Molecular markers for malaria genetic epidemiology: progress and pitfalls.

Over recent years, progress in molecular markers for genotyping malaria parasites has enabled informative studies of epidemiology and transmission dynamics. Results have highlighted the value of these tools for surveillance to support malaria control and elimination strategies. There are many different types and panels of markers available for malaria parasite genotyping, and for end users, the nuances of these markers with respect to 'use case', resolution, and accuracy, are not well defined. This review clarifies issues surrounding different molecular markers and their application to malaria control and elimination. We describe available marker panels, use cases, implications for different transmission settings, limitations, access, cost, and data accuracy. The information provided can be used as a guide for molecular epidemiology and surveillance of malaria.

Serological assays to measure dimeric IgA antibodies in SARS-CoV-2 infections.

Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.

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.

Identification of novel Plasmodium vivax proteins associated with protection against clinical malaria.

As progress towards malaria elimination continues, the challenge posed by the parasite species Plasmodium vivax has become more evident. In many regions co-endemic for P. vivax and Plasmodium falciparum, as transmission has declined the proportion of cases due to P. vivax has increased. Novel tools that directly target P. vivax are thus warranted for accelerated elimination. There is currently no advanced vaccine for P. vivax and only a limited number of potential candidates in the pipeline. In this study we aimed to identify promising P. vivax proteins that could be used as part of a subunit vaccination approach. We screened 342 P. vivax protein constructs for their ability to induce IgG antibody responses associated with protection from clinical disease in a cohort of children from Papua New Guinea. This approach has previously been used to successfully identify novel candidates. We were able to confirm previous results from our laboratory identifying the proteins reticulocyte binding protein 2b and StAR-related lipid transfer protein, as well as at least four novel candidates with similar levels of predicted protective efficacy. Assessment of these P. vivax proteins in further studies to confirm their potential and identify functional mechanisms of protection against clinical disease are warranted.

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.

PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus.

The CYP2D6 enzyme is estimated to metabolize 25% of commonly used pharmaceuticals and is of intense pharmacogenetic interest due to the polymorphic nature of the CYP2D6 gene. Accurate allele typing of CYP2D6 has proved challenging due to frequent copy number variants (CNVs) and paralogous pseudogenes. SNP-arrays, qPCR and short-read sequencing have been employed to interrogate CYP2D6, however these technologies are unable to capture longer range information. Long-read sequencing using the PacBio Single Molecule Real Time (SMRT) sequencing platform has yielded promising results for CYP2D6 allele typing. However, previous studies have been limited in scale and have employed nascent data processing pipelines. We present a robust data processing pipeline "PLASTER" for accurate allele typing of SMRT sequenced amplicons. We demonstrate the pipeline by typing CYP2D6 alleles in a large cohort of 377 Solomon Islanders. This pharmacogenetic method will improve drug safety and efficacy through screening prior to drug administration.

Age-specific patterns of DBLα var diversity can explain why residents of high malaria transmission areas remain susceptible to Plasmodium falciparum blood stage infection throughout life.

Immunity to Plasmodium falciparum is non-sterilising, thus individuals residing in malaria-endemic areas are at risk of infection throughout their lifetime. Here we seek to find a genomic epidemiological explanation for why residents of all ages harbour blood stage infections despite lifelong exposure to P. falciparum in areas of high transmission. We do this by exploring, for the first known time, the age-specific patterns of diversity of variant antigen encoding (var) genes in the reservoir of infection. Microscopic and submicroscopic P. falciparum infections were analysed at the end of the wet and dry seasons in 2012-2013 for a cohort of 1541 residents aged from 1 to 91 years in an area characterised by high seasonal malaria transmission in Ghana. By sequencing the near ubiquitous Duffy-binding-like alpha domain (DBLα) that encodes immunogenic domains, we defined var gene diversity in an estimated 1096 genomes detected in sequential wet and dry season sampling of this cohort. Unprecedented var (DBLα) diversity was observed in all ages with 42,399 unique var types detected. There was a high degree of maintenance of types between seasons (>40% seen more than once), with many of the same types, especially upsA, appearing multiple times in isolates from different individuals. Children and adolescents were found to be significant reservoirs of var DBLα diversity compared with adults. Var repertoires within individuals were highly variable, with children having more related var repertoires compared to adolescents and adults. Individuals of all ages harboured multiple genomes with var repertoires unrelated to those infecting other hosts. High turnover of parasites with diverse isolate var repertoires was also observed in all ages. These age-specific patterns are best explained by variant-specific immune selection. The observed level of var diversity for the population was then used to simulate the development of variant-specific immunity to the diverse var types under conservative assumptions. Simulations showed that the extent of observed var diversity with limited repertoire relatedness was sufficient to explain why adolescents and adults in this community remain susceptible to blood stage infection, even with multiple genomes.

An accurate method for identifying recent recombinants from unaligned sequences.

MOTIVATION: Recombination is a fundamental process in molecular evolution, and the identification of recombinant sequences is thus of major interest. However, current methods for detecting recombinants are primarily designed for aligned sequences. Thus, they struggle with analyses of highly diverse genes, such as the var genes of the malaria parasite Plasmodium falciparum, which are known to diversify primarily through recombination. RESULTS: We introduce an algorithm to detect recent recombinant sequences from a dataset without a full multiple alignment. Our algorithm can handle thousands of gene-length sequences without the need for a reference panel. We demonstrate the accuracy of our algorithm through extensive numerical simulations; in particular, it maintains its effectiveness in the presence of insertions and deletions. We apply our algorithm to a dataset of 17 335 DBLα types in var genes from Ghana, observing that sequences belonging to the same ups group or domain subclass recombine amongst themselves more frequently, and that non-recombinant DBLα types are more conserved than recombinant ones. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/qianfeng2/detREC_program. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SARS-CoV-2 Multi-Antigen Serology Assay.

Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens.

The impact of indoor residual spraying on Plasmodium falciparum microsatellite variation in an area of high seasonal malaria transmission in Ghana, West Africa.

Here, we report the first population genetic study to examine the impact of indoor residual spraying (IRS) on Plasmodium falciparum in humans. This study was conducted in an area of high seasonal malaria transmission in Bongo District, Ghana. IRS was implemented during the dry season (November-May) in three consecutive years between 2013 and 2015 to reduce transmission and attempt to bottleneck the parasite population in humans towards lower diversity with greater linkage disequilibrium. The study was done against a background of widespread use of long-lasting insecticidal nets, typical for contemporary malaria control in West Africa. Microsatellite genotyping with 10 loci was used to construct 392 P. falciparum multilocus infection haplotypes collected from two age-stratified cross-sectional surveys at the end of the wet seasons pre- and post-IRS. Three-rounds of IRS, under operational conditions, led to a >90% reduction in transmission intensity and a 35.7% reduction in the P. falciparum prevalence (p < .001). Despite these declines, population genetic analysis of the infection haplotypes revealed no dramatic changes with only a slight, but significant increase in genetic diversity (He : pre-IRS = 0.79 vs. post-IRS = 0.81, p = .048). Reduced relatedness of the parasite population (p < .001) was observed post-IRS, probably due to decreased opportunities for outcrossing. Spatiotemporal genetic differentiation between the pre- and post-IRS surveys (D = 0.0329 [95% CI: 0.0209 - 0.0473], p = .034) was identified. These data provide a genetic explanation for the resilience of P. falciparum to short-term IRS programmes in high-transmission settings in sub-Saharan Africa.

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.

Evolution of Antimalarial Drug Resistance Markers in the Reservoir of Plasmodium falciparum Infections in the Upper East Region of Ghana.

BACKGROUND: The majority of Plasmodium falciparum infections, constituting the reservoir in all ages, are asymptomatic in high-transmission settings in Africa. The role of this reservoir in the evolution and spread of drug resistance was explored. METHODS: Population genetic analyses of the key drug resistance-mediating polymorphisms were analyzed in a cross-sectional survey of asymptomatic P. falciparum infections across all ages in Bongo District, Ghana. RESULTS: Seven years after the policy change to artemisinin-based combination therapies in 2005, the pfcrt K76 and pfmdr1 N86 wild-type alleles have nearly reached fixation and have expanded via soft selective sweeps on multiple genetic backgrounds. By constructing the pfcrt-pfmdr1-pfdhfr-pfdhps multilocus haplotypes, we found that the alleles at these loci were in linkage equilibrium and that multidrug-resistant parasites have not expanded in this reservoir. For pfk13, 32 nonsynonymous mutations were identified; however, none were associated with artemisinin-based combination therapy resistance. CONCLUSIONS: The prevalence and selection of alleles/haplotypes by antimalarials were similar to that observed among clinical cases in Ghana, indicating that they do not represent 2 subpopulations with respect to these markers. Thus, the P. falciparum reservoir in all ages can contribute to the maintenance and spread of antimalarial resistance.

Competition for hosts modulates vast antigenic diversity to generate persistent strain structure in Plasmodium falciparum.

In their competition for hosts, parasites with antigens that are novel to the host immune system will be at a competitive advantage. The resulting frequency-dependent selection can structure parasite populations into strains of limited genetic overlap. For the causative agent of malaria, Plasmodium falciparum, the high recombination rates and associated vast diversity of its highly antigenic and multicopy var genes preclude such clear clustering in endemic regions. This undermines the definition of strains as specific, temporally persisting gene variant combinations. We use temporal multilayer networks to analyze the genetic similarity of parasites in both simulated data and in an extensively and longitudinally sampled population in Ghana. When viewed over time, populations are structured into modules (i.e., groups) of parasite genomes whose var gene combinations are more similar within than between the modules and whose persistence is much longer than that of the individual genomes that compose them. Comparison to neutral models that retain parasite population dynamics but lack competition reveals that the selection imposed by host immunity promotes the persistence of these modules. The modular structure is, in turn, associated with a slower acquisition of immunity by individual hosts. Modules thus represent dynamically generated niches in host immune space, which can be interpreted as strains. Negative frequency-dependent selection therefore shapes the organization of the var diversity into parasite genomes, leaving a persistence signature over ecological time scales. Multilayer networks extend the scope of phylodynamics analyses by allowing quantification of temporal genetic structure in organisms that generate variation via recombination or other non-bifurcating processes. A strain structure similar to the one described here should apply to other pathogens with large antigenic spaces that evolve via recombination. For malaria, the temporal modular structure should enable the formulation of tractable epidemiological models that account for parasite antigenic diversity and its influence on intervention outcomes.

Networks of genetic similarity reveal non-neutral processes shape strain structure in Plasmodium falciparum.

Pathogens compete for hosts through patterns of cross-protection conferred by immune responses to antigens. In Plasmodium falciparum malaria, the var multigene family encoding for the major blood-stage antigen PfEMP1 has evolved enormous genetic diversity through ectopic recombination and mutation. With 50-60 var genes per genome, it is unclear whether immune selection can act as a dominant force in structuring var repertoires of local populations. The combinatorial complexity of the var system remains beyond the reach of existing strain theory and previous evidence for non-random structure cannot demonstrate immune selection without comparison with neutral models. We develop two neutral models that encompass malaria epidemiology but exclude competitive interactions between parasites. These models, combined with networks of genetic similarity, reveal non-neutral strain structure in both simulated systems and an extensively sampled population in Ghana. The unique population structure we identify underlies the large transmission reservoir characteristic of highly endemic regions in Africa.

Signatures of competition and strain structure within the major blood-stage antigen of Plasmodium falciparum in a local community in Ghana.

The concept of niche partitioning has received considerable theoretical attention at the interface of ecology and evolution of infectious diseases. Strain theory postulates that pathogen populations can be structured into distinct nonoverlapping strains by frequency-dependent selection in response to intraspecific competition for host immune space. The malaria parasite Plasmodium falciparum presents an opportunity to investigate this phenomenon in nature, under conditions of high recombination rate and extensive antigenic diversity. The parasite's major blood-stage antigen, Pf EMP1, is encoded by the hyperdiverse var genes. With a dataset that includes thousands of var DBLα sequence types sampled from asymptomatic cases within an area of high endemicity in Ghana, we address how var diversity is distributed within isolates and compare this to the distribution of microsatellite allelic diversity within isolates to test whether antigenic and neutral regions of the genome are structured differently. With respect to var DBLα sequence types, we find that on average isolates exhibit significantly lower overlap than expected randomly, but that there also exists frequent pairs of isolates that are highly related. Furthermore, the linkage network of var DBLα sequence types reveals a pattern of nonrandom modularity unique to these antigenic genes, and we find that modules of highly linked DBLα types are not explainable by neutral forces related to var recombination constraints, microsatellite diversity, sampling location, host age, or multiplicity of infection. These findings of reduced overlap and modularity among the var antigenic genes are consistent with a role for immune selection as proposed by strain theory. Identifying the evolutionary and ecological dynamics that are responsible for the nonrandom structure in P. falciparum antigenic diversity is important for designing effective intervention in endemic areas.

Population genomics of virulence genes of Plasmodium falciparum in clinical isolates from Uganda.

Plasmodium falciparum causes a spectrum of malarial disease from asymptomatic to uncomplicated through to severe. Investigations of parasite virulence have associated the expression of distinct variants of the major surface antigen of the blood stages known as Pf EMP1 encoded by up to 60 var genes per genome. Looking at the population genomics of var genes in cases of uncomplicated malaria, we set out to determine if there was any evidence of a selective sweep of specific var genes or clonal epidemic structure related to the incidence of uncomplicated disease in children. By sequencing the conserved DBLα domain of var genes from six sentinel sites in Uganda we found that the parasites causing uncomplicated P. falciparum disease in children were highly diverse and that every child had a unique var DBLα repertoire. Despite extensive var DBLα diversity and minimal overlap between repertoires, specific DBLα types and groups were conserved at the population level across Uganda. This pattern was the same regardless of the geographic distance or malaria transmission intensity. These data lead us to propose that any parasite can cause uncomplicated malarial disease and that these diverse parasite repertoires are composed of both upsA and non-upsA var gene groups.

Lack of Geospatial Population Structure Yet Significant Linkage Disequilibrium in the Reservoir of Plasmodium falciparum in Bongo District, Ghana.

Malaria control in West Africa is impeded by the large reservoir of chronic asymptomatic Plasmodium falciparum infections in the human population. This study aimed to assess the extent of diversity in the P. falciparum reservoir in Bongo District (BD), Ghana, at the end of the dry season, the lowest point in malaria transmission over the course of the year. Analysis of the variation in 12 microsatellite loci was completed for 200 P. falciparum isolates collected from a cross-sectional survey of residents of all ages from two catchment areas in BD. Analysis of the multilocus haplotypes showed high levels of genetic diversity (He = 0.74), no population differentiation yet significant linkage disequilibrium (LD) (ISA = 0.0127, P = 0.006) in BD. Multilocus LD was significant between and within catchment areas even though every haplotype in the population was unique and the majority of individuals (84.0%) harbored multiple-clone infections. The linkage structure among multilocus haplotypes was not associated with sampling location. These data provide the first study with deep sampling of the P. falciparum reservoir in an area of seasonal malaria transmission in West Africa. The co-occurrence of high multiplicity of infection (multiple-clone infections) with significant multilocus LD is surprising given the likelihood of high recombination rates in BD. The results suggest that the linkage structure among multilocus haplotypes has not been shaped by geographic separation of parasite populations. Furthermore, the observed LD levels provide a baseline population genetic metric with putatively neutral markers to evaluate the effects of seasonality and malaria control efforts in BD.