Epidemiology and Drug Resistance Pattern of Mycobacterial Isolates among HIV positive and HIV negative TB patients using Conventional and Molecular Methods in South east Ethiopia

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Addis Ababa University


Background:- Tuberculosis is the leading cause of death in hospitalized patients. It has not yet been investigated whether MDR-TB contributes to the reported high mortality. There has also been no study on the molecular epidemiology of TB and M. tuberculosis strain types have not yet been characterized in the area. Nationwide, there have been no previous reports on drug resistance in individuals treated with 4 fixed dose combination (FDC) anti- TB drugs. Shashemene is one of the known locations for high prevalence of TB and HIV in Ethiopia. The TB-HIV co-infection rate in the age group of 15-49 years was estimated to be 47% in the area (MoH, 2004). Objectives:-To determine the prevalence of resistance to the four first line anti-TB drugs with and without HIV co-infected TB patients and to investigate association of TB bacteria strain types with drug resistance. Methods: - A cross-sectional survey on anti-TB drug resistance was conducted in four health centres and one Hospital in and around Shashemene in Ethiopia. Sputum and blood samples were collected from all consenting smear positive pulmonary TB patients visiting the sites between May 2006 and December 2006. Sputum was digested and decontaminated using Petroff s method with 4% NaOH and cultured on Lowenstein Jensen media. Drug sensitivity tests were performed on isolates using the proportion method on Middlebrook 7H10 media and 10% OADC enrichment. Pateints were screened for HIV with rapid assays (Determine®, Capillus® and Unigold®) according to national guidelines. Species were identified with biochemical (Thiophene-2-Carboxylic acid Hydrazide TCH test) and DNA based methods (species specific PCR amplification with RD4 primer). Drug resitance was further characterized using PCR based mutation analysis as a rapid and simple technique for diagnosis of drug resistance in tuberculosis. We described a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC ACC and ACA (Ser Thr) substitutions that confer resistance to isoniazid (INH) in M. tuberculosis clinical isolates. We used PCR-RFLP assay for rpsL43 mutation analysis, in the absence of mutation in AAG AGG position in MboII digest rpsL43 and the fragment was a 210-bp and 60-bp. If a mutation in codon 43 resulted xiii in a 270-bp fragment. Results and Discussion: - A total of 292 (264 new and 28 previously treated) patients were included in this study. Out of these, 82.5% (241/264) were culture positive. Sensitivity test results were available for 217 isolates from new cases and 24 isolates from previously treated patients. Among the isolates, 80% (174/217) of those from new patients and 54.2% (13/24) of those from previously treated patients were sensitive to all drugs tested. Prevalence of MDR-TB among new cases was 0.9% (2 isolates). Resistance to INH, RMP, STM and EMB was 12.9% (28/217), 1.8% (4/217), 16.6% (36/217) and 4.1% (9/217) respectively. In previously treated patients INH, RMP, STM and EMB resistance was 37.5% (9/24), 8.3% (2/24), 41.7% (10/24) and 12.5% (3/24) respectively. There was also no association observed between drug resistance among new cases and HIV. The high prevalence of the katG315 ACC mutant allele among INH resistant and rpsL43 AGG mutant allele among STM resistant M. tuberculosis clinical isolates in the Shashemene area, Southeast Ethiopia, 83.8% (31/37) and 55.56% (20/36) for all isolates was observed. Conclusion: Prevalence of Multi-drug resistant TB in the Shashemene area is comparable with the national report. M. bovis was not isolated from pulmonary TB in the area. Key words: Proportion methods; Multi-drug resistant tuberculosis; Tuberculosis; Human Immunodeficiency Virus; Analysis of TB drug resistant mutants;



Proportion methods, Multi-drug resistant tuberculosis, Tuberculosis, Human Immunodeficiency Virus;, Analysis of TB drug resistant mutants