Retrospective in silico mutation profiling of SARS-CoV-2 structural proteins circulating in Uganda by July 2021: Towards refinement of COVID-19 disease vaccines, diagnostics, and therapeutics.

The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensive in silico analysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or the Alpha, Beta, Delta and Gamma variants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas the Delta dominated the spike sequences (16.5%, 91/550), Gamma was not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

Crimean-Congo Hemorrhagic Fever Outbreak in Refugee Settlement during COVID-19 Pandemic, Uganda, April 2021.

Crimean-Congo hemorrhagic fever (CCHF) was detected in 2 refugees living in a refugee settlement in Kikuube district, Uganda. Investigations revealed a CCHF IgG seroprevalence of 71.3% (37/52) in goats within the refugee settlement. This finding highlights the need for a multisectoral approach to controlling CCHF in humans and animals in Uganda.

Rift Valley Fever Outbreak during COVID-19 Surge, Uganda, 2021.

Rift Valley fever, endemic or emerging throughout most of Africa, causes considerable risk to human and animal health. We report 7 confirmed Rift Valley fever cases, 1 fatal, in Kiruhura District, Uganda, during 2021. Our findings highlight the importance of continued viral hemorrhagic fever surveillance, despite challenges associated with the COVID-19 pandemic.

Whole-genome analysis to determine the rate and patterns of intra-subtype reassortment among influenza type-A viruses in Africa.

Influenza type-A viruses (IAVs) present a global burden of human respiratory infections and mortality. Genome reassortment is an important mechanism through which epidemiologically novel influenza viruses emerge and a core step in the safe reassortment-incompetent live-attenuated influenza vaccine development. Currently, there are no data on the rate, spatial and temporal distribution, and role of reassortment in the evolution and diversification of IAVs circulating in Africa. We aimed to detect intra-subtype reassortment among Africa pandemic H1N1pdm09 (2009-10), seasonal H1N1pdm09 (2011-20), and seasonal H3N2 viruses and characterize the genomic architecture and temporal and spatial distribution patterns of the resulting reassortants. Our study was nested within the Uganda National Influenza Surveillance Programme. Next-generation sequencing was used to generate whole genomes (WGs) from 234 H1N1pdm09 (n = 116) and H3N2 (n = 118) viruses sampled between 2010 and 2018 from seven districts in Uganda. We combined our newly generated WGs with 658 H1N1pdm09 and 1131 H3N2 WGs sampled between 1994 and 2020 across Africa and identified reassortants using an automated Graph Incompatibility Based Reassortment Finder software. Viral reassortment rates were estimated using a coalescent reassortant constant population model. Phylogenetic analysis was used to assess the effect of reassortment on viral genetic evolution. We observed a high frequency of intra-subtype reassortment events, 12 · 4 per cent (94/758) and 20 · 9 per cent (256/1,224), and reassortants, 13 · 3 per cent (101/758) and 38 · 6 per cent (472/1,224), among Africa H1N1pdm09 and H3N2 viruses, respectively. H1N1pdm09 reassorted at higher rates (0.1237-0.4255) than H3N2 viruses (0 · 00912-0.0355 events/lineage/year), a case unique to Uganda. Viral reassortants were sampled in 2009 through 2020, except in 2012. 78 · 2 per cent (79/101) of H1N1pdm09 reassortants acquired new non-structural, while 57 · 8 per cent (273/472) of the H3N2 reassortants had new hemagglutinin (H3) genes. Africa H3N2 viruses underwent more reassortment events involving larger reassortant sets than H1N1pdm09 viruses. Viruses with a specific reassortment architecture circulated for up to five consecutive years in specific countries and regions. The Eastern (Uganda and Kenya) and Western Africa harboured 84 · 2 per cent (85/101) and 55 · 9 per cent (264/472) of the continent's H1N1pdm09 and H3N2 reassortants, respectively. The frequent reassortment involving multi-genes observed among Africa IAVs showed the intracontinental viral evolution and diversification possibly sustained by viral importation from outside Africa and/or local viral genomic mixing and transmission. Novel reassortant viruses emerged every year, and some persisted in different countries and regions, thereby presenting a risk of influenza outbreaks in Africa. Our findings highlight Africa as part of the global influenza ecology and the advantage of implementing routine whole-over partial genome sequencing and analyses to monitor circulating and detect emerging viruses. Furthermore, this study provides evidence and heightens our knowledge on IAV evolution, which is integral in directing vaccine strain selection and the update of master donor viruses used in recombinant vaccine development.

Field evaluation of the performance of seven Antigen Rapid diagnostic tests for the diagnosis of SARs-CoV-2 virus infection in Uganda.

OBJECTIVE: The objective of this study was to evaluate the performance of seven antigen rapid diagnostic tests (Ag RDTs) in a clinical setting to identify those that could be recommended for use in the diagnosis of SARS-CoV-2 infection in Uganda. METHODS: This was a cross-sectional prospective study. Nasopharyngeal swabs were collected consecutively from COVID-19 PCR positive and COVID-19 PCR negative participants at isolation centers and points of entry, and tested with the SARS-CoV-2 Ag RDTs. Test sensitivity and specificity were generated by comparing results against qRT-PCR results (Berlin Protocol) at a cycle threshold (Ct) cut-off of ≤39. Sensitivity was also calculated at Ct cut-offs ≤29 and ≤33. RESULTS: None of the Ag RDTs had a sensitivity of ≥80% at Ct cut-off values ≤33 and ≤39. Two kits, Panbio™ COVID-19 Ag and VivaDiag™ SARS-CoV-2 Ag had a sensitivity of ≥80% at a Ct cut-off value of ≤29. Four kits: BIOCREDIT COVID -19 Ag, COVID-19 Ag Respi-Strip, MEDsan® SARS-CoV-2 Antigen Rapid Test and Panbio™ COVID-19 Ag Rapid Test had a specificity of ≥97%. CONCLUSIONS: This evaluation identified one Ag RDT, Panbio™ COVID-19 Ag with a performance at high viral load (Ct value ≤29) reaching that recommended by WHO. This kit was recommended for screening of patients with COVID -19-like symptoms presenting at health facilities.

Rapid Replacement of SARS-CoV-2 Variants by Delta and Subsequent Arrival of Omicron, Uganda, 2021.

Genomic surveillance in Uganda showed rapid replacement of severe acute respiratory syndrome coronavirus 2 over time by variants, dominated by Delta. However, detection of the more transmissible Omicron variant among travelers and increasing community transmission highlight the need for near-real-time genomic surveillance and adherence to infection control measures to prevent future pandemic waves.

Evaluation of the performance of 25 SARS-CoV-2 serological rapid diagnostic tests using a reference panel of plasma specimens at the Uganda Virus Research Institute.

INTRODUCTION: Serological testing is needed to better understand the epidemiology of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Rapid diagnostic tests (RDTs) have been developed to detect specific antibodies, IgM and IgG, to the virus. The performance of 25 of these RDTs was evaluated. METHODS: A serological reference panel of 50 positive and 100 negative plasma specimens was developed from SARS-CoV-2 PCR and antibody positive patients and pre-pandemic SARS-CoV-2-negative specimens collected in 2016. Test performance of the 25 RDTs was evaluated against this panel. RESULTS: A total of 10 RDTs had a sensitivity ≥98%, while 13 RDTs had a specificity ≥98% to anti-SARS-CoV-2 IgG antibodies. Four RDTs (Boson, MultiG, Standard Q, and VivaDiag) had both sensitivity and specificity ≥98% to anti-SARS-CoV-2 IgG antibodies. Only three RDTs had a sensitivity ≥98%, while 10 RDTs had a specificity ≥98% to anti-SARS-CoV-2 IgM antibodies. Three RDTs (Autobio, MultiG, and Standard Q) had sensitivity and specificity ≥98% to combined IgG/IgM. The RDTs that performed well also had perfect or almost perfect inter-reader agreement. CONCLUSIONS: This evaluation identified three RDTs with a sensitivity and specificity to IgM/IgG antibodies of ≥98% with the potential for widespread antibody testing in Uganda.

Severe COVID-19 in Uganda across Two Epidemic Phases: A Prospective Cohort Study.

Among a prospective cohort of children and adults admitted to a national COVID-19 treatment unit in Uganda from March to December 2020, we characterized the epidemiology of and risk factors for severe illness. Across two epidemic phases differentiated by varying levels of community transmission, the proportion of patients admitted with WHO-defined severe COVID-19 ranged from 5% (7/146; 95% CI: 2-10) to 33% (41/124; 95% CI: 25-42); 21% (26/124; 95% CI: 14-29%) of patients admitted during the peak phase received oxygen therapy. Severe COVID-19 was associated with older age, male sex, and longer duration of illness before admission. Coinfection with HIV was not associated with illness severity; malaria or tuberculosis coinfection was rare. No patients died during admission. Despite low mortality, hospital incidence of severe COVID-19 during the first epidemic peak in Uganda was substantial. Improvements in vaccine deployment and acute care capacity, including oxygen delivery, are urgently needed to prevent and manage severe COVID-19 in sub-Saharan Africa.

Influenza surveillance capacity improvements in Africa during 2011-2017.

BACKGROUND: Influenza surveillance helps time prevention and control interventions especially where complex seasonal patterns exist. We assessed influenza surveillance sustainability in Africa where influenza activity varies and external funds for surveillance have decreased. METHODS: We surveyed African Network for Influenza Surveillance and Epidemiology (ANISE) countries about 2011-2017 surveillance system characteristics. Data were summarized with descriptive statistics and analyzed with univariate and multivariable analyses to quantify sustained or expanded influenza surveillance capacity in Africa. RESULTS: Eighteen (75%) of 24 ANISE members participated in the survey; their cumulative population of 710 751 471 represent 56% of Africa's total population. All 18 countries scored a mean 95% on WHO laboratory quality assurance panels. The number of samples collected from severe acute respiratory infection case-patients remained consistent between 2011 and 2017 (13 823 vs 13 674 respectively) but decreased by 12% for influenza-like illness case-patients (16 210 vs 14 477). Nine (50%) gained capacity to lineage-type influenza B. The number of countries reporting each week to WHO FluNet increased from 15 (83%) in 2011 to 17 (94%) in 2017. CONCLUSIONS: Despite declines in external surveillance funding, ANISE countries gained additional laboratory testing capacity and continued influenza testing and reporting to WHO. These gains represent important achievements toward sustainable surveillance and epidemic/pandemic preparedness.

Early cases of SARS-CoV-2 infection in Uganda: epidemiology and lessons learned from risk-based testing approaches - March-April 2020.

BACKGROUND: On March 13, 2020, Uganda instituted COVID-19 symptom screening at its international airport, isolation and SARS-CoV-2 testing for symptomatic persons, and mandatory 14-day quarantine and testing of persons traveling through or from high-risk countries. On March 21, 2020, Uganda reported its first SARS-CoV-2 infection in a symptomatic traveler from Dubai. By April 12, 2020, 54 cases and 1257 contacts were identified. We describe the epidemiological, clinical, and transmission characteristics of these cases. METHODS: A confirmed case was laboratory-confirmed SARS-CoV-2 infection during March 21-April 12, 2020 in a resident of or traveler to Uganda. We reviewed case-person files and interviewed case-persons at isolation centers. We identified infected contacts from contact tracing records. RESULTS: Mean case-person age was 35 (±16) years; 34 (63%) were male. Forty-five (83%) had recently traveled internationally ('imported cases'), five (9.3%) were known contacts of travelers, and four (7.4%) were community cases. Of the 45 imported cases, only one (2.2%) was symptomatic at entry. Among all case-persons, 29 (54%) were symptomatic at testing and five (9.3%) were pre-symptomatic. Among the 34 (63%) case-persons who were ever symptomatic, all had mild disease: 16 (47%) had fever, 13 (38%) reported headache, and 10 (29%) reported cough. Fifteen (28%) case-persons had underlying conditions, including three persons with HIV. An average of 31 contacts (range, 4-130) were identified per case-person. Five (10%) case-persons, all symptomatic, infected one contact each. CONCLUSION: The first 54 case-persons with SARS-CoV-2 infection in Uganda primarily comprised incoming air travelers with asymptomatic or mild disease. Disease would likely not have been detected in these persons without the targeted testing interventions implemented in Uganda. Transmission was low among symptomatic persons and nonexistent from asymptomatic persons. Routine, systematic screening of travelers and at-risk persons, and thorough contact tracing will be needed for Uganda to maintain epidemic control.

Field evaluation of the performance of a SARS-CoV-2 antigen rapid diagnostic test in Uganda using nasopharyngeal samples.

OBJECTIVES: There is a high demand for SARS-CoV-2 testing to identify COVID-19 cases. Real-time quantitative PCR (qRT-PCR) is the recommended diagnostic test but a number of constraints prevent its widespread implementation, including cost. The aim of this study was to evaluate a low cost and easy to use rapid antigen test for diagnosing COVID-19 at the point of care. METHODS: Nasopharyngeal swabs from suspected COVID-19 cases and low-risk volunteers were tested with the STANDARD Q COVID-19 Ag Test and the results were compared with the qRT-PCR results. RESULTS: In total, 262 samples were collected, including 90 qRT-PCR positives. The majority of samples were from males (89%) with a mean age of 34 years and only 13 (14%) of the positives were mildly symptomatic. The sensitivity and specificity of the antigen test were 70.0% (95% confidence interval (CI): 60-79) and 92% (95% CI: 87-96), respectively, and the diagnostic accuracy was 84% (95% CI: 79-88). The antigen test was more likely to be positive for samples with qRT-PCR Ct values ≤29, with a sensitivity of 92%. CONCLUSIONS: The STANDARD Q COVID-19 Ag Test performed less than optimally in this evaluation. However, the test may still have an important role to play early in infection when timely access to molecular testing is not available but the results should be confirmed by qRT-PCR.

Main Routes of Entry and Genomic Diversity of SARS-CoV-2, Uganda.

We established rapid local viral sequencing to document the genomic diversity of severe acute respiratory syndrome coronavirus 2 entering Uganda. Virus lineages closely followed the travel origins of infected persons. Our sequence data provide an important baseline for tracking any further transmission of the virus throughout the country and region.