Contrasting Epidemiology and Population Genetics of COVID-19 Infections Defined by Multilocus Genotypes in SARS-CoV-2 Genomes Sampled Globally.

Since its emergence in 2019, SARS-CoV-2 has spread and evolved globally, with newly emerged variants of concern (VOCs) accounting for more than 500 million COVID-19 cases and 6 million deaths. Continuous surveillance utilizing simple genetic tools is needed to measure the viral epidemiological diversity, risk of infection, and distribution among different demographics in different geographical regions. To help address this need, we developed a proof-of-concept multilocus genotyping tool and demonstrated its utility to monitor viral populations sampled in 2020 and 2021 across six continents. We sampled globally 22,164 SARS-CoV-2 genomes from GISAID (inclusion criteria: available clinical and demographic data). They comprised two study populations, "2020 genomes" (N = 5959) sampled from December 2019 to September 2020 and "2021 genomes" (N = 16,205) sampled from 15 January to 15 March 2021. All genomes were aligned to the SARS-CoV-2 reference genome and amino acid polymorphisms were called with quality filtering. Thereafter, 74 codons (loci) in 14 genes including orf1ab polygene (N = 9), orf3a, orf8, nucleocapsid (N), matrix (M), and spike (S) met the 0.01 minimum allele frequency criteria and were selected to construct multilocus genotypes (MLGs) for the genomes. At these loci, 137 mutant/variant amino acids (alleles) were detected with eight VOC-defining variant alleles, including N KR203&204, orf1ab (I265, F3606, and L4715), orf3a H57, orf8 S84, and S G614, being predominant globally with > 35% prevalence. Their persistence and selection were associated with peaks in the viral transmission and COVID-19 incidence between 2020 and 2021. Epidemiologically, older patients (≥20 years) compared to younger patients (<20 years) had a higher risk of being infected with these variants, but this association was dependent on the continent of origin. In the global population, the discriminant analysis of principal components (DAPC) showed contrasting patterns of genetic clustering with three (Africa, Asia, and North America) and two (North and South America) continental clusters being observed for the 2020 and 2021 global populations, respectively. Within each continent, the MLG repertoires (range 40−199) sampled in 2020 and 2021 were genetically differentiated, with ≤4 MLGs per repertoire accounting for the majority of genomes sampled. These data suggested that the majority of SARS-CoV-2 infections in 2020 and 2021 were caused by genetically distinct variants that likely adapted to local populations. Indeed, four GISAID clade-defined VOCs - GRY (Alpha), GH (Beta), GR (Gamma), and G/GK (Delta variant) were differentiated by their MLG signatures, demonstrating the versatility of the MLG tool for variant identification. Results from this proof-of-concept multilocus genotyping demonstrates its utility for SARS-CoV-2 genomic surveillance and for monitoring its spatiotemporal epidemiology and evolution, particularly in response to control interventions including COVID-19 vaccines and chemotherapies.

Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests.

The COVID-19 pandemic has resulted in an unprecedented global demand for in vitro diagnostic reagents. Supply shortages and hoarding have impacted testing capacity which has led to inefficient COVID-19 case identification and transmission control, predominantly in developing countries. Traditionally, RNA extraction is a prerequisite for conducting SARS-CoV-2 nucleic acid amplification tests (NAAT); however, simplified methods of sample processing have been successful at bypassing typical nucleic acid extraction steps, enabling extraction-free SARS-CoV-2 NAAT workflows. These methods involve chemical and physical approaches that are inexpensive and easily accessible alternatives to overcome extraction kit supply shortages, while offering acceptable test performance. Here we provide an overview of three main sample preparation strategies that have been shown to facilitate extraction-free SARS-CoV-2 NAATs.

Point-of-Care Diagnostic Tools for Surveillance of SARS-CoV-2 Infections.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a recently emerged and highly contagious virus that causes coronavirus disease 2019 (COVID-19). As of August 24, 2021, there were more than 212 million confirmed COVID-19 cases and nearly 4.4 million deaths reported globally. Early diagnosis and isolation of infected individuals remains one of the most effective public health interventions to control SARS-CoV-2 spread and for effective clinical management of COVID-19 cases. Currently, SARS-CoV-2 infection is diagnosed presumptively based on clinical symptoms and confirmed by detecting the viral RNA in respiratory samples using reverse transcription polymerase chain reaction (RT-PCR). Standard RT-PCR protocols are time consuming, expensive, and technically demanding, which makes them a poor choice for large scale and point-of-care screening in resource-poor settings. Recently developed isothermal nucleic acid amplification tests (iNAAT), antigen and/or serological tests are cost-effective to scale COVID-19 testing at the point-of-care (PoC) and for surveillance activities. This review discusses the development of rapid PoC molecular tools for the detection and surveillance of SARS-CoV-2 infections.

Liposome engraftment and antigen combination potentiate the immune response towards conserved epitopes of the malaria vaccine candidate MSP2.

Merozoite surface protein 2 (MSP2) is a highly abundant, GPI-anchored surface antigen on merozoites of the malaria parasite Plasmodium falciparum. It consists of highly conserved N- and C-terminal domains, and a central polymorphic region that allows all MSP2 alleles to be categorized into the 3D7 or FC27 family. Previously it has been shown that epitope accessibility differs between lipid-bound and lipid-free MSP2, suggesting that lipid interactions modulate the conformation and antigenicity in a way that may better mimic native MSP2 on the merozoite surface. Therefore, we have immunised mice with MSP2 engrafted onto liposomes using a C-terminal tether that mimics the native GPI anchor. To improve the immunogenicity of the formulated antigen, liposomes were supplemented with Pathogen Associated Molecular Pattern molecules, specifically agonists of the Toll-like receptor 4 (TLR4) or TLR2. Induced antibodies were directed mostly towards conserved epitopes, predominantly in the conserved C-terminal region of MSP2. We also found that immunisation with a combination of 3D7 and FC27 MSP2 enhanced antibody responses to conserved epitopes, and that the overall responses of mice immunised with MSP2-engrafted liposomes were comparable in magnitude to those of mice immunised with MSP2 formulated in Montanide ISA720. The antibodies elicited in mice by immunising with MSP2-engrafted liposomes recognised the native form of parasite MSP2 on western blots and were found to be cross-reactive with isolated 3D7 and FC27 merozoites when investigated by ELISA. The liposome-tethered MSP2 induced higher titres of complement-fixing antibodies to 3D7 and FC27 MSP2 than did MSP2 formulated in Montanide ISA720. Our results indicate that liposomal formulation represents a viable strategy for eliciting a strong immune response that favours conserved epitopes in MSP2 and thus a strain-transcendent immune response.

Review of Burden, Clinical Definitions, and Management of COVID-19 Cases.

Our understanding of SARS-CoV-2, the virus responsible for coronavirus disease 2019 (COVID-19), its clinical manifestations, and treatment options continues to evolve at an unparalleled pace. This review sought to summarize the key literature regarding transmission, case definitions, clinical management, and the burden of COVID-19. Our review of the literature showed that SARS-CoV-2 was mainly transmitted via inhalation of respiratory droplets containing the virus and had a mean incubation period of 4-6 days. The commonly reported symptoms were fever (75.3% ± 18.7%) and cough (62.6% ± 17.7%) across the spectrum of clinical disease-mild, moderate, severe, and critical, but with the disease phenotype varying with severity. Categorization of these cases for home care or hospital management needs to be defined, with risk stratification accounting for the age of the patient and the presence of underlying comorbidities. The case definitions varied among countries, which could have contributed to the differences in the case fatality rates among affected countries. The severity and risk of death due to COVID-19 was associated with age and underlying comorbidities. Asymptomatic cases, which constitute 40-80% of COVID-19 cases are a considerable threat to control efforts. The presence of fever and cough may be sufficient to warrant COVID-19 testing, but using these symptoms in isolation will miss a proportion of cases. A clear definition of a COVID-19 case is essential for the management, treatment, and tracking of clinical illness, and to inform the quarantine measures and social distancing that can help control the spread of SARS-CoV-2.

Malaria vaccine candidates displayed on novel virus-like particles are immunogenic and induce transmission-blocking activity.

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.

Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced in Hansenula polymorpha.

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

A framework for assessing local transmission risk of imported malaria cases.

BACKGROUND: A key issue in achieving and sustaining malaria elimination is the need to prevent local transmission arising from imported cases of malaria. The likelihood of this occurring depends on a range of local factors, and these can be used to allocate resources to contain transmission. Therefore, a risk assessment and management strategy is required to identify risk indexes for malaria transmission when imported cases occur. These risks also need to be quantified and combined to give a weighted risk index score. This can then be used to allocate the resources to each administrative region to prevent transmission according to the degree of risk. METHODS: A list of potential risk indexes were generated from a literature review, expert consultation and panel discussion. These were initially classified into 4 first-level indexes including infection source, transmitting conditions, population vulnerability and control capacity. Each of these was then expanded into more detailed second-level indexes. The Delphi method was then used to obtain expert opinion to review and revise these risk indexes over two consecutive rounds to quantify agreement among experts as to their level of importance. Risk indexes were included in the final Transmission Risk Framework if they achieved a weighted importance score ≥ 4. The Analytic Hierarchy Process was then used to calculate the weight allocated to each of the final risk indexes. This was then used to create an assessment framework that can be used to evaluate local transmission risk in different areas. RESULTS: Two rounds of Delphi consultation were conducted. Twenty-three experts were used at each round with 100% recovery rate of participant questionnaires. The coordination coefficients (W) for the two rounds of Delphi consultation were 0.341 and 0.423, respectively (P < 0.05). Three first-level indexes and 13 second-level indexes were identified. The Analytic Hierarchy Process was performed to calculate the weight of the indexes. For the first-level indexes, infection source, transmitting conditions, and control capacity, the index weight was 0.5396, 0.2970 and 0.1634 respectively. For the three top second-level indexes, number of imported malaria cases, Anopheles species, and awareness of timely medical visit of patient, the index weight was 0.3382, 0.2475, and 0.1509 respectively. CONCLUSIONS: An indexed system of transmission risk assessment for imported malaria was established using the Delphi method and the Analytic Hierarchy Process. This was assessed to be an objective and practical tool for assessing transmission risk from imported cases of malaria into China.

Targets of complement-fixing antibodies in protective immunity against malaria in children.

Antibodies against P. falciparum merozoites fix complement to inhibit blood-stage replication in naturally-acquired and vaccine-induced immunity; however, specific targets of these functional antibodies and their importance in protective immunity are unknown. Among malaria-exposed individuals, we show that complement-fixing antibodies to merozoites are more strongly correlated with protective immunity than antibodies that inhibit growth quantified using the current reference assay for merozoite vaccine evaluation. We identify merozoite targets of complement-fixing antibodies and identify antigen-specific complement-fixing antibodies that are strongly associated with protection from malaria in a longitudinal study of children. Using statistical modelling, combining three different antigens targeted by complement-fixing antibodies could increase the potential protective effect to over 95%, and we identify antigens that were common in the most protective combinations. Our findings support antibody-complement interactions against merozoite antigens as important anti-malaria immune mechanisms, and identify specific merozoite antigens for further evaluation as vaccine candidates.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis.

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0-1%. 10 µL of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Agent-based models of malaria transmission: a systematic review.

BACKGROUND: Much of the extensive research regarding transmission of malaria is underpinned by mathematical modelling. Compartmental models, which focus on interactions and transitions between population strata, have been a mainstay of such modelling for more than a century. However, modellers are increasingly adopting agent-based approaches, which model hosts, vectors and/or their interactions on an individual level. One reason for the increasing popularity of such models is their potential to provide enhanced realism by allowing system-level behaviours to emerge as a consequence of accumulated individual-level interactions, as occurs in real populations. METHODS: A systematic review of 90 articles published between 1998 and May 2018 was performed, characterizing agent-based models (ABMs) relevant to malaria transmission. The review provides an overview of approaches used to date, determines the advantages of these approaches, and proposes ideas for progressing the field. RESULTS: The rationale for ABM use over other modelling approaches centres around three points: the need to accurately represent increased stochasticity in low-transmission settings; the benefits of high-resolution spatial simulations; and heterogeneities in drug and vaccine efficacies due to individual patient characteristics. The success of these approaches provides avenues for further exploration of agent-based techniques for modelling malaria transmission. Potential extensions include varying elimination strategies across spatial landscapes, extending the size of spatial models, incorporating human movement dynamics, and developing increasingly comprehensive parameter estimation and optimization techniques. CONCLUSION: Collectively, the literature covers an extensive array of topics, including the full spectrum of transmission and intervention regimes. Bringing these elements together under a common framework may enhance knowledge of, and guide policies towards, malaria elimination. However, because of the diversity of available models, endorsing a standardized approach to ABM implementation may not be possible. Instead it is recommended that model frameworks be contextually appropriate and sufficiently described. One key recommendation is to develop enhanced parameter estimation and optimization techniques. Extensions of current techniques will provide the robust results required to enhance current elimination efforts.

BioStructMap: a Python tool for integration of protein structure and sequence-based features.

Summary: A sliding window analysis over a protein or genomic sequence is commonly performed, and we present a Python tool, BioStructMap, that extends this concept to three-dimensional (3D) space, allowing the application of a 3D sliding window analysis over a protein structure. BioStructMap is easily extensible, allowing the user to apply custom functions to spatially aggregated data. BioStructMap also allows mapping of underlying genomic sequences to protein structures, allowing the user to perform genetic-based analysis over spatially linked codons-this has applications when selection pressures arise at the level of protein structure. Availability and implementation: The Python BioStructMap package is available at https://github.com/andrewguy/biostructmap and released under the MIT License. An online server implementing standard functionality is available at https://biostructmap.burnet.edu.au. Supplementary information: Supplementary data are available at Bioinformatics online.

Structural patterns of selection and diversity for Plasmodium vivax antigens DBP and AMA1.

BACKGROUND: Plasmodium vivax is a significant contributor to the global malaria burden, and a vaccine targeting vivax malaria is urgently needed. An understanding of the targets of functional immune responses during the course of natural infection will aid in the development of a vaccine. Antibodies play a key role in this process, with responses against particular epitopes leading to immune selection pressure on these epitopes. A number of techniques exist to estimate levels of immune selection pressure on particular epitopes, with a sliding window analysis often used to determine particular regions likely to be under immune pressure. However, such analysis neglects protein three-dimensional structural information. With this in mind, a newly developed tool, BioStructMap, was applied to two key antigens from Plasmodium vivax: PvAMA1 and PvDBP Region II. This tool incorporates structural information into tests of selection pressure. RESULTS: Sequences from a number of populations were analysed, examining spatially-derived nucleotide diversity and Tajima's D over protein structures for PvAMA1 and PvDBP. Structural patterns of nucleotide diversity were similar across all populations examined, with Domain I of PvAMA1 having the highest nucleotide diversity and displaying significant signatures of immune selection pressure (Tajima's D > 0). Nucleotide diversity for PvDBP was highest bordering the dimerization and DARC-binding interface, although there was less evidence of immune selection pressure on PvDBP compared with PvAMA1. This study supports previous work that has identified Domain I as the main target of immune-mediated selection pressure for PvAMA1, and also supports studies that have identified functional epitopes within PvDBP Region II. CONCLUSIONS: The BioStructMap tool was applied to leading vaccine candidates from P. vivax, to examine structural patterns of selection and diversity across a number of geographic populations. There were striking similarities in structural patterns of diversity across multiple populations. Furthermore, whilst regions of high diversity tended to surround conserved binding interfaces, a number of protein regions with very low diversity were also identified, and these may be useful targets for further vaccine development, given previous evidence of functional antibody responses against these regions.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Proteome-wide mapping of immune features onto Plasmodium protein three-dimensional structures.

Humoral immune responses against the malaria parasite are an important component of a protective immune response. Antibodies are often directed towards conformational epitopes, and the native structure of the antigenic region is usually critical for antibody recognition. We examined the structural features of various Plasmodium antigens that may impact on epitope location, by performing a comprehensive analysis of known and modelled structures from P. falciparum. Examining the location of known polymorphisms over all available structures, we observed a strong propensity for polymorphic residues to be exposed on the surface and to occur in particular secondary structure segments such as hydrogen-bonded turns. We also utilised established prediction algorithms for B-cell epitopes and MHC class II binding peptides, examining predicted epitopes in relation to known polymorphic sites within structured regions. Finally, we used the available structures to examine polymorphic hotspots and Tajima's D values using a spatial averaging approach. We identified a region of PfAMA1 involving both domains II and III under a high degree of balancing selection relative to the rest of the protein. In summary, we developed general methods for examining how sequence-based features relate to one another in three-dimensional space and applied these methods to key P. falciparum antigens.

Human Immunization With a Polymorphic Malaria Vaccine Candidate Induced Antibodies to Conserved Epitopes That Promote Functional Antibodies to Multiple Parasite Strains.

Background: Overcoming antigenic diversity is a key challenge in the development of effective Plasmodium falciparum malaria vaccines. Strategies that promote the generation of antibodies targeting conserved epitopes of vaccine antigens may provide protection against diverse parasites strains. Understanding differences between vaccine-induced and naturally acquired immunity is important to achieving this goal. Methods: We analyzed antibodies generated in a phase 1 human vaccine trial, MSP2-C1, which included 2 allelic forms of MSP2, an abundant vaccine antigen on the merozoite surface. Vaccine-induced responses were assessed for functional activity against multiple parasite strains, and cross-reactivity of antibodies was determined using competition ELISA and epitope mapping approaches. Results: Vaccination induced cytophilic antibody responses with strain-transcending opsonic phagocytosis and complement-fixing function. In contrast to antibodies acquired via natural infection, vaccine-induced antibodies were directed towards conserved epitopes at the C-terminus of MSP2, whereas naturally acquired antibodies mainly targeted polymorphic epitopes. Functional activity of C-terminal-targeted antibodies was confirmed using monoclonal antibodies that promoted opsonic phagocytosis against multiple parasite strains. Conclusion: Vaccination generated markedly different responses to polymorphic antigens than naturally acquired immunity and targeted conserved functional epitopes. Induction of antibodies targeting conserved regions of malaria antigens provides a promising vaccine strategy to overcome antigenic diversity for developing effective malaria vaccines.

Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetylglucosamine.

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly-N-acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N-acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms.

A portable microfluidic Aptamer-Tethered Enzyme Capture (APTEC) biosensor for malaria diagnosis.

There is a critical need for better biosensors for the detection and diagnosis of malaria. We previously developed a DNA aptamer that recognises the Plasmodium falciparum lactate dehydrogenase (PfLDH) enzyme with high sensitivity and specificity. The aptamer was integrated into an Aptamer-Tethered Enzyme Capture (APTEC) assay as a laboratory-based diagnostic approach. However, a portable equipment-free point-of-care aptamer-mediated biosensor could have a significant impact on malaria diagnosis in endemic regions. Here, we present a new concept for a malaria biosensor whereby aptamers are coated onto magnetic microbeads for magnet-guided capture, wash and detection of the biomarker. A biosensor incorporating three separate microfluidic chambers was designed to enable such magnet-guided equipment-free colorimetric detection of PfLDH. A series of microfluidic biosensor prototypes were optimised to lower rates of inter-chamber diffusion, increase sensitivity, and provide a method for point-of-care sample testing. The biosensor showed high sensitivity and specificity when detecting PfLDH using both in vitro cultured parasite samples and using clinical samples from malaria patients. The high performance of the biosensor provides a proof-of-principle for a portable biosensor that could be adaptable for a variety of aptamer-mediated diagnostic scenarios.

The use of respondent-driven sampling to assess malaria knowledge, treatment-seeking behaviours and preventive practices among mobile and migrant populations in a setting of artemisinin resistance in Western Cambodia.

BACKGROUND: Multi-drug-resistant Plasmodium falciparum threatens malaria elimination efforts in Cambodia and the Greater Mekong Subregion (GMS). Malaria burden in the GMS is higher among certain high-risk demographic groups in Cambodia, especially among migrant and mobile populations (MMPs). This respondent driven sampling (RDS) study was conducted in order to determine malaria knowledge, treatment-seeking behaviours and preventive practices among two MMP groups in Western Cambodia. METHODS: An RDS survey of MMPs was implemented in four purposively-selected communes along the Thai-Cambodia border; two in Veal Veang District and two in Pailin Province, chosen due to their sizeable MMP groups, their convenience of access, and their proximity to Thailand, which allowed for comparison with RDS studies in Thailand. RESULTS: There were 764 participants in Pailin Province and 737 in Veal Veang District. Health messages received in Veal Veang were most likely to come from billboards (76.5%) and family and friends (57.7%), while in Pailin they were most likely to come from sources like radio (57.1%) and television (31.3%). Knowledge of malaria transmission by mosquito and prevention by bed net was above 94% in both locations, but some misinformation regarding means of transmission and prevention methods existed, predominantly in Veal Veang. Ownership of treated bed nets was lower in Pailin than in Veal Veang (25.3% vs 53.2%), while reported use the night before the survey was higher in Pailin than in Veal Veang (57.1% vs 31.6%). Use of private sector health and pharmaceutical services was common, but 81.1% of patients treated for malaria in Pailin and 86.6% in Veal Veang had received a diagnostic test. Only 29.6% of patients treated in Pailin and 19.6% of those treated in Veal Veng reported receiving the indicated first-line treatment. DISCUSSION: Barriers in access to malaria prevention and case management were common among MMPs, with marked variation by site. Resolving both nation-wide and MMP-specific challenges will require targeted interventions that take into account this heterogeneity.