The knowledge of the diversity of strains of
The incidence of TB has continued to increase in many parts of the world [
Thus, identification of strains circulating in a certain geographic region using molecular tools can contribute to the TB control program of that region. Various molecular epidemiology methods allow identifying mycobacterial strains and tracking the transmission of TB in different geographical regions [
The study was conducted in Bahir Dar city and its surrounding districts. Bahir Dar is a city in the highland in northwest of Ethiopia and is the capital city of the Amhara Regional Administrative State. The city is located at geographical coordinates of 11°38′ north in latitude and 37°15′ east in longitude. It has an elevation of 1830 meters above sea level and is characterized by hot and humid weather with an average temperature of 29°C. The population size of the city and its surrounding is 221,991, of which 180,174 (81.2%) are residing in the Bahir Dar city [
Smear positive pulmonary TB (PTB) and extrapulmonary TB (EPTB) patients, who were diagnosed as TB cases between September 2012 and January 2014 at Felegehiwot Referral and GAMBY General Hospitals, were included in this study. High TB patient flow, existence of better diagnostic facilities, and skilled human resource were the major reasons for selecting the specified health facilities. The average TB case flow in these two study hospitals over four years (2010–2013) was 321 as assessed from the respective hospital records. TB patients who visited these health facilities during the study period were enrolled in the study, excluding those below 18 years of age and those who had started treatment prior to launching the study. Children under the age of 18 years were not included as (1) the study was not a pure epidemiological study and its main objective was the identification of strains circulating in the study area, (2) it was not easy to obtain consent from a family member or a guardian for children below 18 years of age, (3) collection of sputum samples can be difficult in children, and (4) systematic differences of MTBC strains comparing the adult population with children were not expected.
A structured questionnaire was used to collect data from all study subjects. These data included patient origin, age, sex, household size, TB category, clinical presentation, and family history of TB infections. Clinical examination of patients suspected to be infected with TB was performed by the attending physicians. Sputum samples submitted for the routine Ziehl Neelsen staining for diagnostic purpose were used for bacteriological examination. Similarly, fine needle aspirates (FNA) collected by a pathologist for the routine diagnosis of TB were used for mycobacterial culture.
Isolation of mycobacteria was made on Lowenstein-Jensen (LJ) medium using the procedure described by the National TB and Leprosy Control Programme Guideline [
All of the 168 isolates were characterized by spoligotyping as previously described by Kamerbeek et al. [
The spoligotype patterns were converted into binary and octal formats and entered into the open source spoligotype database available at the website
The statistical analysis was performed using STATA software version 12 [
Ethical clearance was obtained from Ethical Review Board (Ref. number IRB/05-02/2013) of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University. In addition, permission was obtained from the Research Committee of Bureau of Health, Amhara National Regional State, Ethiopia.
Data generated from 168 subjects were used in the analysis of the demographic and clinical results. Among the study participants, 52.4% were female, 73.8% were in age range of 18–39 years, 84.5% were new cases, 27.4% had a history of TB pertaining to one of their family members, and 67.9% were EPTB patients. Surprisingly, all EPTB cases were identified as TB lymphadenitis (TBLN), of which 67 (60.4%) and 18 (16.2%) were TBLN in cervical and axillary lymph nodes, respectively. Of the 168 isolates, 33.9% and 25.6% originated from South Gondar and West Gojjam, respectively (Table
Demographic and clinical characteristics of the study subjects and their association with spoligotype clustering and major lineages (
Characteristics | Number of isolates, |
Number of isolates clustered versus unique ones | Major lineages by CBN | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Clustered | Unique | Clustering rate |
|
EA | EAI | IO | MA | MB |
|
||
Patient origin (zonal) | 0.502 | 0.822 | |||||||||
North Gondar | 2 (1.19) | 1 | 1 | 0.6 | 1 | 1 | 0 | 0 | 0 | ||
South Gondar | 57 (33.9) | 29 | 28 | 17.3 | 27 | 18 | 3 | 7 | 2 | ||
East Gojjam | 24 (14.3) | 12 | 12 | 7.14 | 13 | 9 | 1 | 1 | 0 | ||
West Gojjam | 43 (25.6) | 29 | 14 | 17.3 | 19 | 18 | 0 | 6 | 0 | ||
Bahir Dar Special | 20 (11.9) | 13 | 7 | 7.74 | 15 | 4 | 0 | 1 | 0 | ||
Awi | 20 (11.9) | 12 | 8 | 7.14 | 10 | 7 | 2 | 1 | 0 | ||
North Wollo | 2 (1.19) | 2 | 0 | 1.19 | 1 | 1 | 0 | 0 | 0 | ||
Age (years) | 0.181 | 0.277 | |||||||||
18–28 | 83 (49.4) | 47 | 36 | 28 | 43 | 30 | 0 | 10 | 0 | ||
29–39 | 41 (24.4) | 28 | 13 | 16.7 | 19 | 16 | 2 | 3 | 1 | ||
40–50 | 20 (11.9) | 13 | 7 | 7.74 | 13 | 4 | 2 | 1 | 0 | ||
>50 | 24 (14.3) | 10 | 14 | 5.95 | 11 | 8 | 2 | 2 | 1 | ||
Sex | 0.175 | 0.674 | |||||||||
Male | 80 (47.6) | 51 | 29 | 30.4 | 41 | 27 | 3 | 9 | 0 | ||
Female | 88 (52.4) | 47 | 41 | 28 | 45 | 31 | 3 | 7 | 2 | ||
Household size | 0.855 | 0.876 | |||||||||
|
85 (50.6) | 49 | 36 | 29.2 | 42 | 31 | 2 | 9 | 1 | ||
>4 | 83 (49.4) | 49 | 34 | 29.2 | 44 | 27 | 4 | 7 | 1 | ||
TB category | 0.943 | 0.93 | |||||||||
Retreatment | 26 (15.5) | 15 | 11 | 8.93 | 15 | 8 | 1 | 2 | 0 | ||
New | 142 (84.5) | 83 | 59 | 49.4 | 71 | 50 | 5 | 14 | 2 | ||
Family TB history | 0.266 | 0.758 | |||||||||
Yes | 46 (27.4) | 30 | 16 | 17.9 | 26 | 14 | 1 | 5 | 0 | ||
No | 122 (72.6) | 68 | 54 | 40.5 | 60 | 44 | 5 | 11 | 2 | ||
Clinical presentation | 0.867 | 0.16 | |||||||||
EPTB | 114 (67.9) | 67 | 47 | 39.9 | 53 | 43 | 6 | 10 | 2 | ||
PTB | 54 (32.1) | 31 | 23 | 18.5 | 33 | 15 | 0 | 6 | 0 | ||
Total | 168 (100) | 98 | 70 | 58.3 | 88 | 58 | 6 | 16 | 2 |
EA: Euro-American; EAI: East-African Indian; IO: Indo-Oceanic; MA:
A total of 168 MTBC isolates were spoligotyped, and 89 (53%) different spoligotype patterns (strains) were identified. Clustering of isolates into strains was observed, and a total of 98 isolates were grouped in 19 (58.3%) different clusters of strains. The dominant strains were SIT289, SIT134, and SIT3411, each consisting of 28 (16.7%), 12 (7.14%), and 8 (4.76%) isolates, respectively. These strains contributed 28.6% (48/168) of all isolates with known spoligotype patterns. Out of the 89 spoligotype patterns (strains), 44 strains associated with 122 isolates matched the preexisting patterns in the SITVIT2 database while the remaining 45 spoligotype patterns associated with 46 isolates were not registered in the international spoligotype SITVIT2 database and thus designated as orphan strains.
Classification of MTBC strains showed the occurrence of the following lineages: Euro-American (86/168; 51.2%), East-African Indian (58/168; 34.5%),
Spoligotype patterns of 44 shared types and their corresponding lineages/sublineages identified from a total of 168
SIT | Isolates with similar pattern | SITVIT2 lineage/sublineage | CBN |
Octal number | Binary format |
---|---|---|---|---|---|
20 | 1 | LAM1 | EA | 677777607760771 |
|
35 | 4 | Ural-1 | EA | 777737777420771 |
|
37 | 1 | T3 | EA | 777737777760771 |
|
41 | 3 | Turkey | EA | 777777404760771 |
|
50 | 3 | H3 | EA | 777777777720771 |
|
51 | 1 | T | EA | 777777777760700 |
|
52 | 1 | T2 | EA | 777777777760731 |
|
53 | 6 | T | EA | 777777777760771 |
|
54 | 3 | Manu2 | EA | 777777777763771 |
|
93 | 1 | LAM5 | EA | 777737607760771 |
|
134 | 12 | H3 | EA | 777777777720631 |
|
137 | 2 | X2 | EA | 777776777760601 |
|
149 | 5 | T3-ETH | EA | 777000377760771 |
|
168 | 3 | H3 | EA | 777777777720671 |
|
205 | 1 | T | EA | 737777777760771 |
|
336 | 1 | X1 | EA | 777776777760731 |
|
699 | 1 | H3 | EA | 677777777720571 |
|
777 | 1 | Ural-1 | EA | 777777777420771 |
|
817 | 1 | Ural-1 | EA | 777777777420731 |
|
1166 | 1 | T | EA | 777377777760771 |
|
1552 | 1 | H1 | EA | 777777774020631 |
|
1688 | 1 | T | EA | 777777403760771 |
|
1877 | 1 | T | EA | 737377777760771 |
|
2007 | 1 | T3 | EA | 777737677760771 |
|
2409 | 1 | T3 | EA | 777737757760771 |
|
3134 | 1 | H3 | EA | 777737377720771 |
|
3411 | 8 | T3-ETH | EA | 777002377760771 |
|
3412 | 2 | T4 | EA | 777003377760771 |
|
21 | 2 | CAS1-Kili | EAI | 703377400001771 |
|
25 | 7 | CAS1-Delhi | EAI | 703777740003171 |
|
26 | 2 | CAS1-Delhi | EAI | 703777740003771 |
|
289 | 28 | CAS1-Delhi | EAI | 703777740003571 |
|
754 | 1 | CAS1-Delhi | EAI | 503777740003771 |
|
952 | 2 | CAS1-Delhi | EAI | 603777740003771 |
|
1200 | 2 | Unknown | EAI | 703777747777771 |
|
1551 | 1 | CAS1-Delhi | EAI | 701777740003771 |
|
2359 | 2 | CAS1-Delhi | EAI | 703677740003171 |
|
343 | 1 | Unknown |
|
700000007175771 |
|
910 | 1 | Unknown |
|
700000007177771 |
|
1729 | 1 | Unknown |
|
700000004177771 |
|
3409 | 1 | AFRI |
|
700020047177771 |
|
665 | 1 | BOV_1 |
|
616773777777600 |
|
982 | 1 | BOV |
|
416773777777600 |
|
523 | 1 | Manu_ancest | IO | 777777777777771 |
|
Spoligotype patterns of 45 orphan strains and their corresponding lineages/sublineages identified from a total of 168
SIT | Isolates with similar pattern | SITVIT2 lineage/sublineage | CBN |
Octal number | Binary format |
---|---|---|---|---|---|
Orphan | 1 | T | EA | 776603777760771 |
|
Orphan | 1 | T | EA | 777737347760771 |
|
Orphan | 1 | T | EA | 276777777760771 |
|
Orphan | 1 | T3-ETH | EA | 777002377420771 |
|
Orphan | 1 | T2 | EA | 777777403760731 |
|
Orphan | 1 | EAI | EA | 777760370000000 |
|
Orphan | 1 | EAI | EA | 777770370000000 |
|
Orphan | 1 | T3-ETH | EA | 777002377760731 |
|
Orphan | 1 | T1-RUS2 | EA | 770002001760771 |
|
Orphan | 1 | EAI | EA | 777760370000000 |
|
Orphan | 1 | T | EA | 276777737760771 |
|
Orphan | 1 | T-H37Rv | EA | 777777444760771 |
|
Orphan | 1 | X1 | EA | 400002757760771 |
|
Orphan | 1 | T2 | EA | 777777403760731 |
|
Orphan | 1 | Manu2 | EA | 577747777767771 |
|
Orphan | 1 | H3 | EA | 777737377720731 |
|
Orphan | 1 | Manu2 | EA | 777777774363771 |
|
Orphan | 1 | LAM3 | EA | 760002007760771 |
|
Orphan | 1 | AFRI | EAI | 700022044037771 |
|
Orphan | 1 | Unknown | EAI | 000022000003771 |
|
Orphan | 1 | CAS | EAI | 700002000000771 |
|
Orphan | 1 | PINI2 | EAI | 400200000000751 |
|
Orphan | 1 | H2 | EAI | 700000004037771 |
|
Orphan | 1 | CAS1-Delhi | EAI | 703602040003571 |
|
Orphan | 1 | PINI2 | EAI | 000022000003771 |
|
Orphan | 1 | H | EAI | 000002004020631 |
|
Orphan | 1 | CAS1-Delhi | EAI | 703677740003571 |
|
Orphan | 1 | CAS | EAI | 703777700001171 |
|
Orphan | 1 | CAS1-Delhi | EAI | 703622040003571 |
|
Orphan | 1 | AFRI |
|
700022007177771 |
|
Orphan | 1 | AFRI |
|
700002044177771 |
|
Orphan | 1 | AFRI |
|
700020044177771 |
|
Orphan | 2 | AFRI |
|
700002004177771 |
|
Orphan | 1 | AFRI |
|
700002004177771 |
|
Orphan | 1 | AFRI |
|
771022044177771 |
|
Orphan | 1 | AFRI |
|
700002004177771 |
|
Orphan | 1 | AFRI |
|
700002007177771 |
|
Orphan | 1 | AFRI |
|
700002007177771 |
|
Orphan | 1 | AFRI |
|
700022004177771 |
|
Orphan | 1 | AFRI |
|
700022047177771 |
|
Orphan | 1 | Manu1 | IO | 773777747777771 |
|
Orphan | 1 | Manu2 | IO | 773777744203771 |
|
Orphan | 1 | Manu1 | IO | 753777747777771 |
|
Orphan | 1 | Manu1 | IO | 717777777777771 |
|
Orphan | 1 | EAI | IO | 717777776003771 |
|
The majority of MTBC strains were identified from the South Gondar Zone (57/168; 33.9%) followed by the West Gojjam Zone (43/168; 25.6%), each with a strain-clustering rate of 17.3% (Table
Map showing the distribution of the three dominant strains in Bahir Dar city and its surrounding zones, northwest Ethiopia. The green coloured portion of the icons in the map shows SIT289 with frequency of 14% (8/57) for South Gondar, 8.33% (2/24) for East Gojjam, 25.9% (11/43) for West Gojjam, 15% (3/20) for Bahir Dar Special Zone, and 15% (3/20) for Awi Zone. The yellow colour shows SIT134 with frequency of 5.26% (3/57) for South Gondar, 4.17% (1/24) for East Gojjam, 6.98% (3/43) for West Gojjam, 10% (2/20) for Bahir Dar Special Zone, and 15% (3/20) for Awi Zone. The red colour shows SIT3411 with frequency of 50% (1/2) for North Gondar, 3.51% (2/57) for South Gondar, 4.17% (1/24) for East Gojjam, 6.98% (3/43) for West Gojjam, and 5% (1/20) for Bahir Dar Special Zone.
The Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), and
Distribution of mycobacterial lineages in the different zones of the Amhara Region, northwest Ethiopia. Five different lineages were identified. The Euro-American (EA) lineage is represented by yellow segments with a frequency of 50% (1/2) for North Gondar, 47.4% (27/57) for South Gondar, 54.2% (13/24) for East Gojjam, 44.2% (19/43) for West Gojjam, 75% (15/20) for Bahir Dar Special Zone, 50% (10/20) for Awi, and 50% (1/2) for North Wollo zone. The East-African Indian (EAI) lineage is represented by red segments with a frequency of 50% (1/2) for North Gondar, 31.6% (18/57) for South Gondar, 37.5% (9/24) for East Gojjam, 41.9% (18/43) for West Gojjam, 20% (4/20) for Bahir Dar Special Zone, 35% (2/20) for Awi, and 50% (1/2) for North Wollo zone. The Indo-Oceanic (IO) lineage is shown by blue segments with the frequency of 5.26% (3/57) for South Gondar, 4.17% (1/24) for East Gojjam, and 10% (2/20) for Awi Zone. The
In the present study, MTBC species were isolated from 168 TB patients from Bahir Dar city itself and the surrounding districts who visited health institutions in Bahir Dar city. The isolates were identified at strain and lineage levels on the basis of spoligotyping. Identification at a higher level of resolution by using variable nucleotide tandem repeat (VNTR) typing, facilitated by mycobacterial interspersed repetitive units- (MIRU-) VNTR, is desirable although this method is not currently available in Ethiopia.
Spoligotyping of 168 mycobacterial isolates revealed 89 distinct patterns, which corresponded to 53% of genotype diversity. The high diversity of spoligotypes strains that we observed in this study was consistent with the 59% reported by Tessema et al. [
The prevalent strains identified in this study were SIT289, SIT134, and SIT3411. All three strains seem to be specific for the Bahir Dar city and its surroundings since they were not reported previously from other sites in Ethiopia [
Five different major lineages, namely, Euro-American, East-African-Indian, Indo-Oceanic,
Screening of the SITVIT2 database also identified 9.52% (16/168) of the isolates as members of
Molecular characterization of MTBC isolates from TB patients in Bahir Dar city and its surroundings was performed using spoligotyping. The high percentage of clustered strains of
The authors declared that they have no competing interests.
Anwar Nuru was involved in the design, data collection and laboratory work, and statistical analysis and interpretation of data and drafted the paper; Gezahegne Mamo was involved in the design, interpretation, and critical revision of the paper; Adane Worku was involved in the spoligotyping of the isolates and interpretation; Aschalew Admasu was involved in data collection, TB culture, and interpretation; Girmay Medhin was involved in the design, statistical analysis, and critical revision of the paper; Rembert Pieper was involved in critical revision of the paper; Gobena Ameni was involved in the design, interpretation, and critical revision of the paper. All authors have read and approved the paper for submission.
This study was jointly funded by the National Institute of Health (NIH, USA) through its H3Africa Consortium Program (Grant Ref. no. U01HG007472-01), Addis Ababa University through its Thematic Research Program, and University of Gondar. The authors are grateful to Dr. Getachew Mulualem and the other technical personnel for collecting samples as well as the institutions which facilitated the conducting of this study. The authors would like to acknowledge David Couvin for comparing the experimental strains with SITVIT2 database and Eyaya Belay for his support in doing the GIS maps.