Genome-wide association study identifies novel candidate malaria resistance genes in Cameroon.

Recent data suggest that only a small fraction of severe malaria heritability is explained by the totality of genetic markers discovered so far. The extensive genetic diversity within African populations means that significant associations are likely to be found in Africa. In their series of multi-site genome-wide association studies (GWAS) across sub-Saharan Africa, the Malaria Genomic Epidemiology Network (MalariaGEN) observed specific limitations and encouraged country-specific analyses. Here, we present findings of a GWAS of Cameroonian participants that contributed to MalariaGEN projects (n = 1103). We identified protective associations at polymorphisms within the enhancer region of CHST15 [Benjamin-Hochberg false discovery rate (FDR) < 0.02] that are specific to populations of African ancestry, and that tag strong eQTLs of CHST15 in hepatic cells. In-silico functional analysis revealed a signature of epigenetic regulation of CHST15 that is preserved in populations in historically malaria endemic regions, with haplotype analysis revealing a haplotype that is specific to these populations. Association analysis by ethnolinguistic group identified protective associations within SOD2 (FDR < 0.04), a gene previously shown to be significantly induced in pre-asymptomatic malaria patients from Cameroon. Haplotype analysis revealed substantial heterogeneity within the beta-like globin (HBB) gene cluster amongst the major ethnic groups in Cameroon confirming differential malaria pressure and underscoring age-old fine-scale genetic structure within the country. Our findings revealed novel insights in the evolutionary genetics of populations living in Cameroon under malaria pressure with new significant protective loci (CHST15 and SOD2) and emphasized the significant attenuation of genetic association signals by fine-scale genetic structure.

Co-infection of Plasmodium falciparum and Schistosoma mansoni is associated with anaemia.

BACKGROUND: Malaria and schistosomiasis persist as major public health challenge in sub-Saharan Africa. These infections have independently and also in polyparasitic infection been implicated in anaemia and nutritional deficiencies. This study aimed at assessing asymptomatic malaria, intestinal Schistosoma infections and the risk of anaemia among school children in the Tono irrigation area in the Kassena Nankana East Municipal (KNEM) in the Upper East Region of Northern Ghana. METHODS: A cross sectional survey of 326 school children was conducted in the KNEM. Kato Katz technique was used to detect Schistosoma eggs in stool. Finger-prick capillary blood sample was used for the estimation of haemoglobin (Hb) concentration and blood smear for malaria parasite detection by microscopy. RESULTS: The average age and Hb concentration were 10.9 years (standard deviation, SD: ± 2.29) and 11.2 g/dl (SD: ± 1.39) respectively with 58.9% (n = 192) being females. The overall prevalence of infection with any of the parasites (single or coinfection) was 49.4% (n = 161, 95% confidence interval, CI [44.0-54.8]). The prevalence of malaria parasite species or Schistosoma mansoni was 32.0% (n = 104) and 25.2% (n = 82), respectively with 7.7% (n = 25) coinfection. The prevalence of anaemia in the cohort was 40.5% (95%CI [35.3-45.9]), of which 44.4% harboured at least one of the parasites. The prevalence of anaemia in malaria parasite spp or S. mansoni mono-infections was 41.8% and 38.6%, respectively and 64.0% in coinfections. There was no statistically significant difference in the odds of being anaemic in mono-infection with malaria (OR = 1.22, 95% CI 0.71-2.11, p = 0.47) or S. mansoni (OR = 1.07, 95% CI 0.58-1.99, p = 0.83) compared to those with no infection. However, the odds of being anaemic and coinfected with malaria parasite species and S. mansoni was 3.03 times higher compared to those with no infection (OR = 3.03, 95% CI 1.26-7.28, p = 0.013). Conclusion The data show a high burden of malaria, S. mansoni infection and anaemia among school children in the irrigation communities. The risk of anaemia was exacerbated by coinfections with malaria parasite(s) and S. mansoni. Targeted integrated interventions are recommended in this focal area of KNEM.

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.

Knowledge, attitude and perception of West Africans towards COVID-19: a survey to inform public health intervention.

BACKGROUND: The first case of the novel coronavirus disease-2019 (COVID-19) in West Africa was first confirmed in Nigeria in February 2020. Since then, several public health interventions and preventive measures have been implemented to curtail transmission of the causative agent, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Therefore, this study was performed to assess the knowledge, attitudes, and perceptions of West Africans towards COVID-19. METHODS: An online survey was conducted between 29 September to 29 October 2020 among West Africans. Thirty-three survey questions were designed to collect sociodemographic data and participants' knowledge, attitude and perception towards COVID-19. The study targeted all West African nationals who were 18 years and above, and willing to participate in the study. Participants were either in-country or abroad. RESULTS: Overall, 1106 respondents (≥18 years) from 16 West African countries, with about 12.1% of them residing outside the West African subregion, participated in the survey. The respondents had an average COVID-19 knowledge score of 67.82 ± 8.31, with knowledge of the disease significantly associated with the country of residence (p = 0.00) and marginally (p = 0.05) so with settlement types (i.e., urban, suburban and rural areas). Most respondents (93.4%) could identify the main COVID-19 symptoms, and 73.20% would consult a healthcare professional if infected with SARS-CoV-2. Also, 75.2% of the respondents are willing to receive the COVID-19 vaccine, whereas 10.40% and 14.40% are unwilling and undecided, respectively. Perceptions of what constitute COVID-19 preventive measures were highly variable. Approximately, 8% of the respondents felt that their government responded excellently in managing the pandemic while a third felt that the response was just good. Also, more than half (54%) opined that isolation and treatment of COVID-19 patients is a way of curbing SARS-CoV-2 spread. CONCLUSIONS: Most West Africans have basic knowledge of COVID-19 and showed a positive attitude, with likely proactive practice towards the disease. However, results showed that these varied across countries and are influenced by the types of settlements. Therefore, the health and education authorities in various countries should develop focused measures capturing people in different settlements to improve their preventative measures when designing public health interventions for COVID-19 and any future epidemics or pandemics.

Plasmodium malariae, current knowledge and future research opportunities on a neglected malaria parasite species.

Plasmodium malariae is often reported as a benign malaria parasite. There are limited data on its biology and disease burden in sub-Saharan Africa (sSA) possibly due to the unavailability of specific and affordable tools for routine diagnosis and large epidemiology studies. In addition, P. malariae occurs at low parasite densities and in co-infections with other species, predominately P. falciparum. The paucity of data on P. malariae infections limits the capacity to accurately determine its contribution to malaria and the effect of control interventions against P. falciparum on its prevalence. Here, we summarise the current knowledge on P. malariae epidemiology in sSA - overall prevalence ranging from 0-32%, as detected by different diagnostic methods; seroprevalence ranging from 0-56% in three countries (Mozambique, Benin and Zimbabwe), and explore the future application of next-generation sequencing technologies as a tool for enriching P. malariae genomic epidemiology. This will provide insights into important adaptive mechanisms of this neglected non-falciparum species, including antimalarial drug resistance, local and regional parasite transmission patterns and genomic signatures of selection. Improved diagnosis and genomic surveillance of non-falciparum malaria parasites in Africa would be helpful in evaluating progress towards elimination of all human Plasmodium species.

Genomic analysis reveals independent evolution of Plasmodium falciparum populations in Ethiopia.

BACKGROUND: Plasmodium falciparum parasite populations in Ethiopia have been experiencing local selective pressures from drugs and immunity, leading to evolutionary adaptation. However, there was a paucity of data on genomic characterization and evolutionary adaptations of P. falciparum isolates from the central area of Ethiopia. METHODS: Whole-genome analysis of 25 P. falciparum isolates from central Ethiopia, specifically from West Arsi, were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles against global isolates from Cambodia, Thailand, DR Congo, and Malawi. RESULTS: A total of 18,517 high-quality single-nucleotide polymorphisms (SNPs) were identified in Ethiopian P. falciparum isolates. About 84% of the Ethiopian P. falciparum isolates had a FWS value > 0.95 showing a dominant single genotype infection in most isolates at the time of collection with little potential for out-crossing as expected in areas with low transmission intensity. Within-host diversity of Ethiopian infections was significantly different from East African (p < 0.001), but not Southeast Asian infections (P > 0.05). A significant population structure has been observed by PCA and population differentiation between Ethiopian parasites and East African (Fst ~ 10%) and Southeast Asian populations (Fst ~ 18%), suggesting limited gene flow and the independent evolution of the Ethiopian parasite population. Moreover, a total of 125 genes under balancing selection was found that include ama1, trap, eba175, and lsa3, previously identified as targets of human host immunity. Recent directional selection analysis using integrated standardized haplotype score (IHS) did not detect any selection signatures in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. However, known drug resistance-conferring mutations analysis showed that at least one SNP marker was fixed in these genes, but not in Pfdhps and PfK13. CONCLUSION: Plasmodium falciparum populations in the central region of Ethiopia was structurally diverged from both Southeast Asian and other East African populations. Malaria infections in Ethiopia had low within-host diversity, and parasites carry fixed chloroquine resistance markers despite the withdrawal of this drug for the treatment of P. falciparum.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana.

BACKGROUND: Anti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana. METHODS: A total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years. RESULTS: At the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons. CONCLUSIONS: Despite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

Machine learning approaches classify clinical malaria outcomes based on haematological parameters.

BACKGROUND: Malaria is still a major global health burden, with more than 3.2 billion people in 91 countries remaining at risk of the disease. Accurately distinguishing malaria from other diseases, especially uncomplicated malaria (UM) from non-malarial infections (nMI), remains a challenge. Furthermore, the success of rapid diagnostic tests (RDTs) is threatened by Pfhrp2/3 deletions and decreased sensitivity at low parasitaemia. Analysis of haematological indices can be used to support the identification of possible malaria cases for further diagnosis, especially in travellers returning from endemic areas. As a new application for precision medicine, we aimed to evaluate machine learning (ML) approaches that can accurately classify nMI, UM, and severe malaria (SM) using haematological parameters. METHODS: We obtained haematological data from 2,207 participants collected in Ghana: nMI (n = 978), SM (n = 526), and UM (n = 703). Six different ML approaches were tested, to select the best approach. An artificial neural network (ANN) with three hidden layers was used for multi-classification of UM, SM, and uMI. Binary classifiers were developed to further identify the parameters that can distinguish UM or SM from nMI. Local interpretable model-agnostic explanations (LIME) were used to explain the binary classifiers. RESULTS: The multi-classification model had greater than 85% training and testing accuracy to distinguish clinical malaria from nMI. To distinguish UM from nMI, our approach identified platelet counts, red blood cell (RBC) counts, lymphocyte counts, and percentages as the top classifiers of UM with 0.801 test accuracy (AUC = 0.866 and F1 score = 0.747). To distinguish SM from nMI, the classifier had a test accuracy of 0.96 (AUC = 0.983 and F1 score = 0.944) with mean platelet volume and mean cell volume being the unique classifiers of SM. Random forest was used to confirm the classifications, and it showed that platelet and RBC counts were the major classifiers of UM, regardless of possible confounders such as patient age and sampling location. CONCLUSION: The study provides proof of concept methods that classify UM and SM from nMI, showing that the ML approach is a feasible tool for clinical decision support. In the future, ML approaches could be incorporated into clinical decision-support algorithms for the diagnosis of acute febrile illness and monitoring response to acute SM treatment particularly in endemic settings.

The biology of unconventional invasion of Duffy-negative reticulocytes by Plasmodium vivax and its implication in malaria epidemiology and public health.

Plasmodium vivax has been largely neglected over the past century, despite a widespread recognition of its burden across region where it is endemic. The parasite invades reticulocytes, employing the interaction between Plasmodium vivax Duffy binding protein (PvDBP) and human Duffy antigen receptor for chemokines (DARC). However, P. vivax has now been observed in Duffy-negative individuals, presenting a potentially serious public health problem as the majority of African populations are Duffy-negative. Invasion of Duffy-negative reticulocytes is suggested to be through duplication of the PvDBP and a novel protein encoded by P. vivax erythrocyte binding protein (EBP) genes. The emergence and spread of specific P. vivax strains with ability to invade Duffy-negative reticulocytes has, therefore, drawn substantial attention and further complicated the epidemiology and public health implication of vivax malaria. Given the right environment and vectorial capacity for transmission coupled with the parasite's ability to invade Duffy-negative individuals, P. vivax could increase its epidemiological significance in Africa. In this review, authors present accruing knowledge on the paradigm shift in P. vivax invasion of Duffy-negative reticulocytes against the established mechanism of invading only Duffy-positive individuals and offer a perspective on the epidemiological diagnostic and public health implication in Africa.

Population genetic analysis of the Plasmodium falciparum circumsporozoite protein in two distinct ecological regions in Ghana.

BACKGROUND: Extensive genetic diversity in the Plasmodium falciparum circumsporozoite protein (PfCSP) is a major contributing factor to the moderate efficacy of the RTS,S/AS01 vaccine. The transmission intensity and rates of recombination within and between populations influence the extent of its genetic diversity. Understanding the extent and dynamics of PfCSP genetic diversity in different transmission settings will help to interpret the results of current RTS,S efficacy and Phase IV implementation trials conducted within and between populations in malaria-endemic areas such as Ghana. METHODS: Pfcsp sequences were retrieved from the Illumina-generated paired-end short-read sequences of 101 and 131 malaria samples from children aged 6-59 months presenting with clinical malaria at health facilities in Cape Coast (in the coastal belt) and Navrongo (Guinea savannah region), respectively, in Ghana. The sequences were mapped onto the 3D7 reference strain genome to yield high-quality genome-wide coding sequence data. Following data filtering and quality checks to remove missing data, 220 sequences were retained and analysed for the allele frequency spectrum, genetic diversity both within the host and between populations and signatures of selection. Population genetics tools were used to determine the extent and dynamics of Pfcsp diversity in P. falciparum from the two geographically distinct locations in Ghana. RESULTS: Pfcsp showed extensive diversity at the two sites, with the higher transmission site, Navrongo, exhibiting higher within-host and population-level diversity. The vaccine strain C-terminal epitope of Pfcsp was found in only 5.9% and 45.7% of the Navrongo and Cape Coast sequences, respectively. Between 1 and 6 amino acid variations were observed in the TH2R and TH3R epitope regions of PfCSP. Tajima's D was negatively skewed, especially for the population from Cape Coast, given the expected historical population expansion. In contrast, a positive Tajima's D was observed for the Navrongo P. falciparum population, consistent with balancing selection acting on the immuno-dominant TH2R and TH3R vaccine epitopes. CONCLUSION: The low frequencies of the Pfcsp vaccine haplotype in the analysed populations indicate a need for additional molecular and immuno-epidemiological studies with broader temporal and geographic sampling in endemic populations targeted for RTS,S application. These results have implications for the efficacy of the vaccine in Ghana and will inform the choice of alleles to be included in future multivalent or chimeric vaccines.

Targeted Next Generation Sequencing for malaria research in Africa: current status and outlook.

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data.

We present a rigorous statistical model that infers the structure of P. falciparum mixtures-including the number of strains present, their proportion within the samples, and the amount of unexplained mixture-using whole genome sequence (WGS) data. Applied to simulation data, artificial laboratory mixtures, and field samples, the model provides reasonable inference with as few as 10 reads or 50 SNPs and works efficiently even with much larger data sets. Source code and example data for the model are provided in an open source fashion. We discuss the possible uses of this model as a window into within-host selection for clinical and epidemiological studies.

Approaches to estimating inbreeding coefficients in clinical isolates of Plasmodium falciparum from genomic sequence data.

BACKGROUND: The advent of whole-genome sequencing has generated increased interest in modelling the structure of strain mixture within clinical infections of Plasmodium falciparum The life cycle of the parasite implies that the mixture of multiple strains within an infected individual is related to the out-crossing rate across populations, making methods for measuring this process in situ central to understanding the genetic epidemiology of the disease. RESULTS: This paper derives a set of new estimators for inferring inbreeding coefficients using whole genome sequence read count data from P. falciparum clinical samples, which provides resources to assess within-sample mixture that connect to extensive literatures in population genetics and conservation ecology. Features of the P. falciparum genome mean that standard methods for inbreeding coefficients and related F-statistics cannot be used directly. After reviewing an initial effort to estimate the inbreeding coefficient within clinical isolates of P. falciparum, several generalizations using both frequentist and Bayesian approaches are provided. A simpler, more intuitive frequentist estimator is shown to have nearly identical properties to the initial estimator both in simulation and in real data sets. The Bayesian approach connects these estimates to the Balding-Nichols model, a mainstay within genetic epidemiology, and a possible framework for more complex modelling. A simulation study shows strong performance for all estimators with as few as ten variants. Application to samples from the PF3K data set indicate significant across-country variation in within-sample mixture. Finally, a comparison with results from a recent mixture model for within-sample strain mixture show that inbreeding coefficients provide a strong proxy for these more complex models. CONCLUSIONS: This paper provides a set of methods for estimating inbreeding coefficients within P. falciparum samples from whole-genome sequence data, supported by simulation studies and empirical examples. It includes a substantially simple estimator with similar statistical properties to the estimator in current use. These methods will also be applicable to other species with similar life-cycles. Implementations of the methods described are available in an open-source R package pfmix. Estimates for the PF3K public data release are provide as part of this resource.

Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification.

BACKGROUND: Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB. METHODS: Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8-12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired (P. falciparum DNA) and contaminating (human) genomes. RESULTS: Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. CONCLUSION: The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.

K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Global Distribution of Founder Variants Associated with Non-Syndromic Hearing Impairment.

The genetic etiology of non-syndromic hearing impairment (NSHI) is highly heterogeneous with over 124 distinct genes identified. The wide spectrum of implicated genes has challenged the implementation of molecular diagnosis with equal clinical validity in all settings. Differential frequencies of allelic variants in the most common NSHI causal gene, gap junction beta 2 (GJB2), has been described as stemming from the segregation of a founder variant and/or spontaneous germline variant hot spots. We aimed to systematically review the global distribution and provenance of founder variants associated with NSHI. The study protocol was registered on PROSPERO, the International Prospective Register of Systematic Reviews, with the registration number "CRD42020198573". Data from 52 reports, involving 27,959 study participants from 24 countries, reporting 56 founder pathogenic or likely pathogenic (P/LP) variants in 14 genes (GJB2, GJB6, GSDME, TMC1, TMIE, TMPRSS3, KCNQ4, PJVK, OTOF, EYA4, MYO15A, PDZD7, CLDN14, and CDH23), were reviewed. Varied number short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) were used for haplotype analysis to identify the shared ancestral informative markers in a linkage disequilibrium and variants' origins, age estimates, and common ancestry computations in the reviewed reports. Asia recorded the highest number of NSHI founder variants (85.7%; 48/56), with variants in all 14 genes, followed by Europe (16.1%; 9/56). GJB2 had the highest number of ethnic-specific P/LP founder variants. This review reports on the global distribution of NSHI founder variants and relates their evolution to population migration history, bottleneck events, and demographic changes in populations linked with the early evolution of deleterious founder alleles. International migration and regional and cultural intermarriage, coupled to rapid population growth, may have contributed to re-shaping the genetic architecture and structural dynamics of populations segregating these pathogenic founder variants. We have highlighted and showed the paucity of data on hearing impairment (HI) variants in Africa, establishing unexplored opportunities in genetic traits.

Comparative Susceptibility of Plasmodium ovale and Plasmodium falciparum Field Isolates to Reference and Lead Candidate Antimalarial Drugs in Ghana.

Malaria treatments resulted in the decline of the deadliest Plasmodium falciparum globally while species, such as P. ovale, infections have been increasingly detected across sub-Saharan Africa. Currently, no experimental drug sensitivity data are available to guide effective treatment and management of P. ovale infections, which is necessary for effective malaria elimination. We conducted a prospective study to evaluate P. ovale epidemiology over 1 year and determined ex vivo susceptibility of the field isolates to existing and lead advanced discovery antimalarial drugs. We report that while P. falciparum dominated both symptomatic and asymptomatic malaria cases, P. ovale in mono or co-infections caused 7.16% of symptomatic malaria. Frontline antimalarials artesunate and lumefantrine inhibited P. ovale as potently as P. falciparum. Chloroquine, which has been withdrawn in Ghana, was also highly inhibitory against both P. ovale and P. falciparum. In addition, P. ovale and P. falciparum displayed high susceptibility to quinine, comparable to levels observed with chloroquine. Pyrimethamine, which is a major drug for disease massive prevention, also showed great inhibition of P. ovale, comparable to effects on P. falciparum. Furthermore, we identified strong inhibition of P. ovale using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drugs currently in clinical phase II testing. We further demonstrated that the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor, KDU691, is highly inhibitory against P. ovale and P. falciparum field isolates. Our data indicated that existing and lead advanced discovery antimalarial drugs are suitable for the treatment of P. ovale infections in Ghana. IMPORTANCE Current malaria control and elimination tools such as drug treatments are not specifically targeting P.ovale. P. ovale can form hypnozoite and cause relapsing malaria. P. ovale is the third most dominant species in Africa and requires radical cure treatment given that it can form liver dormant forms called hypnozoites that escape all safe treatments. The inappropriate treatment of P. ovale would sustain its transmission in Africa where the medical need is the greatest. This is a hurdle for successful malaria control and elimination. Here, we provided experiment data that were lacking to guide P. ovale treatment and disease control policy makers using reference antimalarial drugs. We also provided key experimental data for 2 clinical candidate drugs that can be used for prioritization selection of lead candidate's identification for clinical development.