The genomic epidemiology of SARS-CoV-2 variants of concern in Kenya

The emergence and establishment of SARS-CoV-2 variants of concern presented a major global public health crisis across the world. There were six waves of SARS-CoV-2 cases in Kenya that corresponded with the introduction and eventual dominance of the major SARS-COV-2 variants of concern, excepting the first 2 waves that were both wild-type virus. We estimate that more than 1000 SARS-CoV-2 introductions occurred in the two-year epidemic period (March 2020 - September 2022) and a total of 930 introductions were associated with variants of concern namely Beta (n=78), Alpha(n=108), Delta(n=239) and Omicron (n=505). A total of 29 introductions were associated with A.23.1 variant that circulated in high frequencies in Uganda and Rwanda. The actual number of introductions is likely to be higher than these conservative estimates due to limited genomic sequencing. Our data suggested that cryptic transmission was usually underway prior to the first real-time identification of a new variant, and that multiple introductions were responsible. Following emergence of each VOC and subsequent introduction, transmission patterns were associated with hotspots of transmission in Coast, Nairobi and Western Kenya and follows established land and air transport corridors. Understanding the introduction and dispersal of major circulating variants and identifying the sources of new introductions is important to inform public health control strategies within Kenya and the larger East-African region. Border control and case finding reactive to new variants is unlikely to be a successful control strategy.

COVID-19 mass testing and sequencing: Experiences from a laboratory in Western Kenya.

Background: The Basic Science Laboratory (BSL) of the Kenya Medical Research Institute/Walter Reed Project in Kisumu, Kenya addressed mass testing challenges posed by the emergent coronavirus disease 2019 (COVID-19) in an environment of global supply shortages. Before COVID-19, the BSL had adequate resources for disease surveillance and was therefore designated as one of the testing centres for COVID-19. Intervention: By April 2020, the BSL had developed stringent safety procedures for receiving and mass testing potentially infectious nasal specimens. To accommodate increased demand, BSL personnel worked in units: nucleic acid extraction, polymerase chain reaction, and data and quality assurance checks. The BSL adopted procedures for tracking sample integrity and minimising cross-contamination. Lessons learnt: Between May 2020 and January 2022, the BSL tested 63 542 samples, of which 5375 (8.59%) were positive for COVID-19; 1034 genomes were generated by whole genome sequencing and deposited in the Global Initiative on Sharing All Influenza Data database to aid global tracking of viral lineages. At the height of the pandemic (August and November 2020, April and August 2021 and January 2022), the BSL was testing more than 500 samples daily, compared to 150 per month prior to COVID-19. An important lesson from the COVID-19 pandemic was the discovery of untapped resilience within BSL personnel that allowed adaptability when the situation demanded. Strict safety procedures and quality management that are often difficult to maintain became routine. Recommendations: A fundamental lesson to embrace is that there is no 'one-size-fits-all' approach and adaptability is the key to success.

Temporal lineage replacements and dominance of imported variants of concern during the COVID-19 pandemic in Kenya.

Background: Kenya's COVID-19 epidemic was seeded early in March 2020 and did not peak until early August 2020 (wave 1), late-November 2020 (wave 2), mid-April 2021 (wave 3), late August 2021 (wave 4), and mid-January 2022 (wave 5). Methods: Here, we present SARS-CoV-2 lineages associated with the five waves through analysis of 1034 genomes, which included 237 non-variants of concern and 797 variants of concern (VOC) that had increased transmissibility, disease severity or vaccine resistance. Results: In total 40 lineages were identified. The early European lineages (B.1 and B.1.1) were the first to be seeded. The B.1 lineage continued to expand and remained dominant, accounting for 60% (72/120) and 57% (45/79) in waves 1 and 2 respectively. Waves three, four and five respectively were dominated by VOCs that were distributed as follows: Alpha 58.5% (166/285), Delta 92.4% (327/354), Omicron 95.4% (188/197) and Beta at 4.2% (12/284) during wave 3 and 0.3% (1/354) during wave 4. Phylogenetic analysis suggests multiple introductions of variants from outside Kenya, more so during the first, third, fourth and fifth waves, as well as subsequent lineage diversification. Conclusions: The data highlights the importance of genome surveillance in determining circulating variants to aid interpretation of phenotypes such as transmissibility, virulence and/or resistance to therapeutics/vaccines.

March 2019 dengue fever outbreak at the Kenyan south coast involving dengue virus serotype 3, genotypes III and V.

The first description of a disease resembling dengue fever (DF) was in the 15th century slave trade era by Spanish sailors visiting the Tanzania coast. The disease, then associated with evil spirits is now known to be caused by four serotypes of dengue virus (DENV1-4) that are transmitted by Aedes mosquitoes. Kenya has experienced multiple outbreaks, mostly associated with DENV-2. In this study, plasma samples obtained from 37 febrile patients during a DF outbreak at Kenya's south coast in March 2019 were screened for DENV. Total RNA was extracted and screened for the alpha- and flavi-viruses by real-time polymerase chain reaction (qPCR). DENV-3 was the only virus detected. Shotgun metagenomics and targeted sequencing were used to obtain DENV whole genomes and the complete envelope genes (E gene) respectively. Sequences were used to infer phylogenies and time-scaled genealogies. Following Maximum likelihood and Bayesian phylogenetic analysis, two DENV-3 genotypes (III, n = 15 and V, n = 2) were found. We determined that the two genotypes had been in circulation since 2015, and that both had been introduced independently. Genotype III's origin was estimated to have been from Pakistan. Although the origin of genotype V could not be ascertained due to rarity of these sequences globally, it was most related to a 2006 Brazilian isolate. Unlike genotype III that has been described in East and West Africa multiple times, this was the second description of genotype V in Kenya. Of note, there was marked amino acid variances in the E gene between study samples and the Thailand DENV-3 strain used in the approved Dengvaxia vaccine. It remains to be seen whether these variances negatively impact the efficacy of the Dengvaxia or future vaccines.

The spleen bacteriome of wild rodents and shrews from Marigat, Baringo County, Kenya.

BACKGROUND: There is a global increase in reports of emerging diseases, some of which have emerged as spillover events from wild animals. The spleen is a major phagocytic organ and can therefore be probed for systemic microbiome. This study assessed bacterial diversity in the spleen of wild caught small mammals so as to evaluate their utility as surveillance tools for monitoring bacteria in an ecosystem shared with humans. METHODS: Fifty-four small mammals (rodents and shrews) were trapped from different sites in Marigat, Baringo County, Kenya. To characterize their bacteriome, DNA was extracted from their spleens and the V3-V4 regions of the 16S rRNA amplified and then sequenced on Illumina MiSeq. A non-target control sample was used to track laboratory contaminants. Sequence data was analyzed with Mothur v1.35, and taxomy determined using the SILVA database. The Shannon diversity index was used to estimate bacterial diversity in each animal and then aggregated to genus level before computing the means. Animal species within the rodents and shrews were identified by amplification of mitochondrial cytochrome b (cytb) gene followed by Sanger sequencing. CLC workbench was used to assemble the cytb gene sequences, after which their phylogenetic placements were determined by querying them against the GenBank nucleotide database. RESULTS: cytb gene sequences were generated for 49/54 mammalian samples: 38 rodents (Rodentia) and 11 shrews (Eulipotyphyla). Within the order Rodentia, 21 Acomys, eight Mastomys, six Arvicanthis and three Rattus were identified. In the order Eulipotyphyla, 11 Crucidura were identified. Bacteria characterization revealed 17 phyla that grouped into 182 genera. Of the phyla, Proteobacteria was the most abundant (67.9%). Other phyla included Actinobacteria (16.5%), Firmicutes (5.5%), Chlamydiae (3.8%), Chloroflexi (2.6%) and Bacteroidetes (1.3%) among others. Of the potentially pathogenic bacteria, Bartonella was the most abundant (45.6%), followed by Anaplasma (8.0%), Methylobacterium (3.5%), Delftia (3.8%), Coxiella (2.6%), Bradyrhizobium (1.6%) and Acinetobacter (1.1%). Other less abundant (<1%) and potentially pathogenic included Ehrlichia, Rickettsia, Leptospira, Borrelia, Brucella, Chlamydia and Streptococcus. By Shannon diversity index, Acomys spleens carried more diverse bacteria (mean Shannon diversity index of 2.86, p = 0.008) compared to 1.77 for Crocidura, 1.44 for Rattus, 1.40 for Arvicathis and 0.60 for Mastomys. CONCLUSION: This study examined systemic bacteria that are filtered by the spleen and the findings underscore the utility of 16S rRNA deep sequencing in characterizing complex microbiota that are potentially relevant to one health issues. An inherent problem with the V3-V4 region of 16S rRNA is the inability to classify bacteria reliably beyond the genera. Future studies should utilize the newer long read methods of 16S rRNA analysis that can delimit the species composition.

Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Phylogenetic Variants of Rickettsia africae, and Incidental Identification of "Candidatus Rickettsia Moyalensis" in Kenya.

BACKGROUND: Rickettsia africae, the etiological agent of African tick bite fever, is widely distributed in sub-Saharan Africa. Contrary to reports of its homogeneity, a localized study in Asembo, Kenya recently reported high genetic diversity. The present study aims to elucidate the extent of this heterogeneity by examining archived Rickettsia africae DNA samples collected from different eco-regions of Kenya. METHODS: To evaluate their phylogenetic relationships, archived genomic DNA obtained from 57 ticks a priori identified to contain R. africae by comparison to ompA, ompB and gltA genes was used to amplify five rickettsial genes i.e. gltA, ompA, ompB, 17kDa and sca4. The resulting amplicons were sequenced. Translated amino acid alignments were used to guide the nucleotide alignments. Single gene and concatenated alignments were used to infer phylogenetic relationships. RESULTS: Out of the 57 DNA samples, three were determined to be R. aeschlimanii and not R. africae. One sample turned out to be a novel rickettsiae and an interim name of "Candidatus Rickettsia moyalensis" is proposed. The bonafide R. africae formed two distinct clades. Clade I contained 9% of the samples and branched with the validated R. africae str ESF-5, while clade II (two samples) formed a distinct sub-lineage. CONCLUSIONS: This data supports the use of multiple genes for phylogenetic inferences. It is determined that, despite its recent emergence, the R. africae lineage is diverse. This data also provides evidence of a novel Rickettsia species, Candidatus Rickettsia moyalensis.