ORIGINAL ARTICLE

Genotyping and drug susceptibility testing of Mycobacterium tuberculosis in

Iran: a multi-centre study

S. M. J. Mousavi1, S. Amini2, M. Mirsaeidi3, H. Dabiri1, G. Hamzehloo2, H. Goudarzi1 and M. J. Nasiri1

1) Department of Microbiology, Shahid Beheshti University of Medical Sciences, 2) Regional Tuberculosis Reference Laboratory, Tehran University of Medical
Science, Tehran, Iran and 3) Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Miami, Miller School of Medicine,
Miami, FL, USA

Abstract

Tuberculosis (TB) is a deadly infection and caused 1.4 million deaths in 2018. Assessing the geographic distribution of major lineages of
Mycobacterium tuberculosis can contribute greatly to TB control. Mycobacterial interspersed repetitive unit variable number tandem repeat
(MIRU-VNTR) typing is commonly used to differentiate various lineages of M. tuberculosis. A total of 2747 clinical specimens were
collected consecutively from October 2018 through June 2019. Clinical isolates were identified as M. tuberculosis using standard
biochemical tests. The standard 15-locus MIRU-VNTR typing was used for the genotyping of clinical isolates. Drug susceptibility testing
was performed using the conventional proportion method. From the collected specimens, 100 were culture positive for M. tuberculosis.
Using MIRU-VNTR, 99 different patterns were detected among the 100 isolates. They were distributed in one cluster comprising two
strains and 98 unique patterns. Most of our isolates were similar to New-1 and Delhi/CAS strains. Of the M. tuberculosis isolates, 83
(83.0%) were pan-susceptible and 17 (17.0%) were resistant to at least one drug. Our study showed that MIRU-VNTR is a useful method
for studying the genetic diversity of M. tuberculosis isolates in different regional settings and will help the health authorities to construct a
preventive programme for TB.
© 2020 The Author(s). Published by Elsevier Ltd.

Keywords: Iran, MIRU-VNTR, mycobacterial interspersed repetitive unit variable number tandem repeat, tuberculosis, typing
Original Submission: 2 February 2020; Revised Submission: 24 February 2020; Accepted: 9 July 2020
Article published online: 13 July 2020

Iraq, Turkmenistan, Pakistan and Afghanistan, showed higher

Corresponding author: M.J. Nasiri, Department of Microbiology,
TB incidence rates than Iran [1]. An important threat of TB is
School of Medicine, Shahid Beheshti University of Medical Sciences,
Tehran, Iran. the emergence of drug-resistant strains, especially multidrug-
E-mail: [email protected] resistant TB [2,3].
Genotyping methods have increasingly been applied to
evaluate the epidemiology and transmission dynamics of Myco-
bacterium tuberculosis [4–7]. In addition, these methods could
Introduction be useful in long-term supervision, outbreak studies and tracing
of laboratory contaminations [8–11]. These techniques can also
distinguish between new and old M. tuberculosis infections and
Tuberculosis (TB) is the major cause of morbidity and mortality can detect the source of infection [12,13].
worldwide, especially in developing countries. In 2018, the There are various types of methods for genotyping studies
death rates from TB among human immunodeficiency virus- including IS6110-restriction fragment length polymorphism,
negative and -positive individuals were 1.2 million and spoligotyping and single nucleotide polymorphisms [14]. One of
251 000, respectively [1]. Based on the WHO report in 2018, the commonest genotyping techniques is mycobacterial inter-
the TB incidence rate in Iran was estimated at 14 per 100 000 spersed repetitive unit variable number tandem repeat (MIRU-
population [1]. All neighbouring countries, including Turkey, VNTR), which has been widely used for epidemiological studies

New Microbe and New Infect 2020; 37: 100729

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https://doi.org/10.1016/j.nmni.2020.100729
2 New Microbes and New Infections, Volume 37 Number C, September 2020 NMNI

[5,15]. Recent TB research and programmes around the world VNTR typing
have provided a large database of various M. tuberculosis isolates The DNA was extracted using a previously described method
using MIRU-VNTR typing [16]. In addition, as a result of its high [22]. Molecular typing was performed using the MIRU-VNTR
discriminatory power, reproducibility and affordable cost, typing method. The standard 15-locus MIRU-VNTR typing
MIRU-VNTR is used routinely for genotyping of M. tuberculosis method was performed as described by Supply et al. [23].
strains in many settings [17,18]. The aim of this study was to
analyse the geographical distribution and study the phylogenetic Analysis of VNTR allelic diversity and genetic
relationship of M. tuberculosis strains collected at the Tehran relationships
regional TB reference laboratory (TRTB-RL). All results were entered into Excel® spreadsheets and analysed
by MIRU-VNTRplus software [24]. The allelic diversity of each
VNTR locus was calculated by the Hunter–Gaston discrimi-
Materials and methods natory index, and genetic relationships among the isolates were
estimated using the categorical coefficient and UPGMA [24].
Setting and samples Cluster definition was based on a distance cut-off of 0 and
This cross-sectional study was performed at the TRTB-RL from similar patterns in 15-locus typing [25].
October 2018 through June 2019. TRTB-RL is well-equipped
with biosafety level III facilities for drug susceptibility testing. Database comparison
TRTB-RL acts as a regional centre for TB laboratories, and The 15-locus MIRU-VNTR patterns were compared with the
clinical samples from different provinces of Iran transfer their patterns from the MIRU-VNTRplus database to determine
samples to this laboratory for further identification and drug M. tuberculosis strain lineages and relatedness [26]. Distribution
susceptibility testing. A total of 2747 clinical specimens were of identified lineage was done by similarity search with refer-
consecutively collected from individuals with suspected TB who ence strains.
were referred to the TRTB-RL from different provinces of Iran.
These individuals had clinical signs and symptoms of TB and Results
were undergoing examination for possible active TB. The study
was approved by the ethics committee of Shahid Beheshti
University of Medical Sciences, Tehran, Iran Mycobacterial isolates
(IR.SBMU.MSP.REC.1398.285). During the study period, a total of 2747 specimens from sus-
pected TB cases were collected, of which 1734 (63.1%) were
Microscopy examination and identification of pulmonary (bronchoalveolar lavage fluid, tracheal, sputum) and
M. tuberculosis 1013 (36.9%) were extrapulmonary (urine, abscess, osteo-
Clinical specimens were processed by the standard sodium articular, biopsy, pericardium, cerebrospinal fluid, gastric lavage,
hydroxide method and smears were prepared by the blood and ascites). From the collected specimens, 100 were
Ziehl–Neelsen staining method [19]. After decontamination, culture positive for M. tuberculosis. More than 80.0% of the
specimens were inoculated to the Löwenstein–Jensen medium. isolates were obtained from newly individuals with diagnosed
For the identification of mycobacteria, the slope cultures were TB. All the isolates were defined as M. tuberculosis according to
incubated at 37°C and examined for growth once weekly up to growth rates, pigmentation properties of colonies, production
6 weeks. Bacterial isolates were identified as M. tuberculosis of niacin, nitrate reduction and catalase.
using standard biochemical tests (i.e. production of niacin, ni-
trate reduction, catalase) [19,20]. Drug susceptibility testing
Of 100 M. tuberculosis isolates, 83 (83.0%) were pan-susceptible
Drug susceptibility testing and 17 (17.0%) were resistant to at least one drug. The majority
Drug susceptibility testing for isoniazid, rifampicin and etham-
butol was performed using the conventional proportion TABLE 1. The drug-resistant patterns of the collected isolates
method on Löwenstein–Jensen medium [19]. For pyr-
No. of resistant
azinamide, a modified proportion method was used as Type of resistance isolates
described previously by Heifets et al. [21]. In this modified
Pan-susceptible 83
proportion method we used Middlebrook 7H10 agar-based Mono-resistance (isoniazid-rifampicin-ethambutol- 12 (8-1-3-0)
pyrazinamide)
medium (Becton Dickinson and Co., Cockeysville, MD, USA) Multidrug resistance 5
for preparing the growth conditions.
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NMNI Mousavi et al. Genotyping and drug susceptibility testing of M. tuberculosis in Iran 3

FIG. 1. Genetic relationships of isolates based on the UPGMA tree.

© 2020 The Author(s). Published by Elsevier Ltd, NMNI, 37, 100729

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4 New Microbes and New Infections, Volume 37 Number C, September 2020 NMNI

of isolates were isoniazid mono-resistant (8.0%) followed by comprising two strains and 98 unique patterns. A comparison
ethambutol mono-resistant (3.0%) and rifampicin mono- of the 15-locus MIRU patterns obtained with the international
resistant (1%). Multidrug-resistant TB was observed in five MIRU-VNTRplus database showed that most of our isolates
(5.0%) of the investigated isolates, respectively. The antibiotic- were similar to New-1 and Delhi/CAS strains. The discrimi-
resistance patterns of the collected isolates are presented in natory power of MIRU-VNTR typing for all isolates was high
Table 1. (Hunter–Gaston discriminatory index 0.98). The dendrogram
generated using the UPGMA algorithm based on the MIRU-
VNTR typing VNTRplus database describes the genetic relationships of the
Using MIRU-VNTR, 99 different patterns were detected among 100 isolates (Fig. 1). As shown in the minimum spanning tree
the 100 isolates. They were distributed in one cluster (Fig. 2), there were nine clonal complexes including 25 isolates.

FIG. 2. The minimum spanning tree showed the nine clonal complexes.
© 2020 The Author(s). Published by Elsevier Ltd, NMNI, 37, 100729
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NMNI Mousavi et al. Genotyping and drug susceptibility testing of M. tuberculosis in Iran 5

These 25 isolates had different lineages and most of the isolates Second, as MIRU-VNTR requires isolation of M. tuberculosis by
were from lineages NEW-1 (12 isolates) and Delhi/CAS (eight culture, some isolates were excluded from the study because of
isolates). contamination in the culture media. Third, as a result of the
limited demographic information, the epidemiological links be-
tween patients with the same and similar strains could not be
Discussion
determined.
In conclusion, our study showed that the genetic population
A major problem in TB control programmes is the emergence structure of the included isolates was heterogeneous at the
of drug-resistant TB. Genotyping methods are useful tools for TRTB-RL. It also indicated that MIRU-VNTR is a useful method
studying the genetic diversity of M. tuberculosis isolates and will for studying the genetic diversity of M. tuberculosis isolates in
help the health authorities to construct a preventive pro- different regional settings and can help the health authorities to
gramme for TB. construct a preventive programme for TB.
The results of this study showed that the genetic population
structure of the included isolates was heterogeneous, like other
Author contributions
low-incidence countries, and 99 different patterns were
detected among the 100 isolates [4]. Likewise, these results
were compatible with previous studies about the genetic pop- MJN and SA conceived and designed the study; SMJM and SA
ulation structure of M. tuberculosis in Iran [27–29]. performed the experiments; SMJM, MJN, GH and HG analysed
In our study 17 (17.0%) isolates were resistant to at least one the data; MJN, SMJM wrote the main manuscript text and MJN,
drug and most of the included patients were newly infected MM and HD revised the manuscript.
individuals. Most of the resistant isolates (7 out of 17) belonged
to NEW-1 lineage, which may be a result of a higher potential
Conflict of interest
of mutation or rapid dissemination of this lineage.
It was noteworthy that the incidence rate of TB in Iran was
moderate, whereas in neighbouring countries, including The authors declare no competing interests.
Pakistan, Afghanistan, Turkey, Iraq, Azerbaijan and Armenia,
the rates are two to three times higher than in Iran.
Multidrug-resistant TB rates are also higher in these countries
Acknowledgements
[19,29,30]. Therefore, the genotypic diversity of
M. tuberculosis in Iran might be a result of communication and This study was financially supported by the Research Depart-
trading with neighbouring countries. Tehran is the most ment of the School of Medicine, Shahid Beheshti University of
populous city in Iran and it is the preferred destination for Medical Sciences, Tehran, Iran (Grant number: 15519).
travellers and migrants from neighbouring countries, which
could be the cause of the genetic diversity of TB in this study
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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).