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. Author manuscript; available in PMC: 2018 May 23.
Published in final edited form as: Eur Acad Res. 2016 Dec;4(9):7842–7855.

Molecular identification of Mycobacterium tuberculosis causing Pulmonary Tuberculosis in Sudan

Fatima Abbas Khalid 1,1, Nagla Gasmelseed 2,3, Elena Hailu 4, Muataz Mohamed Eldirdery 5, Markos Abebe 6, Stefan Berg 7, Abraham Aseffa 8
PMCID: PMC5966043  NIHMSID: NIHMS914983  PMID: 29805964

Abstract

Background

Tuberculosis (TB) is a major public health problem in Sudan. However, the strains responsible for the epidemic of TB have been poorly characterized. The aim of this study was to characterize the causative agent of TB isolates circulating in Kassala and Gezira States during 2011–2012.

Method

Ninety two Mycobacterium tuberculosis complex (MTBC) strains were isolated from sputum samples of pulmonary TB patients, attending the Teaching Hospitals in Kassala and Gezira states. Molecular identification was performed using region of difference 9 (RD9) deletion and spoligotyping methods. Spoligotype patterns were compared with those listed in the international SITVIT2 spoligotyping database.

Results

The results determined the circulation of Euro-American, Central Asian, and Indo-Oceanic Lineages. They were clustered in the following clades: Manu2 (64.8%), CAS (22.7%), EAI (4.5), LAM2 (2.3%), H3 (1.1%), T (1.1%), T3-ETH (1.1%), T5-RUS1 (1.1%). Comparison with the international multi-marker database SITVIT2, 76.1% of isolates matched the database, while 23.9% of the isolates were not represented in SITVIT2 (orphans). SIT54 (Manu2) was the most common strain circulating in the two states.

Conclusion

The study showed that a large variety of Mycobacterium tuberculosis (MTB) genotypes were circulating in the two states. Further studies on identification and molecular characterization of mycobacterial are needed to understand the biodiversity, rate of transmission and the associated risk factors of MTB in Sudan.

Keywords: Mycobacterium, Spoligotype, Sudan

BACKGROUND

Tuberculosis continues to be one of the major public health problems worldwide [1]. The problem has become worse after the emergence of drug resistant tuberculosis and the HIV epidemic. In 2014, 9.6 million cases were estimated to have TB with mortality of about 1.5 million per year [2]. Control efforts are threatened by emergence of drug resistance [1, 3]. Sudan accounts for 15% of the TB burden in the WHO Eastern Mediterranean Region with incidence of 94/100,000 [4]. Gezira and Kassala States are classified among the top of states with high TB prevalence in Sudan [5]e The disease is caused by species belonging to Mycobacterium tuberculosis complex (MTBC), which can be characterized by typing of polymorphic genetic loci, including region of difference (RD) typing and spoligotyping that are used as epidemiological tools to discriminate MTB isolates to species and subspecies level. These tools can also be used to understand transmission dynamics that can facilitate control of the disease [6]. Although TB has been a longstanding public health problem, few studies have investigated the genetic diversity of MTBC in Sudan [7]. Molecular typing of MTB would contribute to understanding the population structure of the strains circulating in the country, transmission dynamics, and pathogen biology. Moreover, it could help in control of the epidemics. This study aims at characterizing MTB isolates from pulmonary TB patients from Kassala and Gezira States in Sudan.

METHODS

Sample collection and processing

Sputum samples were collected from newly diagnosed pulmonary TB patients at chest departments in Kassala and Wad Medani Teaching Hospitals during 2011–2012. All patients were diagnosed based on clinical symptoms, chest radiography and bacteriological examination. The collected samples were decontaminated and homogenized with sodium hydroxide and N-acetyl L-cysteine. The samples were concentrated by centrifugation at 4000 rpm for 10 min. The supernatants were poured off. Distilled water was added to sediments, mixed and then centrifuged again. Supernatant was removed, and the pellet were re-suspended in 100µL distilled water and inoculated on slanted Lowenstein-Jensen media. The slants were incubated at 37° C and examined for colony growth weekly for 8 weeks. Any positive growth was analyzed by Ziehl-Neelsen staining to confirm Acid-Fast Bacilli (AFB). AFB isolates were subjected to DNA extraction and molecular analysis.

DNA extraction

Extraction of mycobacterial DNA was performed by boiling method as described by Khosravi [8]. Briefly, a loop full of AFB colonies was taken from Lowenstein-Jensen slants and transferred to 1.5-ml tubes containing 200 µl of distilled water. The re-suspended bacteria were heat-inactivated in a water bath at 85°C for 20 minutes and centrifuged at 10,000× g for 10 minutes. The supernatant was processed for target DNA isolation. Extracted DNA was kept at −20 °C until further analysis by molecular techniques.

Molecular typing

Multiplex PCR was performed to identify MTB lineages. Four primer sets (RD9, RD750, RD239, and TbD1) were employed [9, 10]. PCR was done using a HotStarTaq Master Mix Kit (Qiagen; Prod No 203445, Germany) and performed in a total volume of 20 µL, containing 7.1µl of H2O Qiagen, 10µl of Master mix and 0.3 µL (100mM) for each primer (0.3µl of RDx Flank Rev, 0.3µl of RDx Flank FW and 0.3µl of RDx Internal). Laboratory strain of M. tuberculosis H37Rv and Mycobacterium bovis were used as positive controls. The PCR samples were analyzed by agarose gel electrophoresis.

Spoligotyping was performed following the method described by Kamerbeek [11].

Molecular data analysis

Spoligotype patterns were entered in an Excel spreadsheet, converted into octal codes and were then entered in the SITVIT2 data base of the Institute Pasteur de la Guadeloupe (http://www.pasteur-guadeloupe.fr:8081/SITVITONLINE/) to assign the shared type (SIT) and used the web-based algorithm SPOTCLUST (http://tbinsight.cs.rpi.edu/run_spotclust.html) to assign the lineages of each isolates.

RESULTS

Molecular characterization of isolates determined the circulation of three lineages in Kassala and Gezira states: Euro-American 63 (71.6%), followed by Central Asia (CAS) 17 (19.3%) and Indo-oceanic (IO) 8 (9.1%). 7/8 of IO were isolated from Kassala State. Spoligotype patterns of eighty eight isolates were clustered into 11 families represented as: manu2 57 (64.8%), CAS1-Delhi 19 (21.6%), EAI4 (4.5%), LAM2 2 (2.3%), ill-defined T family 3 (3.3%), and CAS1-Kili 1 (1.1%) (Table1). Absence of hybridization to the spacer was demonstrated in four isolates.

Table 1.

Description of spoligotype patterns of MTB isolates from Kassala and Gezira states

State Spoligotype Patterns Frequency Clade SIT
No %
1111111111111111111111111111111100111111111 26 63.42 Manu2 54
1111111111111101111111111111111100111111111 1 2.4 Manu2 1088
1111111111111111111111111111110100111111111 1 2.44 Manu2 1634
1110000111111111111111000000000000111011111 2 4.88 CAS1-Delhi 289
Kassala 1110000111111111111111000000000000110011111 2 4.88 CAS1-Delhi 25
1110000111111111111111000000000000111111111 1 2.44 CAS1-Delhi 26
1110000111110111111111000000000000111111111 1 2.44 CAS1-Delhi 1343
1100000111111111111111000000000000111111111 1 2.44 CAS1-Delhi 952
1110000110111111111000000000000000011111111 1 2.44 CAS1-Kili 21
1110000111111111111111000000000000111101111 1 2.44 CAS1-Delhi 1401
0000111111100111111100001111111100001111111 2 4.88 LAM2 1588
1111111111111111111111111111111100001111111 1 2.44 T 53
1111111111111100000000001111111100001111111 1 2.44 T5-RUS1 254
Total 41 100
1111111111111111111111111111111100111111111 18 66.7 Manu2 54
1111111111111101111111111111111100111111111 2 7.4 Manu2 1088
Gezira 1111111111110111111111111111111100111111111 1 3.7 Manu2 583
1110000111111111111111000000000000111111111 2 7.4 CAS1-Delhi 26
1110000111110111111111000000000000111111111 1 3.7 CAS1-Delhi 1343
1111111110000000000111111111111100001111111 1 3.7 T3-ETH 149
1111111111111111111111111111110100001111111 1 3.7 H3 50
1111111111111111111001111111111100001111111 1 3.7 H37Rv 451
Total 27 100
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Regarding the frequencies of major shared types, a total of 34 patterns were observed among 88 isolates; 16 patterns corresponding to 19 isolates were described as new patterns not yet reported to the SITVIT2 database (Table2). The remaining 18 patterns (n= 69) matched pre-existing shared types in the SITVIT2 database. As shown in Table1, strains with types SIT54 (50%), SIT26 (3.4%), SIT1088 (3.4%), and SIT1343 (2.3%) were all isolated from both states Kassala and Gezira. However, strains with types SIT21 (1.1%), SIT25 (2.3%), SIT289 (2.3%), SIT952 (1.1%), SIT1401 (1.1%), SIT53 (1.1%), SIT254 (1.1%), SIT1634 (1.1%) and SIT1588 2(2.3%) were isolated from Kassala State. On the other hand, SIT50 (1.1%), SIT149 (1.1%), SIT583 (1.1%) and SIT1690 (1.1%) were isolated from Gezira State.

Table 2.

Description of 16 spoligotype patterns from Kassala and Gezira States not previously described.

State Spoligotype patterns Frequency Clade
1111110111111111111111000000000000111011111 2 CAS1-Delhi
1110100111111111111111000000000000111011111 1 CAS1-Delhi
Kassala 1111100111101111111111000000000000111011111 1 CAS1-Delhi
0010100111111111111111000000000000111011111 1 CAS1-Delhi
1111111111111111111111000000000000111111111 2 EAI
1111111111110111111111000000000000111111111 1 EAI
1111110111101111111111000000000000111011111 1 EAI
1011011111111101111111111111111000111111111 2 Manu2
1110010111111111111111000011111000111111111 1 Manu2
1111000111111111111111111001111000111111111 1 Manu2
Gezira 1111111110111011111101111111111100111111111 1 Manu2
1111111111111111111111111111111000111111111 1 Manu2
1111010111111111111111111111111000111111111 1 Manu2
1111000111111101111111000000000000111111111 1 CAS1-Delhi
1010011111111101111100000000000000111111111 1 CAS1-Delhi
1110000011111011111111100000000000011111111 1 CAS1-Delhi
Total 19
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Sixteen spoligotype patterns were not yet reported in the SITVIT2 database

The isolates which were described as new patterns belonged to three clades; manu2 (7/19), CAS1 Delhi (8/19) and EAI (4/19). Three spoligotype patterns of these isolates were reported twice (773777740003571, 777777740003771 and 557767777743771) (Table 2).

The spoligotype pattern is shown as a series of 1s and 0s with 1 representing hybridization to the spacer and 0 representing absence of hybridization. International names for these spoligotype patterns were assigned by SITVIT2.

DISCUSSION

TB remains a great public health concern in Sudan, and identification of genotypes of the predominant strains that circulates in the country is critical to study disease transmission patterns and epidemiological features. This study describes the population diversity of M. tuberculosis clinical isolates from the two states Kassala and Gezira based on deletion typing and spoligotyping. Four out of 92 isolates showed absence of spoligotype signature. Absence of spoligotyping signature was previously reported [12, 13].This finding might relate to infection by non-tuberculous mycobacteria as shown in the result of large sequence polymorphism (RD9).

The study identified co-circulation of three MTB lineages: Euro-American, Central Asian (CAS) and Indo-Oceanic in the study areas. These lineages have also been reported from TB patients in Ethiopia [14, 15], indicating an epidemiological relationship between the neighboring countries. Sudan and Ethiopia have strong historical relations, and cross boundary movement between tribes of the two countries facilitates epidemiological links. Sudan hosted large numbers of refugees from Ethiopia and Eritrea in Kassala State during the recent war between the two countries. This and previous similar population movements would be expected to contribute to the circulation of shared lineages in these areas.

Cluster analysis of isolates showed the dominance of the manu2 clade (64.8%) followed by CAS1 Delhi (21.9%) as reported previously in Sudan [7, 16]. Manu2 and EAI are characterized by deletion of RD239 and intact TbD1 [10]. Moreover, they are closely related and were probably derived from a last common ancestor [10]. In South Africa, isolation of manu2 was related to mixed infection between Beijing and the Euro-American lineage [17]. Beijing was not detected among isolates in our study. However, the detection of manu2 in Ethiopia was related to co-infection with HIV [18]. Manu is considered ancestral family of principal genetic group1 [19, 20]. It is divided into three groups; manu1 (lack spacer 34), manu2 (lack spacer 33–34), and manu3 (lack spacer 34-36) [21]. Manu2 has been isolated from different countries including neighboring ones [18, 22, 23, 24, 25, 26, 27]. High clustering of manu2 might indicate a high rate of transmission in central and Eastern Sudan and this can potentially accelerate the emergence of drug resistance. The result might be mixed circulation of Manu and East African Indian. Both super families have the same ancestral origins [28]. Dual infections of single patients by distinct strains of M. tuberculosis is increasingly reported in high tuberculosis incidence areas, raising the possibility of false composite spoligotype patterns if performed upon mixed strain samples [29].

Manu2 was determined by different shared types among which SIT54, SIT1634, SIT1088 and SIT1690 were previously isolated from Sudan (Sharafeldein et al 2011, Eldirdery et al 2015). However, SIT583 has not previously been reported in Sudan, but has been reported in France. Shared types of the CAS Delhi clade were represented as SIT25, SIT26, SIT289 which were previously isolated from Sudan [7, 16]. These types are also dominant in Ethiopia (Mengistu et al 2015). CAS1 Kili/SIT21, H3/SIT50, TI/SIT53 patterns were identical to that isolated previously in Sudan [7, 16]. It is interesting to note that no isolates were characterized as Lineage 7, the newly identified M. tuberculosis lineage that is very much defined to the northern highlands of Ethiopia [14]. With significant movement of people across the border to Eastern Sudan, it would not have been a surprise to have found this strain also in Kassala.

The families which were isolated in this study have also been isolated and genotyped by spoligotyping in several other countries. According to the SITVIT Database, MANU2 family/SIT1588 was prevalent in Bulgaria and Germany and SIT1401 was reported in Bangladesh, Pakistan and the United States. SIT952 has been reported from Bangladesh, India, Sweden and the United States. SIT451 has been reported from different African countries, Europe and the Americas. SIT149 has been isolated in Ethiopia and South Africa. SIT54, which is the dominant isolate in Sudan, circulates worldwide. This may be due to open boundaries of Sudan and movement across states.

Isolates with new spoligotypes were related to CAS1 Delhi, manu2 and EAI families. Some of them had identical octal numbers indicating that they are new shared strains (Table 2). Moreover, one of them was already typed in Iraq [30].

CONCLUSION

The study concludes that a large variation of M. tuberculosis genotypes were circulating in the two states. Further studies on identification and molecular characterization of mycobacteria are needed to better understand the biodiversity, rate of transmission and the associated risk factors of MTB in Sudan.

Acknowledgments

The authors would like to thank the staff of chest departments from teaching hospitals in Kassala and Gezira States, who help in collection of samples, and the staff of Armaur Hansen Research Institute, Ethiopia, who facilitate the molecular typing of MTB.

Funding

No fund was received for this study.

Abbreviations

AFB

Acid fast bacilli

MTB

Mycobacterium Tuberculosis

RD

Region of difference

Footnotes

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The study was approved by the Research Ethics Committee at University of Kassala.

Authors' contribution

FA and NG conceptualized the research idea and study design. Data collection (eg sampling, MTB culturing, molecular typing) was completed by FA, NG, EH, and ME. Data analyses were performed by FA, EH and revised by AA and SB. AA and SB closely involved in the design, and review of the manuscript. All authors have read and approved the final version of the manuscript and agreed for publication.

Contributor Information

Fatima Abbas Khalid, Tuberculosis Research Center, University of Kassala, Kassala, Sudan.

Nagla Gasmelseed, Department of Molecular Biology, National Cancer Institute, Sudan; Department of Biology, Faculty of Science University of Hafr Al Batin, Saudi Arabia.

Elena Hailu, Armauer Hansen Research Institute, Addis Ababa, Ethiopia.

Muataz Mohamed Eldirdery, Tropical Medicine Research Institute, National Center for Research, Khartoum, Sudan.

Markos Abebe, Armauer Hansen Research Institute, Addis Ababa, Ethiopia.

Stefan Berg, Animal and Plant Health Agency, New Haw, Surrey, UK.

Abraham Aseffa, Armauer Hansen Research Institute, Addis Ababa, Ethiopia.

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