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ORIGINAL RESEARCH
Acetylator Status Among Newly Diagnosed and
Recurrent Tuberculosis Patients from Kupang,
Eastern Part of Indonesia
Edhyana Sahiratmadja
1
Ika Agus Rini
2
Simeon Penggoam
3
Afandi Charles
2
Ani Melani Maskoen
1,2
Ida Parwati
4
1
Division of Biochemistry and Molecular
Biology, Department of Biomedical
Sciences, Faculty of Medicine, Universitas
Padjadjaran, Bandung, Indonesia;
2
Health
Research Unit, Faculty of Medicine,
Universitas Padjadjaran, Bandung,
Indonesia;
3
Laboratorium Mikrobiologi,
RSUD Prof. Dr. WZ Johannes, Kupang,
Indonesia;
4
Department of Clinical
Pathology, Faculty of Medicine,
Universitas Padjadjaran, Bandung,
Indonesia
Purpose: N-acetyltransferase-2 enzyme in the liver, encoded by NAT2 gene, plays a central
role in metabolizing tuberculosis (TB) drug isoniazid (INH). Low compliance of patients
toward six-month TB therapy and internal host factors, ie comorbid diseases, immune status,
and genetic proles, are factors leading to treatment failure and recurrence of pulmonary TB
infection. This study aimed to explore the NAT2 acetylator status among newly diagnosed
and recurrent pulmonary TB patients in eastern part of Indonesia.
Patients and Methods: Archived DNA of TB patients (n=124) and healthy controls
(n=124) were sequenced, and NAT2 acetylator status was determined, then categorized as
fast, intermediate, or slow acetylators. Pulmonary TB patients who had no previous TB
treatment history were designated as newly diagnosed pulmonary TB, whereas patients with
a history of TB treatment were designated as recurrent pulmonary TB. The demographic,
clinical, and microbiological data between pulmonary TB groups were compared, and
acetylator status was described among groups.
Results: Male was more signicantly prevalent in the recurrent pulmonary TB group
(p=0.025), and anemia was more prevalent in new pulmonary TB (p=0.003). The acetylator
status in pulmonary TB patients compared to healthy controls were rapid (33.9% vs 48.1%),
intermediate (57.8% vs 33.0%), and slow acetylators (8.3% vs 18.9%), respectively.
Interestingly, the rapid and intermediate acetylator were signicantly more prevalent in
pulmonary TB patients than in healthy controls (p=0.023, OR=2.58 (1.12–5.97).
Furthermore, no differences were found in acetylator status between new and recurrent
pulmonary (p=0.776).
Conclusion: Rapid and intermediate acetylators status predominated the pulmonary TB
patients in Kupang, eastern part of Indonesia, postulating different genetic makeup in this
area. As the pulmonary TB patients in Kupang exhibit more rapid acetylator phenotype, the
acetylator status might be relevant to be checked before TB therapy for adjusting treatment
dose to prevent drug resistances.
Keywords: acetylator status, NAT2, Indonesia, tuberculosis, recurrence
Introduction
Pulmonary tuberculosis (TB) has been known for centuries as one of the deadliest
diseases due to a single infection agent, Mycobacterium tuberculosis. Around
10 million people have been infected with TB and there were 1.2 million
deaths related to TB globally in 2018, of which most cases occur in developing
countries, including Indonesia. The estimated incidence rate per year in Indonesia is
placed third globally.
1
Four-drugs regimen, including rifampicin, isoniazid,
Correspondence: Edhyana Sahiratmadja
Division of Biochemistry and Molecular
Biology, Department of Biomedical
Sciences, Faculty of Medicine, Universitas
Padjadjaran, Jl. Raya Bandung – Sumedang
KM 21, Jatinangor, Sumedang, Jawa Barat,
45363, Indonesia
Tel +62 22 779 5594
Fax +62 22 779 5595
Email e.sahiratmadja@unpad.ac.id
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pyrazinamide, and ethambutol, has been used for around
40 years to treat pulmonary TB, yet recurrence and death
numbers due to this disease are still burdening.
2
Low
compliance of patients toward six-month chemotherapy
and internal host factors, ie comorbid diseases, immune
status, and genetic proles, become factors leading to
treatment failure and pulmonary TB recurrence.
3
Based
on pharmacogenetics variation, recently, host-genetic pro-
les have emerged as a promising intervention in deter-
mining the success of treatment for pulmonary TB
patients.
4
One of the genes that play essential roles in TB drug
pharmacokinetics is NAT2 gene, encoding
N-acetyltransferase-2 enzyme in the liver.
4,5
This
enzyme metabolizes INH into less active forms by acet-
ylating the drug molecules, thus determining how much
active drug concentration in the plasma.
4,5
Previous stu-
dies showed three phenotypic host acetylator statuses
were observed due to various polymorphisms in the
NAT2 gene: rapid, intermediate, and slow acetylators.
4
Slow acetylator phenotype is associated with adverse
drug responses such as nausea, drug-induced liver injury,
peripheral neuropathy, and sideroblastic anemia which
may lead to discontinuation of treatment.
6,7
On the
other hand, rapid acetylator phenotype could enhance
drug metabolism in the liver, leading to the lower con-
centration of INH in the plasma below the requirement
to kill the bacteria, thus causing treatment failure and the
emergence of resistance.
8
Therefore, pharmacogenetic
proling of NAT2 gene becomes essential to achieve
successful treatment.
As Indonesia accounts for many TB patients globally,
the pharmacogenetic proling of NAT2 gene is of great
interest. There is a high proportion of TB patients in this
archipelagic country. Studies in Western and Central
Indonesia have been reported recently, resulting in predo-
minantly intermediate acetylators in this area.
9,10
Interestingly, NAT2 gene prole in other populations in
the eastern part of Indonesia has not been explored. East
Nusa Tenggara is a rural province in eastern Indonesia.
This area has an estimated TB incidence of around 18,000
in 2019, with compliance percentage of treatment among
patients just around 66%.
11
Socioeconomic factors may
contribute to patient compliance in these populations.
However, pharmacogenetic proles may also affect anti-
TB drug therapy’s success and the rate of TB recurrence.
12
Therefore, this study aimed to explore NAT2 acetylator
status gene among newly diagnosed and recurrent pulmon-
ary TB patients in eastern part of Indonesia.
Materials and Methods
Study Design
This study was a descriptive and analytical study, with
a retrospective design including pulmonary TB patients
(newly diagnosed pulmonary TB patients and patients
with recurrent TB) and healthy controls from the neigh-
borhood. This study was part of a study exploring the
susceptibility genes among TB patients from Kupang at
the island of Timor, located in a remote area in the eastern
part of Indonesia. Informed consent about the susceptibil-
ity genes study was obtained from all subjects, and DNA
was stored for various genetic studies, including this study.
Our study was conducted in accordance with Declaration
of Helsinki and the study protocol was approved by the
Ethical Committee of Faculty of Medicine Universitas
Padjadjaran Bandung under no. 136/UN6.C2.1.2/KEPK/
PN/2012.
In brief, archived DNA of TB patients (n=124)
recruited in 2011 at Prof W. Z. Johannes General
Hospital, Kupang, were used. Healthy controls (n=124)
were also recruited to explore the NAT2 acetylator status
in the general population. Pulmonary TB patients were
diagnosed according to standard hospital procedure as
directed in WHO guideline, ie history taking of signs and
symptoms of TB infection, physical examinations, chest
radiology examinations, and positive result of three-time
points of sputum examination. Mycobacterial culture was
examined in a part of patients, of which drug susceptibility
test and Mycobacterium tuberculosis spoligotyping test
were performed as described previously.
13
Pulmonary TB patients who had no previous TB treat-
ment history were designated as newly diagnosed pulmon-
ary TB, whereas patients with a history of TB treatment
were designated as recurrent pulmonary TB. Patients with
HIV seropositive or fasting glucose serum of >200 mg/dL
were excluded from this study.
DNA Extraction and NAT2 Gene
Sequencing
Venous blood from pulmonary TB patients and healthy
controls were collected in 3 mL EDTA tubes, then stored
in 4°C before sending to Bandung, West Java, about 4
hours ight from Kupang. In brief, DNA was isolated from
blood according to the manufacturer’s protocol (QIAamp
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DNA Blood Mini Kit, Cat no. 51104, Qiagen). The ampli-
cation of NAT2 gene and the sequencing process was
performed based on our study protocol published
previously.
14
Acetylator Status Assessment
The determination of acetylator status was conducted using
seven single nucleotide polymorphisms in NAT2 gene, ie
rs1801279 for 191G>A, rs1041983 for 282C>T, rs1801280
for 341T>C, rs1799929 for 481C>T, rs1799930 for
590G>A, rs1208 for 803A>G, and rs1799931 for 857G>A.
Sequence data analyses were performed using Bioedit soft-
ware (version 7.2.5.0) and nucleotide BLAST (Genebank
reff. KR231610). The NAT2 genotype identication was
performed by analyzing 7 SNPs of NAT2 according to the
Arylamine N-acetyltransferase Gene Nomenclature website
(http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.
htm). The nucleotide changes at rs1801280 (341T>C),
rs1799930 (590G>A), and rs1799931 (857G>A) were
designated as NAT2*5, NAT2*6, NAT2*7, respectively.
The subjects were then categorized as fast, intermediate, or
slow acetylator phenotype.
Statistical Analyses
Data statistics were performed in Microsoft Excel 365
(Microsoft Corp., Redmond, WA, USA). The demographic,
clinical, and microbiological data were compared between
newly diagnosed and recurrent TB groups, then analysis was
performed using Mann-U Whitney tests (for numerical data)
and chi-square test (for categorical data), and p-value <0.05
was designated as signicant. To determine whether the
SNPs genotypes in NAT2 were in Hardy-Weinberg equili-
brium (HWE), Chi-square tests were conducted and p value
≥ 0.05 indicated that the SNPs genotype were in equilibrium.
The NAT2 haplotype status were presented in numbers (n).
The acetylator status frequencies in different groups (newly
diagnosed TB, recurrent TB, and healthy controls) were
compared using Pearson Chi-square or Fischer’s exact test
as appropriate, then odds ratio (OR) with 95% condence
intervals (95% CI) were also calculated.
Results
Demographic, Clinical, and
Microbiological Prole of Pulmonary TB
Patients
Our study in Kupang recruited initially 124 participants
pulmonary TB, consisting of 99 (79.8%) new and 25
(20.2%) recurrent pulmonary TB patients. However,
patients were excluded (n=9) due to HIV seropositivity
(n=1), high serum glucose suspected for Diabetes Mellitus
(n=6; 4.8%) and incomplete data (n=2). The characteristic
data of both group pulmonary TB patients is shown in
Table 1.
When comparing new and recurrent pulmonary TB
patients, there was a signicant difference in gender.
Male was more prevalent in the recurrent pulmonary TB
group (p = 0.025). Furthermore, no signicant differences
in age, erythrocyte sediments rate, and body mass index
between both groups. Interestingly, hemoglobin concentra-
tion in newly diagnosed pulmonary TB was signicantly
lower compared to recurrent TB (median 10.9 mg/dL vs
12.1 mg/dL; p = 0.018), of which anemia was more pre-
valent in new pulmonary TB (p = 0.003).
In a subset of the patients, drug sensitivity tests were
performed, showing that the number of samples resistant
to all drugs seemed to be higher in group of recurrent TB.
Specically, INH resistance cases were documented higher
in recurrent TB than newly-diagnosed TB patients.
Furthermore, spoligotyping detection showed that non-
Beijing strain of M.tuberculosis predominated infection
in this population, both in newly diagnosed and recurrent
TB groups. Since the test was only performed in a limited
number of patients, no statistical analyses were conducted,
and data is only presented in frequency (Table 1).
NAT2 Polymorphisms and Acetylator
Status of Pulmonary Tuberculosis Patients
The distribution of SNPs in seven rs contributed to NAT2
gene variations among pulmonary TB patients from Kupang,
Nusa Tenggara Timur, Indonesia is described in Table 2.
Four polymorphisms that change the amino acid sequences
(rs1801280, p = 0.616; rs1799930, p = 0.286; rs1208, p =
0.653; and rs1799931, p = 0.332) were in Hardy-Weinberg
equilibrium. Based on those polymorphisms, there were 18
genotypes found in this population, and classied based on
their inferred phenotype as rapid (n=37; 33.9%), intermedi-
ate (n=63; 57.8%), and slow acetylators (n=9; 8.3%). For
comparison, the frequencies of acetylator status in healthy
controls were 48.1%, 33.0%, and 18.9% for rapid, intermedi-
ate, and slow phenotype, respectively. The most common
alleles found in this study were NAT2*4 (wild type; rapid
phenotype), NAT2*6A, NAT2*6B, and NAT2*7A. There
was “unknown” variation detected in this study, which had
not been documented in NAT2 gene bank; that was
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a combination of 282 C>T (rs1041983), 590 G>A
(rs1799930), 625 C>T that inferred as slow phenotype due
to homozygous recessive state. A new polymorphism 625
C>T had not been described elsewhere in NAT2 gene data-
base. However, since it was a non-sense mutation and did not
change the amino acid sequence, this polymorphism might
not change the function of NAT2 enzyme.
Interestingly, the rapid and intermediate acetylator
were signicantly more prevalent in pulmonary TB cases
than in healthy controls (p = 0.023, OR = 2.58 (1.12–
5.97)). However, comparing the frequency of acetylator
status among newly diagnosed and recurrent pulmonary
TB resulted in no signicant difference between both
groups (p = 0.776), as shown in Table 3.
Table 1 Clinical Characteristic of Patients with Newly Diagnosed and Recurrent Pulmonary Tuberculosis from Kupang, Nusa Tenggara
Timur, Eastern Part of Indonesia
Newly Diagnosed Recurrent
Pulmonary TB Pulmonary TB
(n=91) (n=24)
Median (Min-Max) Median (Min-Max) p-value
Age; years 33 (15–74) 35 (15–64) 0.364
BMI
a
; kg/m
2
14.6 (10.1–20.5) 15.6 (11.7–20.8) 0.201
Hb; g/dL 10.9 (7.3–15.5) 12.1 (7–14.8) 0.018*
ESR; mm/h 70 (1–140) 73.5 (4–119) 0.663
N (%) N (%) p-value
Gender
Male 41 (45.1) 17 (70.8) 0.025**
Female 50 7
Nutrional status
c a †
Normal 10 4 0.287
Underweight 56 (84.8) 11 (73.3)
Anemia status ††
Anemia 78 (85.7) 14 (58.3) 0.003**
No Anemia 13 10
ESR group
Normal 17 4 0.820
High (20–25 mm/h) 8 3
Very High (>50 mm/h) 66 (72.5) 17 (70.8)
Drug Sensitivity Test
b
Sensitive - 3(25) n.d.
Resistant to:
Rif, INH, Str, Eth 1 6 (50)
Rif, INH ——————— 1 2
Rif, INH, Str ——— 1 -
Rif, ———— Str, Eth 1 -
Rif, ———— Str, —— 1 -
Str, —— 3 1
Mtb genotypec
c
Beijing 3 4 n.d.
Non-Beijing 13 (81.2) 7 (63.6)
Notes: Data retrieved from
a
81 patients;
b
20 patients,
c
27 patients. † Nutritional Status according to BMI for normal 18–22 and underweight <18. †† Anemia designated as
WHO guideline for men <13 mm/dL and women <12 mm/dL. Statistical analyses p<0.05 designated as signicant for *non-parametric compared median and **chi-square
Abbreviations: BMI, body mass index; Hb, hemoglobin; ESR, erythrocyte sediment rate; TB, tuberculosis; Rif, Rifampicin; INH, isoniazid; Str, Streptomycin; Eth, Ethambutol;
n.d, not determined; Mtb, Mycobacterium tuberculosis.
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Discussion
Recurrent pulmonary TB infection worldwide is reported
in around 13% of all TB cases. Our study has detected that
around 20% of cases are recurrent pulmonary TB, higher
than estimated global data in 2017.
15
The recurrent TB
proportion varies signicantly in different areas; for exam-
ple, in Iran, Zambia, and South Africa, the recurrent TB
proportions are 8.3%, 15.3%, and 26%, respectively.
16–18
Recurrent pulmonary TB could be caused by either reacti-
vation of M.tuberculosis from previous infection or re-
infected from exogenous sources.
19
In this study, recurrent
pulmonary TB is signicantly higher in male patients,
conrming that males become one of the risk factors
contributing to recurrent cases.
20,21
Other risk factors asso-
ciated with recurrent TB cases include smoking, poor
treatment adherence, uncontrolled HIV-comorbid infec-
tion, and specic clinical pictures such as residual cavita-
tion, more signicant area of involved lung tissue, and
positive sputum culture at two months of rst treatment.
3
Previous studies have shown associations between TB
Table 2 Distribution of Single Nucleotide Polymorphisms Contributed to NAT2 Gene Variations Among Pulmonary Tuberculosis
Patients from Kupang, Nusa Tenggara Timur, Eastern Part of Indonesia
rs1801279 rs1041983 rs1801280 rs1799929 rs1799930 rs1208 rs1799931
Genotypes Inferred n 191 G>A 282 C>T 341 T>C 481 C>T 590 G>A 625 C>T 803 A>G 857 G>A
Phenotypes R64Q Y94Y I114T L161L R197Q L209L K268R G286E
NAT2*5 NAT2*6 NAT2*12 NAT2*7
NAT2*4/4 Rapid (WT) 35 GG CC TT CC GG AA GG
NAT2*4/12A Rapid 1 AG
NAT2*4/12M Rapid 1 CT CT AG
NAT2*4/5B Intermediate 4 TC CT AG
NAT2*4/5C Intermediate 1 TC CT AG
NAT2*4/5E Intermediate 1 TC GA AG
NAT2*4/5Q Intermediate 1 TC GA
NAT2*4/5S Intermediate 1 TC GA
NAT2*4/5U Intermediate 1 CT TC CT GA AG
NAT2*4/6A Intermediate 4 CT GA
NAT2*4/6B Intermediate 15 GA
NAT2*4/6J Intermediate 13 CT/TT GA GA
NAT2*4/7A Intermediate 18 GA
NAT2*4/7B Intermediate 3 TT GA
NAT2*6A/6A Slow 6 TT AA
NAT2*7B/7B Slow 2 TT AA
Unknown † Intermediate 1 TC CT GA
Unknown † Slow 1 TT AA CT
Notes: † Unknown is designated as variations that are not registered according to the data base of Arylamine N-acetyltransferase (NATs) for Human NAT2 Alleles
(haplotypes) http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.htm. Intermediate acetylator when the alleles are heterozygous; slow acetylators when the alleles are
in homozygote recessive variant.
Abbreviations: NAT2, N-acetyltransferase 2; WT, wild type.
Table 3 Distribution of Acetylator Status in Newly Diagnosed and Recurrent Pulmonary Tuberculosis Compared to Healthy Controls
from Kupang, Nusa Tenggara Timur, Eastern Part of Indonesia
Group Acetylator Status, n (%) P value* OR (95% CI)
Rapid Intermediate Slow
Healthy Controls 51 (48.1) 35 (33.0) 20 (18.9) 0.023
a
2.58 (1.12–5.97)
Pulmonary TB patients 37 (33.9) 63 (57.8) 9 (8.3)
Newly diagnosed 28 (32.2) 52 (59.8) 7 (8.0) 0.776
b
n.a.
Recurrent 9 (40.9) 11 (50.0) 2 (9.1)
Notes: *rapid and intermediate vs slow acetylator status (chi-square test),
a
Control vs pulmonary TB patients,
b
Newly diagnosed vs recurrent pulmonary TB patients.
Abbreviations: TB, tuberculosis; OR, odds ratio; CI, condence interval; n.a, not available.
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recurrence and drug resistance.
22,23
Our study has revealed
more MDR cases, as dened by simultaneous rifampicin
and isoniazid resistance, documented in recurrent TB cases
despite a small number of tests. These ndings suggest the
importance of using rapid methods to detect drug-
resistance M.tuberculosis to determine which regimens
should be used in recurrent pulmonary TB cases.
Genotype diversity of NAT2 gene has been known to
affect TB therapy response. Different genotypes can inu-
ence how rapidly NAT2 enzyme metabolizes INH drugs in
the liver. Different ethnicities have shown different pro-
portions of rapid, intermediate, and slow phenotypes. The
frequency of slow acetylators varies in different countries,
for example, in Latvia (51.8%), Senegal (49%), and Brazil
(41%),
24–26
which is much higher than in our study popu-
lation. Interestingly, several Asian countries reported slow
acetylator phenotypes in lower frequencies, for example,
China (15.2%) and South Korea (14.4%).
27,28
Moreover,
studies in western and central Indonesia have shown the
frequencies of slow acetylators are around 36% in pul-
monary TB patients with Javanese-Sundanese ethnicity. Of
note, healthy Malay-Indonesian accounts for 38%, similar
to healthy Buginese (42%).
9,10,29
Interestingly, our study
in Kupang, East Nusa Tenggara revealed that only around
8% of pulmonary TB patients were slow acetylator phe-
notypes. Predomination of rapid-intermediate acetylator in
TB patients in Kupang, suggesting that different genetic
background of this population may affect the NAT2 gene
polymorphism diversity. Based on historical perspective,
Kupang people residing in Timor Island, East Nusa
Tenggara, have mixed origin from Austronesian and
Polynesian ancestry.
30,31
This hypothesis should be
explored in other parts of East Nusa Tenggara and other
areas in eastern Indonesia such as Maluku and West
Papua.
Both slow and rapid acetylation could inuence the
drug response in TB patients regarding of the INH meta-
bolism speed in the liver. Slow acetylators are associated
with increased INH levels in plasma and hepatotoxicity
observed in TB patients, conrming the study conducted
in Javanese and Sundanese TB patients.
9
The NAT2*6A
allele is reported as an ultra-slow acetylator that can be
used to determine the effects of NAT2’s genetic poly-
morphism on metabolism and drug toxicity.
32
On the
other hand, a substantial percentage of rapid acetylators
in this population should raise other awareness since
a meta-analysis study showed that rapid acetylators
contribute to microbiological failure, acquired drug resis-
tance, and TB relapse.
8
The rapid acetylator individuals
could exhibit lower INH levels as presented in blood-
stream. This is due to INH molecules that is rapidly
acetylated, thus decreasing INH exposure to MTB in the
affected tissues.
33
Molecular docking study of mutated
MT-K268R model (polymorphsms at 803 A>G in
NAT2*4, NAT2*12 and NAT2*13) has shown less binding
afnity toward INH and increase acetyl-INH score, lead-
ing to more INH acetylation.
34
As we noted that INH drug
resistance was found in this population (especially in peo-
ple with recurrent pulmonary TB), further study with
larger participants might be needed to conrm the associa-
tion of rapid acetylator phenotypes and recurrent TB cases
and drug resistance in Kupang population.
Our study has several limitations among others that the
study was not designed to detect adverse effects such as
hepatotoxicity and treatment failure, especially due to the
nature of the study site which is in a remote area and
laboratory examinations are scarce. Further study needs
to be designed considering the role of pharmacogenetic
background of individuals living in area with possible
different genetic makeup across the islands of Indonesia.
The study on population genetic prole is of great interest.
Furthermore, the sample size is not calculated accordingly
as we had included total respondents who were willing to
participate in the study period. Moreover, some of the
clinical information and other data are sometimes lacking.
Therefore, it is imperative to register the complete infor-
mation in the medical record or registry that would
strengthen the results of the TB treatment with rst-line
drugs in Indonesia.
Conclusion
The slow acetylator status in pulmonary TB in Kupang
population in our study was 8.3%, much lower than pre-
vious study conducted in other parts of Indonesia, suggest-
ing different genetic background in eastern part of
Indonesia. There was no difference in acetylator status
between newly and recurrent pulmonary TB. As majority
of pulmonary TB patients in Kupang are rapid and inter-
mediate acetylator status, these might inuence the emer-
gence of drug resistance and recurrence. The acetylator
status might be relevant as the rationalization in recurrent
TB patients to be checked prior to INH therapy to adjust
treatment dose to prevent further antimicrobial resistance
for successful TB treatment.
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Acknowledgments
The authors thank all the general practitioners and nurses
for examining the patients in Prof W. Z. Johannes General
Hospital, Kupang. We also thank the patients, families and
their neighbors for participating in this study.
Disclosure
The authors declare that they have no conicts of interest
for this work. This study was nancially supported by the
Ministry of Research and Technology, Republic of
Indonesia. The content of this paper is solely the respon-
sibility of the authors and does not necessarily represent
the ofcial views of the funders.
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