Abstract
Background and Objectives
Molecular epidemiology tools are widely used in determining epidemiology of tuberculosis. Spoligotyping is a molecular epidemiology method that is used for characterization and typing of Mycobacterium tuberculosis complex strains. The method is based on polymorphism of the chromosomal DR locus consisting of identical 36-bp DRs alternating with 35-41 unique spacers. The objective of this study was to investigate the prevalence of M. tuberculosis spoligotypes in different provinces of Iran.
Materials and Methods
M. tuberculosis strains were isolated from TB patients of Mycobacteriology Research center (MRC). DNA was extracted from patient’s clinical samples. PCR was performed by using of specific primers for DR region. The amplified DNA was hybridized to the spoligotyping Membrane. Hybridized DNA was detected with ECL detection kit and by exposing ECL Hyperfilm to the membrane. The obtained result was entered to a binary format and was analyzed using SpolDB4 database.
Results
Spoligotyping resulted in 136 different patterns. Out of 1242 M. tuberculosis strains, 1165 strains (93.8%) were classified into 59 clusters and the remaining strains (6.2 %) were singleton.
Conclusions
The results of present study showed that strains of CAS family were more prevalent than other strains in Iran. Other prevalent families were Haarlem, T and Beijing, respectively.
Keywords: Molecular epidemiology, Tuberculosis, Spoligotyping
INTRODUCTION
Tuberculosis (TB) is one of the most urgent health problems in the Middle Eastern countries. Iran, with around 70 million inhabitants, shares geographical borders with four countries with high TB incidence rate, i.e. Pakistan, Afghanistan, Turkmenistan and Iraq. According to the World Health Organization (WHO), the estimated incidence rate of tuberculosis within the country is 21 per 100,000 populations (1). Therefore control and prevention of TB in Iran is the main health concern of national TB program.
Genotyping of M. tuberculosis strains is important for TB control program, because it allows the detection of outbreaks, the tracing of transmission, to monitor species diversity and to identify secondary infections (2, 3).
Large scale genotyping of M. tuberculosis using IS6110 restriction fragment length polymorphism is labor-intensive, time consuming and the results are sometimes difficult to compare among laboratories (2, 4). Based on this knowledge we used an easier and more rapid method in order to differentiate M. tuberculosis strains. Spoligotyping is a PCR based method that permits genotyping of M. tuberculosis complex in a rapid, reliable and cost effective way. The method is based on hybridization of amplified DNA with spacer oligonucleotides. The DR region contains multiple short 36-bp direct repeats (DRs) interspersed with unique spacers, which are 35-41 bp in length (5). The DRs are extremely well conserved among M. tuberculosis complex strains, making spoligotyping a specific method for the genotyping of M. tuberculosis complex members (5). A total of 9 potential super families or clades of M. tuberculosis complex have been identified by spoligotyping method (M. africanum, Beijing, M. bovis, EAI, CAS, T group of families, Haarlem, X and LAM family) (6). In this study, we performed spoligotyping on M. tuberculosis complex strains collected from all over the country, which were isolated between 2010 and 2011, in order to provide preliminary insight into the population structure of M. tuberculosis circulating in the country as well as the distribution of MTB family strains in provinces of Iran.
MATERIALS AND METHODS
M. tuberculosis strains and DNA isolation
A total of 1242 M. tuberculosis strains collected from 24 different provinces of Iran (2010-2011). Clinical specimens were transferred to Mycobacteriology Research Center (MRC). Mycobacterial genomic DNA was extracted from patient’s samples using QIAamp® DNA mini kit (QIAGEN).
Spoligotyping
Spoligotyping was performed as previously described by Kamerbeek et al, (5). The DR region was amplified by PCR using primers DRa (5’-biotin -CCG AGA GGG GAC GGA AAC- 3’) and DRb (5’- GGT TTT GGG TCT GAC GAC-3’), 20-50ng of DNA and 0.5 U of Taq DNA polymerase (Cinnagen, Tehran, Iran). The PCR condition was: 35 cycles of 1 min at 95°C, 1 min at 55°C and 30 sec at 72°C. The first denaturation and final extension steps were held for 10 min. The amplified DNA was hybridized to 43 immobilized oligonucleotides derived from the spacer sequences of MTB H37Rv and M. bovis BCG P3 by reverse line blotting. Hybridized DNA was detected by enhanced chemiluminesence (ECL, Amersham, UK) and by exposing ECL-Hyper film (Amersham) to the membrane for 10 min.
Obtained results were entered in a binary format as excel spreadsheets (Microsoft) and compared with published data (7-10). The strains with spoligotype similar to any pattern of M. tuberculosis strain already found in the database were automatically labeled with an already defined ‘shared type’number. Any spoligotype exhibiting a profile not yet found anywhere in the SpolDB4 database was termed as orphan (not seen) strain.
RESULTS
Spoligotyping
Spoligotyping produced a total of 136 patterns for the 1242 strains. Fifty-nine patterns classified into clusters in the present study (the data are summarized in Table 1 and detailed in Table 2). Fifty nine clusters contained 1165 isolates, which amounted 93.8% of clustering rate (1165/1242).The remainers (n = 77) corresponded to singleton which were not classified into any clusters (Table 3).
Table 1.
Spoligotypes of the 5 most prevalent clades with a Shared Type number in SITVIT databade.
| M.tb Strain | Shared Type | No. of isolates (%) | Spoligo pattern (binary) | Spoligo pattern (octal) |
|---|---|---|---|---|
| H4 | 127 | 255 (20.5%) | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonoooonnnnnnn | 777777777720771 |
| CAS1_DELHI | 26 | 238 (19.1%) | nnnoooonnnnnnnnnnnnnnnoooooooooooonnnnnnnnn | 703777740003771 |
| T1 | 53 | 140 (11.2%) | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 777777777760771 |
| CAS1_DELHI | 25 | 126 (10.1%) | nnnoooonnnnnnnnnnnnnnnoooooooooooonnoonnnnn | 703777740003171 |
| Beijing | 1 | 101 (8.1%) | oooooooooooooooooooooooooooooooooonnnnnnnnn | 000000000003771 |
Table 2.
Spoligotype of other Clustered strains.
| MTB Strains | Shared Type | No. of isolates | Spoligo pattern (binary) | Spoligo pattern (octal) |
|---|---|---|---|---|
| CAS | 22 | 24 | nnnoooonnnnnnnnnnnnoooooooooooooooonnnnnnnn | 703777400001771 |
| CAS | 357 | 20 | nnnoooonnnnnnnnnnnnnnnoooooooooooooonnnnnnn | 703777740000771 |
| MANU 2 | 54 | 19 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoonnnnnnnnn | 777777777763771 |
| H4 | 656 | 17 | nonnnnnnnnnnnnnnnnonnnnnnnnnooonoooonnnnnnn | 577777377420771 |
| H3 | 294 | 15 | nonnnnnnnnnnnnnnnnnnnnnnnnnnnnonoooonnnnnnn | 577777777720771 |
| CAS1_KILI | 21 | 14 | nnnoooonnonnnnnnnnnoooooooooooooooonnnnnnnn | 703377400001771 |
| H4 | 262 | 12 | nnnnnnnoonnnnnnnnnnnnnnnnnnnooonoooonnnnnnn | 774777777420771 |
| Not seen | 10 | noonnnnnnnnnnnnnnnnnnnnnnnnnooonoooonnnnnnn | 477777777420771 | |
| H4 | 777 | 8 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnooonoooonnnnnnn | 777777777420771 |
| Not seen | 8 | nnnoooonnnnnnooooooooooonnnnnnnnoooonnnnnnn | 703740007760771 | |
| H3 | 1908 | 7 | nnnnnnnnnnnnnnnnnnnnnnnnnnooooonoooonnnnnnn | 777777776020771 |
| T1 | 284 | 7 | oooonnnnnnnoonnnnnnnnnnnnnnnnnnnoooonnnnnnn | 37637777760771 |
| CAS | 142 | 6 | nnnoooonnnnnnnnnnnnnnooooooooooooonnnnnnnnn | 703777700003771 |
| CAS | 485 | 6 | nnnoooonnnnnnnnnnnnooooooooooooooonnnnnnnnn | 703777400003771 |
| CAS1_DELHI | 381 | 6 | nnnoooonnnnnnnnnnnnnnnoooooooooooonnooonnnn | 703777740003071 |
| LAM9 | 42 | 6 | nnnnnnnnnnnnnnnnnnnnoooonnnnnnnnoooonnnnnnn | 777777607760771 |
| T1 | 272 | 6 | oooonnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 37777777760771 |
| T1 | 628 | 6 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnoo | 777777777760760 |
| U | 602 | 6 | nnnnnnnnnnnnnnnnnnnnnnnnoooooooooooonnnnnnn | 777777770000771 |
| BOVIS | 595 | 5 | nnonnnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnnooooo | 676777777777600 |
| LAM3 and S/convergent | 4 | 5 | oooooooooooooooooooooooonnnnnnnnoooonnnnnnn | 000000007760771 |
| U | 1188 | 5 | nnnnnnnnnnnnnnnnnnnnnnnnnnnooooooonnnnnnnnn | 777777777003771 |
| Not seen | 5 | nnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnoonnnnnnnnn | 757777777763771 | |
| Not seen | 5 | nnnnnnnnnnnnnnnnnnnnnnnnnnnooooooooonnnnnnn | 777777777000771 | |
| BOVIS | 482 | 4 | nnonnnnnonnnnnnonnnnnnnnnnnnnnnnnnnnnnooooo | 676773777777600 |
| CAS | 486 | 4 | nnnoooonnnnnnnnnnnnnnnooooooooooooooonnnnnn | 703777740000371 |
| T1 | 1166 | 4 | nnnnnnnnnonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 777377777760771 |
| Not seen | 4 | nonoonnnnnnnnnnnnnnnnnnnnnnnooonoooonnnnnnn | 517777777420771 | |
| CAS | 864 | 3 | nnnoooonnnnnnooooooooooooooooooooooonnnnnoo | 703740000000760 |
| CAS | 1089 | 3 | noooooonnnnnnnnnnnnnnnoooooooooooonnnnnnnnn | 403777740003771 |
| CAS | 1093 | 3 | nnnoooonnnnnnnnnnnnnoooooooooooooonnnnnnnnn | 703777600003771 |
| CAS | 1264 | 3 | nnnoooonnnnnnnnnnnnnnnooooooooooooooooooooo | 703777740000000 |
| MANU 2 | 1634 | 3 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonoonnnnnnnnn | 777777777723771 |
| T1 | 520 | 3 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooononnnnn | 777777777760571 |
| T1 | 1144 | 3 | nnnnnnnnnnnnnnnnnnnnooooooonnnnnoooonnnnnnn | 777777600760771 |
| U(CAS_ANCESTOR?) | 27 | 3 | nnnoooonnnnnnnnnnnnnnnoonnnnnnnnnoooonnnnnn | 703777747770371 |
| Not seen | 3 | nnnnnnnnnnnoooonoooooooooooooooooooonnooonn | 777604000000611 | |
| CAS | 599 | 2 | nnnoooonnnnnnnnnnnnooooooooooooooooonnnnnnn | 703777400000771 |
| CAS1_DELHI | 427 | 2 | nnnoooonnnnnooonnnnnnnoooooooooooonnnnnnnnn | 703707740003771 |
| CAS1_DELHI | 428 | 2 | nnnoooonnnnnnnnnnnnnnnoooooooooooonnonnnnnn | 703777740003371 |
| CAS1_DELHI | 1092 | 2 | nnnoooononnnnnnnnnnnnnoooooooooooonnnnnnnnn | 702777740003771 |
| CAS1_DELHI | 1314 | 2 | nnnoooonnonnnnnnnnnnnnoooooooooooonnoonnnnn | 703377740003171 |
| H4 | 361 | 2 | nonnnnnnnnnnonnnnnnnnnnnnnnnooonoooonnnnnnn | 577737777420771 |
| H4 | 597 | 2 | nonnnonnnnnnnnnnnnnnnnnnnnnnooonoooonnnnnnn | 567777777420771 |
| H4 | 764 | 2 | nnnnnnnnnnnnnonnnnnnnnnnnnnnnnonoooonnnnnnn | 777757777720771 |
| H4 | 1568 | 2 | nnnnnnnoonnnnnnnnnonnnnnnnnnooonoooonnnnnnn | 774777377420771 |
| MANU 2 | 1690 | 2 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoononnnnnnn | 777777777762771 |
| T1 | 1626 | 2 | nnnnnnnnnnnnnnnnnnnnnnnnnnonnnnnoooonnnnnnn | 777777776760771 |
| T3 | 37 | 2 | nnnnnnnnnnnnonnnnnnnnnnnnnnnnnnnoooonnnnnnn | 777737777760771 |
| U | 374 | 2 | nnnnnnnnnnnnnnnnnnnnnnononooooooooooooooooo | 777777752000000 |
| Not seen | 2 | nnnnnnnnnnnnonnnnnnnnnnnnnnooooooooonnnnnnn | 777604000000611 | |
| Not seen | 2 | nonnoooooonnnnnnnnnnnnnnnnnnooonoooonnnnnno | 517777777420771 | |
| Not seen | 2 | nonnoooooonnnnnnnnnnnnnnnnnnooonoooonnnnnoo | 517777777420771 | |
| Not seen | 2 | nnnnnnnnnnnnonnnnnnnnnnnnnnooooooooonnnnnnn | 777737777000771 |
Table 3.
Spoligotype of non-clustered strains.
| MTB Strains | Shared Type | No. of isolates | Spoligo pattern (binary) | Spoligo pattern (octal) |
|---|---|---|---|---|
| BOVIS | 694 | 1 | nnonnnnnonnnnooonnnnnnnnnnnnnnnnnnnnnnooooo | 676743777777600 |
| CAS | 1422 | 1 | nnnoooonnnnnnnnnnnoooooooooooooooooonnnnnnn | 703777000000771 |
| CAS | 1616 | 1 | ooooooonnnnnnnnnnnnnnnoooooooooooonnnnnnnnn | 003777740003771 |
| CAS1_DELHI | 289 | 1 | nnnoooonnnnnnnnnnnnnnnoooooooooooonnnonnnnn | 703777740003571 |
| CAS1_DELHI | 754 | 1 | nonoooonnnnnnnnnnnnnnnoooooooooooonnnnnnnnn | 503777740003771 |
| CAS2 | 288 | 1 | nnnooooooonnnnnnnnnnnnoooooooooooonnnnnnnnn | 700377740003771 |
| EAI5 | 236 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnoooononnnnnnnnn | 777777777413771 |
| EAI5orEAI3 | 8 | 1 | noooooooooooonnnnnnnnnnnnnnnoooononnnnnnnnn | 400037777413771 |
| EAI8_MDG | 109 | 1 | noonnnnnnnnnnnnnnnonnnnnnnnnoooononnnnnnnnn | 477777377413771 |
| H3 | 50 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonoooonnnnnnn | 777777777720771 |
| H3 | 511 | 1 | nnnnnnnnnnnnnnnnnnnnnoooooooooonoooonnnnnnn | 777777700020771 |
| H3-T3 | 36 | 1 | nnnnnnnnnnnnonnnnnnnnnnnnnnnnnonoooonnnnnnn | 777737777720771 |
| H4 | 35 | 1 | nnnnnnnnnnnnonnnnnnnnnnnnnnnooonoooonnnnnnn | 777737777420771 |
| LAM11-ZWE | 59 | 1 | nnnnnnnnnnnnnnnnnnnnoooonnoooonnoooonnnnnnn | 777777606060771 |
| LAM6 | 64 | 1 | nnnnnnnnnnnnnnnnnnnnoooonnnnonnnoooonnnnnnn | 777777607560771 |
| LAM9 | 492 | 1 | nnnnnnonnnnnnnnnnnnnoooonnnnnnnnoooonnnnnnn | 773777607760771 |
| LAM9 | 770 | 1 | nnnnnnnnnnnonnnnnnnnoooonnnnnnnnoooonnnnnnn | 777677607760771 |
| MANU 2 | 1192 | 1 | nnnnnnnnnnnnnnnnnnnnonnnnnnnnnnnoonnnnnnnnn | 777777677763771 |
| T1 | 7 | 1 | onnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 377777777760771 |
| T1 | 65 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonoonnnn | 777777777760471 |
| T1 | 102 | 1 | nnnnnnnnnnnnoooonnnnnnnnnnnnnnnnoooonnnnnnn | 777703777760771 |
| T1 | 131 | 1 | nnnnnnnnnnnnoonnnnnnnnnnnnnnnnnnoooonnnnnnn | 777717777760771 |
| T1 | 154 | 1 | nnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 757777777760771 |
| T1 | 205 | 1 | nnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 737777777760771 |
| T1 | 243 | 1 | nnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 737777777760771 |
| T1 | 291 | 1 | nnnnnnnnnnnnnnnnnnnnonnnnnnnnnnnoooonnnnnnn | 777777677760771 |
| T1 | 535 | 1 | nnnnnnnnnnnnnnnnnnnnnooonnnnnnnnoooonnnnnnn | 777777707760771 |
| T1 | 635 | 1 | oooooooooooooooooooooooonnnnonnnoooonnnnnnn | 000000007560771 |
| T1 | 801 | 1 | nnnnnnnoonnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 774777777760771 |
| T1 | 1073 | 1 | nnnnnnnnnnnoonnnnnnnnnnnnnnnnnnnoooonnnnnnn | 777637777760771 |
| T1 | 245 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnooooo | 777777777760600 |
| T1 | 411 | 1 | nooooonnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 407777777760771 |
| T1 | 926 | 1 | nnnnnnonnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 773777777760771 |
| T1 | 1566 | 1 | nnnnoonnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn | 747777777760771 |
| T1 | 1735 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnooonnonoooonnnnnnn | 777777774320771 |
| T1_rus2 | 280 | 1 | nnnnnnoooooooooooonnnnnnnnnnnnnnoooonnnnnnn | 770000777760771 |
| T1_RUS2 | 899 | 1 | nnonnnoooooooooooonnnnnnnnnnnnnnoooonnnnnnn | 670000777760771 |
| T1_RUS2 | 1173 | 1 | nnnnnnoooooooooooonnnnnnnnnnnnnnoooonnnonnn | 770000777760731 |
| T2 | 52 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnoooonnnonnn | 777777777760731 |
| T3 | 1655 | 1 | nnnnnnnnnnnnonoonnnnnnnnnnnnnnnnoooonnnnnnn | 777723777760771 |
| U | 172 | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnooooonnnnnnn | 777777777740771 |
| X1 | 708 | 1 | nnnonnnnnnnnnnnnnonnnnnnnnnnnnnnoooonnnnnnn | 737776777760771 |
| X2 | 302 | 1 | nnnnnnnnnnnnnonnnonnnnnnnnnnnnnnoooonnnnnnn | 777756777760771 |
| Not seen | 1 | nononnnnnooooonnnnnnnnnnnnnnnnnnoooonnnnnnn | 537017777760771 | |
| Not seen | 1 | nonnnnnnooooonnnnnnnnnnnnnnnooonoooonnnnnnn | 576037777420771 | |
| Not seen | 1 | nnnoooonnnnnnnnnnnnnnnnnnnnonnnnoonnnnnnnnn | 703777777363771 | |
| Not seen | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnoooooooooooonnn | 777777777400031 | |
| Not seen | 1 | ooooooooooonnnnnnnnnnnnnnnnnooonoooonnnnnnn | 000177777420771 | |
| Not seen | 1 | nnnnnnnnnonnnnnnnnnnnnnnnnnnnnnnoonnnnnnnnn | 777377777763771 | |
| Not seen | 1 | nnnoooonnnnnnnnnnnnnnnnnnnooononoonnnnnnnnn | 703777776123771 | |
| Not seen | 1 | nnnnnnnnnonononnnnnnnnnnnnnnnoonoooonnnnnnn | 777257777620771 | |
| Not seen | 1 | nnnnnoonnoonnnnoooooonnnnnnnnnnnooooonnnnnn | 763170077760371 | |
| Not seen | 1 | nnnoooonnnnnnnnnnnoooooooooooooooooonnnnnnn | 703777000000771 | |
| Not seen | 1 | nnnoooonnonnnnooooooooooooooooooooooonnnnnn | 703360000000371 | |
| Not seen | 1 | nonnnnnnnonnnonnnnnnnnnnnnnnooonoooononnnnn | 577357777420571 | |
| Not seen | 1 | nnnnnnnnnnnnnoonnnnnnnnnnoonnnnnnnnnnnonnon | 777747774777661 | |
| Not seen | 1 | nnnnnnnnnoooononnnnnnnnnnnooooononnnnnnnnnn | 777027776027771 | |
| Not seen | 1 | nnnnnnnnnnnoonnnnnnnoooonnnnnnnnoooonnnnnnn | 777637607760771 | |
| Not seen | 1 | nnnnnnnnnnnnnnnnnnonnnnnnnnnnnooooonnnnnnnn | 777777377701771 | |
| Not seen | 1 | nnnoooonnnnnnnnnnnnnnoooooooooooooonnnnnnnn | 703777700001771 | |
| Not seen | 1 | nnnoooonnonnnnnnnnnnnooooooooooooooonnnnnnn | 703377700000771 | |
| Not seen | 1 | nnnoooonnnnnnooooooooooooooooooooooonnnnnnn | 703740000000771 | |
| Not seen | 1 | nnnnonnnnonnonnnnnnnnnnnnnnnooonoooonnnnnnn | 757337777420771 | |
| Not seen | 1 | nnnoooonnnnnnnnnnooonnoooooooooooonnnnnnnnn | 703776140003771 | |
| Not seen | 1 | nnnoooonnnnnnooonnnnnnoooooooooooooonnnnnon | 703743740000761 | |
| Not seen | 1 | nnnnonnnoonnonnnnnnnnnnnnnnnooonoooonnnnnnn | 756337777420771 | |
| Not seen | 1 | nnnnnnnnnonnnonnnonnnonnnnnnnnnnoooonoonnnn | 777356737760471 | |
| Not seen | 1 | nnnnnnnnnnnnnnnnnnnnnnnnnnnnooonoonnnnnnnnn | 777777777423771 | |
| Not seen | 1 | nnnooooononoooonnnonnnnnnnnnnnnnoooonnnnnnn | 701207377760771 | |
| Not seen | 1 | nnnnnnonnonnnoonnnnnnnnnnnonnnnnooonononnnn | 773347776761271 | |
| Not seen | 1 | nnnnnnnnnnnnnoooooooooooonnnnnnnoooonnnnnnn | 777740003760771 | |
| Not seen | 1 | nonnnnnooonnnnnnnnnnnnnoonnnooonoooonnnnnnn | 574377763420771 | |
| Not seen | 1 | oooonnnnnnnoonnnnnnnnnnnnnnnnnnnoooonnnonnn | 037637777760731 | |
| Not seen | 1 | oonoooonnnnnnnnnnnnnnnoooooooooooonnnnnnnnn | 103777740003771 | |
| Not seen | 1 | noonnnnnnnnnnnnnnnnnnnnnnnnooooonoooooonnnn | 477777777010071 | |
| Not seen | 1 | nonnnnnnnnnnnnnnnnnnnnnnnnnnooonoooonnnnnoo | 577777777420760 | |
| Not seen | 1 | nnnnnnnnoonnooonnnnnnnoooooooooooonnoonnnnn | 776307740003171 |
Among the 1242 typed isolates, 1165 (93.8%) were classified as shared international types (SITs) according to SITVIT database. The remaining 77 isolates generated 44 new spoligotypes (orphan- not seen) that had not been previously described in the database. Among the 59 clusters, we found 46 minor spoligotypes (including 2 to 9 isolates) and 13 major spoligotypes (> 10 isolates). Isolates ST127 (20.5%; Haarlem family), ST26 (19.1%; CAS family), ST53 (11.2%; T family), ST25 (10.1%; CAS family), and ST1(8.1%; Beijing family) represent almost 70% of the total number of isolates in this study.
The spoligotyping analysis identified the strains in the familes of CAS (n = 471, 37.9%), Haarlem (n = 326, 26.2%), T (n = 195, 15.7%) and Beijing (n = 101, 8.1%). Other spoligotypes belongs to Manu (n = 25, 2%), LAM (n = 13, 1%), U (n = 17, 1.3%), EAI (n = 3, 0.2%), Bovis (n = 10, 0.8%), X (n = 2, 0.16%) and the remianing (n = 77, 6.1%) were orphan.
High spoligotype diversity was documented for CAS, Haarlem and T lineages. Although LAM family was not frequent in this study, a high diversity was also evidenced for this lineage (6 sublineages). Furthermore, 6 M. bovis strains and 4 M. bovis BCG strains were found in this study which classified into 3 clusters.
Geographical distribution of Spoligotypes in Iran
The geographical distribution of M. tuberculosis spoligotypes is shown in Table 4. The most prevalent families were CAS (37.9%) followed by Haarlem (26.2%), T (15.7%), Beijing (8.1%). CAS family strains were predominant in 15 provinces (Khouzestan, Esfahan, Fars, Qazvin, Gilan, Golestan, Hamedan, Hormozgan, Boushehr, Kerman, Kermanshah, Markazi, Tehran, Yazd, Lorestan); strains of Haarlem family were predominant in Qom, Semnan, Kordestan and eastern border provinces, i.e. Khorasan and Sistan -Balouchestan.
Table 4.
geographical distribution of Mycobacterium tuberculosis spoligotypes in Iran.
| Khouzestan | East and West Azarbaijian/Ardebil | Esfahan | Fars | Qazvin | Gilan | Golestan | Hamedan | Hormozgan /Boushehr | Kerman | Kermanshah | Khorasan | Kordestan | Markazi | Mazandaran | Qom | Semnan | Tehran | Yazd | Sistan- Balouchestan | Lorestan | Total | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| BEIJING | 5 | 7 | 6 | 5 | 6 | 1 | 5 | 1 | 12 | 3 | 1 | 10 | 1 | 35 | 3 | 101 | 8.1% | ||||||
| LAM | 7 | 2 | 1 | 1 | 1 | 1 | 2 | 15 | 1.2% | ||||||||||||||
| BOVIS | 1 | 2 | 1 | 2 | 4 | 10 | 0.8% | ||||||||||||||||
| CAS | 67 | 16 | 17 | 16 | 6 | 18 | 23 | 7 | 19 | 10 | 9 | 34 | 4 | 9 | 4 | 18 | 7 | 107 | 6 | 59 | 15 | 471 | 37.9% |
| EAI | 2 | 1 | 3 | 0.2% | |||||||||||||||||||
| HAARLEM | 7 | 10 | 2 | 14 | 2 | 13 | 8 | 5 | 8 | 8 | 2 | 42 | 9 | 3 | 6 | 20 | 8 | 85 | 2 | 65 | 7 | 326 | 26.2% |
| MANU | 2 | 1 | 1 | 1 | 1 | 3 | 14 | 2 | 25 | 2% | |||||||||||||
| T | 13 | 29 | 12 | 1 | 2 | 8 | 3 | 4 | 2 | 4 | 19 | 3 | 13 | 8 | 5 | 47 | 4 | 16 | 2 | 195 | 15.7% | ||
| U | 6 | 1 | 2 | 6 | 2 | 17 | 1.3% | ||||||||||||||||
| X | 1 | 1 | 2 | 0.16% | |||||||||||||||||||
| UNKNOWN | 9 | 3 | 3 | 5 | 2 | 4 | 3 | 4 | 1 | 3 | 23 | 17 | 77 | 6.1% | |||||||||
| TOTAL | 119 | 65 | 42 | 40 | 10 | 39 | 47 | 21 | 38 | 24 | 16 | 117 | 16 | 15 | 26 | 61 | 21 | 324 | 12 | 165 | 24 | 1242 |
Strains of T family were predominant in Mazan daran, East and West Azerbaijan and Ardebil provinces (North western provinces). Distribution of Beijing strains was higher inTehran, Khorasan and Qom provinces.
DISCUSSION
This study aimed to assess the genetic diversity of M. tuberculosis strains collected from 24 provinces of Iran using the spoligotyping method. Although these strains were not representative of all strains presented in Iran, they provided an insight into the population structure of M. tuberculosis spoligotypes in the country.
Previously, the M. tuberculosis isolates were classified into 3 distinct genetic groups by Sreevatsan et al (11): Group I or ancient MTB genotype (CAS, Beijing, EAI) and Group II and III (Haarlem, T, LAM, U, X) which called Modern MTB genotypes. In a similar study, M. tuberculosis isolates belonged to genetic groups II and III failed to hybridize with spacers 33 to 36, suggesting that these spacers and DRs have become deleted from the genome of all these groups (12). In the present study, the prevalence of ancient TB was 47.9% and the prevalence of modern TB was 45.3%. Another study by Merza et al, reported that the prevalence of ancient and modern TB in Iran was almost equal, whereas in Pakistan and Afghanistan, the majority of strains belonged to ancient MTB genotype. In contrast, modern MTB strains were prevalent in Turkey. Therefore they considered Iran as the connecting geographical location between ancient and modern TB (13).
The CAS family which was the most prevalent lineage in our results is essentially localized in Central and Middle Eastern Asia (14). It belongs to ancient TB and its prevalence is steadily decreasing from south Asia to Western Asia. This genotype might be an ancestor of the Beijing lineage since it clusters close to Beijing when analyzed by a combination of MIRU-VNTR and Spoligotyping (14).
The Haarlem family was first isolated from a patient living in Haarlem, the Netherlands (7). Today its widespread distribution in different geographical regions of the world such as Asia, Europe and Africa, has been documented (15, 16). A study conducted in Iran revealed that this family accounts for more than half of all clustered strains among Iranian MDR-TB (Multi-drug resistant tuberculosis) patients (16). In our study, Haarlem family was prevalent in 5 provinces (Khorasan, Kordestan, Sistan-Balouchestan, Qom and semnan) and the prevalence of T family was higher in north-western provinces (East and West Azarbaijan, Ardebil and Mazandaran).
The Beijing genotype was originally described by Van Soolingen et al (21). This family has spread globally during recent years with the highest prevalence found in Asia and the territory of the former Soviet Union (17-19). The frequency of Beijing genotype was higher in Tehran, Qom and Khorasan provinces due to high migration rate from other provinces or countries with high burden of Beijing genotype to these areas. In this study the prevalence of Beijing genotype was 8.1%; in previous studies the prevalence of this genotype in Tehran, Mashhad and Shiraz was 5.5%, 7.1% and 10%s respectively, (6, 17, 20).
Although our results demonstrated that the most prevalent family in Iran is CAS Lineage, this family is not necessarily prevalent in each provinces of Iran.
The high frequency of Beijing strains in provinces with high migration rate should be considered, due to association of this family with drug resistance.
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