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Multi-drug resistance tuberculosis (MDR-TB) in Kassala State, Eastern Sudan Ahmed A Abdul-Aziz, Mogahid M Elhassan, Salma A Abdulsalam, Eman O Mohammed and Mohamed E Hamid Trop Doct 2013 43: 66 originally published online 10 June 2013 DOI: 10.1177/0049475513490421 The online version of this article can be found at: http://tdo.sagepub.com/content/43/2/66

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Short Report

Multi-drug resistance tuberculosis (MDR-TB) in Kassala State, Eastern Sudan

Tropical Doctor 43(2) 66–70 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0049475513490421 tdo.sagepub.com

Ahmed A Abdul-Aziz1, Mogahid M Elhassan2, Salma A Abdulsalam3, Eman O Mohammed4 and Mohamed E Hamid5

Abstract This study investigates the prevalence of drug resistance and mutations in rpoB gene among Mycobacterium tuberculosis isolates from Kassala State, Sudan. In a cross-sectional study during 2011, sputum specimens (n ¼ 90) were examined microscopically and grown cultures were identified by analysing IS6110 insertion sequence. Antimicrobial sensitivity and mutations in the rpoB gene were determined. Of the 90 specimens found which were positive for acid-fast bacilli (AFB), 65 (72.2%) showed growth of mycobacteria. Sixty (66.7%) of these were M. tuberculosis, 5 (5.6%) were rapidly growing mycobacteria and 10 (11.1%) revealed no growth. Of the 60 M. tuberculosis, 31 (51.7%) were drug resistant, including 18 multidrug resistant TB (30%), and 15 (83.3%) demonstrated mutations in the rpoB gene. Isoniazid and rifampicin revealed the highest resistance rates (64.5%, 61.3%, respectively). In conclusion, drug resistance M. tuberculosis in Kassala State was high (30%) and was found to be mainly (83.3%) due to mutations in the rpoB gene. Keywords Mycobacterium tuberculosis, MDR, rpoB gene, Kassala, Sudan

Introduction

Methods

Emerging drug-resistant TB is among the major public health challenges in sub-Saharan African countries. The prevalence of TB in Sudan is estimated to be 209 cases/100,000 of the population and an incidence of 50,000 cases during 2009 was recorded.1 TB drug resistance surveillance in these regions has been limited by laboratory capacity and poor infrastructure.2 Drug resistant TB in Sudan has not been thoroughly surveyed. In an earlier study, Cavanagh3 reported a primary drug resistance to isoniazid, streptomycin and para-aminosalicylic acid at 16% and the acquired drug resistance was 76%. Cure rates have improved considerably since the establishment of the National Tuberculosis Control Program and the introduction of short course chemotherapy but no surveys of drug susceptibility have been reported.4 A recent study by Sharaf Eldin et al.5 in three treatment centres in Sudan reported multi-drug resistance TB (MDR-TB) prevalence at 5% of new cases and 24% of previously treated patients. The objective of this study was to investigate the drug resistance profile among Mycobacterium tuberculosis isolates from Kassala State, Eastern Sudan, and to detect mutations in rpoB gene among resistant strains.

This is a descriptive cross sectional laboratory-based study conducted between February 2011 and December 2011. Different TB centres in the Kassala State of Sudan (Table 1) were enrolled in the study. The study protocol was approved by the Research Committee of the College of Medical Laboratory

1 Senior Laboratory Specialist, College of Medical Laboratory Science, Sudan University of Science and Technology, Sudan 2 Associate Professor, College of Medical Laboratory Science, Sudan University of Science and Technology, Sudan 3 Specialist, Department of Internal Medicine, Armed Forces Hospital Southern Region, Saudi Arabia 4 Laboratory Specialist, Molecular Unit, Tuberculosis Reference Laboratory, National Health Laboratory, Sudan 5 Associate Professor, Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, Saudi Arabia

Corresponding author: Dr Mohamed E Hamid, Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, PO Box 10034, Abha 61321, Saudi Arabia. Email: [email protected]


Abdul-Aziz et al.

67

Table 1. Distribution of drug resistance, the presence of multi-drug resistance and mutations among 60 Mycobacterium tuberculosis isolates from Kassala State, East Sudan. Serial no.

Isolate no.

Hospital

Sex

Age (years)

Patient state

INH

RIF

EMB

STM

MDR (INH/RIF)

RIF mutation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

53 85 3 5 6 7 8 13 14 15 17 21 22 23 29 31 34 55 56 61 62 63 89 75 58 84 4 74 90 10 12 18 47 80 70 86 24 33 35 36 48 49 50 52 66

Aroma Aroma Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala Kassala

Male Male Female Female Female Male Male Male Male Male Male Male Male Male Female Female Female Male Male Female Male Female Female Male Female Male Female Female Male Male Female Female Female Female Male Female Male Female Female Female Female Female Female Male Female

30 42 42 45 22 45 20 18 30 37 37 40 40 52 38 40 35 42 41 26 40 21 17 16 50 19 17 30 56 22 40 22 14 35 30 50 40 18 36 60 15 27 30 32 30

New New New New New New New New New New New New New New New New Old Old Old New New New New New New New New New Old Old New Old New New Old New Old New Old New New New New New New

þ þ þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

þ þ þ -

þ þ þ þ þ þ þ -

þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ þ þ þ þ (continued)


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Table 1. Continued. Serial no.

Isolate no.

Hospital

Sex

Age (years)

Patient state

INH

RIF

EMB

STM

MDR (INH/RIF)

RIF mutation

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

69 82 67 39 42 71 72 88 41 26 37 79 25 43 30

Kassala Kassala Kassala Kassala Kassala Khashm Al Girba Khashm Al Girba Khashm Al Girba Phatou Centre Wager Hospital Wager Hospital Wager Hospital West El-Gash West El-Gash West El-Gash

Male Female Male Male Female Female Female Male Female Female Male Female Male Male Male

21 40 40 37 20 50 45 25 40 25 28 35 52 65 46

New New New Old Old New New New Old New New New Old Old New

þ þ þ þ þ þ þ þ -


þ þ þ þ þ -

þ þ þ þ -


þ þ þ þ þ þ -

EMB, ethambutol; INH, isoniazid; MDR, multidrug resistance; RIF, rifampicin; STM, streptomycin

Science, Sudan University of Science and Technology, and informed consent was obtained from all participants. Patient data and sputum specimens were collected from 90 patients (54 males, 36 females) who were positive for acid-fast bacilli (AFB). On the basis of the World Health Organization (WHO) criteria, of the 90 classified TB cases 18 (20%) were new TB cases and 72 (80%) were considered to be old TB cases. Isolates were obtained in Lowenstein Jensen (LJ) slants and identification of grown cultures was done following standard methods.6 Confirmation of TB suspected isolates was done by amplifying and analysing IS6110 insertion sequence.7 Drug susceptibility testing (DST) was done using the standard proportional method. DST was done on LJ slopes containing streptomycin 8.0 mg/mL, rifampicin (RIF) 40.0 mg/mL, isoniazid (INH) 0.2 mg/mL and ethambutol 2.0 mg/mL.6 The detection of mutations in rpoB gene and identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) was done according to Bobadilla-del-Valle et al.8

Results The results and distribution of TB and resistance among the studied population are shown in Table 1 and Figure 1. Of the 90 sputum specimens which showed AFB: 60 (66.7%) revealed growth that was tentatively identified as M. tuberculosis; five (5.6%)

were rapidly growing mycobacteria; and 10 (11.1%) were found to be contaminated or revealed no growth. The 60 M. tuberculosis isolates were: sensitive to para-nitrobenzoic acid (growth inhibited by PNB); positive for nitrate reduction (reduce nitrate to nitrite); and were negative for catalase at 68 C but positive at room temperature. These 60 strains revealed 123 bp amplicons of IS6119, which is specific for M. tuberculosis complex. On the basis of the WHO criteria, of the 60 confirmed TB cases 46 (76.7%) were new TB cases and 14 (23.3%) were old TB cases. Drug resistance among the 46 new cases was found in 21 (45.7%) of which 10 (47.6%) were MDR. In contrast, drug resistance was found in 10 of the 14 old cases (71.4%) of which five (50%) were MDR. The resistance rates of the 60 M. tuberculosis to first line drugs are summarized in Figure 1. Fifteen (25%) of the 18 MDR strains demonstrated mutations in the rpoB genes by giving a discernible polymerase chain reaction – single-strand conformation polymorphism (PCR-SSCP) pattern. Three strains demonstrated PCR-SSCP patterns similar to that of sensitive standard strain M. tuberculosis H37Rv and, thus, an absence of mutations was recorded.

Discussion The results of this study indicate that the burden of drug resistant TB in Kassala State is serious. Of the isolated M. tuberculosis, 30% (18/60 x100) were found


Abdul-Aziz et al.

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Figure 1. Resistance rates among the 60 patients with Mycobacterium tuberculosis isolated from 90 patients at the Kassala region, East Sudan. EMB, ethambutol; INH, isonicotinic acid hydrazide (isoniazid); MDR, multidrug resistance; RIF, rifampicin; STM, streptomycin; MDR, multidrug resistant TB; XDR-TB, extensively drug-resistant TB.

to be MDR as they were resistant to INH (isonicotinic acid hydrazide [isoniazid]) and rifampicin (RIF; Table 1). Three of the major six hospitals included in the study had cases of MDR strain. Our findings support those of Abdallah and Ali9 who investigated 670 clinically proven TB cases (73.4% pulmonary and 26.6% extra-pulmonary). The results indicate the need for organized detection and treatment strategies for drug resistant TB. RIF and INH are the backbone of the short-course chemotherapy because high bactericidal action and resistance to at least one of them indicates MDR.10 The results of the present study indicated a high MDR rate among isolated M. tuberculosis strains (30%) but comparable to a recent study (51.8%) conducted during 2010 in Khartoum.11 These studies indicate an alarming increase compared with an earlier survey in middle Sudan by Cavanagh3 who reported a primary drug resistance to isoniazid, streptomycin and para-aminosalicylic acid of 16% and an acquired drug resistance of 76%. According to the current definition of MDR, Sharaf Eldin et al.5 recorded 5% MDR-TB among new cases and 24% among previously treated patients. The drug susceptibility testing applied in this study revealed that: 19/60 (31.7%) were resistant to RIF; 20/60 (33.3%) were resistant to INH; and 18 (30%) were resistant to both RIF and INH (¼MDR). RIF resistance can be assumed to be a surrogate marker for MDR-TB, as

more than 90% of RIF isolates are also INH resistant.10 The combined resistance to RIF and INH observed in this study was 90% which is in line with a study from Khartoum State by Elhassan et al.11 They stated that 57.1% of the strains were resistant to RIF, 64.3% to INH and 53.6% were resistant to both drugs (¼MDR) and calculated a combined resistance of 83%.

Conclusion In conclusion, drug resistance M. tuberculosis in Kassala State was high (30%) and 83.3% were found to be due to mutations in the rpoB gene. Three of the major six hospitals in the state recorded cases of MDR. Further systematic surveillance is needed in order to reliably determine the prevalence of MDR in Kassala State and to investigate the reasons for the high TB and MDR rates in the Kassala region. Acknowledgements The authors thank the staff of the participating hospitals at Kassala and the College of Medical Laboratory Science, Sudan University of Science and Technology and Tuberculosis Reference Laboratory for facilitating the achievement of this project.

Declaration of conflicting interests None declared.


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Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References 1. World Health Organization. Global Tuberculosis Control: WHO Report 2010. Geneva: WHO, 2010. 2. Young F, Critchley JA, Johnstone LK, et al. A review of co-morbidity between infectious and chronic disease in Sub Saharan Africa: TB and diabetes mellitus, HIV and metabolic syndrome, and the impact of globalization. Global Hlth 2009; 5: 9. 3. Cavanagh P. The sensitivity to streptomycin, PAS and isoniazid of strains of M. tuberculosis isolated from patients in Khartoum and Wad Medani. Tubercle 1965; 46: 250–255. 4. El-Sony AI, Khamis AH, Enarson DA, et al. Treatment results of DOTS in 1797 Sudanese tuberculosis patients with or without HIV co-infection. Int J Tuberc Lung Dis 2002; 6: 1058–1066. 5. Sharaf Eldin GS, Fadl-Elmula I, Ali MS, et al. Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs. BMC Infect Dis 2011; 11: 219.

6. Kent PT and Kubica GP. Public Health Mycobacteriology. Guide for the Level III Laboratory. Atlanta: Centers for Disease Control, US Department of Health and Human Services, 1985: 31–70. 7. Clarridge JE, Shawar RM, Shinnik TM, et al. Large scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in routine mycobacteriology laboratory. J Clin Microbiol 1993; 31: 2049–2056. 8. Bobadilla-del-Valle M, Ponce-de-Leon A, ArenasHuertero C, et al. rpoB Gene mutations in rifampicinresistant Mycobacterium tuberculosis identified by polymerase chain reaction single-stranded conformational polymorphism. Emerg Infect Dis 2001; 7: 1010–1013. 9. Abdallah TM and Ali AA. Epidemiology of tuberculosis in Eastern Sudan. Asian Pac J Trop Biomed 2012; 2: 999–1001. 10. Resch SC, Salomon JA, Murray M, et al. Cost-effectiveness of treating multidrug-resistant tuberculosis. PLoS Med 2006; 3: e241. 11. Elhassan MM, Saeed SM, Elmekki MA, et al. Detection of multidrug-resistant tuberculosis using PCR compared to the conventional proportional method. Bahrain Med Bull 2012; 34: 1–7.
