MIRU-VNTR analysis of the Mycobacterium tuberculosis isolates from three provinces of Iran

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MIRU-VNTR analysis of the Mycobacterium tuberculosis isolates from three provinces of Iran

Article in Scandinavian Journal of Infectious Diseases · September 2012

DOI: 10.3109/00365548.2012.717233 · Source: PubMed

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Correspondence: M. M. Feizabadi, Department of Microbiology, Tehran University of Medical Sciences, School of Medicine, Tehran, Iran. E-mail: mfeizabadi

@tums.ac.ir

(Received 2 July 2012 ; accepted 25 July 2012 )

ORIGINAL ARTICLE

MIRU-VNTR analysis of the Mycobacterium tuberculosis isolates from three provinces of Iran

SAMIN ZAMANI

¹

, MOLOUD AFLAKI

¹

, ABBAS ALI IMANI FOOLADI

²

,

DAVOOD DARBAN-SAROKHALIL

³

, ZAKARIA BAMERI

⁴

, SEDIGHEH KHAZAEE

⁵

, MOHAMMAD JAVAD NASIRI

¹

& MOHAMMAD MEHDI FEIZABADI

¹

From the

¹

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran,

²

Applied Microbiology Research Centre, Baqiyatallah University of Medical Sciences, Tehran,

³

School of Medicine, Shahrekord University of Medical Sciences, Shahrekord,

⁴

Research Centre for Infectious Diseases and Tropical Medicine, Zahedan University of Medical Sciences, Boo-Ali Hospital, Zahedan, and

⁵

Molecular Pathology Research Centre, Kermanshah University of Medical Sciences, Emam Reza Hospital, Kermanshah, Iran

Abstract

Background: Iran borders 2 high-burden tuberculosis (TB) countries to the east, and has the highest rates of TB in one of its eastern provinces. Limited information is available on the genetic diversity and transmission dynamics of Mycobacterium tuberculosis (MTB) in Iran. To examine the genetic diversity and transmission dynamics of MTB strains we genotyped a collection of isolates from different parts of Iran. Methods: Standard 15-locus variable number tandem repeat (VNTR) typing was applied to genotype 121 MTB clinical isolates collected from 3 provinces of Iran, including Tehran (the capital of Iran), Sistan-Baluchestan (southeast province of Iran, with the highest rate of TB), and Kermanshah (western part of Iran with high TB/human immunodefi ciency virus cases). Antibiotic susceptibility for all isolates was determined using the proportion method. Results: Sixty-six distinct mycobacterial interspersed repetitive unit (MIRU)-VNTR patterns were detected among 121 isolates. Seventy-fi ve strains grouped into 20 clusters, and 46 isolates were unique.

The genetic diversity of strains from Sistan-Baluchestan was higher than that in the other provinces. All isolates from Tehran or Kermanshah that grouped into clusters shared identical patterns with Sistan-Baluchestan. The Hunter – Gaston discriminatory index (HGDI) was 0.972, indicating a high power of discrimination for MIRU-VNTR typing. The MIRU 16 and ETRA loci were designated as highly discriminative. The rates of monoresistance and multidrug resistance were 9.9% and 2.4%, respectively. Conclusions: MIRU-VNTR typing revealed high genetic diversity and suggests the possibility of transmission from Sistan-Baluchestan to other provinces of Iran. This method has potential for genetic analysis and for studying the transmission routes of TB.

Keywords:

Mycobacterium tuberculosis , MIRU-VNTR , Iran

Introduction

Tuberculosis (TB) is considered one of the most important pathogens in the world. The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains and co-infection with human immunodefi ciency virus (HIV)/acquired immunodefi ciency syndrome (AIDS) have made TB a major public health problem [1,2]. In Iran, according to World Health Organization (WHO) reports, the estimated rate of TB was 23 per 100,000

in the year 2010 [3]. The prevalence of TB in some of the countries around Iran is 2- to 3-times higher than this rate [4]. Sistan-Baluchestan is located in the southeast of Iran and has an estimated TB rate of 48.5 per 100,000 population [5]. Bordering high TB burden countries like Pakistan and Afghanistan and the migration of Afghan people have changed the incidence rate of TB in this province and it has the highest incidence in the country [5]. Kermanshah is located in the western part of Iran, bordering Iraq.

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2 S. Zamani et al.

This province has been seriously affected in terms of its economy, unemployment, and stressful living conditions during the 8-y war with Iraq. According to reports, more than half of the HIV-positive cases in the country are detected in this province [6,7].

Also, the rate of clinical TB in HIV-infected patients in this province was found to be signifi cantly higher than the estimated rate of TB in Iran [8]. In Tehran, the capital of Iran, with an estimated population of 8,429,807, the incidence of TB is moderate [4].

Since the discovery of polymorphic DNA, molecular typing of MTB isolates has become a valuable tool for epidemiology and phylogeny of TB by allow- ing investigators to detect unsuspected transmission, identifi cation of false-positive cultures, and distin- guishing between re-infections and relapses [9,10].

Nowadays, a large number of methods for MTB genotyping exist, of which IS6110 -restriction frag- ment length polymorphism (RFLP), spoligotyping, and mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) typing are the most frequently used [11 – 14]. Among these methods, MIRU-VNTR is probably the most popular approach [14 – 17]. These methods are considerably faster to perform and interpret than other methods, require small amounts of DNA, and results can easily be digitized and shared among laboratories [15,17]. Moreover, MIRU-VNTR typing can be used to discriminate IS6110 low copy number isolates, with a resolution surpassing both IS6110 -RFLP and spoligotyping [13].

In the present study, MIRU-VNTR was applied for genotyping and tracking the transmission dyna- mics of MTB strains isolated from patients in 3 prov- inces of Iran (Tehran, Sistan-Baluchestan, and Kermanshah). Several studies have been conducted in Iran, but information on the genetic diversity and transmission dynamics of MTB in these impor- tant provinces is rare. This study focused on the genetic diversity and transmission dynamics of MTB isolates from 3 important provinces using 15-locus MIRU-VNTR.

Materials and methods

Clinical isolates and study population

A total of 121 isolates of MTB cultured from patients from 3 provinces of Iran (Tehran, Sistan- Baluchestan, and Kermanshah) were included in the study. The age of patients ranged from 14 to 83 y and the mean age of the patients was 34 y.

The female-to-male sex ratio was 1:2, with a similar distribution in each of the provinces. Patients were from across the age-range, had clinical signs and symptoms of TB, and were sputum-positive for

acid-fast bacilli. The numbers of MTB isolates from each province were as follows: Tehran n ⫽ 26, Sistan-Baluchestan n ⫽ 66, and Kermanshah, n ⫽ 29.

Isolates were identifi ed by standard biochemical tests, including production of niacin, catalase, and nitrate reduction [18,19]. The extraction of DNA from the clinical isolates was performed by standard protocols, as described previously [19].

Drug susceptibility testing

Susceptibility of isolates to isoniazid (0.2 μ g/ml), rifampin (40 μ g/ml), ethambutol (2 μ g/ml), and streptomycin (10 μ g/ml) was determined using the proportion method [18].

MIRU-VNTR typing

The primers specifi c for the fl anking regions of the VNTRs were used to amplify different VNTR regions of 15 loci as proposed by Supply et al., and the number of VNTR copies was determined by the size of each amplicon [20]. Polymerase chain reactions (PCR) were performed in 25 μ l of reaction mixture contain- ing 2.5 μ l PCR buffer, 1 mM MgCl

₂

, 0.01 pM of each primer, 200 μ M of each dNTP, 1 unit of Taq DNA polymerase (mi Taq, Metabion, Martinsried, Germany), and 2 μ l of DNA template. PCR ampli- fi cation was carried out in a PCR system using the following cycling program: initial denaturation at 94 ° C for 5 min, followed by 30 cycles of 94 ° C for 45 s, 60 ° C for 60 s, and 72 ° C for 90s, with a fi nal extension at 72 ° C for 10 min. To check the reaction for possible contamination, sterile distilled water was used as negative control. The amplicons were run on 1.5% standard agarose gels and analyzed [20].

Analysis of VNTR allelic diversity and genetic relationships

The allelic diversity of each VNTR locus was evalu- ated by the Hunter – Gaston discriminatory index (HGDI), as described previously [21]. Genetic relationships among the isolates were estimated by the unweighted pair group method with arithmetic averages using MIRU-VNTRplus software, evaluat- ing the distance measurement according to the copy numbers of VNTRs, and an UPGMA dendrogram was built [22]. Clusters were determined based on a distance cut-off of 0 and similar patterns in 15 loci. A categorical coeffi cient of 1 and a distance cut-off of ⬍ 0.3, which corresponds to a 7 locus difference, were used to defi ne related strains [23].

The 15-locus MIRU patterns were compared with the patterns from the MIRU-VNTRplus database to determine MTB strain lineages and relatedness.

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Results

Drug susceptibility patterns

Complete antibiotic susceptibility for all isolates was obtained. Briefl y, 106 isolates (87.6%) were pan-susceptible, 12 (9.9%) were monoresistant, and 3 (2.4%) were MDR (2 from Kermanshah and 1 from Tehran).

MIRU-VNTR genotyping

Using MIRU-VNTR, 66 different patterns were detected among the 121 isolates. They were distrib- uted in 20 clusters comprising 75 strains and 46 unique patterns (Table I ). A comparison of the 15-locus MIRU patterns obtained with the international MIRU-VNTRplus database (http://www.

miru-vntrplus.org) showed that none of the strains out of the 186 present in this database matched our profi les. The discriminatory power of MIRU-VNTR

typing for all isolates was high (HGDI ⫽ 0.972).

The largest cluster was composed of 13 isolates.

One cluster with 12 members, 3 clusters with 5 members, 1 cluster with 4 members, 3 clusters with 3 members, and 11 clusters with 2 members were also identifi ed. Five clusters belonged to Sistan- Baluchestan only. Similarly 3 and 2 clusters were found only in Kermanshah and Tehran, respectively.

Five clusters contained isolates from Tehran and Sistan-Baluchestan. Similarly, 5 clusters shared isolates from Sistan-Baluchestan and Kermanshah (Figure 1).

Conversely, isolates from Tehran and Kermanshah were not grouped together in any cluster. The resis- tant strains were not within the clusters and have unique patterns. Depending on the region of origin, high genetic diversity occurred among MTB isolates from Sistan-Baluchestan (HGDI ⫽ 0.978), followed by isolates from Kermanshah (HGDI ⫽ 0.933) and Tehran (HGDI ⫽ 0.907).

Table I. MIRU-VNTR fi ngerprinting results for 121 Mycobacterium tuberculosis isolates.

MIRU pattern ^a Frequency MIRU pattern ^a Frequency

4, 4, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 7, 2, 3, 1, 3, 3, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 7, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 2, 5, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 2, 3, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 2, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 7, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 2, 5, 8, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 9, 2, 7, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 2, 4, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 2, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 2, 4, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 5, 2, 2, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 2, 4, 1, 2, 2, 4, 3, 3, 2, 3, 3 4, 4, 3, 5, 2, 5, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 8, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 7, 2, 4, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 2, 4, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 2, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 2, 3, 5, 2, 3, 1, 2, 2, 2, 3, 3, 2, 3, 3 4, 2, 5, 3, 8, 4, 2, 2, 2, 2, 3, 3, 4, 3, 4 4, 2, 5, 3, 8, 4, 2, 2, 2, 2, 3, 3, 2, 3, 4 2, 2, 3, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 2, 4, 3, 7, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 2, 4, 3, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 5, 2, 4, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 5, 2, 5, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 2, 7, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 2, 7, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3

13 12 5 5 5 4 3 3 3 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3, 9, 2, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 7, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 2, 5, 2, 5, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 8, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 2, 3, 7, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 2, 5, 2, 5, 1, 3, 2, 3, 3, 3, 3, 3, 3 3, 4, 3, 5, 2, 2, 1, 3, 3, 3, 3, 3, 3, 3, 3 3, 4, 3, 7, 2, 3, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 2, 3, 5, 8, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 2, 3, 5, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 3, 5, 2, 2, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 4, 3, 7, 2, 4, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 4, 2, 5, 2, 3, 1, 3, 3, 3, 3, 3, 3, 3, 3 4, 4, 3, 7, 2, 2, 1, 3, 2, 3, 3, 3, 3, 3, 3 4, 4, 2, 5, 2, 3, 1, 3, 2, 3, 3, 3, 3, 3, 3 2, 2, 3, 4, 4, 4, 2, 4, 2, 4, 3, 3, 4, 3, 3 2, 4, 3, 4, 8, 2, 2, 2, 4, 2, 3, 3, 2, 3, 3 2, 4, 3, 4, 8, 4, 2, 2, 4, 2, 3, 3, 2, 3, 3 3, 4, 3, 4, 3, 4, 2, 2, 4, 2, 4, 3, 2, 3, 3 3, 2, 3, 4, 3, 4, 2, 2, 4, 2, 4, 3, 2, 3, 3 3, 4, 2, 3, 3, 5, 1, 4, 4, 4, 3, 3, 4, 4, 3 5, 2, 2, 4, 8, 2, 1, 2, 2, 1, 3, 3, 4, 4, 3 5, 2, 3, 3, 3, 5, 1, 2, 2, 4, 3, 4, 2, 3, 4 5, 2, 2, 4, 8, 2, 1, 2, 2, 4, 3, 3, 4, 3, 3 5, 2, 3, 3, 3, 5, 1, 2, 2, 4, 3, 4, 4, 3, 3 5, 2, 2, 7, 3, 5, 1, 2, 2, 4, 3, 3, 2, 4, 3 3, 4, 2, 3, 3, 5, 1, 2, 3, 4, 3, 2, 4, 3, 3 3, 2, 3, 3, 3, 5, 1, 4, 3, 1, 3, 2, 4, 2, 3 2, 2, 5, 4, 3, 4, 1, 4, 3, 4, 3, 2, 4, 2, 4 2, 9, 2, 4, 4, 5, 1, 2, 3, 1, 3, 2, 4, 2, 3 2, 2, 5, 4, 4, 5, 1, 2, 3, 4, 3, 2, 4, 2, 4 4, 4, 3, 5, 2, 4, 1, 2, 2, 2, 3, 3, 1, 3, 3

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

a MIRU pattern: ETRA, ETRC, MIRU04, ETRE, MIRU10, MIRU16, MIRU26, MIRU40, QUB11b, QUB26, MTUB30, MTUB39, MTUB04, MTUB21, QUB4156.

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4 S. Zamani et al.

Allele frequencies of the sample studied

Based on the discriminatory index, 2 MIRU loci were designated as highly discriminative ( h ⬎ 0.6) in our sample (MIRU 16 and ETRA), 8 were designated as moderately discriminative (0.3 ⱕ h ⱕ 0.6;

MIRU 4, 10, and 40, QUB11b, QUB26, MTUB04, ETRC, and ETRE), and the other MIRU loci (MIRU26, Mtub21, 30, and 39, and QUB4156) were found to be poorly discriminative ( h ⬍ 0.3).

Results of the distribution of the MIRU alleles are shown in Table II .

Phylogenetic analysis

The dendrogram generated using the UPGMA algo- rithm based on the MIRU-VNTR data describes the genetic relationships of the 121 isolates (Figure 1).

Also the distribution of different epidemiological parameters as a function of clustering analysis, char- acteristics such as age, sex, drug resistance, and region of origin, showed no association with strain clustering.

Discussion

MIRU-VNTR typing has become an important methodological achievement towards a better understanding of the molecular epidemiology of TB. In the present study, we used this method to explore the genetic diversity and transmission dynamics of MTB isolates from 3 provinces of Iran.

Genotypes for 121 isolates did not match any strain in the MIRU-VNTRplus database and were considered unrelated to reference strains. This may be due to the lack of suffi cient samples in our study. The method showed high discriminatory power for all isolates, giving an HGDI value of 0.972. This discriminatory power of 0.972 is similar to the discriminatory power reported in previous studies [23 – 27]. No similarity was found among the patterns obtained in this study and those previously reported from East Azarbaijan Province (north-western Iran) and Khuzestan Province (south- western Iran) [28,29]. This study demonstrates high genetic diversity among strains originating from Sistan-Baluchestan; the HGDI was 0.978, which is very high and comparable to that of other surveys [28 – 31]. The high genetic diversity of MTB strains in the Sistan-Baluchestan commu- nity could be a consequence of bordering high TB burden countries, the immigration of Afghans, and reactivation of latent infection. Also, all isolates from Tehran or Kermanshah that grouped into clusters shared identical patterns with isolates from Sistan-Baluchestan. This shows the possibility of

Figure 1. Genetic relationships of the 121 Mycobacterium tuberculosis isolates. Clustered isolates from T: Tehran, K:

Kermanshah, and S: Sistan-Baluchestan. Clusters shared by S with other provinces have been shown as T – S and K – S.

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transmission between provinces. The allelic diversity of the VNTR loci differed considerably per locus. In our samples, loci MIRU 16 and ETRA were highly discriminative, similar to reported loci in previous studies [29,32 – 34]. The loci MIRU 4, 10, and 40, QUB11b, QUB26, MTUB04, ETRC, and ETRE were designated as moderately discriminative. Other studies have identifi ed loci MIRU 4, 10, and 40 and ETRE concordant with our study [25,28,29,31].

Several studies have identifi ed multiple factors associated with TB, including young age and drug resistance [35,36]. However, no association was found between clustering and epidemiological data in our analysis. The rate of pan-susceptible isolates was 106 (87.6%). Since we examined strains from non-selected patients, this rate is higher than that reported in previous studies that used strains from selected patients with a high rate of MDR at TB centres [37,38].

In conclusion, 15-locus MIRU-VNTR genotyping is an easy, reliable, and reproducible method and has potential power for genetic analysis and tracking epidemiological events such as transmission dynamics. This is the fi rst study to reveal the high diversity of MTB strains in different parts of Iran, especially in the province of Sistan-Baluchestan.

It also suggests the possibility of strain transmission from this region to other provinces. Further studies to determine the full spectrum of circulating MTB strains in Iran will lead to a better understanding of the epidemiology of TB.

Acknowledgements

This work was supported by Tehran University of Medical Sciences (Project No. 15356).

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon- sible for the content and writing of the paper.

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