Genetic Diversity of Hepatitis B and Hepatitis C Viruses in Ethiopia

Hepatitis B and C viruses are a major cause of mortality and morbidity worldwide. In 2015 alone, viral hepatitis caused 1.34 million deaths, a number comparable to annual deaths caused by tuberculosis but higher than those caused by either HIV or malaria. However, in contrast to these three infectious diseases, viral hepatitis has received relatively little attention. Worldwide, only 9% of HBV-infected and 20% of HCV-infected persons have access to affordable hepatitis testing. Even more worrisome is the fact that only 8% of those diagnosed with HBV and 7.4% of those diagnosed with HCV had started treatment. This is even more problematic in low-income countries like Ethiopia where more than 80% of the population are living in rural areas with a very limited access to healthcare and where viral hepatitis has never been considered a major health priority. Most importantly, the clinical and treatment outcomes of HBV and HCV infections are largely influenced by the high genetic diversity (genotype, subgenotype, recombinants and quasispecies variants) of these viruses that also display distinct geographical distribution worldwide. In this thesis, therefore, we studied the seroepidemiology of HBV and HCV infection and determined the molecular epidemiology and genetic diversity of HBV and HCV in different geographic regions of Ethiopia. We identified that genotype A (A1) and genotype D (D2 and novel D10) of HBV and genotype 4 ( 4d and 4r ) of HCV are the most prevalent in Ethiopia. We further analyzed HBV and HCV quasispecies variants and their association with patients’ clinical characteristics using ultra-deep sequencing approach and found some important variants

Evaluation of direct colorimetric MTT assay for rapid detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

With the spread of multidrug-resistant tuberculosis (MDR-TB) strains there is an increasing need for new accurate and cost-effective methods for a rapid diagnostic and drug susceptibility testing (DST), particularly in low-income countries where tuberculosis is hyperendemic. A colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) has been suggested as a promising method for DST, especially to rifampicin. In this study, we standardized and evaluated the MTT assay for a rapid direct detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains from sputum specimens using Lowenstein-Jensen (LJ) culture medium as a gold standard. The MTT assay sensitivity, specificity, positive and negative predictive values for rifampicin were 100%, 86%, 100%, 99%, respectively. For isoniazid, the MTT assay had a 100% sensitivity, specificity, positive and negative predictive values. Interestingly, the MTT assay gave interpretable results within two weeks for 94% of the samples compared to 7-14 weeks for LJ media. Overall, an excellent agreement was observed between MTT assay and LJ proportion method (Kappa, 0.91 for rifampicin and 1.00 for isoniazid). In conclusion, the direct colorimetric MTT assay simultaneously detects susceptible and resistant strains of M. tuberculosis within three weeks. It significantly shortens the time required to obtain a DST result and could be a reliable alternative method for rapid detection of drug-resistant TB strains in high-TB-burden resource-limited settings

Identification of HCV Resistant Variants against Direct Acting Antivirals in Plasma and Liver of Treatment Naïve Patients

Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma

Machine-learning based patient classification using Hepatitis B virus full-length genome quasispecies from Asian and European cohorts

Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Vari