Eyasu Makonnen

Evaluation of Patterns of Liver Toxicity in Patients on Antiretroviral and Anti-Tuberculosis Drugs: A Prospective Four Arm Observational Study in Ethiopian Patients

Objectives To evaluate the incidence, type, severity and predictors of antiretroviral and/or anti-tuberculosis drugs induced liver injury (DILI). Methods A total of 1,060 treatment naive patients were prospectively enrolled into four treatment groups: HIV patients receiving efavirenz based HAART alone (Arm-1); TB-HIV co-infected patients with CD4≤200 cells/μL, receiving concomitant rifampicin based anti-TB and efavirenz based HAART (Arm-2); TB-HIV co-infected patients with CD4>200 cells/μL, receiving anti-TB alone (Arm-3); TB patients taking rifampicin based anti-TB alone (Arm-4). Liver enzyme levels were monitored at baseline, 1st, 2nd, 4th, 8th, 12th and 24th weeks during treatment. CD4 and HIV viral load was measured at baseline, 24th and 48th weeks. Data were analyzed using multivariate Cox Proportional Hazards Model. Results A total of 159 patients (15%) developed DILI with severity grades 1, 2, 3 and 4 of 53.5%, 32.7%, 11.3% and 2.5% respectively. The incidence of cholestatic, hepatocellular or mixed pattern was 61%, 15% and 24%, respectively. Incidence of DILI was highest in Arm-2 (24.2%)>Arm-3 (10.8%)>Arm-1 (8.8%)>Arm-4 (2.9%). Concomitant anti-TB-HIV therapy increased the risk of DILI by 10-fold than anti-TB alone (p<0.0001). HIV co-infection increased the risk of anti-TB DILI by 4-fold (pu200a=u200a0.004). HAART associated DILI was 3-fold higher than anti-TB alone, (pu200a=u200a0.02). HAART was associated with cholestatic and grade 1 DILI whereas anti-TB therapy was associated with hepatocellular and grade ≥ 2. Treatment type, lower CD4, platelet, hemoglobin, higher serum AST and direct bilirubin levels at baseline were significant DILI predictors. There was no effect of DILI on immunologic recovery or virologic suppression rate of HAART. Conclusion HAART associated DILI is mainly cholestatic and mild whereas hepatocellular or mixed pattern with high severity grade is more common in anti-tuberculosis DILI. TB-HIV co-infection, disease severity and concomitant treatment exacerbates the risk of DILI.

Genome-wide association and replication study of anti-tuberculosis drugs-induced liver toxicity

BackgroundDrug-induced liver injury (DILI) is a well-recognized adverse event of anti tuberculosis drugs (ATD) possibly associated with genetic variations. The objective of this study was to perform genome-wide association study (GWAS) to identify genetic variants associated with the risk for ATD induced liver toxicity in Ethiopian patients.ResultTreatment-naïve newly diagnosed tuberculosis patients (nu2009=u2009646) were enrolled prospectively and treated with rifampicin based short course anti-tuberculosis therapy. Whole genome genotyping was done using Illumina Omni Express Exome Bead Chip genotyping array with 951,117 single nucleotide polymorphisms (SNPs) on 48 DILI cases and 354 ATD tolerants. Replication study was carried out for 50 SNPs with the lowest P-values (top SNPs) using an independent cohort consisting of 27 DILI cases and 217 ATD tolerants. In the combined analysis, the top SNP identified was rs10946737 (Pu2009=u20094.4u2009×u200910−6, ORu2009=u20093.4, 95xa0% confidence intervalu2009=u20092.2–5.3) in the intron of FAM65B in chromosome 6. In addition, we identified a cluster of SNPs with suggestive genome-wide significance in the intron of ATP/GTP binding protein-like 4 (AGBL4).ConclusionWe identified genetic variants that are potentially associated with ATD induced liver toxicity. Further studies with larger sample sizes are essential to confirm the findings.

High Gastrointestinal Colonization Rate with Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Hospitalized Patients: Emergence of Carbapenemase-Producing K. pneumoniae in Ethiopia.

We investigated the gastrointestinal colonization rate and antibiotic resistance patterns of Extended-Spectrum Beta-Lactamase (ESBL)- producing Escherichia coli and Klebsiella pneumoniae in hospitalized patients admitted at Ethiopia’s largest tertiary hospital. Fecal samples/swabs from 267 patients were cultured on chrome agar. ESBL. Bacterial species identification, verification of ESBL production and antibiotic susceptibility testing were done using Vitek 2 system (bioMérieux, France). Phenotype characterization of ESBL-E.coli and ESBL- K.pneumoniae was done using Neo-Sensitabs™. ESBL positivity rate was much higher in K. pneumoniae (76%) than E. coli (45%). The overall gastrointestinal colonization rate of ESBL producing Enterobacteriaceae (ESBL-E) in hospitalized patients was 52% (95%CI; 46%–58%) of which, ESBL-E. coli and K.pneumoniae accounted for 68% and 32% respectively. Fecal ESBL-E carriage rate in neonates, children and adults was 74%, 59% and 46% respectively. Gastrointestinal colonization rate of ESBL-E.coli in neonates, children and adults was 11%, 42% and 42% respectively. Of all E. coli strains isolated from adults, children and neonates, 44%, 49% and 22% were ESBL positive (p = 0.28). The prevalence of ESBL-K.pneumoniae carriage in neonates, children and adults was 68%, 22% and 7% respectively. All K. pneumoniae isolated from neonates (100%) and 88% of K. pneumoniae isolated from children were ESBL positive, but only 50% of K.pneumoniae isolated from adults were ESBL positive (p = 0.001). Thirteen patients (5%) were carriers of both ESBL-E.coli and ESBL-KP. The overall carrier rate of ESBL producing isolates resistant to carbapenem was 2% (5/267), all detected in children; three with E.coli HL cephalosporinase (AmpC), resistant to ertapenem and two with K. pneumoniae Carbapenemase (KPC) resistant to meropenem, ertapenem and impenem. We report a high gastrointestinal colonization rate with ESBL-E and the emergence of carbapenems-resistant K. pneumoniae in Ethiopia. Urgent implementation of infection control measures, and surveillance are urgently needed to limit the spread within healthcare facilities and further to the community.

In vivo Antihypertensive and Antihyperlipidemic Effects of the Crude Extracts and Fractions of Moringa stenopetala (Baker f.) Cufod. Leaves in Rats.

Background: Moringa stenopetala (Baker f.) Cufod. is a medicinal plant that has been used in Ethiopian traditional medicine as a remedy for treatment of hypertension and diabetes. The aim of this study was to evaluate antihypertensive and antihyperlipidemic effect in fructose induced hypertensive rats. Methods: Rats were randomly divided into control and treatment groups (n = 6). Treatment groups were given daily extracts (250, 500, and 1000 mg/kg) orally with fructose. Whereas, positive, negative and normal control groups were received captopril (20 mg/kg/day with fructose), only fructose (66% w/v ad libitum) and distilled water ad libitum for 15 days, respectively. The blood pressure was measured every 5th day using tail cuff blood pressure analyzer, and on the 16th day the blood was sampled to evaluate antihyperlipidemic effect using clinical chemistry analyzer. Results: The study showed that aqueous and 70% ethanol extracts significantly prevented blood pressure increment in a dose dependent manner comparable to that of the standard drug. Similarly, the extracts suppressed increment in lipid profile (cholesterol, glucose, and triglycerides) compared with negative control. The biochemical test revealed that extracts produced a rise in liver but no effect on kidney function indicators compared with normal control. Conclusion: These findings revealed that both crude extracts of M. stenopetala (Baker f.) Cufod. possess antihypertensive and antihyperlipidemic effect.

HLA-B*57 Allele Is Associated with Concomitant Anti-tuberculosis and Antiretroviral Drugs Induced Liver Toxicity in Ethiopians

Drug-induced liver injury (DILI) is a known adverse effect of both anti-tuberculosis (anti-TB) and antiretroviral (ARV) drugs. Recent studies highlight the implications of genetic predispositions to DILI. We performed a case-control study to identify Human Leukocyte Antigen-B (HLA-B) variant alleles associated with anti-TB and ARV co-treatment induced liver toxicity in Ethiopian TB and HIV co-infected patients. A total of 495 newly diagnosed TB and HIV co-infected patients were enrolled and received rifampicin based anti-TB and efavirenz based ARV therapy. Change in liver enzyme level from baseline was monitored 1st, 2nd, 4th, 8th, 12th, and 24th weeks after treatment initiation to identify patients who developed DILI (cases) and those who did not (treatment tolerants). Genomic DNA from 46 cases and 46 sex and age matched treatment tolerants were genotyped for HLA-B variant alleles using Olerup SSP®HLA-B DNA Typing Kits. The proportion of HLA-B*57 allele carriers in DILI cases (37.0%), particularly in those who developed cholestatic type of DILI (44.8%) was significantly higher compared with those who tolerated the treatment (2.2%). The HLA-B*57 allele frequency was significantly higher in cases (25%) than treatment tolerants (1.1%). In a multivariate logistic analysis, the proportion of patients carrying HLA-B*57 (P = 0.002) and HLA-B*14 (P = 0.014) alleles were significantly higher in DILI cases compared with treatment tolerants. HLA-B*57 was significantly associated with cholestatic (P = 0.001) and mixed (P = 0.017) types of liver toxicity, and mild-to-moderate severity (P = 0.001). Of all HLA-B*57 alleles detected, HLA-B*57:03 accounted 58.3% and HLA-B*57:02 accounted 41.7%. HLA-B*57:01 was not detected. The variant allele frequencies of HLA-B*57:03 (15.2 vs. 0%) and HLA-B*57:02 (9.8 vs. 1.1%) were significantly higher in the DILI cases than treatment tolerants (P < 0.03). We conclude that HLA-B*57 alleles (B*57:03 and B*57:02) confer susceptibility to the development of anti-TB and ARV drugs co-treatment induced liver toxicity, which is mainly of cholestatic type. The possible association of HLA-B*14 with anti-TB and ARV drugs co-treatment induced liver toxicity requires further investigations.

In vitro vasodilatory activity and possible mechanisms of the crude extracts and fractions of Moringa stenopetala (Baker f.) Cufod. leaves in isolated thoracic aorta of guinea pigs

Moringa stenopetala, a plant belonging to the family of Moringaceae, is traditionally used for the treatment of hypertension and diabetes in Ethiopia. This study evaluates the in vitro vasodilatory effect of the extract of M. stenopetala leaves and the possible mechanisms in precontracted isolated thoracic aorta of guinea pigs. A guinea pig was sacrificed by gentle cervical dislocation, and the thoracic aortic ring was removed, cut spirally, and mounted in an organ bath containing Krebs–Henseleit physiological solution maintained at 37°C, and then the solution was aerated with carbogen (95% O2 and 5% CO2). The vasodilatory activity of cumulative doses of M. stenopetala extracts and fractions was evaluated on intact and denuded endothelium of isolated whole, spirally cut thoracic aortic strips of guinea pigs precontracted with potassium chloride (80 mM), epinephrine (1 μM), methylene blue (10 μM), and glibenclamide (10 μM) using polygraph. All extracts showed a relaxant effect in precontracted isolated whole, spirally cut thoracic aortic strips of guinea pigs in a dose-dependent manner, whereas the greater percentage of relaxant effect was shown with the addition of crude extracts in 80 mM of potassium chloride (99.10% and 95.56% for ethanol and aqueous crude extracts, respectively), and 1 μM of epinephrine (82.85% and 90.16% for ethanol and aqueous crude extracts, respectively) in precontracted isolated whole, spirally cut thoracic aortic strips of guinea pigs. Hence, the possible mechanism of relaxation might be mediated through the blockade of receptor-operated calcium influx and L-type voltage-dependent calcium channels. The aqueous extract showed more significant in vitro vasodilatory effect than its fractions and 70% ethanol extract.

In vitro vasodilatory effect of aqueous leaf extract of Thymus serrulatus on thoracic aorta of Guinea pigs

mmol/L) induced precontracted isolated thoracic aorta rings on guinea pigs and the role of aorta endothelium on this action. Methods: Guinea pig thoracic aorta was removed and placed in an organ bath containing Krebs-Henseleit solution and aorta contractions were recorded isometrically. Results: The results revealed that T. serrulatus aqueous leaf extract (0.5-5 mg/mL) significantly (P<0.001) reduced KCl-induced contractions of guinea pig thoracic aorta in both intact (n=5) and denuded (n=5) endothelium in a concentration dependent manner, and the vasodilatory effect of the extract on intact endothelium was significantly (P<0.05) higher than that on denuded endothelium. Glibenclamide (10 µmol/L) significantly (P<0.001) increased the vasodilatory effect of extract in intact endothelium as compared to methylene blue (10 µmol/L), atropine (10 µmol/L) and indomethacin (10 µmol/L). The effect was more obvious on intact than that on denuded endothelium. Conclusions: The present findings demonstrate that T. serrulatus aqueous leaf extract has vasodilatory activity which might result in antihypertensive effect and its vasodilatory effect is endothelium-dependent. This might support the traditional claim of the plant in hypertensive.

N-Acetyltransferase-2 (NAT2) phenotype is influenced by genotype-environment interaction in Ethiopians

Background and objectivesN-acetyltransferase 2 (NAT2) metabolize several drugs including isoniazid. We investigated the effect of genotype, geographical difference, and smoking habit on NAT2 phenotype in Ethiopians.MethodsGenotyping for NAT2 191Gu2009>u2009A, 341xa0Tu2009>u2009C, 590Gu2009>u2009A, and 857Gu2009>u2009A was performed in 163 unrelated healthy Ethiopians (85 living in Ethiopia and 78 living in Sweden). The NAT2 phenotype was determined using caffeine as a probe and log AFMU/(AFMUu2009+u20091Xu2009+u20091xa0U) urinary metabolic ratio (MR) as an index.ResultsThe frequencies of NAT2*4, *5, *6, *7, and *14 haplotypes were 14.1, 48.5, 30.1, 5.5, and 1.8%, respectively. The frequencies of rapid (NAT2*4/*4), intermediate (heterozygous *4), and slow (no *4 allele) acetylator genotypes were 1.8, 24.6, and 73.6%, respectively. The distribution NAT2 MR was bimodal with 70% being phenotypically slow acetylators. NAT2 genotype (pu2009<u20090.0001) and country of residence (pu2009=u20090.004) independently predicted NAT2 phenotype. Controlling for the effect of genotype, ethnic Ethiopians living in Ethiopia had significantly higher NAT2 MR than those living in Sweden (pu2009=u20090.006). NAT2 genotype-phenotype concordance rate was 75%. Distinct country-of-residence-based genotype-phenotype discordance was observed. The proportion of phenotypically determined rapid acetylators was significantly higher and slow acetylators was lower in Ethiopians living in Ethiopia (39% rapid, 61% slow) than in Sweden (20% rapid, 80% slow). Sex and smoking had no significant effect on NAT2 MR.ConclusionsWe report a high prevalence of NAT 2 slow acetylators in Ethiopians and a conditional NAT2 genotype-phenotype discordance implicating a partial phenotype conversion and metabolic adaptation. Gene-environment interactions regulate NAT2 phenotype.

The Next Generation Scientist program: capacity-building for future scientific leaders in low- and middle-income countries

BackgroundScientific and professional development opportunities for early career scientists in low- and middle- income countries (LMICs) are limited and not consistent. There is a disproportionately low number of biomedical and clinical researchers in LMIC’s relative to their high burden of disease, a disparity that is aggravated by emigration of up to 70% of scientists from their countries of birth for education and employment elsewhere. To help address this need, a novel University-accredited, immersive fellowship program was established by a large public-academic-private network. We sought to describe the program and summarize progress and lessons learned over its first 7-years.MethodsHallmarks of the program are a structured learning curriculum and bespoke research activities tailored to the needs of each fellow. Research projects expose the scientists to state-of-the-art methodologies and leading experts in their fields while also ensuring that learnings are implementable within their home infrastructure. Fellows run seminars on drug discovery and development that reinforce themes of scientific leadership and teamwork together with practical modules on addressing healthcare challenges within their local systems. Industry mentors achieve mutual learning to better understand healthcare needs in traditionally underserved settings. We evaluated the impact of the program through an online survey of participants and by assessing research output.ResultsMore than 140 scientists and clinicians from 25 countries participated over the 7-year period. Evaluation revealed strong evidence of knowledge and skills transfer, and beneficial self-reported impact on fellow’s research output and career trajectories. Examples of program impact included completion of post-graduate qualifications; establishment and implementation of good laboratory- and clinical- practice mechanisms; and becoming lead investigators in local programs. There was a high retention of fellows in their home countries (>u200975%) and an enduring professional network among the fellows and their mentors.ConclusionsOur experience demonstrates an example for how multi-sectoral partners can contribute to scientific and professional development of researchers in LMICs and supports the idea that capacity-building efforts should be tailored to the specific needs of beneficiaries to be maximally effective. Lessons learned may be applied to the design and conduct of other programs to strengthen science ecosystems in LMICs.

Toxicological investigation of acute and chronic treatment with Gnidia stenophylla Gilg root extract on some blood parameters and histopathology of spleen, liver and kidney in mice

BackgroundIn southeast Ethiopia, people locally use the roots of Gnidia stenophylla Gilg (Thymelaeaceae) to cure malaria and other diseases with no literature evidence substantiating its safety. The aim of this study was, therefore, to investigate the safety of the aqueous root extract of G. stenophylla after acute (single dose) and repeated sub chronic oral administration in mice.ResultsA single oral administration of the extract at 500, 1000, 2000 and 4000xa0mg/kg body weight did not induce any behavioral change and mortality in both sexes. The oral LD50 of the extract was found to be above 6000xa0mg/kg body weight in mice. Chronic treatment with the extract for 13xa0weeks did not induce any sign of illness and/or death and had no adverse effect on the body weight. Dose-related elevations of erythrocytes, hematocrit, hemoglobin, platelets and neutrophils differential and significant decrease in the number of lymphocyte were observed. Liver sections of mice treated with 800xa0mg/kg body weight, revealed mild inflammations around the portal triads and central veins; whereas the spleen and kidneys appeared normal with no detectable gross morphological and histopathological alteration at both doses.ConclusionsThe results of this study revealed that aqueous root extract of G. stenophylla Gilg at antimalarial dose is safe even when taken for a longer period. At a higher dose, the extract may have a potential to increase some hematological indices but may induce mild hepatotoxicity as a side effect.