XBB.1, BQ1.1 and atypical BA.4.6/XBB.1 recombinants predominate current SARS-CoV-2 Wavelets with flu-like symptoms in Cameroon: A snapshot from genomic surveillance.

As of December 2022, Cameroon had observed a slight resurgence of COVID-19, raising concerns on genomic surveillance of related-SARS-CoV-2 variants under circulation. Following a laboratory-based survey, positive SARS-CoV-2 samples detected from December-2022 through March-2023 were processed for targeted sequencing at the Chantal BIYA International Reference Centre (CIRCB) in Yaoundé-Cameroon. From all positive cases detected, 13 were successfully sequenced (mean age 34 years, 70% female); the majority of the cases were unvaccinated (70%, 9/13) and symptomatic (92%, 12/13); all with flu-like symptoms (100%, 12/12). Following RT-PCR, the median cycle threshold was 22.23 [18-24] for the N gene; and 24.09 [20-26] for the ORF gene, underscoring high viral loads. Phylogenetic analysis of nucleotide sequences identified four major sub-variants in circulation, of which BA.5 (3/13), the recombinants BQ.1.1 (4/13), XBB.1 (4/13) and novel atypical variant of BA.4.6/XBB.1 (2/13). This snapshot surveillance indicates the introduction/emergence and circulation of new Omicron sub-variants, all accompanied by minor/mild symptoms. However, these new sub-variants and recombinants call for continuous genomic surveillance to prevent further resurgence of Covid-19 epidemiological wave.

Boosting pathogen genomics and bioinformatics workforce in Africa.

Next-generation sequencing (NGS) of genomic data has established its fundamental value in public health surveillance, research and development, and precision medicine. In Africa, severe shortages of competent experts in genomics and bioinformatics, few opportunities for research, and inadequate genomic infrastructure have had a knock-on effect on the use of NGS technologies for research and public health practice. Several reasons-ranging from poor funding, inadequate infrastructure for training and practice, to brain drain-might partly account for the scarcity of genomics and bioinformatics expertise in the region. In recognition of these shortcomings and the importance of NGS genomic data, which was amplified during the COVID-19 pandemic in mid-2021, the Africa Centres for Disease Control and Prevention (Africa CDC) through the Africa Pathogen Genomics Initiative began building and expanding Africa's workforce in pathogen surveillance. By the end of 2022, the Africa CDC in collaboration with its partners and centres of excellence had trained 413 personnel, mostly from public health institutions, in 53 (96%) of 55 African Union Member States. Although this training has increased genomics, bioinformatics, and genomic epidemiology literacy, and genomic-informed pathogen surveillance, there is still a need for a strategic and sustainable public health workforce development in Africa.

Amplicon deep sequencing of five highly polymorphic markers of Plasmodium falciparum reveals high parasite genetic diversity and moderate population structure in Ethiopia.

BACKGROUND: Plasmodium falciparum genetic diversity can add information on transmission intensity and can be used to track control and elimination interventions. METHODS: Dried blood spots (DBS) were collected from patients who were recruited for a P. falciparum malaria therapeutic efficacy trial in three malaria endemic sites in Ethiopia from October to December 2015, and November to December 2019. qPCR-confirmed infections were subject to amplicon sequencing of polymorphic markers ama1-D3, csp, cpp, cpmp, msp7. Genetic diversity, the proportion of multiclonal infections, multiplicity of infection, and population structure were analysed. RESULTS: Among 198 samples selected for sequencing, data was obtained for 181 samples. Mean MOI was 1.38 (95% CI 1.24-1.53) and 17% (31/181) of infections were polyclonal. Mean He across all markers was 0.730. Population structure was moderate; populations from Metema and Metehara 2015 were very similar to each other, but distinct from Wondogent 2015 and Metehara 2019. CONCLUSION: The high level of parasite genetic diversity and moderate population structure in this study suggests frequent gene flow of parasites among sites. The results obtained can be used as a baseline for additional parasite genetic diversity and structure studies, aiding in the formulation of appropriate control strategies in Ethiopia.

Insights into SARS-CoV-2 in Angola during the COVID-19 peak: Molecular epidemiology and genome surveillance.

Background: In Angola, COVID-19 cases have been reported in all provinces, resulting in >105,000 cases and >1900 deaths. However, no detailed genomic surveillance into the introduction and spread of the SARS-CoV-2 virus has been conducted in Angola. We aimed to investigate the emergence and epidemic progression during the peak of the COVID-19 pandemic in Angola. Methods: We generated 1210 whole-genome SARS-CoV-2 sequences, contributing West African data to the global context, that were phylogenetically compared against global strains. Virus movement events were inferred using ancestral state reconstruction. Results: The epidemic in Angola was marked by four distinct waves of infection, dominated by 12 virus lineages, including VOCs, VOIs, and the VUM C.16, which was unique to South-Western Africa and circulated for an extended period within the region. Virus exchanges occurred between Angola and its neighboring countries, and strong links with Brazil and Portugal reflected the historical and cultural ties shared between these countries. The first case likely originated from southern Africa. Conclusion: A lack of a robust genome surveillance network and strong dependence on out-of-country sequencing limit real-time data generation to achieve timely disease outbreak responses, which remains of the utmost importance to mitigate future disease outbreaks in Angola.

Unlocking the Potential of Genomic Data to Inform Typhoid Fever Control Policy: Supportive Resources for Genomic Data Generation, Analysis, and Visualization.

The global response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic demonstrated the value of timely and open sharing of genomic data with standardized metadata to facilitate monitoring of the emergence and spread of new variants. Here, we make the case for the value of Salmonella Typhi (S. Typhi) genomic data and demonstrate the utility of freely available platforms and services that support the generation, analysis, and visualization of S. Typhi genomic data on the African continent and more broadly by introducing the Africa Centres for Disease Control and Prevention's Pathogen Genomics Initiative, SEQAFRICA, Typhi Pathogenwatch, TyphiNET, and the Global Typhoid Genomics Consortium.

Targeted and whole-genome sequencing reveal a north-south divide in P. falciparum drug resistance markers and genetic structure in Mozambique.

Mozambique is one of the four African countries which account for over half of all malaria deaths worldwide, yet little is known about the parasite genetic structure in that country. We performed P. falciparum amplicon and whole genome sequencing on 2251 malaria-infected blood samples collected in 2015 and 2018 in seven provinces of Mozambique to genotype antimalarial resistance markers and interrogate parasite population structure using genome-wide microhaplotyes. Here we show that the only resistance-associated markers observed at frequencies above 5% were pfmdr1-184F (59%), pfdhfr-51I/59 R/108 N (99%) and pfdhps-437G/540E (89%). The frequency of pfdhfr/pfdhps quintuple mutants associated with sulfadoxine-pyrimethamine resistance increased from 80% in 2015 to 89% in 2018 (p < 0.001), with a lower expected heterozygosity and higher relatedness of microhaplotypes surrounding pfdhps mutants than wild-type parasites suggestive of recent selection. pfdhfr/pfdhps quintuple mutants also increased from 72% in the north to 95% in the south (2018; p < 0.001). This resistance gradient was accompanied by a concentration of mutations at pfdhps-436 (17%) in the north, a south-to-north increase in the genetic complexity of P. falciparum infections (p = 0.001) and a microhaplotype signature of regional differentiation. The parasite population structure identified here offers insights to guide antimalarial interventions and epidemiological surveys.

Population structure and genetic connectivity of Plasmodium falciparum in pre-elimination settings of Southern Africa.

To accelerate malaria elimination in the Southern African region by 2030, it is essential to prevent cross-border malaria transmission. However, countries within the region are highly interconnected due to human migration that aids in the movement of the parasite across geographical borders. It is therefore important to better understand Plasmodium falciparum transmission dynamics in the region, and identify major parasite source and sink populations, as well as cross-border blocks of high parasite connectivity. We performed a meta-analysis using collated parasite allelic data generated by microsatellite genotyping of malaria parasites from Namibia, Eswatini, South Africa, and Mozambique (N = 5,314). The overall number of unique alleles was significantly higher (P ≤ 0.01) in Namibia (mean A = 17.3 ± 1.46) compared to South Africa (mean A = 12.2 ± 1.22) and Eswatini (mean A = 13.3 ± 1.27, P ≤ 0.05), whilst the level of heterozygosity was not significantly different between countries. The proportion of polyclonal infections was highest for Namibia (77%), and lowest for Mozambique (64%). A was significant population structure was detected between parasites from the four countries, and patterns of gene flow showed that Mozambique was the major source area and Eswatini the major sink area of parasites between the countries. This study showed strong signals of parasite population structure and genetic connectivity between malaria parasite populations across national borders. This calls for strengthening the harmonization of malaria control and elimination efforts between countries in the southern African region. This data also proves its potential utility as an additional surveillance tool for malaria surveillance on both a national and regional level for the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance as countries work towards malaria elimination.

Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.

We propose a novel, non-discriminatory classification of monkeypox virus diversity. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause of the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomic surveillance.

Genomic Epidemiology of SARS-CoV-2 in Seychelles, 2020-2021.

Seychelles, an archipelago of 155 islands in the Indian Ocean, had confirmed 24,788 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the 31st of December 2021. The first SARS-CoV-2 cases in Seychelles were reported on the 14th of March 2020, but cases remained low until January 2021, when a surge was observed. Here, we investigated the potential drivers of the surge by genomic analysis of 1056 SARS-CoV-2 positive samples collected in Seychelles between 14 March 2020 and 31 December 2021. The Seychelles genomes were classified into 32 Pango lineages, 1042 of which fell within four variants of concern, i.e., Alpha, Beta, Delta and Omicron. Sporadic cases of SARS-CoV-2 detected in Seychelles in 2020 were mainly of lineage B.1 (lineage predominantly observed in Europe) but this lineage was rapidly replaced by Beta variant starting January 2021, and which was also subsequently replaced by the Delta variant in May 2021 that dominated till November 2021 when Omicron cases were identified. Using the ancestral state reconstruction approach, we estimated that at least 78 independent SARS-CoV-2 introduction events occurred in Seychelles during the study period. The majority of viral introductions into Seychelles occurred in 2021, despite substantial COVID-19 restrictions in place during this period. We conclude that the surge of SARS-CoV-2 cases in Seychelles in January 2021 was primarily due to the introduction of more transmissible SARS-CoV-2 variants into the islands.

Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.

BACKGROUND: Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. METHODS: A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. RESULTS: Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. CONCLUSIONS: The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

A review of the frequencies of Plasmodium falciparum Kelch 13 artemisinin resistance mutations in Africa.

Artemisinin resistance (AR) emerged in South East Asia 13 years ago and the identification of the resistance conferring molecular marker, Plasmodium falciparum Kelch 13 (Pfk13), 7 years ago has provided an invaluable tool for monitoring AR in malaria endemic countries. Molecular Pfk13 surveillance revealed the resistance foci in the Greater Mekong Subregion, an independent emergence in Guyana, South America, and a low frequency of mutations in Africa. The recent identification of the R561H Pfk13 AR associated mutation in Tanzania, Uganda and in Rwanda, where it has been associated with delayed parasite clearance, should be a concern for the continent. In this review, we provide a summary of Pfk13 resistance associated propeller domain mutation frequencies across Africa from 2012 to 2020, to examine how many other countries have identified these mutations. Only four African countries reported a recent identification of the M476I, P553L, R561H, P574L, C580Y and A675V Pfk13 mutations at low frequencies and with no reports of clinical treatment failure, except for Rwanda. These mutations present a threat to malaria control across the continent, since the greatest burden of malaria remains in Africa. A rise in the frequency of these mutations and their spread would reverse the gains made in the reduction of malaria over the last 20 years, given the lack of new antimalarial treatments in the event artemisinin-based combination therapies fail. The review highlights the frequency of Pfk13 propeller domain mutations across Africa, providing an up-to-date perspective of Pfk13 mutations, and appeals for an urgent and concerted effort to monitoring antimalarial resistance markers in Africa and the efficacy of antimalarials by re-establishing sentinel surveillance systems.

Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples.

BACKGROUND: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS. METHODS: Low parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance. RESULTS: At 100 parasites/µL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. CONCLUSIONS: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

Parasite genetic diversity reflects continued residual malaria transmission in Vhembe District, a hotspot in the Limpopo Province of South Africa.

BACKGROUND: South Africa aims to eliminate malaria transmission by 2023. However, despite sustained vector control efforts and case management interventions, the Vhembe District remains a malaria transmission hotspot. To better understand Plasmodium falciparum transmission dynamics in the area, this study characterized the genetic diversity of parasites circulating within the Vhembe District. METHODS: A total of 1153 falciparum-positive rapid diagnostic tests (RDTs) were randomly collected from seven clinics within the district, over three consecutive years (2016, 2017 and 2018) during the wet and dry malaria transmission seasons. Using 26 neutral microsatellite markers, differences in genetic diversity were described using a multiparameter scale of multiplicity of infection (MOI), inbreeding metric (Fws), number of unique alleles (A), expected heterozygosity (He), multilocus linkage disequilibrium (LD) and genetic differentiation, and were associated with temporal and geospatial variances. RESULTS: A total of 747 (65%) samples were successfully genotyped. Moderate to high genetic diversity (mean He = 0.74 ± 0.03) was observed in the parasite population. This was ascribed to high allelic richness (mean A = 12.2 ± 1.2). The majority of samples (99%) had unique multi-locus genotypes, indicating high genetic diversity in the sample set. Complex infections were observed in 66% of samples (mean MOI = 2.13 ± 0.04), with 33% of infections showing high within-host diversity as described by the Fws metric. Low, but significant LD (standardised index of association, ISA = 0.08, P < 0.001) was observed that indicates recombination of distinct clones. Limited impact of temporal (FST range - 0.00005 to 0.0003) and spatial (FST = - 0.028 to 0.023) variation on genetic diversity existed during the sampling timeframe and study sites respectively. CONCLUSIONS: Consistent with the Vhembe District's classification as a 'high' transmission setting within South Africa, P. falciparum diversity in the area was moderate to high and complex. This study showed that genetic diversity within the parasite population reflects the continued residual transmission observed in the Vhembe District. This data can be used as a reference point for the assessment of the effectiveness of on-going interventions over time, the identification of imported cases and/or outbreaks, as well as monitoring for the potential spread of anti-malarial drug resistance.

Within-household clustering of genetically related Plasmodium falciparum infections in a moderate transmission area of Uganda.

BACKGROUND: Evaluation of genetic relatedness of malaria parasites is a useful tool for understanding transmission patterns, but patterns are not easily detectable in areas with moderate to high malaria transmission. To evaluate the feasibility of detecting genetic relatedness in a moderate malaria transmission setting, relatedness of Plasmodium falciparum infections was measured in cohort participants from randomly selected households in the Kihihi sub-county of Uganda (annual entomological inoculation rate of 27 infectious bites per person). METHODS: All infections detected via microscopy or Plasmodium-specific loop mediated isothermal amplification from passive and active case detection during August 2011-March 2012 were genotyped at 26 microsatellite loci, providing data for 349 samples from 230 participants living in 80 households. Pairwise genetic relatedness was calculated using identity by state (IBS). RESULTS: As expected, genetic diversity was high (mean heterozygosity [He] = 0.73), and the majority (76.5 %) of samples were polyclonal. Despite the high genetic diversity, fine-scale population structure was detectable, with significant spatiotemporal clustering of highly related infections. Although the difference in malaria incidence between households at higher (mean 1127 metres) versus lower elevation (mean 1015 metres) was modest (1.4 malaria cases per person-year vs. 1.9 per person-year, respectively), there was a significant difference in multiplicity of infection (2.2 vs. 2.6, p = 0.008) and, more strikingly, a higher proportion of highly related infections within households (6.3 % vs. 0.9 %, p = 0.0005) at higher elevation compared to lower elevation. CONCLUSIONS: Genetic data from a relatively small number of diverse, multiallelic loci reflected fine scale patterns of malaria transmission. Given the increasing interest in applying genetic data to augment malaria surveillance, this study provides evidence that genetic data can be used to inform transmission patterns at local spatial scales even in moderate transmission areas.

Genomic-informed pathogen surveillance in Africa: opportunities and challenges.

The ongoing COVID-19 pandemic has highlighted the need to incorporate pathogen genomics for enhanced disease surveillance and outbreak management in Africa. The genomics of SARS-CoV-2 has been instrumental to the timely development of diagnostics and vaccines and in elucidating transmission dynamics. Global disease control programmes, including those for tuberculosis, malaria, HIV, foodborne pathogens, and antimicrobial resistance, also recommend genomics-based surveillance as an integral strategy towards control and elimination of these diseases. Despite the potential benefits, capacity remains low for many public health programmes in Africa. The COVID-19 pandemic presents an opportunity to reassess and strengthen surveillance systems and potentially integrate emerging technologies for preparedness of future epidemics and control of endemic diseases. We discuss opportunities and challenges for integrating pathogen genomics into public health surveillance systems in Africa. Improving accessibility through the creation of functional continent-wide networks, building multipathogen sequencing cores, training a critical mass of local experts, development of standards and policies to facilitate best practices for data sharing, and establishing a community of practice of genomics experts are all needed to use genomics for improved disease surveillance in Africa. Coordination and leadership are also crucial, which the Africa Centres for Disease Control and Prevention seeks to provide through its institute for pathogen genomics.