
using Prism Dye terminator kits (ABI) on an ABI 3730 DNA Analyzer
(Applied Biosystems, Foster city, CA, USA).

\selectlanguage{english}\textbf{HIV-1 drug resistance and subtype analysis}

Antiretroviral resistance mutations and subtype analysis were identified
using the HIVdb program of the Stanford University HIV Drug Resistance
Database (\emph{http://hivdb.stanford.edu}; version 9.1, Algorithm Date:
2 June 2022) \textsuperscript{18}. The sum of the average resistance
mutation scores in each patient's HIV-1 polymerase (\emph{pol} ) gene
sequences, comprising protease, reverse transcriptase, and integrase,
was obtained from the Stanford HIV Drug Resistance Database (HIVdb). The
drug resistance intensity was classified as S (susceptible, potential
low-level resistance), I (low or intermediate level resistance), or R
(high-level resistance), based on the Stanford HIVdb algorithm results.

\textbf{Statistical analysis}

Chi-squared test or Fisher's exact test was used to compare categorical
variables, and the Kruskal--Walli's test was used for comparisons
between subtypes. Statistical analysis was performed using the R
Statistical software (v.4.2.1), where \emph{P} values \textless{} 0.05
were considered significant.

\textbf{RESULTS}

This study included data collected from 274 patients from Severance
Hospital and 1,833 patients from 55 other hospitals across South Korea
(Figure 1). The 55 hospitals included 19 in Seoul, 12 in
Gyeonggi-province, 4 in Incheon, 2 in Daejeon, 2 in Daegu, 2 in Busan, 2
in Gwangju, 3 in Gangwon-province, 2 in Gyeongsangbuk-province, and 3
each in Chungcheongnam-province, Gyeongsangnam-province,
Jeollabuk-province, and Jeollanam-province. A total of 2,360 samples
were collected, including 253 follow-up samples.

\textbf{Study population and demographic features}

The demographic and clinical characteristics of the 2,107 patients are
summarized in Supplementary Table 1. Most patients included in the study
were Koreans (92.2\%, 1943/2107) and men (93.3\%, 1966/2107), with a
median age of 37.2 (20.1\selectlanguage{english}-85.5) years. In a subgroup of the Severance
Hospital patients, 81\% were ART-naive and 19\% were ART-treated.
Subtype B infections were predominant in the total study population at
75.7\% (1595/2107). Non-B subtype strains were identified in 24.3\%
(512/2107) of the patients.

Several demographic features differed in the sub-group analysis. Women
were more likely to harbor non-B infections (51.8\%, 73/141) than men
(22.3\%, 439/1966). The difference in the mean age between women and men
was significant (44.5 vs. 40.4 years, \emph{P} \selectlanguage{english}[?] 0.001). Women's
ethnicity was more likely to be non-Korean (26.2\%, 37/141) compared
with men's ethnicity (6.5\%, 127/1966; Supplementary Table 2). Koreans
were more likely to harbor Subtype B infections (79.9\%, 1553/1943) than
non-Korean (25.6\%, 42/164). The difference in the mean age between
Koreans and non-Koreans was significant (40.9 vs. 38.0 years, \emph{P} =
0.001). The rate of harboring high-level-resistance between Koreans and
non-Koreans did not differ significantly (12.5\% vs. 9.8\%, \emph{P} =
0.365; Supplementary Table 3). Moreover, there was no statistically
significant difference in the HIV-1 subtype distribution between
patients from Severance Hospital and those from other hospitals in South
Korea (\emph{P} = 0.544; Supplementary Table 4).

\selectlanguage{english}\textbf{Subtype trends of HIV-1}

Temporal trends showed that the proportion of non-B subtype infection
increased during the observation period from 15.3\% before 2018 to
33.3\% in 2022, stratified by the initial diagnosis of HIV infection
(Figure 2). Among the 512 patients with non-B subtype infections,
CRF01\_AE (64.8\%, 332/512) was the most prevalent, followed by
CRF02\_AG (10.9\%, 56/512), subtype A (10.4\%, 53/512), CRF56\_cpx
(4.7\%, 24/512), subtype C (3.7\%, 19/512), CRF07\_BC (2.3\%, 12/512),
subtype G (1.0\%, 5/512), and other subtypes (2.1\%, 11/512) such as
CRF06\_cpx, CRF55\_01B, and subtype D. The distribution within non-B
subtypes also changed over time. The proportion of CRF01\_AE increased
from 58.4\% to 73.7\% from 2018 to 2022, whereas that of CRF02\_AG
decreased from 11.7\% to 3.0\%.

\textbf{Correlation between DRMs and virus subtypes}

There were 547 (34.3\%) subtype B, 117 (35.2\%) CRF01\_AE, 19 (33.9\%)
CRF02\_AG, 26 (49.1\%) subtype A, 2 (8.3\%) CRF56\_cpx, 7 (36.8\%)
subtype C, 4 (33.3\%) CRF07\_BC, and 9 (56.3\%) other subtypes in the
study cohort. Overall, 731 (34.7\%) HIV-1 \emph{pol} sequences with one
or more DRMs were detected. High-level DRM was detected in 12.3\%
(259/2107) of patients. The DRM profile differed in CRF01\_AE,
CRF02\_AG, and subtype A. The proportion of six mutations, A62V (NRTI),
K219E/Q/N/R (NRTI), E138A/G/K (NNRTI), V179F/D/E (NNRTI), Y181C/V/I/F
(NNRTI), and Y188C/L/H (NNRTI), differed significantly among sub-types
(\emph{P} \textless{} 0.05; Table 1). The percentage of the NRTI
mutation A62V was high in subtype A (32.1\%) and low in subtype B
(0.7\%). NRTI mutations K219E/Q/N/R were observed frequently in
CRF01\_AE (2.4\%) and CRF02\_AG (5.4\%), compared with that in the study
cohort (1.1\%). Moreover, NNRTI mutations, E138A/G/K (10.7\%) and
Y181C/V/I/F (7.1\%), were observed frequently in CRF02\_AG. In contrast,
the PI mutation, M46I/L, was observed only in subtype B (0.9\%) and
CRF01\_AE (0.6\%). The most frequent DRM in all subtypes was V179F/D/E
(11.1\%).

\textbf{Trends in DRM prevalence}

The initial samples for 2,107 patients collected over 5 years were
assessed. The overall prevalence of DRMs, including all DRM types
(low-level, intermediate, and high-level mutations), declined from
39.4\% before 2018 to 31.6\% in 2022 (Figure 3). The prevalence of
high-level resistance mutations, which are more clinically significant,
also declined from 16.7\% to 7.7\% between 2018 and 2022. The DRM trends
in NRTIs and INSTIs decreased from 14.5\% to 6.4\% and 8.0\% to 1.7\%,
respectively, whereas NNRTIs showed a lateral trend from 27.7\% to
26.3\%. The DRM trends in PIs declined steadily from 39.4\% to 31.6\%.

\textbf{Acquired drug-resistant mutation prevalence in ART-treated
patients}

The DRM prevalence was compared between ART-naive (\emph{N} = 222) and
ART-treated groups (\emph{N} = 53) at Severance Hospital (Figure 4) to
evaluate the percentage of transmitted drug resistance mutations (TDRM)
and acquired drug resistance mutations to four classes of antiretroviral
drugs. The NNRTIs resistance was common in ART-naive (26.1\%) and
ART-treated (42.3\%) patients. The prevalence of DRM was higher in
ART-treated patients: NRTIs (36.5\% vs. 6.3\%), PIs (9.6\% vs. 0.9\%),
and INSTIs (26.9\% vs. 2.7\%). These differences were high in dual-class
resistance: NRTI + NNRTI (19.2\% vs. 3.6\%), NRTI + PI (5.8\% vs.
0.5\%), NNRTI + PI (9.6\% vs. 0.5\%), NRTI + INSTI (21.2\% vs. 1.4\%),
and NNRTI + INSTI (15.4\% vs. 1.4\%).

Additionally, 428 samples collected from 172 patients revealed that the
median observation time was 9 months (2--47 months). Comparing the
baseline samples of patients to the follow-up samples, all DRM types
(low-level, intermediate, and high-level mutations) increased from
44.2\% to 51.6\%. The high-level mutations increased from 24.4\% to
28.9\% (Supplementary Table 5).

\textbf{DISCUSSION}

In the current study, the prevalence of HIV-1 subtypes and drug
resistance in South Korea was assessed between 2017 and 2022. The
prevalence of TDR in South Korea reportedly ranges from 2.5\% to 12.0\%;
however, it varies considerably in different
studies\textsuperscript{10-13}. For instance, Jung et al. showed that
TDR increased between 2011 and 2019 (5.1\%\selectlanguage{english}-7.2\%)\textsuperscript{12},
while Park et al. reported no significant increase in TDR between 1999
and 2012 \textsuperscript{11}. These discrepancies might be due to the
analysis of a small fraction of patients with HIV-1 in South Korea, with
a maximum of 141 patients being diagnosed yearly (range: 27\selectlanguage{english}-141). To
date, the current study is the largest-scale study, comprising 2,107
patients, to estimate TDRM in South Korea. More than half of the
patients with HIV-1 diagnosed nationwide between 2017 and 2022 were
included in this analysis. Moreover, while previous DRM studies only
reported the resistance trends of NRTIs, NNRTIs, and
PIs\textsuperscript{11,12}, the present study analyzed INSTI mutations
and is the first to report the trends in INSTI resistance mutations in
South Korea.

In a previous analysis of the HIV-1 subtypes reported between 2005 and
2009, approximately 90\% were subtype B; CRF02\_AG was the most common
among the non-B subtypes, followed by CRF01\_AE and subtype
A1\textsuperscript{14,19}. Meanwhile, a recent study analyzed HIV-1
subtypes between 1999 and 2018 and showed that subtype B infections were
more predominant (78.7\%) than non-B subtypes (21.3\%), which increased
to 27.4\% between 2015 and 2019 in Busan, Korea \textsuperscript{16}.
Lee et al. also reported CRF01\_AE (52.7\%) as the most common non-B
subtype, followed by CRF02\_AG, subtype A1 \textsuperscript{16}.
Similarly, in the present analysis between 2017 and 2022, subtype B
infections were predominant (75.7\%). Non-B subtype strains were
identified in 24.3\% of patients. CRF01\_AE (64.8\%) was the most
prevalent in non-B subtype infections, followed by CRF02\_AG (10.9\%)
and A1 (10.4\%). Although the overall prevalence of subtype B was
similar to that reported by previous studies \textsuperscript{14,16,19},
when stratified by year, the current analysis showed a rapid increase in
non-B subtypes from 20\% to 33.3\% between 2019 and 2022, thus
increasing by 4.4\% each year. This increase in non-B subtypes is driven
primarily by the rapid expansion of the CRF01\_AE population, which
increased from 58.4\% to 73.7\% in the non-B subgroup between 2017 and
2022. However, CRF02\_AG decreased from 11.7\% to 3.0\%, whereas subtype
A1 remained relatively consistent during the same period. These results
are related to the rapid increase in the number of Chinese immigrants,
primarily carrying CRF01\_AE (around 50\%) \textsuperscript{20,21}, from
0.1 million to 1.1 million between 2000 and 2019, accounting for
approximately 50\% of all immigrants \textsuperscript{22}. Moreover,
subgroup analysis showed that patients of Korean ethnicity largely
carried subtype B compared with non-Korean patients (79.9\% vs. 25.6\%).
Women were also more likely to harbor non-B subtypes than men (51.5\%
vs. 22.3\%). These results are consistent with those of a previous
study\textsuperscript{16}.

The current study DRM data showed that 34.7\% of the HIV-1 sequences
harbored DRMs (low-, intermediate-, or high-level). However, only
high-level DRMs, which are clinically significant, were detected in
12.3\% of patients. These results showed a higher prevalence of DRMs
than previous reports in South Korea (range: 2.5\% to
12.0\%)\textsuperscript{10-13}. The criteria for defining DRMs can lead
to differences in estimation of the prevalence of DRM by studies that
include only high-level resistance or all types of DRM. Indeed, the
prevalence of high-level DRMs in the current study is higher than that
of a previous global study \textsuperscript{23}, which conducted a
meta-analysis of 149 studies, including five from upper-income Asian
countries (12.3\% vs. 8.7\%). However, the prevalence results of this
study may be exaggerated as INSTI mutations were also included, unlike
previous studies.

Certain mutations exhibited significantly different percentages in
different sub-types. For instance, the NRTI mutation A62V was observed
in 32.1\% and 31.4\% of patients harboring subtype A and A6,
respectively \textsuperscript{24}; meanwhile, the PI mutation M46I/L was
observed only in 0.9\% and 0.6\% of patients harboring subtype B and
CRF01\_AE, respectively. These results are consistent with a previous
Chinese HIV-1 subgroup analysis \textsuperscript{21}. Moreover, the
prevalence of DRM declined over the 5 years of the study period. All
mutation types declined from 39.4\% to 31.6\%. High-level resistance
mutations also decreased from 16.7\% to 7.7\%, and the prevalence of
NRTI and INSTI DRMs decreased. However, no significant change was
observed in NNRTI or PI. These declining trends in drug resistance are
not common globally. In fact, most previous reports have demonstrated an
overall increase in drug resistance \textsuperscript{1,21,23} with only
a few noting decreases in TDRM prevalence. That is, Tostevin et al.
observed a decrease in DRMs between 2010 and 2013. However, it was
confined to the men who have sex with men population, and the rates
remained stable in those with heterosexually acquired HIV
infections\textsuperscript{25}. Weng et al. observed a stable resistance
prevalence between 2009 and 2015 (slope = -0.086) under a fixed regimen
with zidovudine/lamivudine + efavirenz or nevirapine as the first-line
therapy \textsuperscript{26}. These TDRM trends are affected by
environmental and economic factors, including the COVID-19 pandemic. In
fact, a recent study conducted in Canada revealed that drop-in services
and outreach work by commercial sex workers were reduced due to
COVID-related restrictions \textsuperscript{27}. Access to healthcare
also declined significantly during the COVID-19
pandemic.\textsuperscript{28}. These unusual decreases in TDRM were
observed in countries with national health insurance (NHI) systems,
which fully cover HIV-related medical expenses for NHI subscribers to
ensure the treatment of all patients. These results show that
accessibility to ART can play an essential role in changing TDRM trends.
Additionally, the adoption of pre-exposure prophylaxis treatment since
2018 in South Korea may have affected the TDRM trends.

Additionally, NNRTI resistance was detected in 26.1\% and 42.3\% of
patients in the ART-naive and ART-treated groups, respectively. The
prevalence of DRM was higher for NRTIs (36.5\% vs. 6.3\%), PIs (9.6\%
vs. 0.9\%), and INSTIs (26.9\% vs. 2.7\%) in ART-treated patients. These
differences were more significant in dual-class resistance: NRTI + NNRTI
(19.2\% vs. 3.6\%), NRTI + PI (5.8\% vs. 0.5\%), NNRTI + PI (9.6\% vs.
0.5\%), NRTI + INSTI (21.2\% vs. 1.4\%), and NNRTI + INSTI (15.4\% vs.
1.4\%). Similar results have been demonstrated in previous
studies\textsuperscript{21,29}. These findings underscore the need to
implement surveillance programs for HIV drug resistance in clinical
management.

The current study has certain limitations. First, this was a nationwide
multi-hospital-based, retrospective, observational study. The collection
of clinical and epidemiological information was limited due to
insufficient information provided to the centralized laboratory from
hospitals that requested sample analysis. Second, the analysis comparing
ART-naive and ART-treated groups was performed on a limited number of
patients. However, this was accounted for by performing a subtype
distribution analysis and confirming that patients enrolled at Severance
Hospital were representative of the whole cohort to some extent
(Supplementary Table 4). Third, despite efforts to clearly classify
medical records, clinical information for some patients in the
ART-treated group before 2018 may be inaccurate. GART was adopted as a
routine surveillance test for HIV-1 treatment in 2018 in South
Korea\textsuperscript{9}. Therefore, the GART results before 2018 were
primarily weighted toward ART-treated patients, especially those who
were ART-resistant. This could have exaggerated the prevalence of DRM
before 2018. Fourth, it is difficult to directly compare DRM prevalence
between studies as the algorithm version used in each study differs.
That is, each study used a different mutation analysis program or
version, even if the Stanford University HIV Drug Resistance Database
was applied. In particular, the Stanford University HIV Drug Resistance
Database accumulates more mutation data with each version update.
Therefore, if researchers analyze a previous sequence with a newer
version, the results could be different.

\textbf{CONCLUSIONS}

To date, this is the largest South Korean study on HIV-1 subtype
analysis and drug resistance surveillance, presenting the overall
prevalence of TDRM from 2017 to 2022. The proportion of non-B subtypes
of HIV-1 infections has recently increased rapidly, with CRF01\_AE
becoming the predominant non-B subtype in South Korea. Moreover, an
overall decrease in TDRM was observed in recent years. In particular,
TDRM prevalence decreased in NRTIs and INSTIs. However, considering that
DRMs are detected in more than 20\% of ART-naive patients, routine GART
is strongly recommended before ART treatment. Routine GART surveillance
will also improve HIV prevention and control the development of drug
resistance.

\textbf{Declaration of competing interest}

All authors declare no competing interests.

\textbf{Funding source}

This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.

\textbf{Ethical Approval statement}

This study protocol was reviewed and approved by the institutional
review board of Severance Hospital in Seoul, Korea (IRB No.
4-2019-1076).

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\textbf{FIGURE LEGENDS}

Figure 1. Flow diagram of patient enrollment

Figure 2. Trends in the proportion of HIV-1 B/non-B subtypes and changes
in the distribution of non-B HIV-1 subtypes, stratified by HIV diagnosis
year.

Figure 3. Drug resistance mutation trends over 5 years

Figure 4. Prevalence of DRM between ART-naive and ART-treated patients
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\textbf{Hosted file}

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