Cellular immune response to SARS-CoV-2 and clinical presentation in individuals exposed to endemic malaria.

Ghana and other parts of West Africa have experienced lower COVID-19 mortality rates than other regions. This phenomenon has been hypothesized to be associated with previous exposure to infections such as malaria. This study investigated the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the influence of previous malaria exposure. Blood samples were collected from individuals with asymptomatic or symptomatic COVID-19 (n = 217). A variety of assays were used to characterize the SARS-CoV-2-specific immune response, and malaria exposure was quantified using Plasmodium falciparum ELISA. The study found evidence of attenuated immune responses to COVID-19 among asymptomatic individuals, with elevated proportions of non-classical monocytes and greater memory B cell activation. Symptomatic patients displayed higher P. falciparum-specific T cell recall immune responses, whereas asymptomatic individuals demonstrated elevated P. falciparum antibody levels. Summarily, this study suggests that P. falciparum exposure-associated immune modulation may contribute to reduced severity of SARS-CoV-2 infection among people living in malaria-endemic regions.

Association of symptomatic upper respiratory tract infections with the alteration of the oropharyngeal microbiome in a cohort of school children in Côte d'Ivoire.

Introduction: The oropharyngeal microbiome plays an important role in protection against infectious agents when in balance. Despite use of vaccines and antibiotic therapy to prevent respiratory tract infections, they remain one of the major causes of mortality and morbidity in Low- and middle-income countries. Hence the need to explore other approaches to prevention by identifying microbial biomarkers that could be leveraged to modify the microbiota in order to enhance protection against pathogenic bacteria. The aim of this study was to analyze the oropharyngeal microbiome (OPM) of schoolchildren in Côte d'Ivoire presenting symptoms of upper respiratory tract infections (URTI) for better prevention strategy. Methods: Primary schools' children in Korhogo (n = 37) and Abidjan (n = 39) were followed for six months with monthly oropharyngeal sampling. Clinical diagnostic of URT infection was performed and nucleic acid extracted from oropharyngeal swabs were used for 16S rRNA metagenomic analysis and RT-PCR. Results: The clinical examination of children's throat in Abidjan and Korhogo identified respectively 17 (43.59%) and 15 (40.54%) participants with visible symptoms of URTIs, with 26 episodes of infection in Abidjan and 24 in Korhogo. Carriage of Haemophilus influenzae (12%), Streptococcus pneumoniae (6%) and SARS-CoV-2 (6%) was confirmed by PCR. A significant difference in alpha diversity was found between children colonized by S. pneumoniae and those that were not (p = 0.022). There was also a significant difference in alpha diversity between children colonised with H. influenzae and those who were not (p = 0.017). No significant difference was found for SARS-CoV-2. Sphingomonas, Ralstonia and Rothia were significantly enriched in non-carriers of S. pneumoniae; Actinobacillus was significantly enriched in non-carriers of H. influenzae; Actinobacillus and Porphyromonas were significantly enriched in non-carriers of SARS-CoV-2 (p < 0.001). Discussion: Nearly 40% of children showed clinical symptoms of infection not related to geographical location. The OPM showed an imbalance during H. influenzae and S. pneumoniae carriage. This study provides a baseline understanding of microbiome markers in URTIs in children for future research, to develop targeted interventions aimed at restoring the microbial balance and reducing the symptoms associated with RTIs.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays.

Background: The highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. Methods: To generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. Results: We successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. Significance: This assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

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.

Upper Airway Epithelial Tissue Transcriptome Analysis Reveals Immune Signatures Associated with COVID-19 Severity in Ghanaians.

The immunological signatures driving the severity of coronavirus disease 19 (COVID-19) in Ghanaians remain poorly understood. We performed bulk transcriptome sequencing of nasopharyngeal samples from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected Ghanaians with mild and severe COVID-19, as well as healthy controls to characterize immune signatures at the primary SARS-CoV-2 infection site and identify drivers of disease severity. Generally, a heightened antiviral response was observed in SARS-CoV-2-infected Ghanaians compared with uninfected controls. COVID-19 severity was associated with immune suppression, overexpression of proinflammatory cytokines, including CRNN, IL1A, S100A7, and IL23A, and activation of pathways involved in keratinocyte proliferation. SAMD9L was among the differentially regulated interferon-stimulated genes in our mild and severe disease cohorts, suggesting that it may play a critical role in SARS-CoV-2 pathogenesis. By comparing our data with a publicly available dataset from a non-African (Indians) (GSE166530), an elevated expression of antiviral response-related genes was noted in COVID-19-infected Ghanaians. Overall, the study describes immune signatures driving COVID-19 severity in Ghanaians and identifies immune drivers that could serve as potential prognostic markers for future outbreaks or pandemics. It further provides important preliminary evidence suggesting differences in antiviral response at the upper respiratory interface in sub-Saharan Africans (Ghanaians) and non-Africans, which could be contributing to the differences in disease outcomes. Further studies using larger datasets from different populations will expand on these findings.

Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana.

Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workflow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.

The global transcriptome of Plasmodium falciparum mid-stage gametocytes (stages II-IV) appears largely conserved and gametocyte-specific gene expression patterns vary in clinical isolates.

Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes.

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.

The temporal dynamics of Plasmodium species infection after artemisinin-based combination therapy (ACT) among asymptomatic children in the Hohoe municipality, Ghana.

BACKGROUND: The routine surveillance of asymptomatic malaria using nucleic acid-based amplification tests is essential in obtaining reliable data that would inform malaria policy formulation and the implementation of appropriate control measures. METHODS: In this study, the prevalence rate and the dynamics of Plasmodium species among asymptomatic children (n = 1697) under 5 years from 30 communities within the Hohoe municipality in Ghana were determined. RESULTS AND DISCUSSION: The observed prevalence of Plasmodium parasite infection by polymerase chain reaction (PCR) was 33.6% (571/1697), which was significantly higher compared to that obtained by microscopy [26.6% (451/1697)] (P < 0.0001). Based on species-specific analysis by nested PCR, Plasmodium falciparum infection [33.6% (570/1697)] was dominant, with Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections accounting for 0.1% (1/1697), 0.0% (0/1697), and 0.0% (0/1697), respectively. The prevalence of P. falciparum infection among the 30 communities ranged from 0.0 to 82.5%. Following artesunate-amodiaquine (AS + AQ, 25 mg/kg) treatment of a sub-population of the participants (n = 184), there was a substantial reduction in Plasmodium parasite prevalence by 100% and 79.2% on day 7 based on microscopy and nested PCR analysis, respectively. However, there was an increase in parasite prevalence from day 14 to day 42, with a subsequent decline on day 70 by both microscopy and nested PCR. For parasite clearance rate analysis, we found a significant proportion of the participants harbouring residual Plasmodium parasites or parasite genomic DNA on day 1 [65.0% (13/20)], day 2 [65.0% (13/20)] and day 3 [60.0% (12/20)] after initiating treatment. Of note, gametocyte carriage among participants was low before and after treatment. CONCLUSION: Taken together, the results indicate that a significant number of individuals could harbour residual Plasmodium parasites or parasite genomic DNA after treatment. The study demonstrates the importance of routine surveillance of asymptomatic malaria using sensitive nucleic acid-based amplification techniques.

Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana.

INTRODUCTION: The true nature of the population spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. METHODS: Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from Ghanaian SARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collected from blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection and levels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. RESULTS AND DISCUSSION: This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. CONCLUSION: This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

High burden of asymptomatic malaria and anaemia despite high adherence to malaria control measures: a cross-sectional study among pregnant women across two seasons in a malaria-endemic setting in Ghana.

PURPOSE: Anaemia remains a serious concern among pregnant women, and thus, it is closely monitored from the onset of pregnancy through to delivery to help prevent adverse maternal and neonatal outcomes. In malaria-endemic settings, continuous low-level carriage of P. falciparum parasites is common and its contribution to maternal anaemia should not be underestimated. In this study, we evaluated the impact of adherence to malaria control measures [number of antenatal clinics (ANC) attended, supervised intake of sulphadoxine pyrimethamine (SP), and use of insecticide treated bed nets (ITNs)] on asymptomatic malaria and anaemia outcomes among pregnant women on ANC in hospitals in the Central region of Ghana. METHODS: The study was conducted during two seasons; October-November 2020 (dry season, n = 124) and May-June 2021 (rainy season, n = 145). Among the women, there was a high adherence to the control measures for both seasons (ANC ≥ 3 visits; ~ 82.0%, intake of SP; ~ 80.0% and ITNs use; ~ 75.0%). RESULTS: Asymptomatic P. falciparum carriage was high for both seasons (44.4% for the dry season; 46.9% for the rainy season). Correspondingly, the occurrence of anaemia was high for both seasons (57.3% for the dry season; 68.3% for the rainy season) and was strongly predicted by carriage of P. falciparum parasites. Despite the high adherence to ANC protocols, asymptomatic P. falciparum infection was common and contributed to the high burden of maternal anaemia. CONCLUSIONS: Our findings emphasize the need for improved control measures that can clear asymptomatic/sub-microscopic P. falciparum infection and protect against malaria-induced anaemia among pregnant women attending ANC in malaria endemic-settings.

Genomic variation during culture adaptation of genetically complex Plasmodium falciparum clinical isolates.

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

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.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

A genetic Study of the Ghanaian Population Using 15 Autosomal STR Loci.

Autosomal short tandem repeat (STR) population data collected from a well characterized population are needed to correctly assigning the weight of DNA profiles in the courtroom and widely used for ancestral analyses. In this study, allele frequencies for the 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR® Identifiler® plus kit (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA) were obtained by genotyping 332 unrelated individuals of Ghanaian origin. Statistical tests on STR genotype data showed no significant departure from Hardy-Weinberg equilibrium (HWE). The overall match probability, combined power of exclusion and combined power of discrimination for these loci were 1 in 3.85 × 1017, 0.99999893 and 0.99999998, respectively. Polymorphic information content (PIC) greater than 0.70 was observed for all loci except TH01 and D13S317. These statistical parameters confirm that this combination of loci is valuable for forensic identification and parentage analysis. Our results were also compared with those for 20 other human populations analyzed for the same set of markers. We observed that the Ghanaian population grouped with other African populations in two-dimensional principal coordinate (PCO) and a neighbor-joining (N-J) data mapping and placed closest to Nigerians. This observation reflects cultural similarities and geographical factors, coupled with the long history of migration and trading activities between Ghana and Nigeria. Our report provides what we believe to be the first published autosomal STR data for the general Ghanaian population using 15 loci genotyped using the AmpFlSTR® Identifiler® plus kit methodology. Our data show that the loci tested have sufficient power to be used reliably for DNA profiling in forensic casework and help to elucidate the genetic history of people living in the country.

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.

The COVID-19, tuberculosis and HIV/AIDS: Ménage à Trois.

In December 2019, a novel pneumonic condition, Coronavirus disease 2019 (COVID- 19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), broke out in China and spread globally. The presentation of COVID-19 is more severe in persons with underlying medical conditions such as Tuberculosis (TB), Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) and other pneumonic conditions. All three diseases are of global concern and can significantly affect the lungs with characteristic cytokine storm, immunosuppression, and respiratory failure. Co-infections of SARS-CoV-2 with HIV and Mycobacterium tuberculosis (Mtb) have been reported, which may influence their pathogenesis and disease progression. Pulmonary TB and HIV/AIDS patients could be more susceptible to SARS-CoV-2 infection leading to lethal synergy and disease severity. Therefore, the biological and epidemiological interactions of COVID-19, HIV/AIDS, and TB need to be understood holistically. While data is needed to predict the impact of the COVID-19 pandemic on these existing diseases, it is necessary to review the implications of the evolving COVID-19 management on HIV/AIDS and TB control, including therapy and funding. Also, the impact of long COVID on patients, who may have this co-infection. Thus, this review highlights the implications of COVID-19, HIV/AIDS, and TB co-infection compares disease mechanisms, addresses growing concerns, and suggests a direction for improved diagnosis and general management.

Variants of LRP2, encoding a multifunctional cell-surface endocytic receptor, associated with hearing loss and retinal dystrophy.

Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.3: LRP2: c.5005A > G, p.(Asn1669Asp) and c.149C > G, p.(Thr50Ser). In Family 2, two sisters were found to be compound heterozygous for LRP2 variants, p.(Tyr3933Cys) and an experimentally confirmed c.7715 + 3A > T consensus splice-altering variant. In Family 3, the proband is compound heterozygous for a consensus donor splice site variant LRP2: c.8452_8452 + 1del and p.(Cys3150Tyr). In mouse cochlea, Lrp2 is expressed abundantly in the stria vascularis marginal cells demonstrated by smFISH, single-cell and single-nucleus RNAseq, suggesting that a deficiency of LRP2 may compromise the endocochlear potential, which is required for hearing. LRP2 variants have been associated with Donnai-Barrow syndrome and other multisystem pleiotropic phenotypes different from the phenotypes of the four cases reported herein. Our data expand the phenotypic spectrum associated with pathogenic variants in LRP2 warranting their consideration in individuals with a combination of hereditary hearing loss and retinal dystrophy.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.