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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 1  |  Page : 1-8

High-resolution genotyping of Mycobacterium tuberculosis isolates from syria using mycobacterial interspersed repetitive unit-variable-number tandem repeat


1 Department of Radiation Medicine, Biomedical Section, Atomic Energy Commission, Damascus, Syria
2 Department of Public Health Laboratories, Reference TB Laboratory, Ibn Al-Nafeess Hospital, Ministry of Health, Damascus, Syria

Date of Submission10-Nov-2018
Date of Decision18-Dec-2018
Date of Acceptance26-Dec-2018
Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Maya Habous
Department of Public Health Laboratories, Reference TB Laboratory, Ibn Al-Nafeess Hospital, Ministry of Health, Damascus
Syria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_139_18

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  Abstract 


Background: We aimed to evaluate the utility of 24 loci mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) genotyping method for discrimination of clinical Mycobacterium tuberculosis isolates in Syria. Methods: We studied 68 clinical tuberculosis (TB) isolates originating from unrelated Syrian TB patients from different regions in Syria. Genetic types (consisting of 24 digits) were determined and used to construct a dendrogram. Results: Fifty-six distinct MIRU patterns were revealed, from which 52 patterns were represented by unique isolates. Sixteen isolates were distributed into 4 clusters, 3 of which consisted of isolates belonging to the TUR lineage. Nine MIRUs showed high Hunter-Gaston index (HGI) values (>0.6), with QUB-26 having the highest discriminatory power (HGI = 0.821), followed by MIRU10, MIRU26, MIRU16, and Mtub39. The cumulative HGI value of the 24-MIRU set was 0.985. Interestingly, using the reduced 15-MIRU, set resulted in the same HGI. The TUR lineage was the most frequent in our sample (23.5%), and it appears that it is widespread in Syria as in Turkey. The discriminatory power of MIRU-VNTR among the subset belonging to the TUR lineage was extremely low due to the high clustering rate (62.5%) of TUR isolates, indicating that this method is inappropriate to discriminate isolates of this lineage. On the other hand, the Beijing lineage was not represented in our isolates. Conclusion: We demonstrated the high prevalence of the TUR lineage and the low prevalence of the Beijing lineage among Syrian clinical TB isolates. The MIRU-VNTR method was highly discriminative among non-TUR TB isolates, but it was inappropriate to discriminate isolates of the TUR lineage.

Keywords: High-resolution genotyping, Hunter-Gaston index, mycobacterial interspersed repetitive unit-variable-number tandem repeat, Mycobacterium tuberculosis


How to cite this article:
Zarzour H, Madania A, Ghoury I, Habous M. High-resolution genotyping of Mycobacterium tuberculosis isolates from syria using mycobacterial interspersed repetitive unit-variable-number tandem repeat. Biomed Biotechnol Res J 2019;3:1-8

How to cite this URL:
Zarzour H, Madania A, Ghoury I, Habous M. High-resolution genotyping of Mycobacterium tuberculosis isolates from syria using mycobacterial interspersed repetitive unit-variable-number tandem repeat. Biomed Biotechnol Res J [serial online] 2019 [cited 2019 Mar 25];3:1-8. Available from: http://www.bmbtrj.org/text.asp?2019/3/1/1/254094




  Introduction Top


Tuberculosis (TB) remains one of the most clinically significant infectious diseases worldwide, killing about 2 million people each year and infecting >8 million.[1] In Syria, according to official statistics in 2009, TB is still a public health threat, with about 4000 new TB cases per year. This burden could be higher at the present time due to deterioration in hygiene conditions and health care (especially among refugees) in many parts of the country affected by war activities. Accurate identification and monitoring of Mycobacterium tuberculosis (MTB) strains are required to analyze the transmission of the infection. Various genetic approaches have been applied for the discrimination of MTB isolates, including the standard IS6110-restriction fragment length polymorphism (RFLP) and direct repeat spoligotyping.[2],[3],[4] These methods have their inherent particular disadvantages: IS6110 fingerprinting is laborious, time-consuming, requires large amounts of genomic DNA (gDNA) whereas spoligotyping has a relatively low discrimination power. Furthermore, IS6110 fingerprinting has a low discrimination power among MTB strains harboring a low copy number (fewer than five) of the IS6110 element.

A new polymerase chain reaction (PCR)-based discrimination method relies on the amplification of several independent polymorphic loci of the mycobacterial genome. These loci contain variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) that are located mainly in intergenic regions scattered throughout the MTB genome.[4],[5],[6] This method enumerates the number of tandem repeats at each locus by PCR amplification using primers targeting flanking regions and determining the length of the resultant fragment. Each isolate is typed according to the number of copies of repeated units, yielding a numerical code for each type, where each digit represents the number of repeats at the corresponding locus.[7] Originally, a set of 12 MIRU loci was evaluated for genotyping MTB.[5],[6] To increase the discriminatory power of this method, the number of tested loci was increased to 15 or 24 MIRUs.[8],[9]

Since there is currently no information about MIRU genotypes among MTB in Syria, our aim was to evaluate the discriminatory power of a panel of 24 MIRUs for high-resolution genotyping of MTB isolated from TB patients and to determine which TB lineages are present in Syria.


  Experimental Top


Methods

Clinical isolates and bacterial culture

A total of 68 MTB bacterial isolates were randomly selected from 160 isolates used in a previous study,[10] which were clinical isolates originally cultured from sputum specimens of unrelated pulmonary Syrian TB patients referred to our laboratory. These patients came to us from different regions of Syria for TB diagnosis and drug resistance assessment. The majority of patients came from Damascus (15) and its countryside (10), Aleppo (13, North), and Al-Hasakah (9, North-East). The remaining patients came from various regions, including Latakiah (5, West), Al-Raqqa (5, North), Homs (3, Midland), Deir ez-Zor (3, East), Idleb (2, North), Hama (1, Midland), and Daraa (1, South). The article was approved by the review board of the Atomic Energy Commission of Syria (AECS). Approval number: HTZS - TNBE567.

MTB bacterial strains were cultured following conventional methods.[11] Briefly, sputum specimens were decontaminated with 4% NaOH and cultured on Lőwenstein solid medium by incubation at 37°C for 3–10 weeks, until MTB colonies appeared on the medium surface. Resistance to RIF and INH was tested by culturing bacterial suspensions (0.5 McFarland) on Lőwenstein solid medium containing rifampicin (40 μg/ml) or isoniazid (0.2 μg/ml). An MTB isolate was considered as drug resistant only when the number of colonies emerging on drug-containing medium was at least 1% of the number of colonies emerging on drug-free medium.[11]

Isolation of genomic DNA from Mycobacterium tuberculosis colonies

gDNA was isolated from bacilli following standard phenol/chloroform extraction. Briefly, a 1 mm bacterial colony was resuspended in 500 μl H2O and cells were lysed by adding 50 μl 10% sodium dodecyl sulfate and 15 μl proteinase K (10 mg/ml) and incubating for 1 h at 50°C under shaking. gDNA was extracted once with phenol and 3 times with chloroform and precipitated with ethanol, washed with 75% ethanol, air dried, and dissolved in 200 μl water. gDNA yields varied between 10 and 100 ng/μl.

Variable number tandem repeat polymorphism analysis

MIRU genotyping of each of the 68MTB isolates was performed by monoplex PCR amplification for each of the 24 loci of VNTRs previously identified using primers as previously described.[9]

Each PCR reaction (25 ml final volume) contained following components (final concentrations): buffer (1×), MgCl2 (4 mM), dNTPs (200 mM each), DMSO (5%), primer pair (300 nM each), Taq polymerase (1 unit GoTaq, Promega), and gDNA (30-100 ng). PCR reactions were run in an Eppendorf thermocycler with the following cycling conditions: initial denaturation 95°C for 3 min, then 40 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 120 s (final elongation cycle at 72°C for 5 min). Electrophoresis was performed on a 2% agarose gel containing ethidium bromide for 90 min at 100 V along with 50 bp or 100 pb DNA molecular size ladder. The length of each PCR product was determined, and the number of repeats at each MIRU was calculated according to repeat lengths listed in [Table 1].
Table 1: Primers used in this study

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Phylogenetic analysis

The twenty-four digits MIRU-VNTR allele score obtained for each MTB strain were entered into VNTR-plus software as a character set and used to generate a dendrogram by unweighted pair group using arithmetic averages. To assign our MTB isolates to principal MTBC lineages, we entered our 24-digit MIRU codes into the VNTR-plus database, and a best match analysis against the 186 reference strains of the representative collection was performed. A distance cutoff of ≤0.3 (corresponding to 7 locus difference out of 24 loci) was considered as significant for lineage assignment.

Statistical analysis

Hunter-Gaston index (HGI) was calculated as described previously[12] and was used to evaluate the discriminatory power of the MIRU-VNTR typing for different loci. Mean HGI was calculated as a mean value of HGI values of the 24 loci.

This study was approved by the Ethics Committee of the Ministry of Health and was performed in accordance with all national regulations.


  Results Top


Mycobacterial interspersed repetitive unit genotyping analyses of total isolates

A total of 68 MTB isolates were analyzed by PCR to determine repeat numbers at each of the 24 tested MIRUs. Some MIRUs showed a low or medium degree of diversity while others, as Mtub04, showed a high degree of diversity [Figure 1]. The numbers of repeats at each MIRU were calculated, and a 24-digit number was assigned for each isolate [Table 2]. Based on these data, a dendrogram describing the genetic relatedness of the 68 isolates under study was established [Figure 2]. The 68 isolates were subdivided into 56 distinct patterns, where 52 patterns were represented by unique isolates. Sixteen isolates were distributed into 4 clusters, 3 of which consisted of isolates belonging to the TUR lineage whereas the remaining cluster consisted of 3 isolates of unknown lineage. The clustering rate was 17.6%. For most isolates (44/68, 64.7%), no principal MTBC lineage could be assigned because the distance of each of these isolates from the nearest reference strain was greater than the 0.3 cutoff value. Furthermore, no correlation between MIRU patterns and resistance to either rifampicin or isoniazid were found.
Figure 1: Example of mycobacterial interspersed repetitive unit-variable-number tandem repeat polymorphism analysis by polymerase chain reaction and agarose gel electrophoresis. Mtub04 was analyzed in 13 different tuberculosis isolates (C38–C50), and the number of repeats for each isolate was inferred from amplicon length

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Table 2: Mycobacterial interspersed repetitive unit genotypes for 68 tuberculosis isolates analyzed in this study

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Figure 2: Dendrogram constructed based on mycobacterial interspersed repetitive unit-variable-number tandem repeat genotypes using the unweighted pair group using arithmetic averages method (MIRU-VNTRplus. http://www:miru-vntrplus.org)

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Discriminatory power of mycobacterial interspersed repetitive unit-variable-number tandem repeat typing

HGI values were calculated for each of the 24 MIRU-VNTR loci to estimate their respective power to discriminate between different isolates [Table 3]. HGI values >0.6 are considered highly discriminative while HGI values <0.3 are considered poorly discriminative.[12] Nine MIRUs showed HGI values >0.6, with QUB-26 having the highest discriminatory power (HGI = 0.821), followed by MIRU10 (HGI = 0.758), MIRU26 (HGI = 0.751), MIRU16 (HGI = 0.724), and Mtub39 (HGI = 0.716). On the other hand, nine MIRUs showed HGI values <0.3, with MIRU24 having the lowest discriminatory power (no variability, HGI = 0.000), followed by MIRU20 and Mtub29 (HGI = 0.029). The calculated cumulative HGI of the 24-MIRU set was 0.985 (recommended minimal cumulative value >0.950).[12]
Table 3: Hunter-Gaston index values calculated for 24 mycobacterial interspersed repetitive unit analyzed in this study (68 tuberculosis isolates)

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  Discussion Top


This is the first study describing MIRU-VNTR polymorphism among MTB isolated from TB patients in Syria. Our patients came from different regions in Syria, with about half of them (56%) from Damascus and Aleppo, the two Governorates encompassing together about 60% of the Syrian population. Interestingly, 20% (14/68) of all patients came from Al-Hasakah and Al-Raqqa Governorates, reflecting a higher TB incidence in northern Syria.

The MIRU-VNTR method has been developed by evaluating the usefulness of diverse MIRUs for discriminating MTB isolates and by assessing the discriminatory power of different sets of MIRUs on representative strain collections of the main MTB lineages (12, 15, or 24 loci panels).[9],[13],[14] This method proved to have several advantages: it is easy and fast, does not require complicated apparatus nor expensive software for data analysis, and it offers the ability to compare results easily between laboratories.

In this study, we have evaluated the 24 MIRUs (the 24 loci panel) on a TB collection isolated from Syrian patients. The HGI values of MIRUs belonging to the 15-set [Table 4], upper 15 rows] were close to HGI values calculated for the worldwide extended TB collection,[9] with the exception of MIRU 31 (0.306 in Syria vs. 0.72 worldwide). MIRU 31 also showed diminished HGI value in other.[15] On the other hand, HGI values of the remaining nine MIRUs were generally low, ranging from 0.000 for MIRU 24 to 0.358 for MIRU 23. The HGI values of these MIRUs were also found to be low in previous studies [Table 4], lower 9 rows].
Table 4: Allelic diversity of 24 VNTR loci in M. tuberculosis strains from Syria and other locations

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Our calculated cumulative HGI for the 24-MIRU set was 0.985. Interestingly, the HGI was exactly the same when calculated for the 15-MIRU set. This is a consequence of the fact that dendrograms based either on the 24-MIRU or 15-MIRU sets were identical. Therefore, using the 24-MIRU set did not improve discriminatory power when compared to the 15-MIRU set in our collection of MTB isolates. Our HGI (0.985) was high and comparable to values calculated for TB collections from different countries: 0.950 in Venezuela,[16] 0.987 in Tibet,[17] 0.9735 in India,[18] 0.992 in Iraq,[8] and 0.997 in Bulgaria,[15] indicating that the MIRU-VNTR method is highly valuable for genotyping MTB isolates in Syria.

Using the MIRU-VNTRplus for the identification by similarity search, only 23 (33.8%) isolates could be assigned to a specific TB lineage, the majority of them belonged to the TUR lineage, which also clustered together in the dendrogram [Figure 2]. It is important to note that the 16 isolates of the TUR lineage came from different, geographically distant governorates [Table 2]. This indicates that the TUR lineage is widespread in Syria, as in neighboring Turkey,[19] and it is unlikely that our TUR isolates originated from the same transmission chain. Furthermore, in agreement with a previous study,[20] no isolates belonging to the Beijing lineage were found in our collection, indicating a low prevalence of this lineage in Syria.

Although the cumulative HGI value of the MIRU-NVTR method was high (0.985) when calculated for all 68 Syrian TB isolates, and very high among the subset belonging to non-TUR isolates (HGI = 0.997), the discriminatory power of this method was extremely weak when calculated for the subset belonging to the TUR lineage (HGI = 0.73). This is due to the high clustering rate (62.5%) of TUR isolates. It is obvious that the MIRU-NVTR method is inappropriate to discriminate isolates of the TUR lineage and an alternative method should be used for that purpose, such as IS6110-RFLP.


  Conclusion Top


This study demonstrated the high discriminatory power of the MIRU-VNTR method among clinical MTB of isolates from Syria and validated the use of the reduced 15-MIRU set for epidemiological studies in this country. Furthermore, our results indicate a high prevalence of the TUR lineage and a low prevalence of the Beijing lineage of MTB in Syria.

Acknowledgments

We thank Dr. Ibrahim Othman (General Director of the AECS), Dr. Mohammad M. Al-Nemeh (head of Department of Radiation Medicine in the AECS), for their kind support to complete this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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