Phylogeography and reassortment patterns of human influenza A viruses in sub-Saharan Africa.

The role of sub-Saharan Africa in the global spread of influenza viruses remains unclear due to insufficient spatiotemporal sequence data. Here, we analyzed 222 codon-complete sequences of influenza A viruses (IAVs) sampled between 2011 and 2013 from five countries across sub-Saharan Africa (Kenya, Zambia, Mali, Gambia, and South Africa); these genomes were compared with 1209 contemporaneous global genomes using phylogeographical approaches. The spread of influenza in sub-Saharan Africa was characterized by (i) multiple introductions of IAVs into the region over consecutive influenza seasons, with viral importations originating from multiple global geographical regions, some of which persisted in circulation as intra-subtype reassortants for multiple seasons, (ii) virus transfer between sub-Saharan African countries, and (iii) virus export from sub-Saharan Africa to other geographical regions. Despite sparse data from influenza surveillance in sub-Saharan Africa, our findings support the notion that influenza viruses persist as temporally structured migrating metapopulations in which new virus strains can emerge in any geographical region, including in sub-Saharan Africa; these lineages may have been capable of dissemination to other continents through a globally migrating virus population. Further knowledge of the viral lineages that circulate within understudied sub-Saharan Africa regions is required to inform vaccination strategies in those regions.

Standardized Phylogenetic Classification of Human Respiratory Syncytial Virus below the Subgroup Level.

A globally implemented unified phylogenetic classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. We formulated global consensus of HRSV classification on the basis of the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality curated dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to GenBank and GISAID (https://www.gisaid.org) public sequence databases through March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B and provided guidelines for defining prospective lineages. Our classification demonstrated robustness in its applicability to both complete and partial genomes. We envision that this unified HRSV classification proposal will strengthen HRSV molecular epidemiology on a global scale.

Molecular epidemiology of recurrent zoonotic transmission of mpox virus in West Africa.

Nigeria and Cameroon reported their first mpox cases in over three decades in 2017 and 2018 respectively. The outbreak in Nigeria is recognised as an ongoing human epidemic. However, owing to sparse surveillance and genomic data, it is not known whether the increase in cases in Cameroon is driven by zoonotic or sustained human transmission. Notably, the frequency of zoonotic transmission remains unknown in both Cameroon and Nigeria. To address these uncertainties, we investigated the zoonotic transmission dynamics of the mpox virus (MPXV) in Cameroon and Nigeria, with a particular focus on the border regions. We show that in these regions mpox cases are still driven by zoonotic transmission of a newly identified Clade IIb.1. We identify two distinct zoonotic lineages that circulate across the Nigeria-Cameroon border, with evidence of recent and historic cross border dissemination. Our findings support that the complex cross-border forest ecosystems likely hosts shared animal populations that drive cross-border viral spread, which is likely where extant Clade IIb originated. We identify that the closest zoonotic outgroup to the human epidemic circulated in southern Nigeria in October 2013. We also show that the zoonotic precursor lineage circulated in an animal population in southern Nigeria for more than 45 years. This supports findings that southern Nigeria was the origin of the human epidemic. Our study highlights the ongoing MPXV zoonotic transmission in Cameroon and Nigeria, underscoring the continuous risk of MPXV (re)emergence.

Genomic epidemiology uncovers the timing and origin of the emergence of mpox in humans.

Five years before the 2022-2023 global mpox outbreak Nigeria reported its first cases in nearly 40 years, with the ongoing epidemic since driven by sustained human-to-human transmission. However, limited genomic data has left questions about the timing and origin of the mpox virus' (MPXV) emergence. Here we generated 112 MPXV genomes from Nigeria from 2021-2023. We identify the closest zoonotic outgroup to the human epidemic in southern Nigeria, and estimate that the lineage transmitting from human-to-human emerged around July 2014, circulating cryptically until detected in September 2017. The epidemic originated in Southern Nigeria, particularly Rivers State, which also acted as a persistent and dominant source of viral dissemination to other states. We show that APOBEC3 activity increased MPXV's evolutionary rate twenty-fold during human-to-human transmission. We also show how Delphy, a tool for near-real-time Bayesian phylogenetics, can aid rapid outbreak analytics. Our study sheds light on MPXV's establishment in West Africa before the 2022-2023 global outbreak and highlights the need for improved pathogen surveillance and response.

Informing the pandemic response: the role of the WHO's COVID-19 Weekly Epidemiological Update.

On 31 December 2019, the Municipal Health Commission of Wuhan, China, reported a cluster of atypical pneumonia cases. On 5 January 2020, the WHO publicly released a Disease Outbreak News (DON) report, providing information about the pneumonia cases, implemented response interventions, and WHO's risk assessment and advice on public health and social measures. Following 9 additional DON reports and 209 daily situation reports, on 17 August 2020, WHO published the first edition of the COVID-19 Weekly Epidemiological Update (WEU). On 1 September 2023, the 158th edition of the WEU was published on WHO's website, marking its final issue. Since then, the WEU has been replaced by comprehensive global epidemiological updates on COVID-19 released every 4 weeks. During the span of its publication, the webpage that hosts the WEU and the COVID-19 Operational Updates was accessed annually over 1.4 million times on average, with visits originating from more than 100 countries. This article provides an in-depth analysis of the WEU process, from data collection to publication, focusing on the scope, technical details, main features, underlying methods, impact and limitations. We also discuss WHO's experience in disseminating epidemiological information on the COVID-19 pandemic at the global level and provide recommendations for enhancing collaboration and information sharing to support future health emergency responses.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

TheiaEuk: a species-agnostic bioinformatics workflow for fungal genomic characterization.

Introduction: The clinical incidence of antimicrobial-resistant fungal infections has dramatically increased in recent years. Certain fungal pathogens colonize various body cavities, leading to life-threatening bloodstream infections. However, the identification and characterization of fungal isolates in laboratories remain a significant diagnostic challenge in medicine and public health. Whole-genome sequencing provides an unbiased and uniform identification pipeline for fungal pathogens but most bioinformatic analysis pipelines focus on prokaryotic species. To this end, TheiaEuk_Illumina_PE_PHB (TheiaEuk) was designed to focus on genomic analysis specialized to fungal pathogens. Methods: TheiaEuk was designed using containerized components and written in the workflow description language (WDL) to facilitate deployment on the cloud-based open bioinformatics platform Terra. This species-agnostic workflow enables the analysis of fungal genomes without requiring coding, thereby reducing the entry barrier for laboratory scientists. To demonstrate the usefulness of this pipeline, an ongoing outbreak of C. auris in southern Nevada was investigated. We performed whole-genome sequence analysis of 752 new C. auris isolates from this outbreak. Furthermore, TheiaEuk was utilized to observe the accumulation of mutations in the FKS1 gene over the course of the outbreak, highlighting the utility of TheiaEuk as a monitor of emerging public health threats when combined with whole-genome sequencing surveillance of fungal pathogens. Results: A primary result of this work is a curated fungal database containing 5,667 unique genomes representing 245 species. TheiaEuk also incorporates taxon-specific submodules for specific species, including clade-typing for Candida auris (C. auris). In addition, for several fungal species, it performs dynamic reference genome selection and variant calling, reporting mutations found in genes currently associated with antifungal resistance (FKS1, ERG11, FUR1). Using genome assemblies from the ATCC Mycology collection, the taxonomic identification module used by TheiaEuk correctly assigned genomes to the species level in 126/135 (93.3%) instances and to the genus level in 131/135 (97%) of instances, and provided zero false calls. Application of TheiaEuk to actual specimens obtained in the course of work at a local public health laboratory resulted in 13/15 (86.7%) correct calls at the species level, with 2/15 called at the genus level. It made zero incorrect calls. TheiaEuk accurately assessed clade type of Candida auris in 297/302 (98.3%) of instances. Discussion: TheiaEuk demonstrated effectiveness in identifying fungal species from whole genome sequence. It further showed accuracy in both clade-typing of C. auris and in the identification of mutations known to associate with drug resistance in that organism.

Accelerating bioinformatics implementation in public health.

We have adopted an open bioinformatics ecosystem to address the challenges of bioinformatics implementation in public health laboratories (PHLs). Bioinformatics implementation for public health requires practitioners to undertake standardized bioinformatic analyses and generate reproducible, validated and auditable results. It is essential that data storage and analysis are scalable, portable and secure, and that implementation of bioinformatics fits within the operational constraints of the laboratory. We address these requirements using Terra, a web-based data analysis platform with a graphical user interface connecting users to bioinformatics analyses without the use of code. We have developed bioinformatics workflows for use with Terra that specifically meet the needs of public health practitioners. These Theiagen workflows perform genome assembly, quality control, and characterization, as well as construction of phylogeny for insights into genomic epidemiology. Additonally, these workflows use open-source containerized software and the WDL workflow language to ensure standardization and interoperability with other bioinformatics solutions, whilst being adaptable by the user. They are all open source and publicly available in Dockstore with the version-controlled code available in public GitHub repositories. They have been written to generate outputs in standardized file formats to allow for further downstream analysis and visualization with separate genomic epidemiology software. Testament to this solution meeting the requirements for bioinformatic implementation in public health, Theiagen workflows have collectively been used for over 5 million sample analyses in the last 2 years by over 90 public health laboratories in at least 40 different countries. Continued adoption of technological innovations and development of further workflows will ensure that this ecosystem continues to benefit PHLs.

Rhinovirus dynamics across different social structures.

Rhinoviruses (RV), common human respiratory viruses, exhibit significant antigenic diversity, yet their dynamics across distinct social structures remain poorly understood. Our study delves into RV dynamics within Kenya by analysing VP4/2 sequences across four different social structures: households, a public primary school, outpatient clinics in the Kilifi Health and Demographics Surveillance System (HDSS), and countrywide hospital admissions and outpatients. The study revealed the greatest diversity of RV infections at the countrywide level (114 types), followed by the Kilifi HDSS (78 types), the school (47 types), and households (40 types), cumulatively representing >90% of all known RV types. Notably, RV diversity correlated directly with the size of the population under observation, and several RV type variants occasionally fuelled RV infection waves. Our findings highlight the critical role of social structures in shaping RV dynamics, information that can be leveraged to enhance public health strategies. Future research should incorporate whole-genome analysis to understand fine-scale evolution across various social structures.

Effect of introduction of routine single shot adductor canal block in primary total knee arthroplasty in a rural Australian setting.

BACKGROUND: There is a growing demand for quality osteoarthritis management available in Australia. There has been increasing emphasis on post-total knee replacement pain relief as a significant issue, especially in the context of postoperative recovery protocols which focus on early mobility. METHODS: The aim of this study was to assess if the addition of adductor canal blocks (ACB) reduced postoperative pain and affected mobility post primary total knee arthroplasty (TKR) at Armidale Rural Referral Hospital (ARRH). A retrospective cohort study was performed of unilateral TKR patients before and after the addition of adductor canal blocks to the TKR protocol at ARRH. Forty-seven patients were included in the study who are matched for age and radiographic disease severity. All patients were prescribed post-operative analgesia per the protocol. RESULTS: Patients who received an ACB had a significant difference in opioid consumption in the first 24 h post-operative compared with those who did not, 115.90 mg oral morphine compared to 66.07 mg (P = 0.0001). This was also supported by a significant difference in patient self-rated numerical pain score between groups, no ACB group 3.09/10 and ACB group 1.05/10 (P = 0.0001). The ACB group were able to mobilize further on post-op days 1 and 2 than those who did not receive an ACB with the ACB group (P = 0.0001 and 0.0008). CONCLUSION: This adds to the evidence supporting the use of Adductor Canal Blocks in patients undergoing total knee arthroplasty, of especial significance in a rural population.

Origins and Evolution of Seasonal Human Coronaviruses.

Four seasonal human coronaviruses (sHCoVs) are endemic globally (229E, NL63, OC43, and HKU1), accounting for 5-30% of human respiratory infections. However, the epidemiology and evolution of these CoVs remain understudied due to their association with mild symptomatology. Using a multigene and complete genome analysis approach, we find the evolutionary histories of sHCoVs to be highly complex, owing to frequent recombination of CoVs including within and between sHCoVs, and uncertain, due to the under sampling of non-human viruses. The recombination rate was highest for 229E and OC43 whereas substitutions per recombination event were highest in NL63 and HKU1. Depending on the gene studied, OC43 may have ungulate, canine, or rabbit CoV ancestors. 229E may have origins in a bat, camel, or an unsampled intermediate host. HKU1 had the earliest common ancestor (1809-1899) but fell into two distinct clades (genotypes A and B), possibly representing two independent transmission events from murine-origin CoVs that appear to be a single introduction due to large gaps in the sampling of CoVs in animals. In fact, genotype B was genetically more diverse than all the other sHCoVs. Finally, we found shared amino acid substitutions in multiple proteins along the non-human to sHCoV host-jump branches. The complex evolution of CoVs and their frequent host switches could benefit from continued surveillance of CoVs across non-human hosts.

Effects of packinghouse operations on the flavor of 'Orri' mandarins.

Mandarins have a delicate flavor and are easy to peel and easy to consume. However, they are relatively perishable and suffer from flavor deterioration after harvest. The goal of the current study was to examine the effects of commercial packinghouse operations on the flavor of 'Orri' mandarins. For that purpose, we collected fruit from four different points along a commercial citrus packing line: (1) directly from the harvest bin, (2) after application of a hot (53°C) fungicide treatment for 30 s, (3) after waxing, and (4) after waxing and after the fruit had passed through a hot-air drying tunnel (37°C) for 2 min. The collected fruit were stored for 3 or 6 weeks at 5°C and then kept for five more days under shelf-life conditions at 22°C. The observed results indicate that the hot fungicide treatment had no effect on flavor quality. However, the waxing and waxing +drying treatments resulted in significant increases in ethanol accumulation, lower flavor-acceptability scores, and increased off-flavors. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that the waxing and waxing +drying treatments resulted in particular increases in the levels of alcohol and ethyl ester volatiles; whereas levels of other aroma volatiles (i.e., esters, aldehydes, monoterpenes, and sesquiterpenes) decreased after storage in all fruit samples. Overall, the waxing process in commercial citrus packinghouses increased ethanol and ethyl ester volatile levels and harmed flavor acceptability. These findings demonstrate the need to identify new wax formulations that do not hamper fruit-flavor quality.

Spatial-temporal distribution and sequence diversity of group a human respiratory syncytial viruses in Kenya preceding the emergence of ON1 genotype.

BACKGROUND: Human respiratory syncytial virus (HRSV) is a major cause of severe viral acute respiratory illness and contributes significantly to severe pneumonia cases in Africa. Little is known about its spatial-temporal distribution as defined by its genetic diversity. METHODS: A retrospective study conducted utilizing archived nasopharyngeal specimens from patients attending outpatient clinics in hospitals located in five demographically and climatically distinct regions of Kenya; Coast, Western, Highlands, Eastern and Nairobi. The viral total RNA was extracted and tested using multiplex real time RT-PCR (reverse transcriptase polymerase chain reaction). A segment of the G-gene was amplified using one-step RT-PCR and sequenced by Sanger di-deoxy method. Bayesian analysis of phylogeny was utilized and subsequently median joining methods for haplotype network reconstruction. RESULTS: Three genotypes of HRSVA were detected; GA5 (14.0%), GA2 (33.1%), and NA1 (52.9%). HRSVA prevalence varied by location from 33% to 13.2% in the Highlands and the Eastern regions respectively. The mean nucleotide diversity (Pi[π]) varied by genotype: highest of 0.018 for GA5 and lowest of 0.005 for NA1. A total of 58 haplotypes were identified (GA5 10; GA2 20; NA1 28). These haplotypes were introduced into the population locally by single haplotypes and additional subsidiary seeds amongst the GA2 and the NA1 haplotypes. CONCLUSIONS: HRSVA was found across all the regions throughout the study period and comprised three genotypes; GA5, GA2, and NA1 genotypes. The genotypes were disproportionately distributed across the regions with GA5 gradually increasing toward the Western zones and decreasing toward the Eastern zones of the country.

Comparative analysis of spatial-temporal patterns of human metapneumovirus and respiratory syncytial virus in Africa using genetic data, 2011-2014.

BACKGROUND: Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are leading causes of viral severe acute respiratory illnesses in childhood. Both the two viruses belong to the Pneumoviridae family and show overlapping clinical, epidemiological and transmission features. However, it is unknown whether these two viruses have similar geographic spread patterns which may inform designing and evaluating their epidemic control measures. METHODS: We conducted comparative phylogenetic and phylogeographic analyses to explore the spatial-temporal patterns of HMPV and RSV across Africa using 232 HMPV and 842 RSV attachment (G) glycoprotein gene sequences obtained from 5 countries (The Gambia, Zambia, Mali, South Africa, and Kenya) between August 2011 and January 2014. RESULTS: Phylogeographic analyses found frequently similar patterns of spread of RSV and HMPV. Viral sequences commonly clustered by region, i.e., West Africa (Mali, Gambia), East Africa (Kenya) and Southern Africa (Zambia, South Africa), and similar genotype dominance patterns were observed between neighbouring countries. Both HMPV and RSV country epidemics were characterized by co-circulation of multiple genotypes. Sequences from different African sub-regions (East, West and Southern Africa) fell into separate clusters interspersed with sequences from other countries globally. CONCLUSION: The spatial clustering patterns of viral sequences and genotype dominance patterns observed in our analysis suggests strong regional links and predominant local transmission. The geographical clustering further suggests independent introduction of HMPV and RSV variants in Africa from the global pool, and local regional diversification.

Endemic chikungunya fever in Kenyan children: a prospective cohort study.

BACKGROUND: Chikungunya fever (CHIKF) was first described in Tanzania in 1952. Several epidemics including East Africa have occurred, but there are no descriptions of longitudinal surveillance of endemic disease. Here, we estimate the incidence of CHIKF in coastal Kenya and describe the associated viral phylogeny. METHODS: We monitored acute febrile illnesses among 3500 children visiting two primary healthcare facilities in coastal Kenya over a 5-year period (2014-2018). Episodes were linked to a demographic surveillance system and blood samples obtained. Cross-sectional sampling in a community survey of a different group of 435 asymptomatic children in the same study location was done in 2016. Reverse-transcriptase PCR was used for chikungunya virus (CHIKV) screening, and viral genomes sequenced for phylogenetic analyses. RESULTS: We found CHIKF to be endemic in this setting, associated with 12.7% (95% CI 11.60, 13.80) of all febrile presentations to primary healthcare. The prevalence of CHIKV infections among asymptomatic children in the community survey was 0.7% (95% CI 0.22, 2.12). CHIKF incidence among children < 1 year of age was 1190 cases/100,000-person years and 63 cases/100,000-person years among children aged ≥10 years. Recurrent CHIKF episodes, associated with fever and viraemia, were observed among 19 of 170 children with multiple febrile episodes during the study period. All sequenced viral genomes mapped to the ECSA genotype albeit distinct from CHIKV strains associated with the 2004 East African epidemic. CONCLUSIONS: CHIKF may be a substantial public health burden in primary healthcare on the East African coast outside epidemic years, and recurrent infections are common.

Evolution of respiratory syncytial virus genotype BA in Kilifi, Kenya, 15 years on.

Respiratory syncytial virus (RSV) is recognised as a leading cause of severe acute respiratory disease and deaths among infants and vulnerable adults. Clinical RSV isolates can be divided into several known genotypes. RSV genotype BA, characterised by a 60-nucleotide duplication in the G glycoprotein gene, emerged in 1999 and quickly disseminated globally replacing other RSV group B genotypes. Continual molecular epidemiology is critical to understand the evolutionary processes maintaining the success of the BA viruses. We analysed 735 G gene sequences from samples collected from paediatric patients in Kilifi, Kenya, between 2003 and 2017. The virus population comprised of several genetically distinct variants (n = 56) co-circulating within and between epidemics. In addition, there was consistent seasonal fluctuations in relative genetic diversity. Amino acid changes increasingly accumulated over the surveillance period including two residues (N178S and Q180R) that mapped to monoclonal antibody 2D10 epitopes, as well as addition of putative N-glycosylation sequons. Further, switching and toggling of amino acids within and between epidemics was observed. On a global phylogeny, the BA viruses from different countries form geographically isolated clusters suggesting substantial localized variants. This study offers insights into longitudinal population dynamics of a globally endemic RSV genotype within a discrete location.

Genomic epidemiology and evolutionary dynamics of respiratory syncytial virus group B in Kilifi, Kenya, 2015-17.

Respiratory syncytial virus (RSV) circulates worldwide, occurring seasonally in communities, and is a leading cause of acute respiratory illness in young children. There is paucity of genomic data from purposively sampled populations by which to investigate evolutionary dynamics and transmission patterns of RSV. Here we present an analysis of 295 RSV group B (RSVB) genomes from Kilifi, coastal Kenya, sampled from individuals seeking outpatient care in nine health facilities across a defined geographical area (∼890 km2), over two RSV epidemics between 2015 and 2017. RSVB diversity was characterized by multiple virus introductions into the area and co-circulation of distinct genetic clusters, which transmitted and diversified locally with varying frequency. Increase in relative genetic diversity paralleled seasonal virus incidence. Importantly, we identified a cluster of viruses that emerged in the 2016/17 epidemic, carrying distinct amino-acid signatures including a novel nonsynonymous change (K68Q) in antigenic site ∅ in the Fusion protein. RSVB diversity was additionally marked by signature nonsynonymous substitutions that were unique to particular genomic clusters, some under diversifying selection. Our findings provide insights into recent evolutionary and epidemiological behaviors of RSVB, and highlight possible emergence of a novel antigenic variant, which has implications on current prophylactic strategies in development.