Yalemtsehay Mekonnen

Molecular typing of Mycobacterium tuberculosis complex isolated from pulmonary tuberculosis patients in central Ethiopia

BackgroundIdentification of the types of strains of Mycobacterium tuberculosis (M. tuberculosis) complex causing tuberculosis (TB) could contribute to TB control program of specific geographic region as well as it could add knowledge onto the existing literature on TB worldwide. The objective of the present study was to identify the species and strains of M. tuberculosis complex causing pulmonary tuberculosis in central Ethiopia.MethodsA health institution- based cross-sectional study was conducted on 338 smear positive TB cases visiting three hospitals between October 2012 and September 2013. Morning and spot sputum samples were collected before the starting of treatment regimens. Thus, a total of 338 pooled sputum samples collected from these cases. Samples were cultured on Löwenstein Jensen media and the isolates were identified by the region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping.ResultOf the total isolates 98.6% of the isolates were identified to be M. tuberculosis while the remaining 1.4% were identified as M. africanum. Further, typing of M. tuberculosis using spoligotyping lead to the identification of 90 different strains of M. tuberculosis. Of these strains, 32 were clustered consisting of more than one isolate while the remaining 58 strains were unique consisting of single isolate. Thus, 79.3% (223/281) of the isolates were found in the clustered while only 20.6% (58/281) of the strains were unique. Forty-five of the spolgotyping patterns were registeredin the SITVIT2 or SpolDB4 database in while the remaining 45 were notfound in the database and hence were orphan strains. The dominant strains were SIT53, SIT149, and SIT54, consisting of 43, 37 and 34 isolates, respectively. Classification of the spoligotype patterns using TB-insight RUN TB-Lineage showed that 86.8, 6.4, 5, 1.4% ofthe isolatesbelonged to the Euro-American lineage, East-African-Indian, Indo-oceanic and M. africanum, respectively.ConclusionThe identification of clustered and new strains using spolygotyping in present study does not give conclusive finding as spoligotyping has low discriminatory power. Thus, further identification of these isolates using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VENTR) and or whole genome sequencing (WGS) recommended.

Mycobacterium tuberculosis in central Ethiopia: drug sensitivity patterns and association with genotype

Drug resistance tuberculosis (TB) and the emergence of multidrug resistant (MDR) isolates are significant concerns regarding TB control programs in several countries. This study was undertaken to evaluate the drug sensitivity of Mycobacterium tuberculosis and to assess its association with strains and lineages of M. tuberculosis. A total of 279 M. tuberculosis strains isolated from Central Ethiopia were tested for their drug sensitivity patterns to first line TB drugs using the conventional proportion method on Löwenstein Jensen media. The association between drug sensitivity and strain type was assessed on 263 isolates of the 279 isolates. Of the 268 M. tuberculosis isolates obtained from new cases, 209 (78%) were susceptible to first line TB drugs, and 59 (22.2%) bacterial isolates were resistant to at least one of the first line drugs. The highest mono-resistance (7.5%) pertained to streptomycin (STM). Remarkably, seven of eleven isolates (63.6%) previous treatment for TB were resistant to at least one of the first line drugs. The prevalence of MDR-TB was 1.5% (4/268) for newly identified TB cases, all of which were members of the Euro-American Lineage. There was no statistically significant association (P > 0.05) between drug sensitivity, and either strains, sub-lineages or main lineages of M. tuberculosis. A significant proportion of M. tuberculosis was resistant to at least one first line anti-TB drug. Moreover, the frequencies of resistance to either isoniazid or rifampicin were high compared to data that were previously reported in some part of the country.

Evaluation of the GenoType MTBDRplus assay for detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates in central Ethiopia

Objective/Background: Multidrug-resistant tuberculosis (MDR-TB) is growing globally and becoming a major challenge for national TB control programs. Therefore, rapid identification of MDR strains of Mycobacterium tuberculosis and monitoring their transmission could contribute significantly to the control of TB. The GenoType MTBDRplus assay has been recommended by the World Health Organization to identify rifampicin (RIF)- and isoniazid (INH)-resistant M. Tuberculosis isolates. This study was carried out to evaluate the performance of the GenoType MTBDRplus assay for the detection of RIF- and INH-resistant M. Tuberculosis isolates in central Ethiopia. Methods: A total of 279 M. Tuberculosis strains isolated from active TB cases in central Ethiopia were evaluated for their drug sensitivity by the conventional drug-susceptibility test (DST) and compared with data derived from the GenoType MTBDRplus assay. The DST served as the gold standard for evaluating the GenoType MTBDRplus assay. Results: The sensitivity and specificity of the GenoType MTBDRplus assay for the detection of RIF-resistant M. Tuberculosis isolates were 80.0% and 99.6%, respectively. Its sensitivity and specificity for the detection of INH-resistant M. Tuberculosis isolates were 82.7% and 99.6%, respectively, whereas they were 75.0% and 100%, respectively, for the detection of MDR M. Tuberculosis strains. The concordances of the GenoType MTBDRplus assay and the conventional DST for the detection of RIF and INH susceptibility were 80% (8/10) and 86.2% (25/29), respectively. Furthermore, the concordance of the two tests for the detection of MDR M. Tuberculosis strains was 75%. Specific mutations were detected in 55.6% (5/9) of the RIF-resistant isolates, with the highest mutation rate (33.3%) for the rpoB gene (Codon S531L). For INH-resistant isolates, the highest mutation rate (88.8%) related to a katG mutation (Codon S315T1). Conclusion: The findings of this study revealed that the GenoType MTBDRplus assay has high sensitivity and specificity for the detection of RIF and INH resistance. These preliminary data support the notion that the assay should be considered as an alternative to the DST for the characterization of MDR in M. Tuberculosis isolates and the control of TB.

In vitro evaluation of the antibacterial activities of the methanol, aqueous and n-hexane extracts of Ocimum lamiifolium from Ethiopia

Ocimum lamiifolium (local name Dama Kesse, Amharic) is a medicinal plant in Ethiopia. Its leaves are squeezed and sniffed to treat coughs and colds. They are also used to treat eye infections and to stop nose bleedings. In the present study, leaves of O. lamiifolium were collected from their growing habitats. Dried leaf powders were extracted using methanol, distilled water and n-hexane. 25, 50, and 100 mg/ml doses of the extracts made in Tween 80 (2%) were screened for their antimicrobial activities against Staphylococcus aureus, Escherchia coli, Pseudomonas aeruginosa and Shigella boydii using disk diffusion assay. The inhibition zones due to the methanolic extract ranged from 0 (in S. aureus due to 25 mg/ml) to 12 mm (in E. coli due to 100 mg/ml). Inhibition zones due to the aqueous extract ranged from 8 mm in S. aureus and S. boydii to 12 mm in S. boydii at concentrations of 25 and 100 mg/ml, respectively. The n-hexane extract at 25 mg/ml resulted in inhibition zone that ranges from 7 mm (against S. aureus) to 11 mm (against E. coli) at 50 and 100 mg/ml doses. The minimum inhibitory concentration of S. boydii and E. coli was 10 mg/ml due to all the extracts. The minimum inhibitory concentrations on S. aureus were 10, 20 and 50 mg/ml due to the aqueous, n-hexane and methanolic extracts, respectively. P. aeruginosa was minimally inhibited at 10 mg/ml due to the methanol and aqueous extracts and 15 mg/ml due to the n-hexane extract. The methanol, aqueous, and n-hexane extracts of O. lamiifolium leaf extracts inhibited the test bacteria with significantly higher levels of inhibition zones than the negative control (T80). The positive controls (Tetracycline and Chloramphenicol) also showed significantly higher inhibition zones than the 100 mg/ml concentration of the extracts and T80 except that Chloramphenicol failed to inhibit S. aureus and P. aeruginosa. However, combination of Chloramphenicol with plant extracts raised their inhibition zones from zero to 23 and 25 mm in S. aureus and P. aeruginosa, respectively.

Composition and hepatoprotective activity of essential oils from Ethiopian thyme species (Thymus serrulatus and Thymus schimperi)

ABSTRACT The chemical composition of six essential oils (EOs) of Thymus serrulatus and Thymus schimperi from six localities in Ethiopia, namely Ofla, Alamata, Yilmana Densa, Tarmaber, Butajira and Bale was determined. The in vivo hepatoprotective potential of three of the EOs was evaluated. GC-MS and NMR were used to determine chemical composition and GC-FID to quantify the main compounds. The number of EO components ranged from 17 to 41. The EOs were of two chemotypes with either thymol (Ofla, Alamata, Tarmaber and Bale) or carvacrol (Yilmana Densa and Butajira) being the main compound. Hepatoprotection of EOs was tested against paracetamol-induced hepatotoxicity in Wistar albino rats. Hepatoprotective properties of the oils were demonstrated by reduced serum levels of hepatic marker enzymes (AST, ALT and ALP) and normal hepatocytes structure. The most effective one was the thymol type EO from Alamata.

Acute oral toxicity study of Thymus serrulatus and Thymus schimperi from Ethiopia

Thymus serrulatus and Thymus schimperi both endemic to Ethiopia are used by the public as tea and food additives. They are claimed to have some sort of toxicity. However, no toxicity test has been conducted to date. So the present study aimed to test the acute oral toxicities of their Essential Oils (EOs). T. serrulatus was collected from Ofla (Ofl), Alamata (Ala), and Yilmana Densa (Yil) and T. schimperi from Tarmaber (Tar), Butajira (Buta), and Bale (Bal). The control group (Group I) mice were administered with calculated amounts of 0.1% Tween-80 in normal saline. Experimental group (Groups II to VI), on the other hand, were delivered with 2000 μL/Kg body weight of Ofl, Ala, Yil, Tar, Buta and Bal EOs respectively. Treated and control mice were observed, and changes were recorded for 14 days. On the 14th day, after mice were humanely killed by heart puncture method, their organs were weighed, organ to body weight ratios were calculated and packed cell volumes (PCVs) were determined. Growth rate decrease was observed in mice treated with Yil and Buta EOs (carvacrol chemotypes) than in those treated with the thymol chemotypes (Ofl, Ala, Tar, and Bal). The organ to body weight ratios of the control group were either significantly higher than or comparable to that of the treatment groups implying that the EOs had no any inflammatory effects on the organs. The % PCVs of mice treated with the EOs were either significantly higher than or comparable to the control mice. The median lethal dose (LD50) of each EO was between 2000 μL/kg to 5000 μL/kg body weight. The LD50 values of the dry weights of thyme were calculated based on their EO yields that were approximated to be around 278g /kg bw (Bal), 313g /kg bw (Yil, Tar, and Buta) and 500g /kg bw (Ofl and Ala). Since the aerial parts, not the EOs, of thyme are used in the form of tea and food additives (not their EOs), this value is so high that these plants are not toxic. However, cautions should be taken for vulnerable groups. Key words : Acute Toxicity, Thymus serrulatus, Thymus schimperi, Essential oils