PLOS ONE

RESEARCH ARTICLE

Drug-resistant Mycobacterium tuberculosis

among Nepalese patients at a tuberculosis
referral center
Arun Bahadur Chand ID1,2,3*, Ajaya Basnet2, Bhagwan Maharjan3, Ganesh Rai2, Yadav
Prasad Joshi4, Lok Raj Bhatt1, Bindu Sen5, Shiba Kumar Rai2,6
1 Department of Microbiology, KIST Medical College and Teaching Hospital, Lalitpur, Nepal, 2 Department
of Medical Microbiology, Shi-Gan International College of Science and Technology, Kathmandu, Nepal,
3 German Nepal Tuberculosis Project, Kathmandu, Nepal, 4 Department of Public Health, Manmohan
Memorial Institute of Health Sciences, Kathmandu, Nepal, 5 Department of Dentistry, KIST Medical College
and Teaching Hospital, Lalitpur, Nepal, 6 Department of Microbiology, Nepal Medical College Teaching
Hospital, Kathmandu, Nepal
a1111111111
* [email protected]
a1111111111
a1111111111
a1111111111
a1111111111 Abstract

Background
OPEN ACCESS Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resis-
tance, is caused by chromosomal mutations that restrict treatment options and complicate
Citation: Chand AB, Basnet A, Maharjan B, Rai G,
Joshi YP, Bhatt LR, et al. (2024) Drug-resistant tuberculosis management. This study sought to investigate the prevalence of pre-exten-
Mycobacterium tuberculosis among Nepalese sively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well
patients at a tuberculosis referral center. PLoS ONE
as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains.
19(5): e0301210. https://doi.org/10.1371/journal.
pone.0301210

Editor: Divakar Sharma, Lady Hardinge Medical

Methods
College, INDIA A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal
Received: September 21, 2023 Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identi-
Accepted: March 12, 2024 fied pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any
fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and
Published: May 6, 2024
at least one additional group A drug. Mutation status was determined by comparing bands
Copyright: © 2024 Chand et al. This is an open
on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for
access article distributed under the terms of the
Creative Commons Attribution License, which low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Life-
permits unrestricted use, distribution, and science GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet.
reproduction in any medium, provided the original SPSS version 17.0 was used for data analysis.
author and source are credited.

Data Availability Statement: The data relevant to

Results
this study are available from Dryad at DOI: 10.
5061/dryad.1ns1rn927. Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57
Funding: The author(s) received no specific (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains,
funding for this work. 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent
Competing interests: The authors have declared mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the
that no competing interests exist gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene

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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

(100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the
gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs.

Conclusions
Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific muta-
tion bands present in gyrA and rrs genes.

Introduction
Tuberculosis (TB) stands as the second most prevalent infectious cause of death worldwide,
claiming countless lives [1]. This disease, caused by the Mycobacterium tuberculosis complex
(MTBC), spreads from person to person through airborne transmission [2,3]. Alarming statis-
tics from 2019 reveal that a staggering 10 million individuals were afflicted by tuberculosis glob-
ally, resulting in 1.4 million deaths and 465,000 cases of MDR/RR-TB [4]. South-East Asia bears
a substantial burden, accounting for 43% of the global impact [5]. In Nepal, around 117,000
people are affected by tuberculosis, which is also the seventh leading cause of death [6].
The development of antimicrobial resistance in M. tuberculosis stems from spontaneous
mutations in various chromosomal genes, leading to altered interactions between anti-tubercu-
losis drugs and their intended targets [7]. For instance, resistance to fluoroquinolone (FLQ)
arises from mutations in the gyrA or gyrB gene [8], while amikacin and kanamycin resistance are
linked to mutational changes in the rrs gene and conformational alterations in the aminoglyco-
side acetyltransferase gene (eis) [9,10]. In developing countries with limited resources and high
tuberculosis burdens, routine testing for resistance to fluoroquinolone gyrA, gyrB, and second-
line injectable drugs (SLID) is often not feasible, potentially contributing to the spread of drug
resistant M. tuberculosis strains [11]. Consequently, delayed drug susceptibility testing and the
use of inappropriate regimens may heighten the risk of second-line drug (SLD) resistance [12].
The emergence of pre-extensive drug resistant (pre-XDR) and extensively drug resistant
(XDR) M. tuberculosis isolates globally underscores the importance of molecular genotyping,
which can aid in understanding tuberculosis transmission dynamics and identifying prevalent
genotypes in specific geographic areas. Moreover, timely detection of pre-XDR-TB cases
among multi-drug resistant (MDR)-TB patients is crucial for preventing treatment failure and
implementing effective measures to curb the progression to XDR-TB [13].
The German Nepal Tuberculosis Project (GENETUP) conducted a survey in 2012 to gather
information on pre-XDR-TB and XDR-TB. However, there was insufficient data available on
these conditions and the specific gene responsible for drug-resistant tuberculosis in Nepal. As
a result, the objective of this study was to determine both the prevalence of pre-XDR-TB and
XDR-TB and the pattern of particular mutation bands related to FLQ and SLID resistance in
clinical isolates obtained from Nepalese patients diagnosed with multidrug - or rifampicin-
resistant M. tuberculosis.

Materials and methods

Study design and population
This cross-sectional study was conducted at the German Nepal Tuberculosis Project (GEN-
ETUP), National Reference Laboratory, Kathmandu, Nepal, from June 2017 to June 2018 on
patients diagnosed with MDR/RR-TB.

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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

A total of 171 confirmed patients with MDR/RR-TB from various provinces across Nepal
were selected for the study, including diverse genders, encompassing both males and females
and age groups ranging from 12 to 75 years. MDR-TB cases were identified using both conven-
tional drug susceptibility tests and the genotype MTBDRplus line probe assay, while RR-TB
cases were diagnosed using GeneXpert. Sputum samples from 11 different sites across the
country, including Rapid Test Center, Baglung Hospital, Team Hospital (Province: 4), Bhim
Hospital, Lumbini Zonal Hospital (Province: 5), TB Nepal Provincial Hospital (Province: 6),
Seti Zonal Hospital, Mahakali Zonal Hospital, Dandeldhura Hospital, and Bayelpata Hospital
(Province: 7), were collected at the GENETUP National Reference Laboratory in Kathmandu,
Nepal (Province: 3). The collected samples underwent SLD resistance testing, which included
microscopic examination, decontamination, microbiological culture, and a line probe assay
test at GENETUP National Reference Laboratory, Kathmandu, Nepal. The study included
MDR/RR-TB Nepalese patients enrolled in the SLD resistance program of the National Tuber-
culosis Program, excluding those who had started SLD treatment (FLQs and aminoglycosides/
cyclic peptides) during sample collection.

Ethical approval
This study received approval from the Institutional Review Committee (IRC) of Shi-Gan
Health Foundation in Kathmandu, Nepal (Reference number: 2074/12/10). Patient details
were collected with the consent of each participant or their parent/guardian. The privacy of
the data was protected by assigning a unique laboratory identity number to each participant.

Data collection
Patient socio-demographic data was collected from the hospital record section, while informa-
tion on pre-XDR-TB and XDR-TB cases, along with their specific mutation bands responsible
for FLQ and SLID resistance, was gathered from the laboratory. Once complete and detailed
information was obtained, it was verified, anonymized, and then entered and curated using
Microsoft Excel 2010. Missing or ambiguous records were addressed through communication
with healthcare workers involved in the process.

Specimen processing and laboratory diagnosis

Out of a total of 171 cases, 126 were from direct sputum samples and 45 were from culture posi-
tive isolates. The GenoType MTBDRsl assay was performed to detect drug susceptibility test as
well as MTBC and Mycobacterium other than tuberculosis (MOTT) following the standard pro-
tocol provided by the manufacturing company Hain Lifescience GmbH, Nehren, Germany [14].
Decontamination. After collecting the samples, they were transferred into appropriately
labeled 50ml falcon tubes, with a volume of 3-5ml each, in preparation for further processing.
The specimens underwent processing using a standard N-acetyl-L-cysteine-NaOH-sodium
citrate digestion decontamination solution [15]. Subsequently, a DNA extraction was per-
formed using 500μl of the re-suspended sample.

Molecular analysis
a. DNA extraction. DNA was extracted from both direct sputum specimens and cultured isolates
of bacteria grown on solid Lowenstein-Jensen medium, using the GenoLyse1 kit. The extrac-
tion followed the protocol provided by Hain Lifescience GmbH, Nehren, Germany [14].

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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

b. Amplification and hybridization. The amplification master mixture (45μl) was prepared in a
PCR hood in a DNA-free room. Subsequently, the DNA sample (5μl) was added in a sepa-
rate area within a class II biosafety cabinet. The amplification master mixture was prepared
and the hybridization process was carried out according to the standard protocol outlined
in the manufacturer’s instructions (Hain Lifescience GmbH, Nehren, Germany) [14].
c. Interpretation of results. The assessment of resistance to second-line anti-TB drugs relied on
analyzing the wild type and mutation band patterns. The examination of the gyrA and gyrB
genes aimed to identify resistance to FLQs (e.g., ofloxacin or moxifloxacin), while the analy-
sis of the rrs gene targeted cross-resistance to aminoglycosides/cyclic peptides antibiotics
such as kanamycin, amikacin, capreomycin, and viomycin. Furthermore, the eis gene was
examined to detect low-level kanamycin resistance [14].

Statistical analysis
We utilized SPSS software (IBM SPSS version 17.0) for conducting statistical analyses.
Descriptive statistics [mean (± SD), n, and %] were employed to depict the study variables. P-
values < 0.05 were considered as statistically significant.

Results
Socio-demographic details of the studied sample
Among a total of 171 samples, 160 (93.57%) were positive for MTBC and 11 (6.43%) were neg-
ative for MTBC (MOTT). The median [Interquartile (Q1-Q3)] age of patients with MTBC was
30 (22–40.75) years. There were 91 (56.88%) males with MTBC. In 160 MTBC positive cases,
126 (78.75%) [96 (76.19%) were smear positive and 30 (23.81%) were smear negative cases]
were direct sputum specimens. Of 96 (76.19%) smear positives, 95 (98.96%. 95/96) were
MTBC positive and 1 (1.04%, 1/96) was MTBC negative (MOTT) while in smear negative
cases, 22 (73.22%, 22/30) were MTBC positives and 8 (26.67%, 8/30) were negatives (MOTT).
However, 43 (95.5%, 43/45) of 45 cultures positive isolates tested positive for MTBC, while 2
(4.44%, 2/45) tested negative (MOTT) (Fig 1).

Distribution of pre-XDR-TB and XDR-TB drug resistance cases

Among 160 MTBC positive cases, 56 (35.0%) were FLQ resistance pre-XDR-TB, 1 (0.63%) was
SLID resistance pre-XDR-TB, and 10 (6.25%) were XDR-TB cases (Fig 2).

Frequencies of mutations responsible for pre-XDR-TB and XDR-TB

Among 160 MTBC positive cases, 93 (58.13%) were susceptible to FLQ and SLID whereas 56
(35.0%) were resistance to FLQ (pre-XDR-TB) and 1 (0.63%) has resistance to SLID (pre-
XDR-TB). The most frequently observed mutations were at codon MUT1 (23.21%, 13/56) and
MUT3C (57.14%, 32/56) of gyrA gene (Table 1).
Among 160 MTBC positive cases, 10 (6.25%) were resistance to FLQ and SLID (XDR-TB
according to the previous definition). The most frequently observed mutations were at codon
MUT1 (30%, 3/10) and MUT3C (30%, 3/10) of gyrA gene and at codon MUT1 (30%, 3/10) of
the rrs gene (Table 2).

Pre-XDR-TB and XDR-TB based on age and gender of the patients

In a total of 171 MDR/RR-TB cases, 6.43% (11/171) were MOTT, 35.63% (57/160) were pre-
XDR-TB and 6.25% (10/160) were XDR-TB cases. In 160 MTBC positive cases, 66 (41.25%)

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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

Fig 1. Age and gender based distribution of the patients infected with M. tuberculosis complex. In 160 MTBC
positive cases, 91(56.88%) were male and 69(43.13%) were female participants. The highest number of male
participants belonged to age 20–29 years whereas the age range for females was 20–29 years and 30–39 years.
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Fig 2. Diagram shows the distribution of pre-XDR-TB and XDR-TB drug resistance cases. This pie chart shows the prevalence of
drug resistance patterns observed in cases of pre-XDR-TB and XDR-TB.
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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

Table 1. Frequencies [n (%)] of mutations responsible for either FLQ or SLID resistance.
Drug Mutation band gyrA gyrB FLQ drug
n = 56 n=0 status
gyrA MUT1 13(23.21) 0(0) R
FLQ gyrA MUT2 2(3.57) 0(0) R
gyrA MUT3A 1(1.79) 0(0) R
gyrA MUT3B 3(5.36) 0(0) R
gyrA MUT3C 32(57.14) 0(0) R
gyrA MUT3D 1(1.79) 0(0) R
gyrA MUT1 + MUT3A 1(1.79) 0(0) R
gyrA MUT 3B + MUT3C 1(1.79) 0(0) R
gyrA WT missing 2(3.57) 0(0) R
Drug Mutation band Rrs eis SLID status
n=1 n=0
SLID rrs MUT1 1(100) 0(0) R

WT missing- Wild type probe missing FLQ- Fluoroquinolones, SLID- Second line injectable drug, R- Resistance.

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were resistance to FLQ, the higher resistance was found in males 41 (25.63%) than females 25
(15.63%). In 11 (6.88%) SLID resistance cases, males 7 (4.38%) had more resistance cases than
females 4 (2.50%). The age group 16–59 years had the highest FLQ resistance of 61 (38.18%)
and the highest SLID resistance of 10 (6.25%). Fluoroquinolone resistance pre-XDR-TB 52
(91.23%, 52/57), SLID resistance pre-XDR-TB 1 (1.75%, 1/57), and XDR-TB 9 (90%, 9/10)
resistance were all highest in the 16–59 years age group (Table 3).
The proportion of pre-XDR-TB patients in the children, adult, and elderly age groups is dif-
ferent from the proportion of XDR-TB patients. The proportion of pre-XDR-TB by gender is
different from the proportion of XDR-TB (Table 3).

Distribution of pre-XDR-TB and XDR-TB at province level

In a total of 160 MTBC positive cases, the Bagmati and Gandaki provinces had the highest
prevalence of pre-XDR-TB 23 (14.38%) and XDR-TB 5 (3.13%) cases respectively while the
Koshi Province had the lowest prevalence of pre-XDR-TB 4 (2.50%) (Fig 3).

Discussion
Tuberculosis, caused by MTBC, is an ancient disease [16]. Rapid molecular assays have
emerged as a superior and more reliable method for detecting drug-resistant TB, surpassing

Table 2. Frequencies [n (%)] of mutations responsible both FLQ and SLID resistance.
Drug Mutation band for FLQ and SLID FLQ and SLID status Total isolates with mutation
gyrA gyrB rrs eis n = 10
MUT1 WT missing WT WT missing R 1(10)
FLQ MUT1 WT MUT1 WT R 3(30)
and
SLID MUT3C WT WT WT missing R 1(10)
MUT3C WT MUT1 WT R 3(30)
MUT3C WT MUT2 WT R 1(10)
MUT3C+ MUT1 WT MUT1 WT R 1(10)

WT- Wild type probe, WT missing- Wild type probe missing, R- Resistance, FLQ-Fluoroquinolone, SLID- Second-line injectable drug.

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Table 3. Distribution [n (%)] of pre-XDR-TB and XDR-TB based on age and gender of the patients.
Demographics pre-XDR-TB XDR-TB
n = 57 n = 10 Pearson Chi-Square(pre XDR or XDR TB)
FLQ SLID FLQ SLID
n = 56 n=1 n = 10 n = 10
Age group (years) � 15: Children 2(3.51) 0(0) 0(0) 0(0) 0.55
16–59: Adults 52(91.23) 1(1.75) 9(90) 9(90) 0.74
�60: Elderly 2(3.51) 0(0) 1(10) 1(10) 0.36
Gender Male 35(61.40) 1 (1.75) 6(60) 6(60) 0.85
Female 21(36.8) 0(0) 4(40) 4(40)

SLID–Second line injectable drug, pre-XDR–Pre-extensively drug resistant, XDR–Extensively drug resistant.

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conventional culture techniques in terms of time and specificity. A notable example is Geno-
TypeMTBDRsl version 2.0, an enhanced version of GenoTypeMTBDRsl version 1.0 that con-
tains fewer mutations. This advanced test can detect molecular FLQ resistance involving gyrA
and gyrB, as well as SLID resistance involving the rrs and eis genes [14]. Precisely diagnosing
MDR-TB, pre-XDR-TB, and XDR-TB is crucial for interrupting transmission and selecting
appropriate treatment options, including injectable drugs and FLQs. A thorough understand-
ing of the drug resistant pattern plays a vital role in effectively managing pre-XDR-TB and
XDR-TB cases.
This study revealed a higher prevalence of pre-XDR-TB (35.63%) and XDR-TB (6.25%).
Similarly, a previous study conducted by GENETUP in 2012 reported a prevalence of 28% for

Fig 3. Pattern for pre-XDR-TB and XDR-TB cases. This line chart shows the pattern of pre-XDR-TB and XDR-TB across the provinces of Nepal,
highlighting the provinces with the highest and lowest prevalence rates of pre-XDR-TB and XDR-TB.
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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

pre-XDR-TB and 8% for XDR-TB [17]. In contrast, previous studies have shown divergent
rates, with India reporting a pre-XDR-TB rate of 56%, China at 34%, and Bangladesh at 16%
[13,18,19]. Similarly, multi-center studies of MDR-TB patients across eight nations and Poland
found a comparable rate of XDR-TB, with 6.7% and 6.4%, respectively [20,21]. The inappro-
priate use of anti-TB drugs has contributed to the increased prevalence of pre-XDR-TB and
XDR-TB patients, leading to the emergence of drug-resistant TB [22]. Specifically, the com-
mon use of FLQ in medical treatment has been linked to the increased prevalence of pre-
XDR-TB. In this study, the prevalence of the gyrA MUT3C region associated with FLQ resis-
tance was found to be the highest (57.14%, 32/56), while the rrs MUT1 region linked to SLID
resistance showed a prevalence of 100% (1/1). For the specific mutation bands responsible for
pre-XDR-TB, the MUT1 (30%, 3/10) and MUT3C (30%, 3/10) regions, associated with FLQ
and SLID, respectively, had a considerable number of total isolates with specific mutations
responsible for XDR-TB. A previous study in South Africa also observed frequent mutations
in gyrA MUT1, gyrA MUT3C, and rrs MUT1, which align with the findings of our analysis
regarding the frequency of specific bands on mutation regions [23].
Herein, the prevalence of FLQ resistance in pre-XDR-TB cases is significantly higher
(98.25%, 56/57) compared to SLID resistance (1.75%, 1/57) in pre-XDR-TB cases. A study con-
ducted in India also highlights the alarming global increase in FLQ resistance tuberculosis [11].
Another study by Ahmad et al. reported lower rate (52.7%) of FLQ resistance in MDR-TB
patients [24]. In Delhi, India, a study revealed a notable prevalence of FLQ resistance among M.
tuberculosis isolates, including drug-sensitive and multidrug-resistant strains [25]. The mecha-
nism by which FLQ works is by blocking DNA gyrase, a crucial enzyme for bacterial DNA syn-
thesis. Mycobacterium tuberculosis develops resistance to FLQ mainly due to mutations in DNA
gyrase, which is composed of two A and two B subunits encoded by the genes gyrA and gyrB,
respectively [26]. High level resistance to FLQ is often associated with mutations in the gyrA
gene, while low-level resistance can be attributed to mutations in gyrB. In addition, another con-
tributing mechanism involves efflux pumps that expel the drug from bacterial cells [27,28]. The
inappropriate use of antimicrobial drugs, as well as the use of ineffective drug formulations and
premature treatment interruption, can lead to drug resistant. This resistant can then be trans-
mitted, particularly in crowded environments such as prisons and hospitals [29].
It Is interesting to note that a higher number of pre-XDR-TB and XDR-TB cases were diag-
nosed in the adult (16–59 years) age group. Independent studies in Bangladesh and India also
reported an increased prevalence of pre-XDR-TB among the 21–30 and 18–25 age groups,
respectively [13,18]. Moreover, the prevalence of FLQ and SLID resistance TB was notably
higher in individuals aged 16–59, especially among males compared to females. The previous
study on FLQ and SLID resistance did not specifically explore variations in drug resistance
patterns with age and gender [30]. However, it is possible that within this age group, particu-
larly among males, increased mobility, extensive environmental exposure, and outdoor activi-
ties might contribute to the higher prevalence of drug-resistant tuberculosis.
The study emphasizes the importance of timely detection of pre-XDR-TB and XDR-TB cases
through efficient diagnostic tools to improve treatment strategies and align with global efforts to
eradicate tuberculosis. However, a limitation of the study is the small data size specific to pre-
XDR-TB, XDR-TB, and diagnostic facilities in GENETUP, as well as the ability of the MTBDRsl
line probe assay to identify restricted mutations responsible for pre-XDR-TB, XDR-TB.

Conclusions
This study found a higher prevalence of pre-XDR-TB and XDR-TB compared to previous
studies. Notably, the prevalence of FLQ resistant cases was significantly higher than that of

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PLOS ONE Drug-resistant Mycobacterium tuberculosis among Nepalese Patients at a Tuberculosis Referral Center

SLID within pre-XDR-TB. The most prevalent mutations were found in the gyrA gene at
codons MUT1 and MUT3C and in the rrs gene at codon MUT1 were associated with SLID.
This study highlights the importance of early diagnosis and prompt treatment for drug-resis-
tant tuberculosis in Nepal.

Author Contributions
Conceptualization: Arun Bahadur Chand, Bhagwan Maharjan, Bindu Sen.
Data curation: Arun Bahadur Chand, Bhagwan Maharjan, Lok Raj Bhatt, Bindu Sen.
Formal analysis: Arun Bahadur Chand, Ajaya Basnet, Yadav Prasad Joshi.
Investigation: Arun Bahadur Chand, Lok Raj Bhatt, Bindu Sen.
Methodology: Arun Bahadur Chand, Ajaya Basnet, Bhagwan Maharjan, Lok Raj Bhatt,
Bindu Sen.
Project administration: Arun Bahadur Chand, Yadav Prasad Joshi.
Resources: Arun Bahadur Chand.
Software: Arun Bahadur Chand, Yadav Prasad Joshi, Lok Raj Bhatt.
Supervision: Arun Bahadur Chand, Bhagwan Maharjan, Ganesh Rai, Yadav Prasad Joshi,
Shiba Kumar Rai.
Validation: Arun Bahadur Chand, Bhagwan Maharjan, Ganesh Rai, Shiba Kumar Rai.
Visualization: Arun Bahadur Chand.
Writing – original draft: Arun Bahadur Chand, Lok Raj Bhatt, Bindu Sen.
Writing – review & editing: Arun Bahadur Chand, Ajaya Basnet, Bhagwan Maharjan,
Ganesh Rai, Yadav Prasad Joshi, Shiba Kumar Rai.

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