Genomic epidemiology of variants circulating in Ghana
A total of 1077 COVID-19 PCR positive samples from ten regions in Ghana between June 2020 and September 2021 were successfully sequenced. Of these, 44.4 % (479/1077) were near full-length genomes (missingness (N) < 500), 47.8 % (515/1077) had 500-4000 unresolved nucleotide assignments, and only 7.7 % (83/1077) had 4000-8913 missingness (N). Of 1077 high quality samples, sampling contribution from the different regions in Ghana, was as follows: Ashanti (4.4 %, n=47), Bono East (1.2 %, n=13), Central (11.9 %, n=129), Eastern (4.8 %, n=52), Greater Accra (43.2 %, n=465), Northern (5.1 %, n=55), Upper East (0.4 %, n=4), Upper West (1.1%, n=12), Volta (9.6 %, n=103) and Western (8.4 %, n=91) (Fig. 1a). With the highest number of active COVID-19 cases (GHS, 2021), the Greater Accra region had the highest number of sequenced samples. Only 15.7 % (169/1077) of all the samples were collected in 2020, while the remaining were collected in 2021 (n=908/1077). Of the 908 samples collected in 2021, 11.7 % (n=106) were from travellers arriving in the country through the Kotoka International Airport.
Overall, the Alpha (20.8 %, 224/1077), Delta Plus (18.7 %, 201/1077), B.1.1.318 (16.7 %, 180/1077), Delta (14.8 %, 159/1077), B.1.1 (9.0 %, 97/1077), and Eta (4.4 %, 47/1077) made up the top viral lineages within the sequenced SARS-CoV-2 genomes in Ghana over the period (Fig. 1b). Since the B.1.1.318 had the third-highest frequency, it is considered a variant under monitoring in Ghana. In 2020, the Ashanti, Central, Eastern, Greater Accra and Western regions had the highest numbers of confirmed cases of COVID-19 in Ghana. Different regions showed variations in relative frequencies of the variants. Genomes which clustered within the B.1.1 lineage dominated samples from Greater Accra (68.8 %, 11/16), Volta region (55.6 %, 10/18), Western region (51.2 %, 22/43) and Central region (44.4 %, 20/45). B.1.623 (27.8 %, 5/18) co-dominated with B.1.1 (27.8 %, 5/18) in the Ashanti region whereas B.1.1.359 (48.3 %, 14/29) dominated alongside B.1.1 (34.5 %, 10/29) in the Eastern region (Fig. 1c).
In 2021, there was a marked shift in the circulating variants and occurrence of regional specific outbreaks, with Eta dominating in Northern and middle belt regions, while B.1.1.318 dominated the major cities. The highest frequencies of Eta variants were observed in the Northern (23.6 %, 13/55), Bono (30.8%, 4/13) and Eastern (13%, 3/23) regions. The city of Tamale in the Northern region is the gateway, and central trading hub with Ghana's northern neighbours, whilst the Bono region harbours major interaction routes with Ivory Coast in the western corridor of Ghana. Meanwhile, a third of all the variants detected in 2021 were B.1.1.318 (30%, 176/802), and Greater Accra, where the capital city and the major international airport are located, had 80% (140/176) of all the genomes. (Fig. 1d). These data suggest that Eta and B.1.1.318 variants, which dominated transmission in these areas in April-May 2021, could have been introduced through these major land borders. Interestingly, the B.1. 1, B.1.359, B.1.1., and B.1.623 that dominated Ghana in 2020 became supplanted by the modal variants responsible for most transmissions in all the regions. It is worth noting that in regions where more than 50 samples were sequenced in 2021, there was penetration or transmission of all the VOCs, including the Central region, Greater Accra, and Volta Region (Fig. 1d).
Importation of SARS-CoV-2 variants into Ghana by travellers
One hundred and six of the sequenced samples (9.8%, 106/1077) were obtained from quarantined travellers identified as COVID-19 positive at the KIA. Of this number, Alpha accounted for 44.3% (n=47) of the genomes while the other VOCs accounted for lower proportions; Beta (6.6 %, n=7), Delta (6.6 %, n=7), and Delta Plus (0.9 %, n=1) (Fig. 2a). The VOIs such as Eta (4.7 %, n=5), Kappa and the local variant-under-monitoring, B.1.1.318, 3.8 % (n=4) were detected at low proportions. Importantly, the VOC Alpha was identified in travellers entering Ghana from all over the World, including other African countries, in January and March 2021 (Fig. 2b and c). Furthermore, VOCs were detected in travellers from several of Ghana's neighbouring countries, demonstrating that these variants were already in those countries even though not reported or detected (Fig. 2c). In most cases, VOCs and VOIs were identified amongst quarantined travellers before their detection within local samples. Travellers from Nigeria, Dubai, and the UK accounted for most detections of Alpha, Beta, Delta, Eta, and Delta-plus variants. Interestingly, the Beta and Kappa variants did not become dominant in Ghana; instead, B.1.1.318, which is likely to have originated from Nigeria, and detected in a traveller from Gabon, became dominant in Ghana.
Temporal trends of SARS-CoV-2 variant detection and frequency
Ghana was one of the last African countries to detect COVID-19 cases in March 2020, and the waves of COVID-19 in Ghana have lagged slightly behind other African countries and significantly behind the rest of the World (Fig. 3a). Previous work from our group described the viral genome dynamics between March and June 2020, when Ghana was largely closed to international travel (Ngoi et al., 2021). WHO (2021) reported that different variants rose to dominance at different times and during different infection waves across the country (Fig. 3a and 3b). Variants that cluster closely to B.1.1 were first detected in June 2020 (44.4%, 4/9) and peaked in July 2020 (58.7 %, 27/46). The B.1.1 lineage remained dominant in August (59.3 %, n=16/27), and October 2020 (55.2 %, n=16/29) but in September 2020, B.1.1.359 (55.6 %, n=10/18) became dominant while in December 2020 B.1.623 (33.3 %, n=9/27) was dominant alongside B.1.1 (29.6 %, n=8/27). The Alpha VOC, first detected in local samples from 1st January 2021, quickly supplanted the B.1.1 lineage as the most dominant circulating lineage (72.3 %, n=102/141) in January. Alpha maintained high frequency (60.0 %, n=42/70) in February 2021, followed by the VOI, Eta (20.0 %, n=14/70) (Fig. 3b & 3c). Alpha was still dominant in the March samples (70.9%, 22/31) but was almost completely supplanted in April 2021, as B.1.1.318 rose to prominence. Sequences clustering to B.1.1.318 had been detected as early as February 2021 (4.3 %, n=3/70) but became dominant in April 2021 (63.3 %, n=38/60) alongside Eta (23.3 %, n=14/60). May 2021 represented the peak of B.1.1.318 (82.3 %, n=79/96) and the emergence of the Delta (3.1 %, n=3/96) and Delta Plus (2.1 %, n=2/96) variants in local circulation. Delta and Delta-plus variants increased in proportion over time, overtaking the B.1.1.318 in June 2020. Delta-plus variants surged in July and now dominate all samples, with Delta being the only other variant with a significant presence in Ghana as of September 2021 (Fig. 3c).
Clinical presentation of the COVID-19 cases
Although most of the samples were from asymptomatic individuals who reported for COVID-19 testing, a good number of genomes (121/971) were from individuals who presented at treatment centres with mild/moderate or severe/critical symptoms. Majority of the individuals classified as mild/moderate (69.4 %, 84/121) presented with fever/chills, cough, pains, sore throat, diarrhoea, runny nose, nausea/vomiting, loss of smell, loss of taste or headache. Our data shows that less than a third of individuals presenting at the hospital were classified as severe/critical (30.5%, 37/121) with difficulty breathing (shortness of breath), hypoxia or multiorgan system dysfunction. The variant type was significantly associated clinical presentations (Pearson chi-square test, X2 = 23.4, df=5, P< .001), with all the cases of Alpha (100%, 16/16), as well as the vast majority of patients with Eta (10/11, 90.9 %) and B.1.1.318 (10/11, 90.9 %), presenting with mild/moderate symptoms. On the contrary, about half of cases infected with Delta (20/41, 48.8 %) and Delta-plus (10/19, 52.6 %) variants exhibited severe/critical COVID-19 (Fig. 3d).
Genetic diversity and evolutionary relationships of the SARS-CoV-2 variants
Amongst the many individual lineages represented in the data presented here, Delta, Delta Plus, Alpha, B.1.1.318, B.1.1.359, B.1.1, and Eta were the most evolved, with the highest genetic diversity (Fig. 4a). These variants exhibited a local variation in the number of mutations from sample to sample, with Delta, Alpha and B.1.1.318 presenting a mean ~30 (spread/range of 20-45) mutations in the majority of the genomes (Fig. 4a). The VOC delta plus, a subline of the Delta, had the highest mean (~35) and presented a range of mutations from 25 to 45 mutations across all the samples (Fig. 4a). It is worth noting that this level of genetic diversity in the 200 Delta plus samples mainly was attributed to the AY.39 (174/200) and AY.37 (15/200) lineages. Most of the other lineages with a small range of mutations were reported in 2020 and occurred spontaneously in very few samples hence the relatively low genetic diversity (Fig. 4a). The high level of genetic diversity in most VOCs, including the B.1.1318, is probably indicative of Ghana's local evolution and consequential adaptation compared to the other variants that did not gain prominence in the Ghanaian population (Fig. 4a).
A snapshot of the evolutionary relationship of these VOCs in Ghana shows an exciting relationship of variants through space and time throughout the epidemic (Fig. 4b). Using a phylogenetic tree, we outline the transmission events, how the VOCs were introduced, and how they gained prominence coinciding with the COVID-19 waves in Ghana. The outbreak of the COVID-19 pandemic started in mid-November 2019, but then the tree shows that the earliest lineages in Ghana are dated June 2020, although most VOCs were introduced in 2021 (Fig. 4b). The phylogenetic analysis of the genomes from Ghana shows similarities to VOCs around the World, with all the VOCs having the same common ancestor (Wuhan). Still, as they diverge, they share uniquely more recent ancestors; for example, we show that the recently classified sub-lineages of Delta share several recent ancestors in the same clade (Fig. 4b). The root-to-tip divergence of the VOCs as a function of sampling time show a molecular clock of the various VOCs, and with strong evidence, the variants are evolving in a clocklike manner (R2 =0.68) (Fig. 4c). The variants in Ghana are gaining 26.54 mutations per year, and of particular interest is the B.1.1.318 that did not gain prominence worldwide, but its molecular clock is similar to most of the VOCs in Ghana (Fig. 4c). Mutational fitness of the B.1.1.318 lineage showed that ten samples had spike mutations that were likely to confer viral fitness (mutational fitness >1) (Fig. 4c).
Mutational analysis of the amino acid substitutions
The most abundant mutation in all the samples was the Spike D614G (98%, 957/971), followed by ORF1b: P314L (92%, 900/971) (Fig. 5a). For many of the genes, one or more mutations were occurred in more than 100 samples, although spike protein dominated the profile (Fig. 5a). Interestingly, some variants with different evolutionary lineages had similar amino acid substitutions, mainly spike glycoprotein. The Eta variant had the highest (three) individual mutations (Q52R, Q677H and F888L) in the spike protein compared to other VOCs, contributing to its adaptability in Africa. Compared to other VOCs, the mutations unique to the Alpha variant were S13I, R567K, A570D, and T716I. The only mutation unique to the B.1.1.318 on the spike protein was the D1127G compared to other VOCs and VOIs. (Fig. 5b). Within these samples, the Delta and Delta plus shared fourteen mutations (T19R, G142D, R158G, A222V, L452R, T478K, E484Q, D614G, S680F, P681R, D950N, K1191N, G1219V and C1253F), but the Delta plus had P26L as a unique mutation (Fig. 5b). The unique mutations were fewer than shared mutations, thus explaining the increased abundance of some mutations among the VOCs. Those with the highest frequency in the spike protein among Delta plus, Delta, B.1, B.1.1, B.1.1.318, Alpha, Beta and Eta were fitness substitutions D614G and P681R/H (Fig. 5b). Alpha and B.1.1.318 had the P681H substitution while P681R was present in Delta, Eta and Delta-plus variants. Immune escape mutation E484K was present in B.1.1.318, Beta, and Eta, while Delta-plus variants frequently presented with E484Q mutation.