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Background: Significant effort and resources have been invested to control malaria transmission in sub-Saharan Africa, but it remains a major public health problem. For the parasite to be transmitted, the female Anopheles vector must survive 10-14 days following an infective bite to allow Plasmodium gametocytes to develop into infectious sporozoites. The goal of this study was to assess factors associated with wild-caught Anopheles survival and infection following host-seeking and indoor resting., Methods: The study was conducted between January 2020 to March 2022 in a longitudinal cohort of 75 households in 5 villages including a total of 755 household members in Bungoma County, Kenya. Monthly adult mosquito collection was conducted by attenuated aspiration in all enrolled households, and mosquitoes were reared for 7 days. The daily mortality rate was determined through day 7. All mosquitoes were morphologically identified. Female Anopheles were dissected, and species-level members of the Anopheles gambiae complex were resolved by molecular methods. The abdomens of all samples were processed for Plasmodium falciparum oocyst detection by PCR., Results: Within a 25-month period, the total numbers of non-Anopheles and Anopheles mosquitoes collected indoors were 12,843 and 712, respectively. Anopheles gambiae and An. funestus were the major vectors, though their distributions varied between different villages; 61.2% (n = 436/712) of the Anopheles mosquitoes survived up to day 7, with the lowest mortality rate recorded on day 5 of captivity. The survival rate also varied between the different Anopheles species. Six hundred eighty-three of 712 mosquito abdomens were tested for P. falciparum; 7.8% (53/683) tested positive for P. falciparum, with An. funestus having a higher (10%) prevalence than An. gambiae s.s. (6.0%, p = 0.095, Pearson Chi-square test). The proportion of household members sleeping under a bednet the night before mosquito collection varied across time and village. Anopheles funestus survival times were refractory to household ITN usage, and An. gambaie s.s. survival was reduced only under very high (100%) ITN usage., Conclusions: Despite ITN usage, mosquitoes still acquired blood meals and P. falciparum infections. Survival differed across species and was inversely correlated with high ITN usage in the household but not oocyst development., Competing Interests: Declarations Ethics approval and consent to participate Ethical approval was granted by Moi University Institutional Research and Ethics Committee (Formal Approval No. 0001863) and Duke University Institutional Review Board (Pro00082000). Written informed consent was obtained the household heads and participants prior to data and mosquito collection. All study methods were carried out in accordance with ethical guidelines and regulations set out by both Moi University and Duke Ethical Review Boards. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Mosquitoes are important vectors for human diseases, transmitting pathogens that cause a range of parasitic and viral infections. Mosquito blood-feeding is heterogeneous, meaning that some human hosts are at higher risk of receiving bites than others, and this heterogeneity is multifactorial. Mosquitoes integrate specific cues to locate their hosts, and mosquito attraction differs considerably between individual human hosts. Heterogeneous mosquito biting results from variations in both host attractiveness and availability and can impact transmission of vector-borne diseases. However, the extent and drivers of this heterogeneity and its importance for pathogen transmission remain incompletely understood. Here, we review methods and recent data describing human characteristics that affect host-seeking behavior and host preferences of mosquito disease vectors, and the implications for vector-borne disease transmission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Molecular epidemiologic studies of malaria parasites and other pathogens commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n = 270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with <1.5 genomes/μL (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with ≥75 genomes/μL) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, the majority of haplotypes can be successfully recovered from DBS while limiting the false positive rate., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lapp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding biting bias in a natural setting can help target interventions to interrupt transmission. In a 15-month cohort in western Kenya, we detected P. falciparum in indoor-resting Anopheles and human blood samples by qPCR and matched mosquito bloodmeals to cohort participants using short-tandem repeat genotyping. Using risk factor analyses and discrete choice models, we assessed mosquito biting behavior with respect to parasite transmission. Biting was highly unequal; 20% of people received 86% of bites. Biting rates were higher on males (biting rate ratio (BRR): 1.68; CI: 1.28-2.19), children 5-15 years (BRR: 1.49; CI: 1.13-1.98), and P. falciparum-infected individuals (BRR: 1.25; CI: 1.01-1.55). In aggregate, P. falciparum-infected school-age (5-15 years) boys accounted for 50% of bites potentially leading to onward transmission and had an entomological inoculation rate 6.4x higher than any other group. Additionally, infectious mosquitoes were nearly 3x more likely than non-infectious mosquitoes to bite P. falciparum-infected individuals (relative risk ratio 2.76, 95% CI 1.65-4.61). Thus, persistent P. falciparum transmission was characterized by disproportionate onward transmission from school-age boys and by the preference of infected mosquitoes to feed upon infected people., (© 2024. The Author(s).)

Measuring vector-human contact in a natural setting can inform precise targeting of interventions to interrupt transmission of vector-borne diseases. One approach is to directly match human DNA in vector bloodmeals to the individuals who were bitten using genotype panels of discriminative short tandem repeats (STRs). Existing methods for matching STR profiles in bloodmeals to the people bitten preclude the ability to match most incomplete profiles and multi-source bloodmeals to bitten individuals.We developed bistro, an R package that implements 3 preexisting STR matching methods as well as the package's namesake, bistro, a new algorithm described here. bistro employs forensic analysis methods to calculate likelihood ratios and match human STR profiles in bloodmeals to people using a dynamic threshold. We evaluated the algorithm's accuracy and compared it to existing matching approaches using a publicly-available panel of 188 single-source and 100 multi-source samples containing DNA from 50 known human sources. Then we applied it to match 777 newly field-collected mosquito bloodmeals to a database of 645 people.The R package implements four STR matching algorithms in user-friendly functions with clear documentation. bistro correctly matched 99% (187/188) of profiles in single-source samples, and 62% (224/359) of profiles from multi-source samples, resulting in a sensitivity of 0.75 (vs < 0.51 for other algorithms). The specificity of bistro was 0.9998 (vs. 1 for other algorithms). Furthermore, bistro identified 79% (720/906) of all possible matches for field-derived mosquitoes, yielding 1.4x more matches than existing algorithms.bistro identifies more correct bloodmeal-human matches than existing approaches, enabling more accurate and robust analyses of vector-human contact in natural settings. The bistro R package and corresponding documentation allow for straightforward uptake of this algorithm by others., Competing Interests: Conflict of interest: The authors declare no conflicts of interest.

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum. We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p = 0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p = 0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p = 0.022) epitope types. The association of symptomatic malaria with reduced hazard of homologous reinfection was strongest for rare epitope types. Symptomatic malaria provides more durable protection against reinfection with parasites bearing homologous epitope types. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Markwalter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human movement impacts the spread and transmission of infectious diseases. Recently, a large reservoir of Plasmodium falciparum malaria was identified in a semi-arid region of northwestern Kenya historically considered unsuitable for malaria transmission. Understanding the sources and patterns of transmission attributable to human movement would aid in designing and targeting interventions to decrease the unexpectedly high malaria burden in the region. Toward this goal, polymorphic parasite genes (ama1, csp) in residents and passengers traveling to Central Turkana were genotyped by amplicon deep sequencing. Genotyping and epidemiological data were combined to assess parasite importation. The contribution of travel to malaria transmission was estimated by modelling case reproductive numbers inclusive and exclusive of travelers. P. falciparum was detected in 6.7% (127/1891) of inbound passengers, including new haplotypes which were later detected in locally-transmitted infections. Case reproductive numbers approximated 1 and did not change when travelers were removed from transmission networks, suggesting that transmission is not fueled by travel to the region but locally endemic. Thus, malaria is not only prevalent in Central Turkana but also sustained by local transmission. As such, interrupting importation is unlikely to be an effective malaria control strategy on its own, but targeting interventions locally has the potential to drive down transmission., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Countries in the Greater Mekong Subregion have committed to eliminate Plasmodium falciparum malaria by 2025. Subclinical malaria infections that can be detected by highly sensitive polymerase chain reaction (PCR) testing in asymptomatic individuals represent a potential impediment to this goal, although the extent to which these low-density infections contribute to transmission is unclear. To understand the temporal dynamics of subclinical malaria in this setting, a cohort of 2,705 participants from three epidemiologically distinct regions of Myanmar was screened for subclinical P. falciparum and P. vivax infection using ultrasensitive PCR (usPCR). Standard rapid diagnostic tests (RDTs) for P. falciparum were also performed. Individuals who tested positive for malaria by usPCR were followed for up to 12 weeks. Regression analysis was performed to estimate whether the baseline prevalence of infection and the count of repeated positive tests were associated with demographic, behavioral, and clinical factors. At enrollment, the prevalence of subclinical malaria infection measured by usPCR was 7.7% (1.5% P. falciparum monoinfection, 0.3% mixed P. falciparum and P. vivax, and 6.0% P. vivax monoinfection), while P. falciparum prevalence measured by RDT was just 0.2%. Prevalence varied by geography and was higher among older people and in those with outdoor exposure and travel. No difference was observed in either the prevalence or count of subclinical infection by time of year, indicating that even in low-endemicity areas, a reservoir of subclinical infection persists year-round. If low-density infections are shown to represent a significant source of transmission, identification of high-risk groups and locations may aid elimination efforts.

Background: Screening malaria-specific antibody responses on protein microarrays can help identify immune factors that mediate protection against malaria infection, disease, and transmission, as well as markers of past exposure to both malaria parasites and mosquito vectors. Most malaria protein microarray work has used serum as the sample matrix, requiring prompt laboratory processing and a continuous cold chain, thus limiting applications in remote locations. Dried blood spots (DBS) pose minimal biohazard, do not require immediate laboratory processing, and are stable at room temperature for transport, making them potentially superior alternatives to serum. The goals of this study were to assess the viability of DBS as a source for antibody profiling and to use DBS to identify serological signatures of low-density Plasmodium falciparum infections in malaria-endemic regions of Myanmar., Methods: Matched DBS and serum samples from a cross-sectional study in Ingapu Township, Myanmar were probed on protein microarrays populated with P. falciparum antigen fragments. Signal and trends in both sample matrices were compared. A case-control study was then performed using banked DBS samples from malaria-endemic regions of Myanmar, and a regularized logistic regression model was used to identify antibody signatures of ultrasensitive PCR-positive P. falciparum infections., Results: Approximately 30% of serum IgG activity was recovered from DBS. Despite this loss of antibody activity, antigen and population trends were well-matched between the two sample matrices. Responses to 18 protein fragments were associated with the odds of asymptomatic P. falciparum infection, albeit with modest diagnostic characteristics (sensitivity 58%, specificity 85%, negative predictive value 88%, and positive predictive value 52%)., Conclusions: Malaria-specific antibody responses can be reliably detected, quantified, and analysed from DBS, opening the door to serological studies in populations where serum collection, transport, and storage would otherwise be impossible. While test characteristics of antibody signatures were insufficient for individual diagnosis, serological testing may be useful for identifying exposure to asymptomatic, low-density malaria infections, particularly if sero-surveillance strategies target individuals with low previous exposure as sentinels for population exposure., (© 2021. The Author(s).)

Ultrasensitive PCR used in low-transmission malaria-endemic settings has revealed a much higher burden of asymptomatic infections than that detected by rapid diagnostic tests (RDTs) or standard PCR, but there is limited evidence as to whether this is the case in higher transmission settings. Using dried blood spots (DBS) collected among 319 schoolchildren in Bagamoyo, Tanzania, we found good correlation (Pearson's R = 0.995) between Plasmodium falciparum parasite densities detected by a DNA-based 18s rRNA real-time PCR (qPCR) and an RNA-based ultrasensitive reverse transcriptase (RT)-PCR (usPCR) for the same target. Whereas prevalence by usPCR was higher than that found by qPCR (37% versus 32%), the proportion of additionally detected low-density infections (median parasite density < 0.050 parasites/µL) represented an incremental increase. It remains unclear to what extent these low-density infections may contribute to the infectious reservoir in different malaria transmission settings.

Rapid diagnostic tests (RDTs) are critical to the success of malaria elimination campaigns. These tests are rapid, user-friendly, and field-deployable to resource-limited regions. However, RDTs demonstrate poor sensitivity because they can only tolerate a small (5 μL) volume of blood, which limits the amount of protein biomarker delivered to the test. We have developed the Antibody-free Dual-biomarker Rapid Enrichment Workflow (AnDREW) for purifying histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (PLDH) from large volume (150 μL) blood samples. We used Zn(II)NTA and aptamer-conjugated magnetic beads to capture HRP2 and PLDH, respectively. Both biomarkers were then eluted into RDT-compatible volumes using ethylene diamine tetraacetic acid (EDTA). We optimized both bead conjugates individually by enzyme-linked immunosorbent assays (ELISAs) and then combined the optimized capture and elution assays for both biomarkers to produce the AnDREW. The AnDREW-enhanced RDTs exhibited a 11-fold and 9-fold improvement in analytical sensitivity for detection of HRP2 and PLDH, respectively, when compared to unenhanced RDTs. Moreover, the limit of detection for PLDH was improved 11-fold for the AnDREW-enhanced RDTs (3.80 parasites/μL) compared to unenhanced RDTs (42.31 parasites/μL). Importantly, the AnDREW utilizes a pan-specific PLDH aptamer and improves upon existing methods by eluting both biomarkers without complexed antibodies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although preventive chemotherapy has been instrumental in reducing schistosomiasis incidence worldwide, serious challenges remain. These problems include the omission of certain groups from campaigns of mass drug administration, the existence of persistent disease hotspots, and the risk of recrudescent infections. Central to these challenges is the fact that the diagnostic tools currently used to establish the burden of infection are not sensitive enough, especially in low-endemic settings, which results in underestimation of the true prevalence of active Schistosoma spp infections. This central issue necessitates that the current schistosomiasis control strategies recommended by WHO are re-evaluated and, possibly, adapted. More targeted interventions and novel approaches have been used to estimate the prevalence of schistosomiasis, such as establishing infection burden by use of precision mapping, which provides high resolution spatial information that delineates variations in prevalence within a defined geographical area. Such information is instrumental in guiding targeted intervention campaigns. However, the need for highly accurate diagnostic tools in such strategies is a crucial factor that is often neglected. The availability of highly sensitive diagnostic tests also opens up the possibility of applying strategies of sample pooling to reduce the cost of control programmes. To interrupt the transmission of, and eventually eliminate, schistosomiasis, better local targeting of preventive chemotherapy, in combination with highly sensitive diagnostic tools, is crucial., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The emergence of artemisinin-resistant Plasmodium falciparum in the Greater Mekong Subregion threatens both the efficacy of artemisinin-based combination therapy (ACT), the first-line treatment for malaria, and prospects for malaria elimination. Monitoring of ACT efficacy is essential for ensuring timely updates to elimination policies and treatment recommendations. In 2014-2015, we assessed the therapeutic efficacies of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) for the treatment of uncomplicated P. falciparum at three study sites in Rakhine, Shan, and Kachin states in Myanmar. Patients presenting with uncomplicated P. falciparum malaria were enrolled, treated, and followed up for 28 days for AL or 42 days for DP. Both AL and DP demonstrated good therapeutic efficacy at all three study sites. The 28-day cure rate for AL was > 96% across all study sites, and the 42-day cure rate for DP was 100%. Parasitemia on day 3 was detected in 0%, 3.3%, and 3.6% of participants treated with AL at the Rakhine, Shan, and Kachin sites, respectively. No participants treated with DP were parasitemic on day 3. No evidence of P. falciparum k13 mutations was found at the Rakhine study site. A high prevalence of k13 mutations associated with artemisinin resistance was observed at the Kachin and Shan state study sites. These results confirm that ACT efficacy has been resilient in therapeutic efficacy study (TES) sentinel sites in Myanmar, despite the presence at some sites of k13 mutations associated with resistance. Studies are ongoing to assess whether this resilience persists.

Infectious diseases claim millions of lives each year. Robust and accurate diagnostics are essential tools for identifying those who are at risk and in need of treatment in low-resource settings. Inorganic complexes and metal-based nanomaterials continue to drive the development of diagnostic platforms and strategies that enable infectious disease detection in low-resource settings. In this review, we highlight works from the past 20 years in which inorganic chemistry and nanotechnology were implemented in each of the core components that make up a diagnostic test. First, we present how inorganic biomarkers and their properties are leveraged for infectious disease detection. In the following section, we detail metal-based technologies that have been employed for sample preparation and biomarker isolation from sample matrices. We then describe how inorganic- and nanomaterial-based probes have been utilized in point-of-care diagnostics for signal generation. The following section discusses instrumentation for signal readout in resource-limited settings. Next, we highlight the detection of nucleic acids at the point of care as an emerging application of inorganic chemistry. Lastly, we consider the challenges that remain for translation of the aforementioned diagnostic platforms to low-resource settings.

Accurate and sensitive point-of-care diagnostic tools are critical for schistosomiasis control and elimination. The existing ultrasensitive lateral flow assay for the detection of Schistosoma circulating anodic antigen (CAA) has demonstrated excellent sensitivity but is time-consuming and requires significant laboratory infrastructure that limits its applicability at the point of care. To address this challenge, we sought to develop an alternative sample preparation method to concentrate CAA from large-volume urine samples requiring little-to-no laboratory equipment. The developed method relies on electrostatic interactions between the negatively-charged CAA biomarker and positively-charged poly(amidoamine) (PAMAM) dendrimers functionalized to the surface of magnetic particles. After CAA capture on the surface of the PAMAM-functionalized magnetic beads, the supernatant was removed, and CAA was eluted into a small-volume, high-salt elution buffer. This concentrated eluate was subsequently applied to the existing lateral flow assay. The PAMAM-functionalized magnetic bead-based CAA concentration method was extensively characterized for its robustness, evaluated on a set of endemic urine samples, and compared to spin filter-based concentration methods. The novel bead-based sample preparation method used only disposable laboratory materials, resulted in a 200-fold improvement in CAA limits of detection, and performed just as well as infrastructure-intensive and high-cost spin filter methods. Additionally, the functionalized beads were robust to variations in sample pH and storage conditions. The PAMAM-functionalized magnetic bead-based CAA concentration method represents a promising step toward ultrasensitive schistosomiasis diagnosis at the point of care.

Background: Rapid diagnostic tests based on histidine-rich protein 2 (HRP2) detection are the primary tools used to detect Plasmodium falciparum malaria infections. Recent conflicting reports call into question whether α-HRP2 antibodies are present in human host circulation and if resulting immune complexes could interfere with HRP2 detection on malaria RDTs. This study sought to determine the prevalence of immune-complexed HRP2 in a low-transmission region of Southern Zambia., Methods: An ELISA was used to quantify HRP2 in patient sample DBS extracts before and after heat-based immune complex dissociation. A pull-down assay reliant on proteins A, G, and L was developed and applied for IgG and IgM capture and subsequent immunoprecipitation of any HRP2 present in immune complexed form. A total of 104 patient samples were evaluated using both methods., Results: Immune-complexed HRP2 was detectable in 17% (18/104) of all samples evaluated and 70% (16/23) of HRP2-positive samples. A majority of the patients with samples containing immune-complexed HRP2 had P. falciparum infections (11/18) and were also positive for free HRP2 (16/18). For 72% (13/18) of patients with immune-complexed HRP2, less than 10% of the total HRP2 present was in immune-complexed form. For the remaining samples, a large proportion (≥ 20%) of total HRP2 was complexed with α-HRP2 antibodies., Conclusions: Endogenous α-HRP2 antibodies form immune complexes with HRP2 in the symptomatic patient population of a low-transmission area in rural Southern Zambia. For the majority of patients, the percentage of HRP2 in immune complexes is low and does not affect HRP2-based malaria diagnosis. However, for some patients, a significant portion of the total HRP2 was in immune-complexed form. Future studies investigating the prevalence and proportion of immune-complexed HRP2 in asymptomatic individuals with low HRP2 levels will be required to assess whether α-HRP2 antibodies affect HRP2 detection for this portion of the transmission reservoir.

A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase ( p LDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM ( p LDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify p LDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; p LDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35-52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria.

Predicting antibody pair performance in a sandwich format streamlines development of antibody-based diagnostics and laboratory research tools, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFAs). We have evaluated panels of monoclonal antibodies against the malarial parasite biomarker Plasmodium falciparum histidine rich protein 2 (HRP2), including 9 new monoclonal antibodies, using biolayer interferometry (BLI) and screened antibody pairs in a checkerboard ELISA. This study showed BLI predicts antibody pair ELISA performance for HRP2. Pairs that included capture antibodies with low off-rate constants and detection antibodies with high on-rate constants performed best in an ELISA format., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Dried blood spots are commonly used for sample collection in clinical and non-clinical settings. This method is simple, and biomolecules in the samples remain stable for months at room temperature. In the field, blood samples for the study and diagnosis of malaria are often collected on dried blood spot cards, so development of a biomarker extraction and analysis method is needed., Methods: A simple extraction procedure for the malarial biomarker Plasmodium falciparum histidine-rich protein 2 (HRP2) from dried blood spots was optimized to achieve maximum extraction efficiency. This method was used to assess the stability of HRP2 in dried blood spots. Furthermore, 328 patient samples made available from rural Zambia were analysed for HRP2 using the developed method. These samples were collected at the initial administration of artemisinin-based combination therapy and at several points following treatment., Results: An average extraction efficiency of 70% HRP2 with a low picomolar detection limit was achieved. In specific storage conditions HRP2 was found to be stable in dried blood spots for at least 6 months. Analysis of patient samples showed the method to have a sensitivity of 94% and a specificity of 89% when compared with microscopy, and trends in HRP2 clearance after treatment were observed., Conclusions: The dried blood spot ELISA for HRP2 was found to be sensitive, specific and accurate. The method was effectively used to assess biomarker clearance characteristics in patient samples, which prove it to be ideal for gaining further insight into the disease and epidemiological applications.

In many diagnostic assays, specific biomarker extraction and purification from a patient sample is performed in microcentrifuge tubes using surface-functionalized magnetic beads. Although assay binding times are known to be highly dependent on sample viscosity, sample volume, capture reagent, and fluid mixing, the theoretical mass transport framework that has been developed and validated in engineering has yet to be applied in this context. In this work, we adapt this existing framework for simultaneous mass transfer and surface reaction and apply it to the binding of biomarkers in clinical samples to surface-functionalized magnetic beads. We discuss the fundamental fluid dynamics of vortex mixing within microcentrifuge tubes as well as describe how particles and biomolecules interact with the fluid. The model is solved over a wide range of parameters, and we present scenarios when a simplified analytical expression would be most accurate. Next, we review of some relevant techniques for model parameter estimation. Finally, we apply the mass transfer theory to practical use-case scenarios of immediate use to clinicians and assay developers. Throughout, we highlight where further characterization is necessary to bridge the gap between theory and practical application., (Copyright © 2017. Published by Elsevier B.V.)

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2(+) (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.