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Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Abstract Introduction Despite the global vaccination campaign to prevent HPV-related morbidity, HPV vaccination uptake remains unacceptably low in the developing world, like Ethiopia. For strong interventional measures, compiled data in the field is required which is otherwise missed in the Ethiopian context. Therefore, this systematic review aimed to provide an estimate of the HPV vaccination uptake, mothers‘ willingness to vaccinate their adolescent girls, and associated factors in Ethiopia. Methods Articles were systematically searched using comprehensive search strings from PubMed/Medline, SCOPUS, and grey literature from Google Scholar. Two reviewers assessed study eligibility, extracted data, and assessed the risk of bias independently. Meta-analysis was performed using STATA v 14 to pool the vaccination uptake and mothers‘ willingness toward HPV vaccination in Ethiopia. Results We included 10 articles published between 2019 and 2022 covering reports of 3,388 adolescent girls and 2,741 parents. All the included articles had good methodological quality. The pooled estimate of the proportion of good knowledge about HPV vaccination and the agreement of girls to get the vaccine was 60% (95%CI: 59–62) and 65% (95%CI: 64–67), respectively. The pooled estimate of vaccination uptake of at least one dose of HPV vaccine among girls was 55% (95%CI: 53–57). Positive attitudes to the vaccine, higher maternal education, and having knowledge about HPV and its vaccine were reported as statistically significant predictors. On the contrary, not having adequate information about the vaccine and concerns about possible side effects were reported as reasons to reject the vaccine. Likewise, the pooled estimate of mothers who were knowledgeable about HPV vaccination, who had a positive attitude, and willing to vaccinate their children were 38% (95%CI: 36–40) 58% (95%CI: 56–60), and 74% (95%CI: 72–75), respectively. Conclusions Knowledge about the HPV vaccine among girls and their vaccination uptake is suboptimal that falls short of the 2030 WHO targets. Therefore, stakeholders need major efforts in rolling out vaccination programs and monitoring their uptake. Social mobilization towards primary prevention of HPV infection should focus on adolescents. The existing strategies need to address the predictors of uptake by educating girls and parents.

Following an interseasonal rise in mainly pediatric respiratory syncytial virus (RSV) cases in Germany in 2021, an exceptionally high number of adult cases was observed in the subsequent respiratory season of 2022/2023. The aim of this study was to compare the clinical presentation of RSV infections in the pre- and post-SARS-CoV-2 pandemic periods. Additionally, the local epidemiology of the RSV fusion protein was analyzed at a molecular genetic and amino acid level. RSV detections in adults peaked in calendar week 1 of 2023, 8 weeks earlier than the earliest peak observed in the three pre-pandemic seasons. Although the median age of the adult patients was not different (66.5 vs. 65 years), subtle differences between both periods regarding comorbidities and the clinical presentation of RSV cases were noted. High rates of comorbidities prevailed; however, significantly lower numbers of patients with a history of lung transplantation (p = 0.009), chronic kidney disease (p = 0.013), and immunosuppression (p = 0.038) were observed in the 2022/2023 season. In contrast, significantly more lower respiratory tract infections (p < 0.001), in particular in the form of pneumonia (p = 0.015) and exacerbations of obstructive lung diseases (p = 0.008), were detected. An ICU admission was noted for 23.7% of all patients throughout the study period. Sequence analysis of the fusion protein gene revealed a close phylogenetic relatedness, regardless of the season of origin. However, especially for RSV-B, an accumulation of amino acid point substitutions was noted, including in antigenic site Ø. The SARS-CoV-2 pandemic had a tremendous impact on the seasonality of RSV, and the introduction of new vaccination and immunization strategies against RSV warrants further epidemiologic studies of this important pathogen.

Abstract With the widespread use of Integrase strand transfer inhibitors (INSTIs), surveillance of HIV-1 pretreatment drug resistance is critical in optimizing antiretroviral treatment efficacy. However, despite the introduction of these drugs, data concerning their resistance mutations (RMs) is still limited in Ethiopia. Thus, this study aimed to assess INSTI RMs and polymorphisms at the gene locus coding for Integrase (IN) among viral isolates from ART-naive HIV-1 infected Ethiopian population. This was a cross-sectional study involving isolation of HIV-1 from plasma of 49 newly diagnosed drug-naive HIV-1 infected individuals in Addis-Ababa during the period between June to December 2018. The IN region covering the first 263 codons of blood samples was amplified and sequenced using an in-house assay. INSTIs RMs were examined using calibrated population resistance tool version 8.0 from Stanford HIV drug resistance database while both REGA version 3 online HIV-1 subtyping tool and the jumping profile Hidden Markov Model from GOBICS were used to examine HIV-1 genetic diversity. Among the 49 study participants, 1 (1/49; 2%) harbored a major INSTIs RM (R263K). In addition, blood specimens from 14 (14/49; 28.5%) patients had accessory mutations. Among these, the M50I accessory mutation was observed in a highest frequency (13/49; 28.3%) followed by L74I (1/49; 2%), S119R (1/49; 2%), and S230N (1/49; 2%). Concerning HIV-1 subtype distribution, all the entire study subjects were detected to harbor HIV-1C strain as per the IN gene analysis. This study showed that the level of primary HIV-1 drug resistance to INSTIs is still low in Ethiopia reflecting the cumulative natural occurrence of these mutations in the absence of selective drug pressure and supports the use of INSTIs in the country. However, continues monitoring of drug resistance should be enhanced since the virus potentially develop resistance to this drug classes as time goes by.

Background: Viral meningitis/encephalitis (ME) is a rare but potentially harmful disease. The prompt identification of the respective virus is important to guide not only treatment but also potential public health countermeasures. However, in about 40% of cases, no virus is identified despite an extensive diagnostic workup. The aim of the present study was to analyze demographic, seasonal, and routine cerebrospinal fluid (CSF) parameters in cases of viral ME and assess their utility for the prediction of the causative virus. Methods: Demographic data, season, and routine CSF parameters (total leucocytes, CSF cell differentiation, age-adjusted CSF/serum albumin ratio, and total immunoglobulin ratios) were retrospectively assessed in cases of viral ME. Results: In total, 156 cases of acute viral ME (74 female, median age 40.0 years) were treated at a tertiary-care hospital in Germany. Specific viral infections were detected in 93 (59.6%) cases. Of these, 14 (9.0%) cases were caused by herpes simplex virus (HSV), 36 (23.1%) by varicella-zoster virus (VZV), 27 (17.3%) by enteroviruses, 9 (5.8%) by West Nile virus (WNV), and 7 (4.5%) by other specific viruses. Additionally, 64 (41.0%) cases of ME of unknown viral etiology were diagnosed. Cases of WNV ME were older, predominantly male, showed a severe disruption of the blood–CSF–barrier, a high proportion of neutrophils in CSF, and an intrathecal total immunoglobulin M synthesis in the first CSF sample. In a multinominal logistic regression analysis, the accuracy of these CSF parameters together with age and seasonality was best for the prediction of WNV (87.5%), followed by unknown viral etiology (66.7%), VZV (61.8%), and enteroviruses (51.9%). Conclusions: Cases with WNV ME showed a specific pattern of routine CSF parameters and demographic data that allowed for their identification with good accuracy. These findings might help to guide the diagnostic workup in cases with viral ME, in particular allowing the timely identification of cases with ME due to WNV.

Abstract Background Human papillomavirus (HPV) genotypes differ by geographic location. With the advent of HPV vaccination and HPV-based cervical screening tests in Ethiopia, a nationwide dataset on the genotype distribution of HPV among women has paramount importance in the fight against cervical cancer. However, there is limited data in this regard in the northwest part of the country. Therefore, this study aimed to identify the genotype distribution of high-risk HPVs among women presenting with cervical abnormalities. Methods A health facility-based cross-sectional study was conducted at Felege Hiwot Comprehensive Specialized Hospital (FHCSH), Bahir Dar–Ethiopia. Women aged ≥ 30 years who visited the hospital gynecology unit from 01 March 2019 to 30 October 2021 were included. Following general and pelvic examinations, a senior gynecologist collected cervical punch biopsies for histopathological examinations and cervical swabs for HR-HPV detection using the Abbott Alinity m system (Abbott Molecular, Des Plaines, IL, USA). Extended genotyping was carried out with the INNO-LiPA HPV Genotyping Extra II assay (INNO-LiPA; Fujirebio Europe, Ghent, Belgium) as per the manufacturer protocols at the Institute of Virology, Leipzig University Hospital, Germany. Results We included 355 women with a mean age of 46.4 ± 11.4 years. The majority of the participants, 277 (79.4%) were sexually active before the age of 18 years and 180 (51.6%) had multiple sexual partners. Forty-eight (13.5%) of the participants were HIV positive. The proportion of HR-HPV was 53.0% (n = 188; 95%CI: 47.8–58.1%). From these samples, 13 different HR-HPV types with a total of 258 sequences were identified. The detection of HR-HPV increased significantly with an increase in the age of the participants. The predominant identified HR-HPV was HPV16, 50.4% followed by HPV31 (9.7%), HPV33 (8.5%), HPV39, and HPV68 each (5.8%) and HPV18 (4.7%). Of the total HR-HPV-positive women, 23.9% (45/188) were infected with multiple HR-HPV types. All HPV16, HPV18, HPV35, and HPV45 genotypes (as a single or in coinfections) were found to be associated with either high-grade lesions or cervical cancer. Conclusions HR-HPV infection was reportedly higher among women in the present study area. Based on our findings, we strongly recommend the nonavalent HPV vaccine for immunization and any HPV-based screening method to take into consideration the predominant genotypes circulating in the country. The role of multiple HPV infections in high-grade cervical lesions entails further study in Ethiopia.

Abstract Background Hepatitis B virus (HBV) infection is a particular concern in human immunodeficiency virus (HIV) infected individuals. In Ethiopia, detailed clinical and virological descriptions of HBV prevailing during HIV co-infection and symptomatic liver disease patients are lacking. The aim of this study was to investigate HBV virological characteristics from Ethiopian HBV/HIV co-infected and HBV mono-infected individuals. Methods A total of 4105 sera from HIV positive individuals, liver disease patients, and blood donors were screened serologically for HBV. The overlapping polymerase/surface genome region of HBV from 180 infected individuals was extracted, amplified, and sequenced for genotypic analysis. Results The HBsAg seroprevalence was detected 43% in liver disease patients, 8.4% in blood donors, and 6.7% in HIV/HBV co-infected individuals. The occult HBV prevalence was 3.7% in HIV/HBV co-infected individuals and 2.8% in blood donors with an overall prevalence rate of 3.4%. A phylogenetic analysis showed three HBV genotypes; A (61.1%), D (38.3%) and E (0.6%). Genotype A belongs to subtypes A1 (99.1%) and A9 (0.9%), but genotype D showed heterogeneous subtypes; D2 (63.8%) followed by D4 (21.7%), D1 (8.7%), D3 (4.3%), and D10 (1.4%). Conclusions The HIV/HBV co-infected individuals and blood donors showed lower HBsAg seroprevalence compared to liver diseases patients. Occult HBV prevalence showed no difference between HIV/HBV co-infected and blood donor groups. This study demonstrated predominance distribution of HBV subtypes A1 and D2 in northwest Ethiopia. The observed virological characteristics could contribute for evidence-based management of viral hepatitis in Ethiopia where antiretroviral therapy guidelines do not cater for viral hepatitis screening during HIV co-infection.

Abstract Background Cervical cancer is caused by infection with high-risk human papillomaviruses (HR-HPVs). It is one of the leading causes of cancer-related deaths in Ethiopia and globally. To develop efficient vaccination and HPV-based cervical cancer screening approaches, data on genotype distribution of HPVs is crucial. Hence, the study was aimed to review HPV genotype distribution in Ethiopia. Methods Research articles were systematically searched using comprehensive search strings from PubMed/Medline and SCOPUS. Besides, Google Scholar was searched manually for grey literature. The last search was conducted on 18 August 2021. The first two authors independently appraised the studies for scientific quality and extracted the data using Excel sheet. The pooled HPV genotype distribution was presented with descriptive statistics. Results We have included ten studies that were reported from different parts of the country during 2005 and 2019. These studies included 3633 women presented with different kinds of cervical abnormalities, from whom 29 different HPV genotypes with a sum of 1926 sequences were reported. The proportion of high-risk, possible/probable high-risk and low-risk HPVs were at 1493 (77.5%), 182 (9.4%) and 195 (10.1%), respectively. Of the reported genotypes, the top five were HPV 16 (37.3%; 95% CI 35.2.1–39.5%), HPV 52 (6.8%; 95% CI 5.8–8.0%), HPV 35 (4.8%; 95% CI 3.9–5.8%), HPV 18 (4.4%; 95% CI 3.5–5.3%) and HPV 56 (3.9%: 95% CI 3.1–4.9%). Some of other HR-HPV groups include HPV 31 (3.8%), HPV 45 (3.5%), HPV 58 (3.1%), HPV 59(2.3%), and HPV 68 (2.3%). Among the high-risk types, the combined prevalence of HPV 16/18 was at 53.7% (95% CI 51.2–56.3%). HPV 11 (2.7%: 95% CI 2.1–3.5%), HPV 42 (2.1%: 95% CI 1.5–2.8%) and HPV 6 (2.1%: 95% CI 1.4–2.7%) were the most common low-risk HPV types. Conclusions We noted that the proportion of HR-HPV types was higher and HPV 16 in particular, but also HPV 52, HPV 35 and HPV 18, warrant special attention in Ethiopian’s vaccination and HPV based cervical screening program. Additional data from other parts of the country where there is no previous HPV genotype report are needed to better map the national HPV genotypes distribution of Ethiopia.

Introduction and Objectives: Chronic hepatitis D infection contributes substantially to the progression of chronic liver disease, especially in most low and middle-income countries, where hepatitis B virus-related chronic liver disease is endemic. Therefore, this study aimed to determine the magnitude and genotype of hepatitis delta virus (HDV) among patients with chronic hepatitis B (CHB)-related liver diseases in Ethiopia. Patients and Methods: In this cross-sectional study, 323 known HBsAg positive individuals comprising 220 patients with CHB-related liver diseases [121 advanced liver diseases (hepatocellular carcinoma /HCC/ and non-HCC) and 99 chronic hepatitis (CH)], and 103 symptomless blood donors (BD) were enrolled. An ELISA kit was employed to determine HDV infection, and quantitative real-time PCR was used to detect HDV RNA. In addition, a non-coding genomic RNA region was sequenced for genotyping and phylogenetic analysis. Results: Irrespective of the stage of liver disease, the overall magnitude of HDV was 7.7% (25/323). The frequency of anti-HDV increases with the severity of liver disease, 1.9%, 4%, 10%, and 21.3% among BD, CH, non-HCC, and HCC patients, respectively. HDV RNA has been detected in 1.54 %(5/323) cases with a mean viral load of 4,010,360 IU/ml. All isolates were found to be HDV genotype 1. Conclusions: The magnitude of HDV infection increased with the severity of liver disease, indicating HDV infection is more common among patients with CHB-related liver diseases in Ethiopia.

Following the extensive non-pharmaceutical interventions (NPIs) and behavioral changes in the wake of the SARS-CoV-2 pandemic, an interseasonal rise in respiratory syncytial virus (RSV) cases was observed in Germany in 2021. The aim of this study was to characterize the local molecular epidemiology of RSV infections in comparison to the three pre-pandemic seasons. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of RSV infections. RSV detections peaked in calendar week 40 of 2021, 18 weeks earlier than the usual peak observed in the three pre-pandemic seasons. Sequence analysis revealed a close phylogenetic relatedness regardless of the season of origin. A significantly higher amount of pediatric cases (88.9% of all cases, p < 0.001) was observed for season 2021/2022. For the pediatric cases, significant differences were observed for an increased number of siblings in the household (p = 0.004), a lower rate of fever (p = 0.007), and a reduced amount of co-infections (p = 0.001). Although the mean age of the adult patients was significantly younger (47.1 vs. 64.7, p < 0.001), high rates of comorbidities, lower respiratory tract infections and intensive care unit admissions prevailed. The NPIs in the wake of the SARS-CoV-2 pandemic had a tremendous impact on the epidemiologic characteristics and seasonality of RSV and warrant further epidemiologic studies of this important pathogen.

Abstract Background The development of pretreatment drug resistance (PDR) is becoming an obstacle to the success of antiretroviral therapy (ART). Besides, data from developing settings including Ethiopia is still limited. Therefore, this study was aimed to assess HIV-1 genetic diversity and PDR mutations among ART-naive recently diagnosed HIV-1 infected individuals in Addis Ababa, Ethiopia. Methods Institutional based cross-sectional study was conducted from June to December 2018 in Addis Ababa among ART-naive recently diagnosed individuals. Partial HIV-1 pol region covering the entire protease (PR) and partial reverse transcriptase (RT) regions of 51 samples were amplified and sequenced using an in-house assay. Drug resistance mutations were examined using calibrated population resistance (CPR) tool version 6.0 from the Stanford HIV drug resistance database and the International Antiviral Society-USA (IAS-USA) 2019 mutation list. Results According to both algorithms used, 9.8% (5/51) of analyzed samples had at least one PDR Mutation. PDR mutations to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) were the most frequently detected (7.8% and 9.8%, according to the CPR tool and IAS-USA algorithm, respectively). The most frequently observed NNRTIs-associated mutations common to both algorithms were K103N (2%), Y188L (2%), K101E (2%), and V106A (2%), while E138A (2%) was observed according to IAS-USA only. Y115F and M184V (mutations that confer resistance to NRTIs) dual mutations were detected according to both criteria in a single study participant (2%). PDR mutation to protease inhibitors was found to be low (only G73S; 2% according to the CPR tool). Phylogenetic analysis showed that 98% (50/51) of the study participants were infected with HIV-1C virus while one individual (2%) was infected with HIV-1A1 virus. Conclusions This study showed an increased level of PDR and persistence HIV-1C clade homogeneity after 15 years of the rollout of ART and 3 decades of HIV-1C circulation in Ethiopia, respectively. Therefore, we recommend routine baseline genotypic drug resistance testing for all newly diagnosed HIV infected patients before initiating treatment. This will aid the selection of appropriate therapy in achieving the 90% of patients having an undetectable viral load in consonance with the UN target.

BackgroundDespite the availability of effective vaccines and treatments for hepatitis B virus (HBV), it continues to be a major public health problem in sub-Saharan Africa including Ethiopia. Routine screening for HBV in pregnant women is widely recommended, but there is lack of screening for HBV during pregnancy in Ethiopia. Therefore, this study aimed to assess viral load, and genetic diversity among pregnant women in the Amhara National Regional State, Ethiopia.Materials and methodsHepatitis B surface antigen (HBsAg) testing was performed on 1846 pregnant women, 85 of who tested positive were included in this study. HBV DNA was isolated from 85 positive sera, and the partial surface/polymerase gene was amplified and sequenced. HBV genotypes, sub-genotypes, serotypes and mutations in surface genes and polymerase were studied.ResultsOut of 85 pregnant women`s HBsAg positive sera, 59(69.4%) had detectable viral DNA. The median viral load was 3.4 log IU/ml ranging from 2.6 to7.6 and 46 samples were successfully sequenced and genotyped. Genotypes A and D were identified in 39 (84.8%) and 7 (15.2%); respectively. All genotype A isolates were further classified into sub-genotype A1 and serotype adw2 (84.8%) whereas genotype D isolates were further classified into three sub genotypes; 2 (4.3%) D2, 1(2.2%) D4, and 4 (8.7%) D10 with serotypes ayw2 (10.9%), and ayw3 (4.3%). There were 19 (41.3%) surface gene mutations in the major hydrophilic region (MHR). Six (13.1%) of them were discovered in MHR`s `a'-determinant region. Six polymerase gene mutations (13%) were identified.ConclusionGenotype A was the predominant genotype in the Amhara National Regional State. The surface and polymerase gene mutations identified in this study may lead to immune therapy failure, diagnostics escape and drug resistance. Thus, the data generated in this study will contribute to the planning of HBV diagnosis, vaccination and treatment, and most importantly to the prevention of vertical transmission of HBV in Ethiopia. Therefore, further molecular studies on HBV are warranted and continuous surveillance is important for patient management and for the prevention and control of HBV infection in the country.

IntroductionCervical cancer is an important public health problem in Ethiopia. However, the disease is not well characterized and studied in various parts of the country. This study was designed to describe the histopathological profile of cervical biopsies and to identify risk factors associated with high-grade cervical lesions and cancer (CIN2+C).MethodsA cross-sectional study was conducted at Felege Hiwot Compressive Specialized Hospital (FHCSH) between 1 March 2019 and 30 October 2021. A structured questionnaire was used to collect data on the participants' demographic, reproductive and gynecologic history. From women presented with different degrees of cervical lesions, a senior gynecologist collected cervical swabs using (Digene HC2 DNA collection device: Qiagen, Hilden, Germany) for detection of high-risk Human papillomaviruses (HR-HPV) and punch biopsy for histopathological examinations. HR-HPVs were detected using the Abbott Alinity m system following the manufacturer protocol at the Institute of Virology, Leipzig University Hospital, Germany. Collected data entered and analyzed using SPSS version 25. A logistic regression model was used for both bivariable & multivariable analysis in order to determine the association between independent variables and CIN2+C. Statistical significance was set at a p-value ResultsIn this study, 335 women were included; the mean age was at 46.5±11.4 years. Most were living in rural settings, 221(66%) and had no formal education, 259 (77.3%). More than half of the participants, 193(57.6%) were unaware of cervical cancer. The prevalence of HIV infection and previous history of cervical screening were 44(13.1%) and 93(27.8%), respectively. HR-HPVs were detected in 178(54.3%) of the participants. The majority of biopsies, 140(41.8%; 95%CI: 36.6-47.1%), were diagnosed as cervical carcinoma. Normal histology, cervicitis, cervical intraepithelial neoplasia (CIN)-1, CIN-2, and CIN-3 accounted for 74(22.1%), 30(9.0%), 40(11.9%), 12(3.6%), and 12(3.6%), respectively. High-grade lesions and cancer (CIN2+C) together accounted 164(49.0%; 95%CI: 43.6-54.2). Cervical cancer increased steadily with the age of the participants (pConclusionsThe findings of this study revealed that cervical cancer continues to be an important women's health challenge in northwest Ethiopia. Postmenopausal women, who had no screening history within a five-year period and those women who tested positive for HR-HPV need special attention. It is important to increase the awareness of women about cervical cancer and actions for early detection of precancerous lesions should be expanded.

Background: The hepatitis B and D virus (HBV/HDV) hepatocyte entry inhibitor bulevirtide (BLV) has been available in Europe since July 2020, after the registrational trial MYR202. Real-life data on the efficacy and safety of BLV are sparse. Methods: We have analysed the course of treatment with BLV (2 mg/day) plus tenofovir disoproxil fumarate (TDF) (245 mg/day) in patients with chronic hepatitis delta (CHD). Virologic (≥2 log reduction in HDV RNA or suppression of HDV RNA below the lower limit of detection) and biochemical (normalisation of serum ALT) treatment responses after 24 weeks were defined according to the MYR202 trial. Results: Seven patients were recruited (four with liver cirrhosis Child–Pugh A). After 24 weeks, a virologic response was observed in five of seven and a biochemical response was seen in three of six patients with elevated serum ALT at baseline. Extended treatment data > 48 weeks were available in three cases: two presented with continuous virologic and biochemical responses and in one individual an HDV-RNA breakthrough was observed. Adverse effects were not recorded. Conclusions: The first real-life data of the approved dosage of 2 mg of BLV in combination with TDF confirm the safety, tolerability, and efficacy of the registrational trial MYR202 for a treatment period of 24 weeks and beyond.

Enteroviruses are associated with various diseases accompanied by rare but severe complications. In recent years, outbreaks of enterovirus D68 and enterovirus A71 associated with severe respiratory infections and neurological complications have been reported worldwide. Since information on molecular epidemiology in respiratory samples is still limited, the genetic diversity of enteroviruses was retrospectively analysed over a 4-year period (2013–2016) in respiratory samples from paediatric patients. Partial viral major capsid protein gene (VP1) sequences were determined for genotyping. Enteroviruses were detected in 255 (6.1%) of 4187 specimens. Phylogenetic analyses of 233 (91.4%) strains revealed 25 different genotypes distributed to Enterovirus A (39.1%), Enterovirus B (34.3%), and Enterovirus D (26.6%). The most frequently detected genotypes were enterovirus D68 (26.6%), coxsackievirus A6 (15.9%), and enterovirus A71 (7.3%). Enterovirus D68 detections were associated with lower respiratory tract infections and increased oxygen demand. Meningitis/encephalitis and other neurological symptoms were related to enterovirus A71, while coxsackievirus A6 was associated with upper respiratory diseases. Prematurity turned out as a potential risk factor for increased oxygen demand during enterovirus infections. The detailed analysis of epidemiological and clinical data contributes to the non-polio enterovirus surveillance in Europe and showed high and rapidly changing genetic diversity of circulating enteroviruses, including different enterovirus D68 variants.

Abstract Background Although hepatitis B virus (HBV) is hyperendemic and heterogeneous in its genetic diversity in Ethiopia, little is known about hepatitis D virus (HDV) circulating genotypes and molecular diversity. Methods A total of 321 hepatitis B surface antigen (HBsAg) positives (125 HIV co-infected, 102 liver disease patients and 94 blood donors) were screened for anti-HDV antibody. The anti-HDV positive sera were subjected to Real time PCR for HDV-RNA confirmation. The non coding genome region (spanning from 467 to 834 nucleotides) commonly used for HDV genotyping as well as complete HDV genome were sequenced for genotyping and molecular analysis. Results The anti-HDV antibody was found to be 3.2% (3) in blood donors, 8.0% (10) in HIV co-infected individuals and 12.7% (13) in liver disease patients. None of the HIV co-infected patients who revealed HBV lamivudine (3TC) resistance at tyrosine-methionine/isoleucine-aspartate-aspartate (YM(I)DD) reverse transcriptase (RT) motif with concomitant vaccine escape gene mutants was positive for anti-HDV antibody. The HDV viremia rate was 33.3%, 30.0% and 23.1% in respect to the above study groups. All the six isolates sequenced were phylogenetically classified as HDV genotype 1 (HDV-1) and grouped into two monophyletic clusters. Amino acid (aa) residues analysis of clathrin heavy chain (CHC) domain and the isoprenylation signal site (Py) at 19 carboxyl (C)-terminal amino acids (aa 196–214) and the HDV RNA binding domain (aa 79–107) were highly conserved and showed a very little nucleotide variations. All the sequenced isolates showed serine at amino acid position 202. The RNA editing targets of the anti-genomic HDV RNA (nt1012) and its corresponding genomic RNA (nt 580) showed nucleotides A and C, respectively. Conclusions The low seroprevalence and viraemic rates of HDV in particular during HIV-confection might be highly affected by HBV drug resistance selected HBsAg mutant variants in this setting, although HDV-1 sequences analysis revealed clade homogeneity and highly conserved structural and functional domains. Thus, the potential role of HBV drug resistance associated polymerase mutations and concomitant HBsAg protein variability on HDV viral assembly, secretion and infectivity needs further investigation.

[This corrects the article DOI: 10.1371/journal.pone.0205119.].

We recently reported complex hepatitis B virus (HBV) drug resistant and concomitant vaccine escape hepatitis B surface antigen (HBsAg) variants during human immunodeficiency virus (HIV) co-infection and antiretroviral therapy (ART) exposure in Ethiopia. As a continuation of this report using the HBV positive sera from the same study participants, the current study further analyzed the HBV basal core promoter (BCP)/precore (PC) genes variability in patients with HBV drug resistance (at tyrosine-methionine-aspartate-aspartate (YMDD) reverse transcriptase (RT) motifs) and HIV co-infection in comparison with HBV mono-infected counterparts with no HBV drug resistant gene variants.A total of 143 participants of HBV-HIV co-infected (n = 48), HBV mono-infected blood donors (n = 43) and chronic liver disease (CLD) patients (n = 52) were included in the study. The BCP/PC genome regions responsible for HBeAg expression from the EcoRI site (nucleotides 1653-1959) were sequenced and analyzed for the BCP/PC mutant variants.Among the major mutant variants detected, double BCP mutations (A1762T/G1764A) (25.9%), Kozak sequences mutations (nt1809-1812) (51.7%) and the classical PC mutations such as A1814C/C1816T (15.4%), G1896A (25.2%) and G1862T (44.8%) were predominant mutant variants. The prevalence of the double BCP mutations was significantly lower in HIV co-infected patients (8.3%) compared with HBV mono-infected blood donors (32.6%) and CLD patients (36.5%). However, the Kozak sequences BCP mutations and the majority of PC mutations showed no significant differences among the study groups. Moreover, except for the overall BCP/PC mutant variants, co-prevalence rates of each major BCP/PC mutations and YMDDRT motif associated lamivudine (3TC)/entecavir (ETV) resistance mutations showed no significant differences when compared with the rates of BCP/PC mutations without YMDD RT motif drug resistance gene mutations. Unlike HIV co-infected group, no similar comparison made among HBV mono-infected blood donors and CLD patients since none of them developed the YMDD RT motif associated 3TC/ETV resistance mutations. However, HBV mono-infected blood donors and CLD patients who had no any drug resistance gene variants developed comparable G1862T (60.6% vs. 65.1%) and G1896A (24.2% vs. 11.6%) PC gene mutations.No correlation observed between the BCP/PC genome variability and the YMDD RT motif associated HBV drug resistance gene variants during HIV co-infection. Nevertheless, irrespective of HIV co-infection status, the higher records of the BCP/PC gene variability in this study setting indicate a high risk of potential HBeAg negative chronic HBV infection in Northwest Ethiopia.

BACKGROUND:Using HIV proviral DNA as a template may be suitable for initial detection of transmitted drug resistance mutations (TDRMs) as it is easy to handle and less expensive compared to RNA. However, existing literatures which are mainly focused on HIV-1B subtypes DNA extracted from PBMCs revealed controversial findings ranging from the detection of significantly lower or higher mutations in proviral DNA compared to historic viral RNA. Thus, to verify whether viral RNA or proviral DNA has improved sensitivity in detecting transmitted genotypic drug resistance mutations paired viral RNA and proviral DNA (which is directly extracted from stored whole blood) samples were tested from Ethiopian antiretroviral naive HIV-1C infected subjects. METHODS:In the present comparative study the frequency of TDR mutations was assessed in paired samples of viral RNA and proviral DNA (extracted directly from stored whole blood) of HIV-1C infected treatment naïve patients and interpreted using the 2009 WHO drug resistance surveillance mutation lists, Stanford University drug resistance data base and International Antiviral Society-USA mutation lists. RESULTS:High agreement in rate of TDR between the two compartments was observed using the WHO mutation lists. While mutations G190A and E138A were concurrently found in both compartments, others such as G73S on PR and A62V, M184I, M230I on RT were identified in proviral DNA only. All signature mutations seen in viral RNA were not missed in proviral DNA. CONCLUSIONS:The concordance of major genotype drug resistance mutation between RNA and proviral DNA in treatment naïve patients suggests that proviral DNA might be an alternative approaches for an initial assessment of drug resistance prior to initiation of antiretroviral therapy using the WHO mutations lists in resource-limited countries. However, the clinical importance of TDRMs observed only in proviral DNA in terms of being a risk factor for virologic failure and whether they limit future treatment options needs additional investigation using more sensitive sequencing approaches such as Next Generation Sequencing (NGS).

The emergence, accumulation and spread of HIV-1 drug resistance strains in Africa could compromise the effectiveness of HIV treatment programs. This study was aimed at determining the incidence of virological failure and acquired drug resistance mutations overtime and identifying the most common mutational pathways of resistance in a well characterized HIV-1C infected Ethiopian cohort.A total of 320 patients (220 ART naïve and 100 on first lines ART) were included and followed. ART initiation and patients' monitoring was based on the WHO clinical and immunological parameters. HIV viral load measurement and genotypic drug resistance testing were done at baseline (T0-2008) and after on average at a median time of 30 months on ART at three time points (T1-2011, T2-2013, T3-2015).The incidence of virological failure has increased overtime from 11 at T1 to 17 at T2 and then to 30% at T3. At all time point's almost all of the patients with virological failure and accumulated drug resistance mutations had not met the WHO clinical and immunologic failure criteria and continued the failing regimen. A steep increase in the incidence and accumulation of major acquired NRTI and NNRTI drug resistance mutations have been observed (from 40% at T1 to 64% at T2 and then to 66% at T3). The most frequent NRTIs drug resistance associated mutations are mainly the lamivudine-induced mutation M184V which was detected in 4 patients at T1 and showed a 2 fold increase in the following time points (T2: n = 8) and at (T3: n = 12) and the thymidine analogue mutations (such as D67N, K70R and K219E) which were not-detected at baseline T0 and T1 but were increased progressively to 10 at T2 and to 17 at T3. The most frequent NNRTIs associated mutations were K103N, V106M and Y188C.An upward trend in the incidence of virological failure and accumulation of NRTI and NNRTI associated acquired antiretroviral drug resistance mutations are observed. The data suggest the need for virological monitoring, resistance testing for early detection of failure and access for TDF and PI containing drugs. Population-level and patient targeted interventions to prevent the spread of mutant variants is warranted.

Abstract Background Helminth infections as well as structural alternations in the long-terminal repeat (LTR) regions of HIV-1 are known to contribute to elevated HIV RNA level and enhance HIV-1 diseases progression. However, the impact of helminths infections on the occurrences of triple NF-κB and genetic variability in LTR region of HIV-1C isolates is not known. We aimed to examine the presence of genetic variability in the LTR region of HIV-1C isolates during chronic HIV-helminth co-infection. Methods HIV-1C infected Ethiopians with (n = 22) and without (n = 20) helminth infection were included. The LTR region of HIV was amplified and sequenced. Sequences were aligned with reference set from the Los Alamos HIV database. Phylogenetic analysis and frequency of polymorphic changes was performed by the neighbour-joining method using Geneious Basic software. Results All LTR sequences from patients with or without of helminth co-infection clustered with HIV-1 subtype C with two distinct subclusters (C and C’). The enhancer element was found to have three copies of 10-base pair binding sites for NF-κBs which is an evidence for predominance of triple NF-κB sites (94%) in HIV-1C isolates irrespective of helminths co-infection and subclusters. Moreover, irrespective of helminth co-infection and C/C’ subclusters high sequences similarity in LTR was observed. There was no significant difference in plasma HIV RNA level between HIV-1 C and C’ subclusters. Conclusions Despite the small sample size, the predominance of triple NF-κB binding sites and high sequence similarities in LTR region irrespective of helminths infection suggest the natural occurrence of the three NF-κB binding sites in HIV-1C isolates without the influence of secondary infection. Thus, the higher HIV-1C viraemia in helminth co-infected individuals is more likely a result of immune activation rather than LTR sequence variation. Moreover, the lack of significant difference in plasma HIV RNA level between HIV-1 C and C’ subcluster may show the lack of functional differences among the two groups.

The emergence of HIV-1 drug resistance mutations has mainly been linked to the duration and composition of antiretroviral treatment (ART), as well as the level of adherence. This study reports the incidence and pattern of acquired antiretroviral drug resistance mutations and long-term outcomes of ART in a prospective cohort from Northwest Ethiopia. Two hundred and twenty HIV-1C infected treatment naïve patients were enrolled and 127 were followed-up for up to 38 months on ART. ART initiation and patients' monitoring was based on the WHO clinical and immunological parameters. HIV viral RNA measurement and drug resistance genotyping were done at baseline (N = 160) and after a median time of 30 (IQR, 27-38) months on ART (N = 127). Viral suppression rate (HIV RNA levels ≤ 400 copies/ml) after a median time of 30 months on ART was found to be 88.2% (112/127), which is in the range for HIV drug resistance prevention suggested by WHO. Of those 15 patients with viral load >400 copies/ml, six harboured one or more drug resistant associated mutations in the reverse transcriptase (RT) region. Observed NRTIs resistance associated mutations were the lamivudine-induced mutation M184V (n = 4) and tenofovir associated mutation K65R (n = 1). The NNRTIs resistance associated mutations were K103N (n = 2), V106M, Y181S, Y188L, V90I, K101E and G190A (n = 1 each). Thymidine analogue mutations and major drug resistance mutations in the protease (PR) region were not detected. Most of the patients (13/15) with virologic failure and accumulated drug resistance mutations had not met the WHO clinical and/or immunological failure criteria and continued the failing regimen. The incidence and pattern of acquired antiretroviral drug resistance mutations is lower and less complex than previous reports from sub Saharan Africa countries. Nevertheless, the data suggest the need for virological monitoring and resistance testing for early detection of failure. Moreover, adherence reinforcement will contribute to improving overall treatment outcomes.

BACKGROUND:Successful therapy of chronic hepatitis B with nucleos(t)ide analogues (NUCs) has been defined by undetectable HBV-DNA determined with conventional PCR (lower limit of detection (LLD) 60-80 IU/mL) in clinical registration trials. However, current EASL guidelines recommend highly sensitive real-time PCR (LLD