Use of in silico approaches, synthesis and profiling of Pan-filovirus GP-1,2 preprotein specific antibodies.

Intermolecular interactions of protein-protein complexes play a principal role in the process of discovering new substances used in the diagnosis and treatment of many diseases. Among such complexes of proteins, we have to mention antibodies; they interact with specific antigens of two genera of single-stranded RNA viruses belonging to the family Filoviridae-Ebolavirus and Marburgvirus; both cause rare but fatal viral hemorrhagic fever in Africa, with pandemic potential. In this research, we conduct studies aimed at the design and evaluation of antibodies targeting the filovirus glycoprotein precursor GP-1,2 to develop potential targets for the pan-filovirus easy-to-use rapid diagnostic tests. The in silico research using the available 3D structure of the natural antibody-antigen complex was carried out to determine the stability of individual protein segments in the process of its formation and maintenance. The computed free binding energy of the complex and its decomposition for all amino acids allowed us to define the residues that play an essential role in the structure and indicated the spots where potential antibodies can be improved. Following that, the study involved targeting six epitopes of the filovirus GP1,2 with two polyclonal antibodies (pABs) and 14 monoclonal antibodies (mABs). The evaluation conducted using Enzyme Immunoassays tested 62 different sandwich combinations of monoclonal antibodies (mAbs), identifying 10 combinations that successfully captured the recombinant GP1,2 (rGP). Among these combinations, the sandwich option (3G2G12* - (rGP) - 2D8F11) exhibited the highest propensity for capturing the rGP antigen.

Long-term non-progression and risk factors for disease progression among children living with HIV in Botswana and Uganda: A retrospective cohort study.

OBJECTIVES: We utilize a large retrospective study cohort derived from electronic medical records to estimate the prevalence of long-term non-progression (LTNP) and determine the factors associated with progression among children infected with HIV in Botswana and Uganda. METHODS: Electronic medical records from large tertiary HIV clinical centers in Botswana and Uganda were queried to identify LTNP children 0-18 years enrolled between June 2003 and May 2014 and extract demographic and nutritional parameters. Multivariate subdistribution hazard analyses were used to examine demographic factors and nutritional status in progression in the pre-antiretroviral therapy era. RESULTS: Between the two countries, 14,246 antiretroviral therapy-naïve children infected with HIV were enrolled into clinical care. The overall proportion of LTNP was 6.3% (9.5% in Botswana vs 5.9% in Uganda). The median progression-free survival for the cohort was 6.3 years, although this was lower in Botswana than in Uganda (6.6 vs 8.8 years; P <0.001). At baseline, the adjusted subdistribution hazard ratio (aHRsd) of progression was increased among underweight children (aHRsd 1.42; 95% confidence interval [CI]: 1.32-1.53), enrolled after 2010 (aHRsd 1.32; 95% CI 1.22-1.42), and those from Botswana (aHRsd 2; 95% CI 1.91-2.10). CONCLUSIONS: In our study, the prevalence of pediatric LTNP was lower than that observed among adult populations, but progression-free survival was higher than expected. Underweight, year of enrollment into care, and country of origin are independent predictors of progression among children.

A Cross-Sectional Study on Knowledge and Attitudes About Organ Donation and Transplantation in an Urban Population in a Low-Income Country.

BACKGROUND: Uganda's Health Sector Development Plan (2015/16 through 2019/2020) noted that most referrals for treatment abroad were for organ transplant services that cost the government >5.6 million USD. The government of Uganda has invested in building the capacity for organ donation and transplantation services by training human resources personnel and setting up infrastructure in Kampala, where these services can be accessed. However, there is no information on the readiness of communities and the scientific community to embrace (communities) or undertake (science) organ transplantation in the country. We set out to assess knowledge and attitudes about organ donation and transplantation among the urban population in Kampala. METHODS: We conducted a cross-sectional survey among 395 participants from the urban population of Kampala at Garden City Mall, Wandegeya market, and Nakawa market from 28 May through 7 June 2021. We asked about knowledge of organ donation and transplantation, collected sociodemographic data, and performed a sentiment analysis of participants' attitudes toward organ donation and transplantation. RESULTS: The M:F ratio of participants was 1:1; the majority (55.9%) of participants were Baganda, two-thirds of participants knew about organ donation, and 90% of participants did not know of any government policy on organ donation and transplantation. Radio/television was the most common source of information, and the kidney was the most frequently transplanted organ. Overall, there were 94.3% and 93.2% positive sentiments toward organ transplantation and organ donation, respectively. The need for stricter laws governing organ donation and transplantation, corruption, and fear were the main negative sentiments expressed by participants. CONCLUSIONS: Sensitization of the community is required regarding government policy on organ donation and transplantation, and this should be communicated through radio/television and social media. There was a positive attitude toward organ donation and transplantation.

Continental concerted efforts to control the seventh outbreak of Ebola Virus disease in Uganda: The first 90 days of the response.

On 20th September 2022, Uganda declared the 7th outbreak of Ebola virus disease (EVD) caused by the Sudan Ebola strain following the confirmation of a case admitted at Mubende Regional Referral Hospital. Upon confirmation, the Government of Uganda immediately activated the national incident management system to initiate response activities. Additionally, a multi-country emergency stakeholder meeting was held in Kampala; convening Ministers of Health from neighbouring Member States to undertake cross-border preparedness and response actions. The outbreak spanned 69 days and recorded 164 cases (142 confirmed, 22 probable), 87 recoveries and 77 deaths (case fatality ratio of 47%). Nine out of 136 districts were affected with transmission taking place in 5 districts but spilling over in 4 districts without secondary transmission. As part of the response, the Government galvanised robust community mobilisation and initiated assessment of medical counter measures including therapeutics, new diagnostics and vaccines. This paper highlights the response actions that contributed to the containment of this outbreak in addition to the challenges faced with a special focus on key recommendations for better control of future outbreaks.

Safety and feasibility of autologous adipose-derived stromal vascular fraction in the treatment of keloids: a phase one randomized controlled pilot trial.

INTRODUCTION: Autologous adipose-derived stromal vascular fraction (SVF) has been described to have therapeutic benefits in the treatment of keloids. However, most of the evidence on its efficacy is based on observational studies the majority of which are conducted in high-income countries and yet the highest burden of keloids is in low- and middle-income countries (LMICs). OBJECTIVES: We set out to determine the safety and feasibility of using autologous adipose derived stromal vascular fraction in the treatment of keloids in LMICs. METHODS: In this phase II randomized controlled pilot clinical trial conducted in the Plastic Surgery Unit of Kirruddu National Referral Hospital in Kampala Uganda, 8 patients were assigned a 1:1 ratio to either SVF or triamcinolone acetonide (TAC) arms. In the SVF arm, a median (Inter quartile range) amount of stromal cell infiltration of 2.7×106 (11×106) was administered, while the controls received 10 mg/ml TAC at a ratio of 1:1 TAC to keloid volume. Primary endpoints were adverse event development based on the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 tool and feasibility assessment based on ≥ 70% recruitment feasibility and ≥ 80% interventional feasibility rates. RESULTS: The participants' mean age was 27.9 (±6.5) years, with a female predilection of 5 (63%). Overall, no adverse events were reported in the SVF arm, while ulceration in a single patient in the TAC arm, which was a grade II adverse event, was reported. Recruitment feasibility of 80% and interventional feasibility with 100% completion were reported. CONCLUSION: Based on our findings, an autologous adipose-derived stromal vascular fraction is feasible and safe for the treatment of keloids in LMICs.

Autologous adipose-derived stromal vascular fraction (SVF) in scar treatment among patients with keloids and hypertrophic scars: a systematic review and meta-analysis of current practices and outcomes.

BACKGROUND: Keloids and hypertrophic scars are some of the most common skin conditions globally, associated with poor treatment response and high recurrence rates. Autologous adipose-derived stromal vascular fraction (SVF) is increasingly recognized as an emerging therapy albeit limited literature on its outcome in scar treatment. This review aimed to describe the current practices and outcomes of adipose-derived stromal Vascular Fraction in scar treatment. METHODS: This systematic review assessed articles describing the use of SVF in scar treatment published between 2000 and 2023. Article searches of Medline/PubMed, Cochrane Library, and Embase databases using Mesh terms and the Boolean operators ("AND", "OR") by two independent researchers were done whilst following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Clinical studies assessing SVF in scar treatment with a primary outcome measure being an improvement in scar characteristics including the thickness, scar assessment scores were included. RESULTS: Among the 1425 studies identified in the search, 20 studies met the inclusion criteria with a total of 493 patients included. Eight of these were clinical trials with the rest being observational studies. Follow-up ranged from 3 months to 24 months. In all studies, there was an improvement in scar characteristics following single-dose treatment with SVF or its equivalent. All studies reported SVF to be safe. CONCLUSION: The review found that autologous adipose-derived SVF is a clinically effective therapy for keloids and scar treatment.

Adipose-derived stromal vascular fraction (SVF) in scar treatment: a systematic review protocol.

BACKGROUND: Autologous adipose-derived stromal vascular fraction (SVF) is an emerging therapy that is being pioneered as a potential treatment for keloids and hypertrophic scars. Up to this point, there isn't a cure for keloids and hypertrophic scars yet they comprise the commonest benign skin disorders. Despite published studies reporting potential therapeutic benefits of SVF, their use and efficacy on scar improvement are not clearly described. The aim of this review is to describe the clinical practice involved in harvesting, processing, utilization of SVF, and associated efficacy in scar treatment. METHODS: We shall include published clinical articles evaluating the efficacy of SVF on improving scar characteristics and assessment scores among adults with keloids or hypertrophic scars. Article search of Medline/PubMed, Cochrane Library and Embase using Mesh terms of "scars" and "stromal vascular fraction" combined with the Boolean operators ("AND", "OR") will be performed by two independent researchers following the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) statement. The primary outcome measure will be the mean difference in the Scar characteristics including Scar assessment scores, scar thickness among others. DATA SYNTHESIS: Descriptive data synthesis and mean differences between treatment arms will be calculated for the primary outcome of the scar assessment scores. In case more than three studies provide consistent characteristics of the scar assessment scores, a meta-analysis will be conducted. DISCUSSION: Evidence obtained from the systematic review will form the foundation upon which further clinical trials research will be conducted in evaluating the efficacy of autologous adipose-derived stromal vascular fraction in keloid and hypertrophic scar. The systematic review has been submitted to the PROSPERO database and is currently under review.

Safety and efficacy of hydroxychloroquine for treatment of non-severe COVID-19 among adults in Uganda: a randomized open label phase II clinical trial.

BACKGROUND: Several repurposed drugs such as hydroxychloroquine (HCQ) have been investigated for treatment of COVID-19, but none was confirmed to be efficacious. While in vitro studies have demonstrated antiviral properties of HCQ, data from clinical trials were conflicting regarding its benefit for COVID-19 treatment. Drugs that limit viral replication may be beneficial in the earlier course of the disease thus slowing progression to severe and critical illness. DESIGN: We conducted a randomized open label Phase II clinical trial from October-December 2020. METHODS: Patients diagnosed with COVID-19 using RT-PCR were included in the study if they were 18 years and above and had a diagnosis of COVID-19 made in the last 3 days. Patients were randomized in blocks, to receive either HCQ 400 mg twice a day for the first day followed by 200 mg twice daily for the next 4 days plus standard of care (SOC) treatment or SOC treatment alone. SARS COV-2 viral load (CT values) from RT-PCR testing of samples collected using nasal/orapharyngeal swabs was performed at baseline, day 2, 4, 6, 8 and 10. The primary outcome was median time from randomization to SARS COV-2 viral clearance by day 6. RESULTS: Of the 105 participants enrolled, 55 were assigned to the intervention group (HCQ plus SOC) and 50 to the control group (SOC only). Baseline characteristics were similar across treatment arms. Viral clearance did not differ by treatment arm, 20 and 19 participants respectively had SARS COV-2 viral load clearance by day 6 with no significant difference, median (IQR) number of days to viral load clearance between the two groups was 4(3-4) vs 4(2-4): p = 0.457. There were no significant differences in secondary outcomes (symptom resolution and adverse events) between the intervention group and the control group. There were no significant differences in specific adverse events such as elevated alkaline phosphatase, prolonged QTc interval on ECG, among patients in the intervention group as compared to the control group. CONCLUSION: Our results show that HCQ 400 mg twice a day for the first day followed by 200 mg twice daily for the next 4 days was safe but not associated with reduction in viral clearance or symptom resolution among adults with COVID-19 in Uganda. TRIAL REGISTRATION:  NCT04860284.

Exome Sequencing Reveals a Putative Role for HLA-C*03:02 in Control of HIV-1 in African Pediatric Populations.

Human leucocyte antigen (HLA) class I molecules present endogenously processed antigens to T-cells and have been linked to differences in HIV-1 disease progression. HLA allelotypes show considerable geographical and inter-individual variation, as does the rate of progression of HIV-1 disease, with long-term non-progression (LTNP) of disease having most evidence of an underlying genetic contribution. However, most genetic analyses of LTNP have occurred in adults of European ancestry, limiting the potential transferability of observed associations to diverse populations who carry the burden of disease. This is particularly true of HIV-1 infected children. Here, using exome sequencing (ES) to infer HLA allelotypes, we determine associations with HIV-1 LTNP in two diverse African pediatric populations. We performed a case-control association study of 394 LTNPs and 420 rapid progressors retrospectively identified from electronic medical records of pediatric HIV-1 populations in Uganda and Botswana. We utilized high-depth ES to perform high-resolution HLA allelotyping and assessed evidence of association between HLA class I alleles and LTNP. Sixteen HLA alleles and haplotypes had significantly different frequencies between Uganda and Botswana, with allelic differences being more prominent in HLA-A compared to HLA-B and C allelotypes. Three HLA allelotypes showed association with LTNP, including a novel association in HLA-C (HLA-B∗57:03, aOR 3.21, Pc = 0.0259; B∗58:01, aOR 1.89, Pc = 0.033; C∗03:02, aOR 4.74, Pc = 0.033). Together, these alleles convey an estimated population attributable risk (PAR) of non-progression of 16.5%. We also observed novel haplotype associations with HLA-B∗57:03-C∗07:01 (aOR 5.40, Pc = 0.025) and HLA-B∗58:01-C∗03:02 (aOR 4.88, Pc = 0.011) with a PAR of 9.8%, as well as a previously unreported independent additive effect and heterozygote advantage of HLA-C∗03:02 with B∗58:01 (aOR 4.15, Pc = 0.005) that appears to limit disease progression, despite weak LD (r 2 = 0.18) between these alleles. These associations remained irrespective of gender or country. In one of the largest studies of HIV in Africa, we find evidence of a protective effect of canonical HLA-B alleles and a novel HLA-C association that appears to augment existing HIV-1 control alleles in pediatric populations. Our findings outline the value of using multi-ethnic populations in genetic studies and offer a novel HIV-1 association of relevance to ongoing vaccine studies.

Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings.

Background: In January 2020, a previously unknown coronavirus strain was identified as the cause of a severe acute respiratory syndrome (SARS-CoV-2). The first viral whole-genome was sequenced using high-throughput sequencing from a sample collected in Wuhan, China. Whole-genome sequencing (WGS) is imperative in investigating disease outbreak transmission dynamics and guiding decision-making in public health. Methods: We retrieved archived SARS-CoV-2 samples at the Integrated Biorepository of H3Africa Uganda, Makerere University (IBRH3AU). These samples were collected previously from individuals diagnosed with coronavirus disease 2019 (COVID-19) using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). 30 samples with cycle thresholds (Cts) values <25 were selected for WGS using SARS-CoV-2 ARTIC protocol at Makerere University Molecular Diagnostics Laboratory. Results: 28 out of 30 (93.3%) samples generated analyzable genomic sequence reads. We detected SARS-CoV-2 and lineages A (22/28) and B (6/28) from the samples. We further show phylogenetic relatedness of these isolates alongside other 328 Uganda (lineage A = 222, lineage B = 106) SARS-CoV-2 genomes available in GISAID by April 22, 2021 and submitted by the Uganda Virus Research Institute. Conclusions: Our study demonstrated adoption and optimization of the low-cost ARTIC SARS-CoV-2 WGS protocol in a resource limited laboratory setting. This work has set a foundation to enable rapid expansion of SARS-CoV-2 WGS in Uganda as part of the Presidential Scientific Initiative on Epidemics (PRESIDE) CoV-bank project and IBRH3AU.

Unmapped exome reads implicate a role for Anelloviridae in childhood HIV-1 long-term non-progression.

Human immunodeficiency virus (HIV) infection remains a significant public health burden globally. The role of viral co-infection in the rate of progression of HIV infection has been suggested but not empirically tested, particularly among children. We extracted and classified 42 viral species from whole-exome sequencing (WES) data of 813 HIV-infected children in Botswana and Uganda categorised as either long-term non-progressors (LTNPs) or rapid progressors (RPs). The Ugandan participants had a higher viral community diversity index compared to Batswana (p = 4.6 × 10-13), and viral sequences were more frequently detected among LTNPs than RPs (24% vs 16%; p = 0.008; OR, 1.9; 95% CI, 1.6-2.3), with Anelloviridae showing strong association with LTNP status (p = 3 × 10-4; q = 0.004, OR, 3.99; 95% CI, 1.74-10.25). This trend was still evident when stratified by country, sex, and sequencing platform, and after a logistic regression analysis adjusting for age, sex, country, and the sequencing platform (p = 0.02; q = 0.03; OR, 7.3; 95% CI, 1.6-40.5). Torque teno virus (TTV), which made up 95% of the Anelloviridae reads, has been associated with reduced immune activation. We identify an association between viral co-infection and prolonged AIDs-free survival status that may have utility as a biomarker of LTNP and could provide mechanistic insights to HIV progression in children, demonstrating the added value of interrogating off-target WES reads in cohort studies.

Ebola Virus Disease and Current Therapeutic Strategies: A Review.

The Ebola virus disease is a severe hemorrhagic fever that affects humans and other primates. Ebola virus, the causative agent of the disease, is transmitted to humans from wild animals and is highly contagious and aggressive with an estimated fatality rate to be around 50%. Since 1976, 11 outbreaks of Ebola virus disease have been reported in total, affecting mostly sub-Saharan Africa, while the most recent ongoing outbreak in the Democratic Republic of the Congo has more than 3000 reported cases and 72 deaths. Although an effective vaccine against Ebola virus disease has become available, no targeted treatment with proven efficacy upon infection is developed. Herein, we review the epidemiology of Ebola virus and the current situation in terms of prevention, diagnosis, and treatment of the disease.

Variations in Trim5α and Cyclophilin A genes among HIV-1 elite controllers and non controllers in Uganda: a laboratory-based cross-sectional study.

BACKGROUND: Tripartite Motif Containing 5 alpha (TRIM5α), a restriction factor produced ubiquitously in cells and tissues of the body plays an important role in the immune response against HIV. TRIM5α targets the HIV capsid for proteosomal destruction. Cyclophilin A, an intracellular protein has also been reported to influence HIV infectivity in a cell-specific manner. Accordingly, variations in TRIM5α and Cyclophilin A genes have been documented to influence HIV-1 disease progression. However, these variations have not been documented among Elite controllers in Uganda and whether they play a role in viral suppression remains largely undocumented. Our study focused on identifying the variations in TRIM5α and Cyclophilin A genes among HIV-1 Elite controllers and non-controllers in Uganda. RESULTS: From the sequence analysis, the rs10838525 G > A mutation in exon 2 of TRIM5α was only found among elite controllers (30%) while the rs3824949 in the 5'UTR was seen among 25% of the non-controllers. In the Cyclophilin A promoter, rs6850 was seen among 62.5% of the non-controllers and only among 10% elite controllers. Furthermore, rs17860048 in the Cyclophillin A promoter was predominantly seen among elite controllers (30%) and 12.5% non-controllers. From gene expression analysis, we noted that the respective genes were generally elevated among elite controllers, however, this difference was not statistically significant (TRIM5α p = 0.6095; Cyclophilin A p = 0.6389). CONCLUSION: Variations in TRIM5α and Cyclophillin A promoter may influence HIV viral suppression. The rs10838525 SNP in TRIM5α may contribute to viral suppression among HIV-1 elite controllers. The rs6850 in the cyclophillin A gene may be responsible for HIV-1 rapid progression among HIV-1 non-controllers. These SNPs should be investigated mechanistically to determine their precise role in HIV-1 viral suppression.

Identity and validity of conserved B cell epitopes of filovirus glycoprotein: towards rapid diagnostic testing for Ebola and possibly Marburg virus disease.

BACKGROUND: Ebolavirus and Marburgvirus are genera of the virus family Filoviridae. Filoviruses cause rare but fatal viral hemorrhagic fevers (VHFs) in remote villages of equatorial Africa with potential for regional and international spread. Point-of-care (POC) rapid diagnostic tests (RDTs) are critical for early epidemic detection, reponse and control. There are 2 RDTs for Zaire ebolavirus (EBOV), but not other Ebolavirus spp. or Marburg marburgvirus (MARV). We validate 3 conserved B cell epitopes of filovirus glycoprotein (GP) using ebola virus diseases (EVD) survivor samples, towards devising pan-filovirus RDTs. METHODS: In-silico Immuno-informatics:- (a) multiple and basic local alignments of amino-acid sequences of filovirus (4 Ebolavirus spp. & MARV) Gp1, 2 and epitope prediction and conservation analyses within context of ClusterW, BLAST-P and the immune epitope database analysis resource (IEDB-AR); alongside (b) in-vitro enzyme immuno-assays (EIAs) for SUDV Gp1, 2 antigen and host-specific antibodies (IgM and IgG) among 94 gamma irradiated EVD survivor serum and 9 negative controls. RESULTS: Linear B cell epitopes were present across the entire length of all Gp1, 2, most lying in the region between amino acids positioned 350 and 500. Three seperate epitopes 97/80_GAFFLYDRLAST, 39_YEAGEWAENCY and 500_CGLRQLANETTQALQLFLRATTELR (designated UG-Filo-Peptide- 1, 2 and 3 respectively) were conserved within all studied filovirus species Gp1, 2. Gp1, 2 host specific IgM levels were comparably low (av. ODs < 0.04 [95% CI: 0.02837 to 0.04033]) among the 9 negative controls and 57 survivor samples analyzed. Host specific IgG levels, on the other hand, were elevated (av. ODs > 1.7525 [95% CI: 0.3010 to 3.1352]) among the 92 survivor samples relative to the 9 negative controls (av. ODs < 0.2.321 [95% CI: -0.7596 to 0.5372]). Filovirus Gp1, 2 antigen was not detected [av. ODs < 0.20] within EVD survivor serum relative to recombinant protein positive controls [av. ODs = 0.50]. CONCLUSIONS: These conserved B cell epitopes of filovirus Gp1, 2 and their derivative antibodies are promising for research and development of RDTs for EVD, with potential for extension to detect MVD.

Whole-Exome Sequencing Reveals Uncaptured Variation and Distinct Ancestry in the Southern African Population of Botswana.

Large-scale, population-based genomic studies have provided a context for modern medical genetics. Among such studies, however, African populations have remained relatively underrepresented. The breadth of genetic diversity across the African continent argues for an exploration of local genomic context to facilitate burgeoning disease mapping studies in Africa. We sought to characterize genetic variation and to assess population substructure within a cohort of HIV-positive children from Botswana-a Southern African country that is regionally underrepresented in genomic databases. Using whole-exome sequencing data from 164 Batswana and comparisons with 150 similarly sequenced HIV-positive Ugandan children, we found that 13%-25% of variation observed among Batswana was not captured by public databases. Uncaptured variants were significantly enriched (p = 2.2 × 10-16) for coding variants with minor allele frequencies between 1% and 5% and included predicted-damaging non-synonymous variants. Among variants found in public databases, corresponding allele frequencies varied widely, with Botswana having significantly higher allele frequencies among rare (<1%) pathogenic and damaging variants. Batswana clustered with other Southern African populations, but distinctly from 1000 Genomes African populations, and had limited evidence for admixture with extra-continental ancestries. We also observed a surprising lack of genetic substructure in Botswana, despite multiple tribal ethnicities and language groups, alongside a higher degree of relatedness than purported founder populations from the 1000 Genomes project. Our observations reveal a complex, but distinct, ancestral history and genomic architecture among Batswana and suggest that disease mapping within similar Southern African populations will require a deeper repository of genetic variation and allelic dependencies than presently exists.