Comparison of molecular surveillance methods to assess changes in the population genetics of Plasmodium falciparum in high transmission.

A major motivation for developing molecular methods for malaria surveillance is to measure the impact of control interventions on the population genetics of Plasmodium falciparum as a potential marker of progress towards elimination. Here we assess three established methods (i) single nucleotide polymorphism (SNP) barcoding (panel of 24-biallelic loci), (ii) microsatellite genotyping (panel of 12-multiallelic loci), and (iii) varcoding (fingerprinting var gene diversity, akin to microhaplotyping) to identify changes in parasite population genetics in response to a short-term indoor residual spraying (IRS) intervention. Typical of high seasonal transmission in Africa, multiclonal infections were found in 82.3% (median 3; range 1-18) and 57.8% (median 2; range 1-12) of asymptomatic individuals pre- and post-IRS, respectively, in Bongo District, Ghana. Since directly phasing multilocus haplotypes for population genetic analysis is not possible for biallelic SNPs and microsatellites, we chose ~200 low-complexity infections biased to single and double clone infections for analysis. Each genotyping method presented a different pattern of change in diversity and population structure as a consequence of variability in usable data and the relative polymorphism of the molecular markers (i.e., SNPs < microsatellites < var). Varcoding and microsatellite genotyping showed the overall failure of the IRS intervention to significantly change the population structure from pre-IRS characteristics (i.e., many diverse genomes of low genetic similarity). The 24-SNP barcode provided limited information for analysis, largely due to the biallelic nature of SNPs leading to a high proportion of double-allele calls and a view of more isolate relatedness compared to microsatellites and varcoding. Relative performance, suitability, and cost-effectiveness of the methods relevant to sample size and local malaria elimination in high-transmission endemic areas are discussed.

Background malaria incidence and parasitemia during the three-dose RTS,S/AS01 vaccination series do not reduce magnitude of antibody response nor efficacy against the first case of malaria.

BACKGROUND: RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group. METHODS: To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria (to exclude the effect of naturally acquired immunity) using data from three study areas (Kintampo, Ghana; Lilongwe, Malawi; Lambaréné, Gabon) from the 2009-2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and background malaria incidence. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01. RESULTS: We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by background incidence or parasitemia during the primary vaccination series. CONCLUSIONS: We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that control-group immunity is likely a major reason for lower efficacy in high transmission settings, not reduced immune responses to RTS,S/AS01. This may be reassuring for implementation in high transmission settings, though further studies are needed.

Evaluation of KIR3DL1/KIR3DS1 allelic polymorphisms in Kenyan children with endemic Burkitt lymphoma.

Endemic Burkitt lymphoma (eBL) is a fast-growing germinal center B cell lymphoma, affecting 5-10 per 100,000 children annually, in the equatorial belt of Africa. We hypothesize that co-infections with Plasmodium falciparum (Pf) malaria and Epstein-Barr virus (EBV) impair host natural killer (NK) and T cell responses to tumor cells, and thus increase the risk of eBL pathogenesis. NK cell education is partially controlled by killer immunoglobulin-like receptors and variable expression of KIR3DL1 has been associated with other malignancies. Here, we investigated whether KIR3D-mediated mechanisms contribute to eBL, by testing for an association of KIR3DL1/KIR3DS1 genotypes with the disease in 108 eBL patients and 99 healthy Kenyan children. KIR3DL1 allelic typing and EBV loads were assessed by PCR. We inferred previously observed phenotypes from the genotypes. The frequencies of KIR3DL1/KIR3DL1 and KIR3DL1/KIR3DS1 did not differ significantly between cases and controls. Additionally, none of the study participants was homozygous for KIR3DS1 alleles. EBV loads did not differ by the KIR3DL1 genotypes nor were they different between eBL survivors and non-survivors. Our results suggest that eBL pathogenesis may not simply involve variations in KIR3DL1 and KIR3DS1 genotypes. However, considering the complexity of the KIR3DL1 locus, this study could not exclude a role for copy number variation in eBL pathogenesis.

Performance of SNP barcodes to determine genetic diversity and population structure of Plasmodium falciparum in Africa.

Panels of informative biallelic single nucleotide polymorphisms (SNPs) have been proposed to be an economical method to fast-track the population genetic analysis of Plasmodium falciparum in malaria-endemic areas. Whilst used successfully in low-transmission areas where infections are monoclonal and highly related, we present the first study to evaluate the performance of these 24- and 96-SNP molecular barcodes in African countries, characterised by moderate-to-high transmission, where multiclonal infections are prevalent. For SNP barcodes it is generally recommended that the SNPs chosen i) are biallelic, ii) have a minor allele frequency greater than 0.10, and iii) are independently segregating, to minimise bias in the analysis of genetic diversity and population structure. Further, to be standardised and used in many population genetic studies, these barcodes should maintain characteristics i) to iii) across various iv) geographies and v) time points. Using haplotypes generated from the MalariaGEN P. falciparum Community Project version six database, we investigated the ability of these two barcodes to fulfil these criteria in moderate-to-high transmission African populations in 25 sites across 10 countries. Predominantly clinical infections were analysed, with 52.3% found to be multiclonal, generating high proportions of mixed-allele calls (MACs) per isolate thereby impeding haplotype construction. Of the 24- and 96-SNPs, loci were removed if they were not biallelic and had low minor allele frequencies in all study populations, resulting in 20- and 75-SNP barcodes respectively for downstream population genetics analysis. Both SNP barcodes had low expected heterozygosity estimates in these African settings and consequently biased analyses of similarity. Both minor and major allele frequencies were temporally unstable. These SNP barcodes were also shown to identify weak genetic differentiation across large geographic distances based on Mantel Test and DAPC. These results demonstrate that these SNP barcodes are vulnerable to ascertainment bias and as such cannot be used as a standardised approach for malaria surveillance in moderate-to-high transmission areas in Africa, where the greatest genomic diversity of P. falciparum exists at local, regional and country levels.

Malaria Transmission Intensity and Parasitemia during the Three-Dose RTS,S/AS01 Vaccination Series do not Reduce Magnitude of Antibody Response nor Efficacy Against the First Case of Malaria.

Background: RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group. Methods: To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria to exclude the delayed malaria effect using data from three study areas (Kintampo, Ghana; Lilongwe, Malawi; Lambaréné, Gabon) from the 2009-2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and malaria transmission intensity. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01. Results: We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by transmission intensity or parasitemia during the primary vaccination series. Conclusions: We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that delayed malaria is likely the main reason for lower efficacy in high transmission settings, not reduced immune responses. This may be reassuring for implementation in high transmission settings, though further studies are needed.

Measuring changes in Plasmodium falciparum census population size in response to sequential malaria control interventions.

Here we introduce a new endpoint "census population size" to evaluate the epidemiology and control of Plasmodium falciparum infections, where the parasite, rather than the infected human host, is the unit of measurement. To calculate census population size, we rely on a definition of parasite variation known as multiplicity of infection (MOIvar), based on the hyper-diversity of the var multigene family. We present a Bayesian approach to estimate MOIvar from sequencing and counting the number of unique DBLα tags (or DBLα types) of var genes, and derive from it census population size by summation of MOIvar in the human population. We track changes in this parasite population size and structure through sequential malaria interventions by indoor residual spraying (IRS) and seasonal malaria chemoprevention (SMC) from 2012 to 2017 in an area of high-seasonal malaria transmission in northern Ghana. Following IRS, which reduced transmission intensity by > 90% and decreased parasite prevalence by ~40-50%, significant reductions in var diversity, MOIvar, and population size were observed in ~2,000 humans across all ages. These changes, consistent with the loss of diverse parasite genomes, were short lived and 32-months after IRS was discontinued and SMC was introduced, var diversity and population size rebounded in all age groups except for the younger children (1-5 years) targeted by SMC. Despite major perturbations from IRS and SMC interventions, the parasite population remained very large and retained the var population genetic characteristics of a high-transmission system (high var diversity; low var repertoire similarity) demonstrating the resilience of P. falciparum to short-term interventions in high-burden countries of sub-Saharan Africa.

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.

SARS-CoV-2 Molecular Evolutionary Dynamics in the Greater Accra Region, Ghana.

To assess dynamics of SARS-CoV-2 in Greater Accra Region, Ghana, we analyzed SARS-CoV-2 genomic sequences from persons in the community and returning from international travel. The Accra Metropolitan District was a major origin of virus spread to other districts and should be a primary focus for interventions against future infectious disease outbreaks.

Determinants of yam farmers' adaptation practices to climate variability in the Ejura Sekyedumase municipality, Ghana.

This study assessed the factors affecting yam farmers' adaptation practices to climate variability in the Ejura Sekyedumase Municipality, Ghana. Primary data was collected through 160 household surveys and 8 key informant interviews. Binary logistic regression was used to assess the factors affecting the yam farmers' choice of adaptation practices. Findings revealed that the yam farmers perceived climate variability through changes in the onset and patterns of rainfall, increasing windstorms, and rising temperatures. The yam farmers perceived the adverse effects of climate variability on their yam production to include a reduction in yam yields (98%), decreased income of farmers (97%), and reduced soil fertility (97%). Findings also showed that the yam farmers utilized key adaptation practices including changing the planting period of yam (88%), planting early-maturing yam seeds/varieties (86%), and intercropping yam with other crops (84%). The key adaptation practices implemented by the farmers were significantly affected by socioeconomic factors including age, years of stay in the community, household size, access to extension services, marital status, education, farming experience, access to climate information, estimated farm size, and estimated farm income (p < 0.05). The empirical findings of this study reinforce the importance of policymakers devising suitable climate variability adaptation policies for yam farmers in Ghana while taking into cognizance the socioeconomic factors that could affect their adoption of such adaptation practices.

Neutral vs. non-neutral genetic footprints of Plasmodium falciparum multiclonal infections.

At a time when effective tools for monitoring malaria control and eradication efforts are crucial, the increasing availability of molecular data motivates their application to epidemiology. The multiplicity of infection (MOI), defined as the number of genetically distinct parasite strains co-infecting a host, is one key epidemiological parameter for evaluating malaria interventions. Estimating MOI remains a challenge for high-transmission settings where individuals typically carry multiple co-occurring infections. Several quantitative approaches have been developed to estimate MOI, including two cost-effective ones relying on molecular data: i) THE REAL McCOIL method is based on putatively neutral single nucleotide polymorphism loci, and ii) the varcoding method is a fingerprinting approach that relies on the diversity and limited repertoire overlap of the var multigene family encoding the major Plasmodium falciparum blood-stage antigen PfEMP1 and is therefore under selection. In this study, we assess the robustness of the MOI estimates generated with these two approaches by simulating P. falciparum malaria dynamics under three transmission conditions using an extension of a previously developed stochastic agent-based model. We demonstrate that these approaches are complementary and best considered across distinct transmission intensities. While varcoding can underestimate MOI, it allows robust estimation, especially under high transmission where repertoire overlap is extremely limited from frequency-dependent selection. In contrast, THE REAL McCOIL often considerably overestimates MOI, but still provides reasonable estimates for low and moderate transmission. Regardless of transmission intensity, results for THE REAL McCOIL indicate that an inaccurate tail at high MOI values is generated, and that at high transmission, an apparently reasonable estimated MOI distribution can arise from some degree of compensation between overestimation and underestimation. As many countries pursue malaria elimination targets, defining the most suitable approach to estimate MOI based on sample size and local transmission intensity is highly recommended for monitoring the impact of intervention programs.

Plasmodium malariae structure and genetic diversity in sub-Saharan Africa determined from microsatellite variants and linked SNPs in orthologues of antimalarial resistance genes.

Plasmodium malariae, a neglected human malaria parasite, contributes up to 10% of malaria infections in sub-Saharan Africa (sSA). Though P. malariae infection is considered clinically benign, it presents mostly as coinfections with the dominant P. falciparum. Completion of its reference genome has paved the way to further understand its biology and interactions with the human host, including responses to antimalarial interventions. We characterized 75 P. malariae isolates from seven endemic countries in sSA using highly divergent microsatellites. The P. malariae infections were highly diverse and five subpopulations from three ancestries (independent of origin of isolates) were determined. Sequences of 11 orthologous antimalarial resistance genes, identified low frequency single nucleotide polymorphisms (SNPs), strong linkage disequilibrium between loci that may be due to antimalarial drug selection. At least three sub-populations were detectable from a subset of denoised SNP data from mostly the mitochondrial cytochrome b coding region. This evidence of diversity and selection calls for including P. malariae in malaria genomic surveillance towards improved tools and strategies for malaria elimination.

Malaria Transmission Intensity Likely Modifies RTS, S/AS01 Efficacy Due to a Rebound Effect in Ghana, Malawi, and Gabon.

BACKGROUND: RTS,S/AS01 is the first malaria vaccine to be approved and recommended for widespread implementation by the World Health Organization (WHO). Trials reported lower vaccine efficacies in higher-incidence sites, potentially due to a "rebound" in malaria cases in vaccinated children. When naturally acquired protection in the control group rises and vaccine protection in the vaccinated wanes concurrently, malaria incidence can become greater in the vaccinated than in the control group, resulting in negative vaccine efficacies. METHODS: Using data from the 2009-2014 phase III trial (NCT00866619) in Lilongwe, Malawi; Kintampo, Ghana; and Lambaréné, Gabon, we evaluate this hypothesis by estimating malaria incidence in each vaccine group over time and in varying transmission settings. After estimating transmission intensities using ecological variables, we fit models with 3-way interactions between vaccination, time, and transmission intensity. RESULTS: Over time, incidence decreased in the control group and increased in the vaccine group. Three-dose efficacy in the lowest-transmission-intensity group (0.25 cases per person-year [CPPY]) decreased from 88.2% to 15.0% over 4.5 years, compared with 81.6% to -27.7% in the highest-transmission-intensity group (3 CPPY). CONCLUSIONS: These findings suggest that interventions, including the fourth RTS,S dose, that protect vaccinated individuals during the potential rebound period should be implemented for high-transmission settings.

Targeting the Plasmodium falciparum's Thymidylate Monophosphate Kinase for the Identification of Novel Antimalarial Natural Compounds.

Recent reports of resistance to artemisinin-based combination drugs necessitate the need to discover novel antimalarial compounds. The present study was aimed at identifying novel antimalarial compounds from natural product libraries using computational methods. Plasmodium falciparum is highly dependent on the pyrimidine biosynthetic pathway, a de novo pathway responsible for the production of pyrimidines, and the parasite lacks the pyrimidine salvage enzymes. The P. falciparum thymidylate monophosphate kinase (PfTMPK) is an important protein necessary for rapid DNA replication; however, due to its broad substrate specificity, the protein is distinguished from its homologs, making it a suitable drug target. Compounds from AfroDB, a database of natural products originating from Africa, were screened virtually against PfTMPK after filtering the compounds for absorption, distribution, metabolism, excretion, and toxicity (ADMET)-acceptable compounds with FAF-Drugs4. Thirteen hits with lower binding energies than thymidine monophosphate were selected after docking. Among the thirteen compounds, ZINC13374323 and ZINC13365918 with binding energies of -9.4 and -8.9 kcal/mol, respectively, were selected as plausible lead compounds because they exhibited structural properties that ensure proper binding at the active site and inhibitory effect against PfTMPK. ZINC13374323 (also called aurantiamide acetate) is known to exhibit anti-inflammatory and antiviral activities, and ZINC13365918 exhibits antileishmanial activity. Furthermore, aurantiamide acetate, which is commercially available, is a constituent of Artemisia annua, the herb from which artemisinin was derived. The compound also shares interactions with several residues with a potent thymidine analog inhibitor of PfTMPK. The anti-plasmodial activity of aurantiamide acetate was evaluated in vitro, and the mean half-maximal inhibitory concentration (IC50) was 69.33 µM when synchronized P. falciparum 3D7 culture was used as compared to IC50 > 100 µM with asynchronized culture. The significance of our findings within the context of malaria treatment strategies and challenges is discussed.

Indoor residual spraying with a non-pyrethroid insecticide reduces the reservoir of Plasmodium falciparum in a high-transmission area in northern Ghana.

High-malaria burden countries in sub-Saharan Africa are shifting from malaria control towards elimination. Hence, there is need to gain a contemporary understanding of how indoor residual spraying (IRS) with non-pyrethroid insecticides when combined with long-lasting insecticidal nets (LLINs) impregnated with pyrethroid insecticides, contribute to the efforts of National Malaria Control Programmes to interrupt transmission and reduce the reservoir of Plasmodium falciparum infections across all ages. Using an interrupted time-series study design, four age-stratified malariometric surveys, each of ~2,000 participants, were undertaken pre- and post-IRS in Bongo District, Ghana. Following the application of three-rounds of IRS, P. falciparum transmission intensity declined, as measured by a >90% reduction in the monthly entomological inoculation rate. This decline was accompanied by reductions in parasitological parameters, with participants of all ages being significantly less likely to harbor P. falciparum infections at the end of the wet season post-IRS (aOR = 0.22 [95% CI: 0.19-0.26], p-value < 0.001). In addition, multiplicity of infection (MOI var ) was measured using a parasite fingerprinting tool, designed to capture within-host genome diversity. At the end of the wet season post-IRS, the prevalence of multi-genome infections declined from 75.6% to 54.1%. This study demonstrates that in areas characterized by high seasonal malaria transmission, IRS in combination with LLINs can significantly reduce the reservoir of P. falciparum infection. Nonetheless despite this success, 41.6% of the population, especially older children and adolescents, still harboured multi-genome infections. Given the persistence of this diverse reservoir across all ages, these data highlight the importance of sustaining vector control in combination with targeted chemotherapy to move high-transmission settings towards pre-elimination. This study also points to the benefits of molecular surveillance to ensure that incremental achievements are not lost and that the goals advocated for in the WHO's High Burden to High Impact strategy are realized.

Perspectives on returning individual and aggregate genomic research results to study participants and communities in Kenya: a qualitative study.

BACKGROUND: A fundamental ethical challenge in conducting genomics research is the question of what and how individual level genetic findings and aggregate genomic results should be conveyed to research participants and communities. This is within the context of minimal guidance, policies, and experiences, particularly in Africa. The aim of this study was to explore the perspectives of key stakeholders' on returning genomics research results to participants in Kenya. METHODS: This qualitative study involved focus group discussions (FGDs) and in-depth interviews (IDIs) with 69 stakeholders. The purposively selected participants, included research ethics committee (REC) members (8), community members (44), community resource persons (8), and researchers (9). A semi-structured interview guide was used to facilitate discussions. Six FGDs and twenty-five (IDIs) were conducted among the different stakeholders. The issues explored in the interviews included: (1) views on returning results, (2) kind of results to be returned, (3) value of returning results to participants, and (4) challenges anticipated in returning results to participants and communities. The interviews were audio-recorded, transcribed verbatim, and coded in Nvivo 12 pro. Thematic and content analysis was conducted. RESULTS: Participants agreed on the importance of returning genomic results either as individual or aggregate results. The most cited reasons for returning of genomic results included recognizing participants' contribution to research, encouraging participation in future research, and increasing the awareness of scientific progress. Other aspects on how genomic research results should be shared included sharing easy to understand results in the shortest time possible and maintaining confidentiality when sharing sensitive results. CONCLUSIONS: This study identified key stakeholders' perspectives on returning genomic results at the individual and community levels in two urban informal settlements of Nairobi. The majority of the participants expect to receive feedback about their genomic results, and it is an obligation for researchers to see how to best fulfil it.

Global Analysis of Plasmodium falciparum Dihydropteroate Synthase Variants Associated with Sulfadoxine Resistance Reveals Variant Distribution and Mechanisms of Resistance: A Computational-Based Study.

The continual rise in sulfadoxine (SDX) resistance affects the therapeutic efficacy of sulfadoxine-pyrimethamine; therefore, careful monitoring will help guide its prolonged usage. Mutations in Plasmodium falciparum dihydropteroate synthase (Pfdhps) are being surveilled, based on their link with SDX resistance. However, there is a lack of continuous analyses and data on the potential effect of molecular markers on the Pfdhps structure and function. This study explored single-nucleotide polymorphisms (SNPs) in Pfdhps that were isolated in Africa and other countries, highlighting the regional distribution and its link with structure. In total, 6336 genomic sequences from 13 countries were subjected to SNPs, haplotypes, and structure-based analyses. The SNP analysis revealed that the key SDX resistance marker, A437G, was nearing fixation in all countries, peaking in Malawi. The mutation A613S was rare except in isolates from the Democratic Republic of Congo and Malawi. Molecular docking revealed a general loss of interactions when comparing mutant proteins to the wild-type protein. During MD simulations, SDX was released from the active site in mutants A581G and A613S before the end of run-time, whereas an unstable binding of SDX to mutant A613S and haplotype A437A/A581G/A613S was observed. Conformational changes in mutant A581G and the haplotypes A581G/A613S, A437G/A581G, and A437G/A581G/A613S were seen. The radius of gyration revealed an unfolding behavior for the A613S, K540E/A581G, and A437G/A581G systems. Overall, tracking such mutations by the continuous analysis of Pfdhps SNPs is encouraged. SNPs on the Pfdhps structure may cause protein-drug function loss, which could affect the applicability of SDX in preventing malaria in pregnant women and children.

Genomic approaches for monitoring transmission dynamics of malaria: A case for malaria molecular surveillance in Sub-Saharan Africa.

Transmission dynamics is an important indicator for malaria control and elimination. As we move closer to eliminating malaria in Sub-Saharan Africa (sSA), transmission indices with higher resolution (genomic approaches) will complement our current measurements of transmission. Most of the present programmatic knowledge of malaria transmission patterns are derived from assessments of epidemiologic and clinical data, such as case counts, parasitological estimates of parasite prevalence, and Entomological Inoculation Rates (EIR). However, to eliminate malaria from endemic areas, we need to track changes in the parasite population and how they will impact transmission. This is made possible through the evolving field of genomics and genetics, as well as the development of tools for more in-depth studies on the diversity of parasites and the complexity of infections, among other topics. If malaria elimination is to be achieved globally, country-specific elimination activities should be supported by parasite genomic data from regularly collected blood samples for diagnosis, surveillance and possibly from other programmatic interventions. This presents a unique opportunity to track the spread of malaria parasites and shed additional light on intervention efficacy. In this review, various genetic techniques are highlighted along with their significance for an enhanced understanding of transmission patterns in distinct topological settings throughout Sub-Saharan Africa. The importance of these methods and their limitations in malaria surveillance to guide control and elimination strategies, are explored.

Trends and predictive factors for treatment failure following artemisinin-based combination therapy among children with uncomplicated malaria in Ghana: 2005-2018.

BACKGROUND: Since the introduction of artemisinin-based combination therapy (ACT) in Ghana in 2005 there has been a surveillance system by the National Malaria Control Programme (NMCP) and the University of Ghana Noguchi Memorial Institute for Medical Research (UG-NMIMR) to monitor the therapeutic efficacy of ACTs for the treatment of uncomplicated malaria in the country. We report trends and determinants of failure following treatment of Ghanaian children with artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) combinations. METHODS: Per protocol analyses as well as cumulative incidence of day 28 treatment failure from Kaplan Meier survival analyses were used to describe trends of failure over the surveillance period of 2005-2018. Univariable and multivariable cox regression analyses were used to assess the determinants of treatment failure over the period. RESULTS: Day 28 PCR-corrected failure, following treatment with ASAQ, significantly increased from 0.0% in 2005 to 2.0% (95% CI: 1.1-3.6) in 2015 (p = 0.013) but significantly decreased to 0.4% (95% CI: 0.1-1.6) in 2018 (p = 0.039). Failure, following treatment with AL, decreased from 4.5% (95% CI: 2.0-9.4) in 2010 to 2.7% (95% CI: 1.4-5.1) in 2018, though not statistically significant (p = 0.426). Risk of treatment failure, from multivariable cox regression analyses, was significantly lower among children receiving ASAQ compared with those receiving AL (HR = 0.24; 95% CI: 0.11-0.53; p < 0.001); lower among children with no parasitaemia on day 3 compared with those with parasitaemia on day 3 (HR = 0.02; 95% CI: 0.01-0.13; p < 0.001); and higher among children who received ASAQ and had axillary temperature ≥ 37.5 °C on day 1 compared with those with axillary temperature < 37.5 °C (HR = 3.96; 95% CI: 1.61-9.75; p = 0.003). CONCLUSIONS: Treatment failures for both ASAQ and AL have remained less than 5% (below WHO's threshold of 10%) in Ghana since 2005. Predictors of treatment failure that need to be considered in the management of uncomplicated malaria in the country should include type of ACT, day 3 parasitaemia, and day 1 axillary temperature of patients being treated.

Immediate pools of malaria infections at diagnosis combined with targeted deep sequencing accurately quantifies frequency of drug resistance mutations.

Antimalarial resistance surveillance in sub-Saharan Africa is often constrained by logistical and financial challenges limiting its breadth and frequency. At two sites in Ghana, we have piloted a streamlined sample pooling process created immediately by sequential addition of positive malaria cases at the time of diagnostic testing. This streamlined process involving a single tube minimized clinical and laboratory work and provided accurate frequencies of all known drug resistance mutations after high-throughput targeted sequencing using molecular inversion probes. Our study validates this method as a cost-efficient, accurate and highly-scalable approach for drug resistance mutation monitoring that can potentially be applied to other infectious diseases such as tuberculosis.

Network-driven analysis of human-Plasmodium falciparum interactome: processes for malaria drug discovery and extracting in silico targets.

BACKGROUND: The emergence and spread of malaria drug resistance have resulted in the need to understand disease mechanisms and importantly identify essential targets and potential drug candidates. Malaria infection involves the complex interaction between the host and pathogen, thus, functional interactions between human and Plasmodium falciparum is essential to obtain a holistic view of the genetic architecture of malaria. Several functional interaction studies have extended the understanding of malaria disease and integrating such datasets would provide further insights towards understanding drug resistance and/or genetic resistance/susceptibility, disease pathogenesis, and drug discovery. METHODS: This study curated and analysed data including pathogen and host selective genes, host and pathogen protein sequence data, protein-protein interaction datasets, and drug data from literature and databases to perform human-host and P. falciparum network-based analysis. An integrative computational framework is presented that was developed and found to be reasonably accurate based on various evaluations, applications, and experimental evidence of outputs produced, from data-driven analysis. RESULTS: This approach revealed 8 hub protein targets essential for parasite and human host-directed malaria drug therapy. In a semantic similarity approach, 26 potential repurposable drugs involved in regulating host immune response to inflammatory-driven disorders and/or inhibiting residual malaria infection that can be appropriated for malaria treatment. Further analysis of host-pathogen network shortest paths enabled the prediction of immune-related biological processes and pathways subverted by P. falciparum to increase its within-host survival. CONCLUSIONS: Host-pathogen network analysis reveals potential drug targets and biological processes and pathways subverted by P. falciparum to enhance its within malaria host survival. The results presented have implications for drug discovery and will inform experimental studies.