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. 2019 Oct;8(3):335–346.

Introducing the Best Six Loci in Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) Typing for Mycobacterium Tuberculosis Genotyping

Mahdis Ghavidel 1,2, Keyvan Tadayon 3, Nader Mosavari 4, Kimiya Nourian 5, Hamid Reza BahramiTaghanaki 6, Gholam Reza Mohammadi 7, Mohammad Rashtibaf 8, Kiarash Ghazvini 1,2,*
PMCID: PMC7103078  PMID: 32274406

Abstract

Background:

Tuberculosis (TB) still remains endemic worldwide making epidemiological studies essential to mitigating efforts implicated in identifying its source, controlling, and preventing the spread of dangerous strains amongst humans such as Mycobacterium tuberculosis (Mtb).

Methods:

In this study, we sought to determine the 6 Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) loci with high discriminatory powers for Mtb genotyping as well as the loci with the highest and the lowest discriminatory powers for MIRU-VNTR. To conduct our search, we used several databases such as science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed using key words including: Mycobacterium tuberculosis, MIRU–VNTR, Allele diversity, Genetic diversity and human patient. Finally, 56 articles were selected after filtering out titles, abstracts and full texts.

Results:

Loci with high discriminatory powers included MIRU10 and MIRU26, while MIRU2, MIRU20, MIRU24 and ETRD had poor discriminatory powers. According to previous data in the literature, the loci MIRU10, MIRU26, MIRU40, QUB 26, QUB 11b and Mtub21 have high discriminatory powers.

Conclusion:

Therefore, these loci recommended for genotyping Mtb to save time and cost and to ensure the production of reliable results.

Key Words: Discriminatory power, Genotyping, MIRU-VNTR, Mycobacterium tuberculosis

Introduction

In spite of recent efforts to control and eliminate TB, this highly infectious disease still remains the second leading cause of death worldwide (1, 2). In 2018, WHO predicted that 10 million patients (ranging from 9 to 11.1 million) were stricken by TB. Mtb is a member of the TB complex which causes TB and can be transmitted via aerosolization of bodily fluids from coughing, sneezing or speaking (2).

To effectively control TB, preventive policies based on transmission routes are required. Molecular epidemiology of Mtb involves monitoring special strains like multi-drug-resistant (MDR) Mtb during periods of increasing prevalence, inspecting regions of latest and potential outbreaks, locating the source and route of transmission and transmission gene sequence, discovering hidden strains and tracking circulating immigrant strains (3).

Various molecular methods are used in epidemiological studies about Mtb strains. Each method has a particular specificity and sensitivity. Some of these approaches include IS6110, IS6110-restriction fragment length polymorphism (RFLP), MIRU-VNTR, Spoligotyping, whole genome sequencing (WGS), Random Amplification of Polymorphic DNA PCR (RAPD_PCR), Repetitive element sequence-based PCR (rep-PCR), Pulsed-field gel electrophoresis (PFGE), Next Generation Sequencing (NGS) and finally, a combination of two or more techniques listed above will be applied. Among current typing methods, a test has to be chosen according to its feasibility, cost-benefit and discriminatory powers (4-7)

In a survey comparing PFGE, 24 locus MIRU-VNTR and IS6110-RFLP, it was revealed that the 24 locus MIRU-VNTR method was the preferred method due to a high power of discrimination and time management during epidemiological investigations (7).

This study was performed to review published applications of the Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) method in Mtb genotyping and to introduce the best 6 loci for MIRU-VNTR in typing Mtb isolated from human patients along with determining the loci with highest and lowest discriminatory powers for MIRU- VNTR.

Materials and Methods

To identify the best loci of the 6 loci in MIRU-VNTR method for Mtb genotyping, the literature search was performed using several databases including science direct, Embase (Elsevier), Web of Science, Scopus, ISC and Medline via PubMed. Chosen keywords were: Mtb, MIRU–VNTR, Allele diversity, genetic diversity and human patient. Inclusion and exclusion criteria were determined by the following:

1. Isolation of Mtb from human patients.

2. Investigating the genetic diversity among Mtb just based on MIRU-VNTR.

3. Excluding the studies where lineage determination was based on MIRU-VNTR where Allele diversity “h” was not measured for each locus.

4- Excluding the studies in which MIRU-VNTR ability in cluster analysis and Hunter-Gaston discriminatory index (HGDI) for this method was investigated and h was not estimated for each of their locus.

Several articles were excluded from our study since allele diversity (h) was not mentioned for each locus. Screening the articles was done in 3 steps: 1. Title screening, 2. Abstract evaluation, 3. Full text evaluation based on these criteria.

Results

A total number of 228 articles were found collectively amongst the databases. As the title screening was performed, 90 articles were removed. Abstract screening resulted in 82 more studies to be omitted during the search. Finally, after full text screening, 56 articles remained. In the remaining articles, genotyping for Mtb using MIRU-VNTR was investigated from 2002 to 2019. Allele diversity (h) was evaluated amongst the 56 articles for each locus separately (Table 1).

Table 1.

Fifty-six studies after full-text evaluation that MIRU-VNTR were used.

No. Authors reference year Geographical region Continent Locus of MIRU-VNTR Numbers of locus High power Low power
1 Cowan et al (8) 2002 United States (Michigan) America ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU40 MIRU2,20
2 Sola et al (9) 2003 Different regions such as; USA, Thailand, Sicily, Guadeloupe and Russia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 15 ETRA, MIRU40 ,26,10 MIRU2,20, 27
3 Sun et al (10) 2004 Singapore Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU26,10, ETRE MIRU2,20
4 Kremer et al (11) 2005 China Asia ETR A-E MIRU10,16,26,39,40 QUB 11a,11b,26,1895 14 QUB 11b,11a MIRU10 MIRU16 ETRC
5 Kovalev et al (12) 2005 Russian Federation Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU26, ETRE MIRU24,27
6 Asgarzade et al (13) 2007 Iran Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU26, 40 MIRU16,39
7 Çavuşoğlu et al (14) 2007 Turkey Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU16,40,26 MIRU24,27
8 Maes et al (15) 2008 Venezuela America ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 MIRU40,26, ETRB MIRU20,2, 24
9 Alonso-Rodríguez et al (16) 2008 Spain Europe ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 QUB, 26,11b, MIRU40, 10 ETRD
10 Yun et al (17) 2009 Korea Asia ETRA-F MIRU2,10,16,20,23,24,26,27,39,40 6 MIRU26, ETRE,F MIRU24,20 ETRD
11 Stavrum et al (18) 2009 South Africa Africa ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 QUB 11b ETRD
12 Shamputa et al (19) 2010 South Korea Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 QUB 11b,26, Mtub4 MIRU2,24 ,23 ETRB,D
13 Noguti et al (20) 2010 Brazil America ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU40,23,10 MIRU24,39 ETRD
14 Jafarian et al (21) 2010 Iran Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU 26, 10,16 MIRU2, 24 ETRD
15 Zhang et al (22) 2011 Cambodia Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 ETRD,Mtub 39, QUB26 Mtub 34, MIRU2,20
16 Asgarzad et al (23) 2011 Iran Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 15 MIRU10,26,40 MIRU39,2,4
17 Bidovec-Stojkovi et al (24) 2011 Slovenia Europe ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 QUB 26, 11b, MIRU40 ,10 MIRU24,39
18 Cerezo et al (25) 2012 Colombia America ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU10, 40 MIRU24,39
19 Chatterjee et al (26) 2013 India Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU26, 10 MIRU2,20
20 Zamani et al (27) 2013 Iran Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 MIRU16 ETRA MIRU26, Mtub 21, 30, 39 QUB4156
21 Joseph et al (28) 2013 India Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 ETRA,B MIRU40 MIRU2,20
21 Yasmin et al (29) 2014 Pakistan Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 MIRU26 QUB26 MIRU10 MIRU2,20,27,24
23 Ali et al (30) 2014 Pakistan Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 QUb26 ,MIRU10,26 ETRD
24 Chaoui et al (31) 2014 Morocco Africa ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU40,23,10 MIRU24,39
25 Zheng et al (32) 2014 China Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 QUB11b,26 Mtub 21 MIRU26 MIRU24 Mtub 34
26 Vasconcellos et al (33) 2014 Brazil America ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 QUB 4156, 11b MIRU10 MIRU24,39
27 Rindi et al (34) 2014 Italy Europe ETRA,D,E MIRU10,16,26,40 Mtub21 QUB 11b, 26 VNTR 42,43,47,52,53 15 QUB 26 MIRU 40 QUB 11b MIRU 04 ETRE
28 Bouklata et al (35) 2015 Morocco Africa ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 QUB 26, RU40,26 MIRU20,27
29 Devi et al (36) 2015 India Asia ETR A-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26,4156 24 Qub26, 11b MIRU10 QUB 4156 MIRU26 Mtub 21 MIRU2,20, 27
30 Zamani et al (37) 2016 Iran Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 MIRU 40 MIRU 10 QUB 4156
31 Hoza et al (38) 2016 Tanzania Africa ETRA,C-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,39 QUB 11b,26,4156 22 MIRU26,10,16 ETRAE QUB 26, 4156 MIRU27,2, 20
32 Cheng et al (39) 2016 China Asia ETR A-E MIRU10,16,20,26,27,39,40 Mtub31 QUB 11a,11b 15 QUB11a ETRB,C
33 Bhembe et al (40) 2017 South Africa (Eastern Cape) Africa ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 ETRE, MIRU27,24 MIRU40
34 Zhang et al (41) 2017 China Asia ETR A-E MIRU 10,16,23,26,27,39,40 Mtub21,30,39 15 Mtub 21, MIRU26,10 ETRC,B
35 Liu et al (42) 2017 China Asia ETRA-E MIRU10,16,23,26,27,39,40 Mtub21,30,39 15 MIRU26 Mtub 21 MIRU27,23 ETRD
36 Pasechnik et al (43) 2017 West Siberia Asia ETRA-D MIRU2,10,16,20,23,24,26,27,39,40 15 MIRU26 MIRU24 ETRB
37 Pan et al (44) 2017 China Asia ETRA,D,E MIRU10,16,26,39,40 Mtub4,21,24,30,39 QUB11a,11b,18,26,3232,1895,4156 VNTR3820, 4120 22 VNTR3820 QUB3232 QUB4156 Mtub 24
38 Khosravi et al (45) 2017 Iran Asia ETR D,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU 10, 26 MIRU2,20
39 Ravansalar et al (46) 2017 Iran Asia ETRA-F MIRU10,16,26,39,40 QUB 11b 12 MIRU10, 26 ETRF ETRD
40 Shah et al (47) 2017 Nepal Asia ETRA-F MIRU2,10,16,20,23,24,26,27,39,40 Mtub21,30,39 QUB 11b,11b, 26, 4156 24 QUB 26 MIRU10 ETR B,C
41 Rasoaha et al (48) 2017 Madagascar Africa ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 MIRU26 QUB 11b Mtub 21 QUB 26 ETRD,C Mtub4
42 Chen et al (49) 2017 Asian countries (Cambodia, Singapore and Taiwan) Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 Mtub 21 QUB 11b MIRU2 QUB 4156
43 Li et al (50) 2018 China Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 Mtub4 MIRU40,10 Mtub21 MIRU27
44 Xu et al (51) 2018 China Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB 11b,26,4156 15 QUB 11b Mtub21 MIRU26 ETRC MIRU16 QUB 4156
45 Esteves et al (52) 2018 Brazil America ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 QUB11,26 VNTR42,1955,47,52,53,49 24 QUB26 QUB11 VNTR42 MIRU39,24 ETRD
46 Augusto et al (6) 2018 Brazil America ETRD,E MIRU2,10,16,20,23,24,26,27,39,40 12 MIRU16, 10,26 MIRU20,24
47 Riyahi Zaniani et al (53) 2018 Iran Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 MIRU 10 QUB 26 Mtub 4,34 ETRD, MIRU 20, Mtub 29
48 Azimi et al (54) 2018 Iran Asia ETRA,C-E MIRU10,16,26,40 Mtub4,21,30,39 QUB11b,26,4156 15 MIRU26, 10 Mtub 21 QUB 26 ETR D,E
49 Lil et al (3) 2018 China Asia ETRA-E MIRU16,23,26,27,39,40 Mtub21,30,39 15 ETRE, MIRU10, 26,39,40 Mtub 21 ETRB, C MIRU16,23
50 Mansoori et al (55) 2018 Iran Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 MIRU 10, 16, 26, MIRU 2, 20 ETRD
51 Chawla et al (56) 2018 India Asia ETRD,E MIRU2,10,16,20,23,24,26,27,39 40 12 MIRU 39,10,26 MIRU 2,20
52 Shi et al (57) 2018 China Asia ETRA-F MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,38,39 QUB 11b,26, 4156 26 QUB 11b Mtub 21 MIRU26 MIRU24,2, 20
53 Ei et al (58) 2018 Myanmar Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 QUB 26 QUB 11b MIRU26 Mtu34 MIRU20
54 Weerasekera et al (59) 2019 Sri Lanka Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 Mtub 21,39 QUB 11b MIRU20,2 ,16
55 Zarzour et al (60) 2019 Syria Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 QUB 26 MIRU10,26 MIRU24,20, Mtub 29
56 Li et al (61) 2019 China Asia ETRA-E MIRU2,10,16,20,23,24,26,27,39,40 Mtub4,21,29,30,34,39 QUB 11b,26, 4156 24 QUB11b MIRU 26 Mtub21 MIRU2, 20,24
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Mycobacterium tuberculosis genotyping was performed on 56 studies using the MIRU-VNTR technique; 39 of which were conducted in Asia, seven in America, six in Africa, three in Europe and one in a different country. The location and number of studies are shown in Figure 1. Each individual study employed a different number of loci. The results revealed that MIRU10 and MIRU26 had the highest discriminatory powers while MIRU2, MIRU20, MIRU24 and ETRD had the lowest discriminatory powers, respectively.

Figure 1.

Figure 1

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Location and numbers of studies was shown in the world from 2002 to 2019. Two studies were performed in different regions: Studies No. 2 and 42

Table 2 shows both the number of studies in which MIRU10, MIRU26, QUB26, MIRU40, QUB11b and Mtub21 was reported to be the loci with the highest discriminatory powers (h>0.6), including the range of h for the remaining loci.

Table 2.

Six MIRU-VNTR loci for Mycobacterium tuberculosis based on the number of studies and the reported allele diversity (h) range for each locus.

locus Number of studies h range
MIRU10 28 0.61 to 0.82
MIRU26 32 0.61 to 0.81
QUB26 18 0.604 to 0.89
MIRU40 17 0.604 to 0.76
QUB11b 17 0.72 to 0.84
Mtub21 12 0.64 to 0.83
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MIRU2 and MIRU20 (each in 21 studies), MIRU24 (17 studies) and ETRD (13 studies) were suggested as the loci with the lowest discriminatory powers (h<0.3).

Discussion

An alarming 62% of emerging TB cases occurred in the South-East Asia and Western Pacific regions, followed by Africa, which accounts for 25% of new cases. Countries such as India, China, Indonesia, the Philippines, Pakistan, Bangladesh, Nigeria and South Africa hold a prevalence rate greater than 60% for TB. It is believed that the surge in TB research in Asia could be that TB is highly prevalent in this continent based on 2018 WHO report (2). Of the 56 studies extracted, 39 studies were conducted within Asia. More specifically, 11 were performed in China, ten in Iran, four in India, two in Pakistan, two in Korea and only one in Nepal, Singapore, Myanmar, Turkey, Cambodia, Sri Lanka, Syria, Russia, Siberia, Taiwan, and Cambodia. Among the 39 studies conducted in Asia, MIRU26 and MIRU10 was reported having high discriminatory power loci. Among the 7 studies conducted in the Western Hemisphere, four studies were carried out in Brazil, and one study was performed in Michigan, Venezuela and Colombia. Both MIRU40 and MIRU10 had high power discriminatory power loci in these ancients.

Among the six studies performed in Africa, four studies were conducted in South Africa and Morocco, respectively. One study, performed in Madagascar and Tanzania, reported that QUB26 and MIRU26 had high discriminatory power loci in this continent. Several investigations in this study used PCR-based techniques such as spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units–variable-number of tandem repeats (MIRU-VNTR) analyses. Although spoligotyping benefits from genetic diversity, it can minimize Mtb clonal diversity. This method uses one direct repeat (DR) which incorporates identical alternate and variable spacers and the results are represented as a single digit pattern. The basis of MIRU-VNTR depends on the (variable) number of tandem repeat elements called mycobacterial interspersed repetitive units (MIRU). The results are represented as a code, and offers a low discriminatory power when used alone. The MIRU-VNTR method analyzes DNA segments which involves tandem repeat sequences and the copy number which differs amongst various strains. This method depends on PCR efficiency, specifically the quantity of repeats which is based on the size of the amplified product. The results are illustrated as characters that range from 15 –24 characters, in which each character represents the number of repeats at a single locus (3, 62).

These results are evaluated in comparison to a strain database on the web-based tool MIRU-VNTR plus. Therefore, MIRU-VNTR typing is considered the gold-standard for genotypic analysis of Mtb (7, 9). The discriminatory power of these methods was found to be different which was determined by the Hunter and Gaston Index factor. Several studies reported different Hunter-Gaston Index for MIRU-VNTR ranging from 0.951 to 0.999 (10, 13, 45, 47, 48, 53-56).

The allele diversity index (h) is used to describe the discriminatory power of MIRU-VNTR loci. If the index is greater than 0.6 (h>0.6), the discriminatory power of the locus is high. If the index lies between 0.3 and 0.6 (0.3<h <0.6), the locus has medium discriminatory power, however, if the index is less than 0.3 (0.3>h), the discriminatory power is considered weak. Our results suggest that among the 24 defined loci for MIRU-VNTR introduced by the MIRU-VNTR plus database, the MIRU26, MIRU10, MIRU40, QUB26, QUB11b and Mtub21 loci had the highest discriminatory powers, in contrast to the MIRU2, MIRU20, MIRU24 and ETRD loci which yielded low discriminatory powers.

Mycobacterium bovis (M. bovis) is the causative agent of TB in humans. When comparing the discriminatory powers of loci between Mtb and M. bovis, the loci QUB 11b and QUB 3232 have the highest discriminatory powers (for M. bovis), whereas ETRD (for both strains) and MIRU10 (for M. bovis) had the lowest discriminatory powers. Therefore, it can be concluded from our study that QUB 11b in both Mtb and M. bovis has a high discriminatory power while ETRD for both strains is considered a low power locus. However, a difference exists between the two strains in the MIRU10 locus which has a high discriminatory power in Mtb but low in M. bovis (63).

While the 24 loci MIRU-VNTR has been introduced as the best typing method for Mtb based on multiple comparisons between various molecular techniques, it is, therefore, highly recommended to include the following loci, QUB26, QUB11b, MIRU10, MIRU26, MIRU40 and Mtub21, provided that MIRU-VNTR is done with less than 24 loci, in order to obtain the best results when genotyping Mtb isolates. In this regard, data extraction becomes inexpensive and time efficient.

Among typing methods, MIRU-VNTR is considered to be one of the best. Further, the 6 loci including MIRU10, MIRU26, MIRU40, QUB 26, QUB 11b and Mtub21, all of which have high discriminatory powers, are recommended in Mtb genotyping to save time and cost. Indeed, epidemiological studies are critical for disease surveillance of TB as they enable us to track circulating strains on a global scale, as well as for identifying important risk factors necessary for implementing control measures in vulnerable populations worldwide.

Acknowledgements

We thank Antimicrobial Resistance Research Center and Mashhad University of Medical Sciences The authors of this study declare no conflicts of interest.

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