Browsing by Author "Lakew Mekuria (PhD)"

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    Antagonistic and Antibiotic Effects of Endophytic and Rhizobacteria of Sugarcane Plant at Wonji Sugar Estate, Ethiopia
    (Addis Ababa University, 2013-06-06) Tilahun Sefinew; Lakew Mekuria (PhD); Assefa Fassil (PhD)
    The thesis targeted to identify the endophytic, (leaf and root) and rhizobacteria of three sugarcane sample plants taken from different sites of Wonji sugar estates plantation. Accordingly, leaf and root samples cleared of surface bacteria by chemical sterilization, crushed using sterile mortar and pestle and the extract plated on nutrient agar media. The soil samples were also sieved and serially diluted from 10-1 -10-6 and 100µl of the solution spread plated on nutrient agar media. A total of 40 representative isolates were identified based on standard morphological characterization. These isolates were sub cultured for purity and preserved on nutrient agar slant for future use in different testes: Antagonism, Antibiotic effect and development of serological methods of screening of the isolates. The identification of the isolates was done in two steps. First, grouping of the isolates by morphological, physiological and Biochemical tests and second, sorting of the groups that had more than one genera in to the specific genus was done by Busnson’s method. The result showed 11 genera, the dominant of which were Shigella, Bacillus and Klebsiella. Antagonstic interactions between all the isolates revealed 22 of them were susceptible to 18 isolates that had inhibitory effects. The test for antibiotic effect of isolates against four human pathogenic bacteria: E. coli, Salmonella thyphimurium, Staphylococcus aureus and Streptococcus pneumonae showed 18 isolates with anti-biotic effects ranging from 10-17mm clear zone diameter. This was less sensitive compared to the positive control antibiotics, chloranphenicol and ampicillin. The serological method development for screening of the isolates was started by SDS-PAGE fractionation of the proteins and induction of antibodies in swiss albino mice shall be tested as soon as the conjugated antibodies arrive. The potential use of the identified isolates; in sustainable agriculture, environmental protection and animal and human benefits are discussed in detail.
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    Drug Resistance Patterns of Tuberculosis Among Re-Treatment Cases in St Peter’s Tb Specialized Hospital, Addis Ababa
    (Addis Ababa University, 2003-07) W/ Meskel Dawit; Abate Getahun (PhD); Aseffa Abraham (PhD); Lakew Mekuria (PhD); Goshu Solomon (PhD)
    Tuberculosis (TB) is a major public health problem worldwide. About 3.8 million TB cases were reported to WHO in the year 2001 from the 8.5 million estimated new cases. In Ethiopia the estimated incidence of all cases of tuberculosis has reached 292 per 100000 populations. This figure placed the country to rank 10th among the 22 high burden countries. The resurgence of tuberculosis has been accompanied by high frequency of drug resistant strains from all over the world. In most TB patients drug resistance predominantly arises as a result of multiple interruptions of treatment. To avoid this problem fixed-dose combinations (FDCs) tablets are now recommended by WHO and IUATLD. However, in FDC formulations the bioavailability of the component drugs, and especially of rifampicin, may be reduced. Simple, rapid and inexpensive methods of detecting drug resistant tuberculosis are also essential for effective treatment. This study has the objectives of assessing the prevalence of drug resistance among re-treatment cases after introduction of Three Fixed Dose Combination (3FDC) therapy for tuberculosis; evaluation of MTT assay for direct detection of rifampicin resistance and analysis of the RFLP pattern of the different TB strains. Single sputum samples were collected from 100 smear positive re-treatment TB cases who attended St Peter’s TB specialized Hospital between 21 December 2001 and 15 October 2002. The sputum samples were cultured on Lowenstein- Jensen (LJ) media and 7H9 broth. Drug sensitivity was done on 7H10 agar using the proportion method. Broth media (7H9) supplemented with PANTA and Oleic acid Dextrose Catalase (OADC) were used for MTT assay. Formazan production was quantified by measuring the optical density (OD) at 570nm. Relative optical density unit (RODU) was calculated and resistance was defined as RODU> 0.5 and susceptibility as RODU< 0.2. Among the 89 culture positive isolates, 75 were tested for drug sensitivity pattern. Totally 58.7% of the isolates were resistant to one or more drugs. Isolates resistant and partially resistant to isoniazid were found to be 42.7% and 6.7% respectively. Resistance to rifampicin was 33.3%. Isolates resistant and partially resistant to streptomycin were found to be 21.3% and 12% respectively. The percentage of isolates resistant to ethambutol was 9.3% while 25.3% were partially resistant. Multidrug resistant isolates (MDR) were observed in 29.3% of the patients. Patients who had a history of treatment with 3FDC had a statistically significant higher rate of resistance to isoniazid (P< 0.05) and rifampicin (P< 0.05) compared to those treated with loose drugs. Direct MTT assay identified 30.7% isolates as rifampicin resistant and 69.3% as susceptible within three weeks of time. Comparing MTT assay to the proportion method resulted in 97.3% matching. RFLP analysis of ten isolates showed the presence of eight unique patterns. Two isolates showed the dominant type of RFLP profile existing in Ethiopia. Contrary to our expectation, patients treated with 3FDC regimen had more rifampicin and isoniazid resistant isolates than those treated with loose drugs. This is a good indicator for a more systematic study to evaluate the efficacy of the 3FDC drug formulation. There has been a marked increase in drug resistant tuberculosis. Therefore, nationwide drug resistance surveillance with a larger number of samples is needed to monitor drug resistance tuberculosis in the country
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    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
    (Addis Ababa University, 2007-05) Arega Desta; Aseffa Abraham (PhD); Lakew Mekuria (PhD); Engers Howard (PhD); Gedamu Lashitew (Professor)
    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;
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    Molecular Characterization of Superoxide Dismutase Gene From Leishmania Aethiopica
    (Addis Ababa University, 2003-06) Genetu Abebe; Aseffa Abraham (PhD); Lakew Mekuria (PhD)
    This study was perfOlmed with the aim of characterizing the genes that code for superoxide dismutase in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species typing, and molecular characterization of the SOD genes. A total of 20 suspected cutaneous leishmaniasis patients were enrolled in the study. Clinical and epidemiological infOlmation was collected from all the subjects including age, sex, address, duration of the lesion, and site of lesion. Skin slit was taken from the active lesion(s) and was subjected to ill vitro parasite culture on NNN medium. Promastigote stage of Leishmania was then isolated and characterized. Species typing was done on the isolate using isoenzyme electrophoresis and PCR-RFLP. Total genomic DNA was extracted from the promastigotes and then the SOD genes were amplified using polymerase chain reaction. The amplified fragments were then sequenced and the sequence information was analyzed using vector NTI software. The study subjects came from different parts of the countLy including Addis Ababa. The mean age of the patients was 25.6 year with a range of 7 to 65 years. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results of one isolate (1093/02) confilmed that the isolate is L. aethiopica. The SODB genes amplified from promastigotes of L. aethiopica were similar in size as the SODB genes of other ·Leishmania species. Nucleotide sequences of LaethFeSODBI showed 94.9% and 73% identity with LcFeSODB I and LmFeSODBI respectively. On the other hand, predicted amino acid sequence comparison indicated that LaethFeSODB 1 had 89.8% and 90.8% identity with LcFeSODB 1 and LmFeSODB I respectively. The high degree of amino acid sequence similarity of L. aetlziopica SODB genes with those of other species has an important implication in the use of new antileishmanial drugs that are targeting SODs of other Leishmania species to treat cutaneous leishmaniasis in Ethiopia. Key words: Leishmallia aethiopica, superoxide dismutase, gene, Ethiopia