1. Introduction

Tuberculosis (TB) remains one of the world’s deadliest communicable diseases. In 2013, an estimated 9.0 million people developed TB and 1.5 million died from the disease, 360 000 of whom were HIV-positive. The death toll from the disease is still unacceptably high. World health organization (WHO) report 2013 shows higher global new TB cases and deaths, reflecting an increased improved case detection rates. Worldwide, the proportion of new cases with multidrug-resistant TB (MDR-TB) was 3.5% in 2013 and has not changed compared with previous years. However, much higher levels of resistance and poor treatment outcomes are of major concern in some parts of the world ^[2].

Globally in 2013, 5% of TB cases were estimated to have MDR-TB (3.5% of new and 20.5% of previously treated TB cases). Drug resistance surveillance data show that an estimated 480 000 people developed MDR-TB in 2013 and 210 000 mortality. Extensively drug-resistant TB (XDR-TB) has been reported by 100 countries in 2013. On average, an estimated 9% of people with MDR-TB have XDR-TB ^[3].

Sudan harbors a high TB incidence among the WHO east Mediterranean countries. The indicator of the TB incidence in Sudan is the average annual risk of infection (ARI), where the ARI reaches 1% corresponds to an approximately incidence of 50 cases of pulmonary smear positive TB/10000 population. The prevalence of TB infection is probably very different in different regions of Sudan ^[4].

Advances in molecular techniques have enabled development of a variety of genotyping methods for the differentiation of clinical isolates of Mycobacterium tuberculosis ^{[5, 6]}. In particular, repetitive and insertion sequences have proven useful for studying both the epidemiology and the phylogeny of M. tuberculosis [7-15]^[7] and regularly updated genetic diversity databases are available for this pathogen ^{[16, 17, 18]}. The chromosomal locus, containing a large number of direct repeats (DRs) interspersed with unique spacer sequences, is the target of spoligotyping (i.e., spacer oligonucleotide typing) technique ^[19]. This method has been widely applied to study the molecular epidemiology and evolutionary genetics of TB. Since the technique is PCR-based, it requires less DNA than conventional molecular typing. The purpose of the current study was to genotype M. tuberculosis isolate from patients attended the hospitals and National Reference TB laboratory in Khartoum and Eastern Sudan using spoligotyping technique.

2. Materials and Methods

Scientific and ethical approval of the study was obtained from National centre for research ethical committee (Sudan). Sputum samples were collected from new cases and previously treated Pulmonary Tuberculosis patients who attended the hospitals and National Reference TB laboratory and consecutively screened for acid fast bacilli (AFB) using Ziehl-Neelsen (ZN) smear microscopy, all smear positive patients were enrolled after obtained their written consent.

The collected samples were decontaminated using NaOH, GXT DNA/RNA Extraction kit was used for DNA extraction as described by manufacturer (Hain Life Science Company) ^[19]. PCR reaction was carried in 25 µl volume, 5 µl of DNA, 2 µl of each primer (DRa: 5’-GGT TTT GGG TCT GAC GAC-3’, DRb: 5’-CCG AGA GGG GAC GGA AAC-3’), 12.5 µl of Q master mix and 3.5 µ µl of QH₂O. Denaturation was done at 96°C for 15 min, followed by 30 cycles of 96°C for 1 min, 55°C for 1 min, 72°C for 30 sec, and 72°C for 10 min and soaked in 4°C ^[20].

A total of 75 M. tuberculosis isolates were genotyped by commercially available spoligotyping kit according to the manufacturer instructions (Isogen Bioscience B.V., Maarsen, Netherlands) and as described by Kamerbeek method ^[20]. Briefly 25 µl of the amplified PCR product was diluted in 150 µl of 2X SSPE–0.1% SDS and denatured by heat. The diluted samples were pipetted into the parallel channels in the miniblotter apparatus. To avoid any possibility of artifact hybridization spots on the commercial membranes, appropriate controls included DNA from M. bovis and M. tuberculosis H37Rv and autoclaved purified water for adequate number of negative controls in each experiment. Hybridization was done for one hour at 60°C. After hybridization, the membrane was washed twice in 250 ml of 2X SSPE/0.5% SDS for 10 min each time at 60°C and then incubated in 1:14000-diluted streptavidin-peroxidase conjugate for 60 min at 42°C. The membrane was washed 2x times, for 10 min each time, in 250 ml of 2X SSPE/0.5% SDS at 42°C and rinsed with 250 ml of 2X SSPE for 5 min at room temperature. Detection of hybridizing DNA was done by using chemiluminescent ECL (Amersham) detection liquid, followed by exposure to X-ray film (Hyperfilm ECL; Amersham) in accordance with the instructions of the manufacturer (Amersham Pharmacia, Uppsala, Sweden). For repeated use of membranes, the membranes were stripped by washing 2x times for 30 min each time in 1% SDS at 80°C and then incubated for 15 min in 20 mM EDTA (pH 8) at room temperature. Membranes were sealed in plastic and stored at 4°C until further use. Results were double checked visually by an experienced operator to eliminate any systematic artifact caused by using commercial membranes. The spoligotype patterns were entered in an Excel spreadsheet and compared to the international database (SpoIDB4) and SITVIT2 (Institute Pasteur de Guadeloupe) to determine the specific MTB complex strain.

3. Results

A total of 32 distinct spoligo patterns were obtained from the 75 sputum samples. (76%) of mycobacterium had patterns that matched those in the database, while (24%) of isolates had unique pattern. The most prevalent clustered spoligotypes in this study were Manu2/SIT54 37(49.3%) followed by CAS1 Delhi/ SIT25 6(8%), Table 1.

Table 1. Frequency and percentage of spoligotyping patterns

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Spoligotypes that did not match any existing pattern in the database were defined as orphans, out of 18 orphan observed in this study, 8 (44.44%) isolates were identified as having identical patterns to Manu2/ SIT54 (7 isolates lacked a single spacer and one lacked 2 spacers), on the other hand 2 (11.11 %) orphan had similar pattern of CAS1 Delhi/ SIT25 (one lacked a single spacer and other gained 2 spacers), most differences between the patterns occur in 34-43 spacers region, the other orphans (8 patterns) were still unique.

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Figure 1. Spoligotyping autorad showing the genotyping result (1- Manu2 /SIT 54. 2- Orphan. 3- CASI-Delhi /SIT25)

4. Discussion

The present study provides and determines the M. tuberculosis genotyping using spoligotyping technique. This is the third study in Sudan after two studies were done previously by Sharaf eldien et al ^{[21, 22]}. The Manu2/SIT54, CAS1 Delhi/ SIT25, CAS1 Kili /SIT21, H3/SIT50, TI/SIT53, CAS/SIT142, H3/SIT316 patterns were identical to that isolated previously in Sudan by Sharaf eldein et al in 2011 ^[21], the other spoligotype SIT (29/U, 40/T4, 240/U (like LAM), 289/CAS1 Delhi, 294/H3, 1690/H3, 1787/CAS1 Delhi, 1634/Manu2) were not previously reported in Sudan. Cluster analysis revealed that Manu2 clade was the dominant clade 40(53.3%) included SIT54, SIT1634 and SIT1690. Casi Delhi clade represents 8(10.7) of the total clades which were isolated in this study and it include SIT25, SIT289 and SIT1787, compared to previous study was done in Sudan CASI- Delhi was dominant spoligotypes pattern ^[22], this difference between the finding of this study and the previous studies may be due to the time difference between the two studies where the samples of study were collected in 2005 and in this study samples were collected in 2013 during this period tuberculosis were spread larger due to population movement. The dominance of Manu2/SIT54 in this study may be due to the location of Khartoum as the capital of the country, with intense migration from the rural areas and neighboring countries. Increase the possibility of spread of some M. tuberculosis strains from an area to another. A possible cause of the spread of Manu2/SIT54 in Kassala and Al-Gaddarif could be the movement of population from Ethiopia which a highly endemic country. Belay et al., ^[23] study in Ethiopia revealed that Manu2 lineage strains were dominant patterns ^[23]. The Mycobacteria isolated in this study genotyped by spoligotyping were reported in some countries according to the SITVIT Database 4 (spolDB4.0 - Institute Pasteur, Guadeloupe).

The families which were isolated in this study was isolated and Genotyped by spoligotyping in some countries according to the SITVIT Database 4 (spolDB4.0 - Institute Pasteur, Guadeloupe); MANU2 family/SIT54 was prevalent in North America, India and Russia, also found predominantly in Madagascar, Egypt, Georgia, Armenia and Thailand. CAS1Delhi family SIT25 was prevalent in North America, United Kingdom, France and India also predominantly in Djibouti, Morocco, Afghanistan, New Zealand and Ethiopia. CAS1_Delhi family/ SIT21 was prevalent in Madagascar, Zambia, North America and predominantly in Tanzania and South Africa. H3 family/ SIT316 was prevalent in Central African Republic, Cameroon and Nigeria, also found predominantly in Taiwan and USA. CAS/SIT142 was prevalent in USA, Bangladesh and India. T1family/ SIT53 were Prevalent in Zambia, Finland, Republic Of Côte d'Ivoire and Italy. H3 family/ SIT50 was Prevalent in Austria, Czech Republic, Italy, Côte d'Ivoire and South Africa. CAS1 Delhi family SIT1787 was prevalent in Pakistan and France. U (likely LAM) SIT was prevalent in the United States, Portugal, Spain and France. U/SIT 240 prevalent in United States, BRAZIL and Italy. CAS1 Delhi/SIT 289 prevalent in Bangladesh, Nepal and United States. H3/SIT 294 prevalent in Indonesia, Brazil and United States. Manu2/SIT 1690 was prevalent in Brazil and United States. Manu2/SIT 1634 was prevalent in Indonesia and United States.

Acknowledgement

This study was funded by International Atomic Energy Agency (IAEA), National Center for Research and National TB Control Program. Also Extends thanks to Tropical Medicine Research Institute (TMRI) staff, National Reference Laboratory Staff and (Kassala & Al Gadarif) Hospitals Staff. Finally I am particularly grateful to the all participators patients in this study and we hope to all of them fully health and wellness.

Competing Interests

The authors have no competing interests.

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