Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence. Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield™) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls. Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), 1000-fold (P. vivax) and 10-fold (P. cynomolgi). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/µL for P. knowlesi and 0.002 parasites/µL for both P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/µL); Divis et al. real-time 18S rRNA (0.0002 parasites/µL); Lubis et al. hemi-nested SICAvar (1.1 parasites/µL) and Lee et al. nested 18S rRNA (11 parasites/µL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were 0.02 and 0.20 parasites/µL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi. Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

Characterisation of Plasmodium vivax lactate dehydrogenase dynamics in P. vivax infections.

Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of P. vivax. It is widely used as a diagnostic biomarker and a measure of total-body parasite biomass in vivax malaria. However, the dynamics of PvLDH remains poorly understood. Here, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated key biological parameters characterising in vivo PvLDH dynamics based on longitudinal data of parasitemia and PvLDH concentration collected from P. vivax-infected humans, with the estimates informed by the ex vivo data as prior knowledge in a Bayesian hierarchical framework. We found that the in vivo accumulation rate of intraerythrocytic PvLDH peaks at 10-20 h post-invasion (late ring stage) with a median estimate of intraerythrocytic PvLDH mass at the end of the life cycle to be 9.4 × 10-3ng. We also found that the median estimate of in vivo PvLDH half-life was approximately 21.9 h. Our findings provide a foundation with which to advance our quantitative understanding of P. vivax biology and will facilitate the improvement of PvLDH-based diagnostic tools.

Interdisciplinary evidence for early domestic horse exploitation in southern Patagonia.

The introduction of domestic horses transformed Indigenous societies across the grasslands of Argentina, leading to the emergence of specialized horse cultures across the Southern Cone. However, the dynamics of this introduction are poorly chronicled by historic records. Here, we apply archaeozoological and biomolecular techniques to horse remains from the site of Chorrillo Grande 1 in southern Argentina. Osteological and taphonomic analyses suggest that horses were pastorally managed and used for food by Aónikenk/Tehuelche hunter-gatherers before the onset of permanent European settlement, as early as the mid-17th century. DNA-based sex identifications suggest consumption of both male and female horses, while ceramic residue also shows use of guanaco products. Sequential isotope analyses on horse dentition reveal an origin in southern Patagonia and movement of these animals between the Río Coig and Río Gallegos basins. These results reinforce emerging evidence for rapid Indigenous-mediated dispersal of horses in the Americas and demonstrate that horses catalyzed rapid economic and social transformations.

Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets.

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

Serological evaluation of risk factors for exposure to malaria in a pre-elimination setting in Malaysian Borneo.

Malaysia has reported no indigenous cases of P. falciparum and P. vivax for over 3 years. When transmission reaches such low levels, it is important to understand the individuals and locations where exposure risks are high, as they may be at greater risk in the case of a resurgence of transmission. Serology is a useful tool in low transmission settings, providing insight into exposure over longer durations than PCR or RDT. We ran blood samples from a 2015 population-based survey in northern Sabah, Malaysian Borneo on a multiplex bead assay. Using supervised machine learning methods, we characterised recent and historic exposure to Plasmodium falciparum and P. vivax and found recent exposure to P. falciparum to be very low, with exposure to both species increasing with age. We performed a risk-factor assessment on environmental, behavioural, demographic and household factors, and identified forest activity and longer travel times to healthcare as common risk-factors for exposure to P. falciparum and P. vivax. In addition, we used remote-sensing derived data and geostatistical models to assess environmental and spatial associations with exposure. We created predictive maps of exposure to recent P. falciparum in the study area and showed 3 clear foci of exposure. This study provides useful insight into the environmental, spatial and demographic risk factors for P. falciparum and P. vivax at a period of low transmission in Malaysian Borneo. The findings would be valuable in the case of resurgence of human malarias in the region.

No Association between the Plasmodium vivax crt-o MS334 or In9pvcrt Polymorphisms and Chloroquine Failure in a Pre-Elimination Clinical Cohort from Malaysia with a Large Clonal Expansion.

Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raises concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motif lengths at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from a low endemic setting in Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Among a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none of the variants were associated with CQ treatment failure (all P > 0.05). Multi-locus genotypes (MLGs) at 9 neutral microsatellites revealed a predominant P. vivax strain (MLG6) accounting for 52% of Day 0 infections. The MLG6 strain comprised equal proportions of CQS and CQR infections. Our study reveals complexity in the genetic basis of CQ resistance in the Malaysian P. vivax pre-elimination setting and suggests that the proposed pvcrt-o MS334 and In9pvcrt markers are not reliable markers of CQ treatment efficacy in this setting. Further studies are needed in other endemic settings, applying hypothesis-free genome-wide approaches, and functional approaches to understand the biological impact of the TGAAGH repeats linked to CQ response in a cross are warranted to comprehend and track CQR P. vivax.

Factors affecting implementation of tuberculosis contact investigation and tuberculosis preventive therapy among children in Sabah, East Malaysia: A qualitative study.

Contact investigation and TB preventive treatment of children under five years of age who are close contacts of a TB case is a key component of TB prevention. However, the uptake of TB preventive treatment is low in many high-TB burden settings. This study explores factors affecting the implementation of TB contact investigation and preventive treatment among children in Malaysia's city of Kota Kinabalu, Sabah State. This study was conducted in three primary health clinics between 2019 and 2020. We purposively sampled 34 parents and guardians of child contacts eligible for TB preventive treatment, and 25 healthcare providers involved in the management of child contacts. We conducted thematic analysis of semi-structured interviews and focus group discussions to illicit factors affecting implementation and uptake of TB contact investigation and TB preventive therapy. Six main themes emerged from the analyses-four of these relating to contact investigation and two relating to TB preventive therapy. Factors affecting TB contact investigation were addressed under system related factors (external factors, stakeholder collaboration, healthcare workers' and clients' concerns), clinic related factors (perceived performance, clinic schedule, and space), healthcare worker related factors (cooperation, commitment, knowledge, misconception, counselling and communication) and patient and contact related factors (cooperation and commitment). Factors affecting TB preventive treatment delivery were addressed under guardian related factors (cooperation, commitment, knowledge and misconception) and treatment related factors (child-friendly form and adverse effects). To address gaps and barriers identified in our study, we recommend developing system capacity to maintain routine contact investigation and preventive treatment in the context of external program risks, providing training to healthcare workers to address misconceptions, safeguarding vulnerable clients against the risk of detention and deportation while accessing care, ensuring public and private services are provided regardless of migration status, and improving processes and resources for contact investigation and preventive treatment.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Early dispersal of domestic horses into the Great Plains and northern Rockies.

The horse is central to many Indigenous cultures across the American Southwest and the Great Plains. However, when and how horses were first integrated into Indigenous lifeways remain contentious, with extant models derived largely from colonial records. We conducted an interdisciplinary study of an assemblage of historic archaeological horse remains, integrating genomic, isotopic, radiocarbon, and paleopathological evidence. Archaeological and modern North American horses show strong Iberian genetic affinities, with later influx from British sources, but no Viking proximity. Horses rapidly spread from the south into the northern Rockies and central plains by the first half of the 17th century CE, likely through Indigenous exchange networks. They were deeply integrated into Indigenous societies before the arrival of 18th-century European observers, as reflected in herd management, ceremonial practices, and culture.

Evaluation of a point-of-care haemozoin assay (Gazelle device) for rapid detection of Plasmodium knowlesi malaria.

Plasmodium knowlesi is the major cause of zoonotic malaria in Southeast Asia. Rapid and accurate diagnosis enables effective clinical management. A novel malaria diagnostic tool, Gazelle (Hemex Health, USA) detects haemozoin, a by-product of haem metabolism found in all Plasmodium infections. A pilot phase refined the Gazelle haemozoin identification algorithm, with the algorithm then tested against reference PCR in a larger cohort of patients with P. knowlesi mono-infections and febrile malaria-negative controls. Limit-of-detection analysis was conducted on a subset of P. knowlesi samples serially diluted with non-infected whole blood. The pilot phase of 40 P. knowlesi samples demonstrated 92.5% test sensitivity. P. knowlesi-infected patients (n = 203) and febrile controls (n = 44) were subsequently enrolled. Sensitivity and specificity of the Gazelle against reference PCR were 94.6% (95% CI 90.5-97.3%) and 100% (95% CI 92.0-100%) respectively. Positive and negative predictive values were 100% and 98.8%, respectively. In those tested before antimalarial treatment (n = 143), test sensitivity was 96.5% (95% CI 92.0-98.9%). Sensitivity for samples with ≤ 200 parasites/µL (n = 26) was 84.6% (95% CI 65.1-95.6%), with the lowest parasitaemia detected at 18/µL. Limit-of-detection (n = 20) was 33 parasites/µL (95% CI 16-65%). The Gazelle device has the potential for rapid, sensitive detection of P. knowlesi infections in endemic areas.

Quantification of parasite clearance in Plasmodium knowlesi infections.

BACKGROUND: The incidence of zoonotic Plasmodium knowlesi infections in humans is rising in Southeast Asia, leading to clinical studies to monitor the efficacy of anti-malarial treatments for knowlesi malaria. One of the key outcomes of anti-malarial drug efficacy is parasite clearance. For Plasmodium falciparum, parasite clearance is typically estimated using a two-stage method, that involves estimating parasite clearance for individual patients followed by pooling of individual estimates to derive population estimates. An alternative approach is Bayesian hierarchical modelling which simultaneously analyses all parasite-time patient profiles to determine parasite clearance. This study compared these methods for estimating parasite clearance in P. knowlesi treatment efficacy studies, with typically fewer parasite measurements per patient due to high susceptibility to anti-malarials. METHODS: Using parasite clearance data from 714 patients with knowlesi malaria and enrolled in three trials, the Worldwide Antimalarial Resistance Network (WWARN) Parasite Clearance Estimator (PCE) standard two-stage approach and Bayesian hierarchical modelling were compared. Both methods estimate the parasite clearance rate from a model that incorporates a lag phase, slope, and tail phase for the parasitaemia profiles. RESULTS: The standard two-stage approach successfully estimated the parasite clearance rate for 678 patients, with 36 (5%) patients excluded due to an insufficient number of available parasitaemia measurements. The Bayesian hierarchical estimation method was applied to the parasitaemia data of all 714 patients. Overall, the Bayesian method estimated a faster population mean parasite clearance (0.36/h, 95% credible interval [0.18, 0.65]) compared to the standard two-stage method (0.26/h, 95% confidence interval [0.11, 0.46]), with better model fits (compared visually). Artemisinin-based combination therapy (ACT) is more effective in treating P. knowlesi than chloroquine, as confirmed by both methods, with a mean estimated parasite clearance half-life of 2.5 and 3.6 h, respectively using the standard two-stage method, and 1.8 and 2.9 h using the Bayesian method. CONCLUSION: For clinical studies of P. knowlesi with frequent parasite measurements, the standard two-stage approach (WWARN's PCE) is recommended as this method is straightforward to implement. For studies with fewer parasite measurements per patient, the Bayesian approach should be considered. Regardless of method used, ACT is more efficacious than chloroquine, confirming the findings of the original trials.

Allelic contribution of Nrxn1α to autism-relevant behavioral phenotypes in mice.

Copy number variations (CNVs) in the Neurexin 1 (NRXN1) gene, which encodes a presynaptic protein involved in neurotransmitter release, are some of the most frequently observed single-gene variants associated with autism spectrum disorder (ASD). To address the functional contribution of NRXN1 CNVs to behavioral phenotypes relevant to ASD, we carried out systematic behavioral phenotyping of an allelic series of Nrxn1 mouse models: one carrying promoter and exon 1 deletion abolishing Nrxn1α transcription, one carrying exon 9 deletion disrupting Nrxn1α protein translation, and one carrying an intronic deletion with no observable effect on Nrxn1α expression. We found that homozygous loss of Nrxn1α resulted in enhanced aggression in males, reduced affiliative social behaviors in females, and significantly altered circadian activities in both sexes. Heterozygous or homozygous loss of Nrxn1α affected the preference for social novelty in male mice, and notably, enhanced repetitive motor skills and motor coordination in both sexes. In contrast, mice bearing an intronic deletion of Nrxn1 did not display alterations in any of the behaviors assessed. These findings demonstrate the importance of Nrxn1α gene dosage in regulating social, circadian, and motor functions, and the variables of sex and genomic positioning of CNVs in the expression of autism-related phenotypes. Importantly, mice with heterozygous loss of Nrxn1, as found in numerous autistic individuals, show an elevated propensity to manifest autism-related phenotypes, supporting the use of models with this genomic architecture to study ASD etiology and assess additional genetic variants associated with autism.

Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

Background: The interaction between iron deficiency and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. Methods: We retrieved samples and associated data from 55 participants enrolled in malaria VIS, and 171 malaria patients and 30 healthy controls enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. Results: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline iron status (ferritin) was associated with post-treatment increases in liver transaminase levels. In Malaysian malaria patients, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. Hepcidin normalised by day 28; however, ferritin and sTfR both remained elevated 4 weeks following admission. Conclusion: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency.

A molecular barcode and web-based data analysis tool to identify imported Plasmodium vivax malaria.

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.

Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum.

Background: Plasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets. Methods: Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH. Results: Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels. Conclusion: Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria.

Assessing seroprevalence and associated risk factors for multiple infectious diseases in Sabah, Malaysia using serological multiplex bead assays.

Background: Infectious diseases continue to burden populations in Malaysia, especially among rural communities where resources are limited and access to health care is difficult. Current epidemiological trends of several neglected tropical diseases in these populations are at present absent due to the lack of habitual and efficient surveillance. To date, various studies have explored the utility of serological multiplex beads to monitor numerous diseases simultaneously. We therefore applied this platform to assess population level exposure to six infectious diseases in Sabah, Malaysia. Furthermore, we concurrently investigated demographic and spatial risk factors that may be associated with exposure for each disease. Methods: This study was conducted in four districts of Northern Sabah in Malaysian Borneo, using an environmentally stratified, population-based cross-sectional serological survey targeted to determine risk factors for malaria. Samples were collected between September to December 2015, from 919 villages totaling 10,100 persons. IgG responses to twelve antigens of six diseases (lymphatic filariasis- Bm33, Bm14, BmR1, Wb123; strongyloides- NIE; toxoplasmosis-SAG2A; yaws- Rp17 and TmpA; trachoma- Pgp3, Ct694; and giardiasis- VSP3, VSP5) were measured using serological multiplex bead assays. Eight demographic risk factors and twelve environmental covariates were included in this study to better understand transmission in this community. Results: Seroprevalence of LF antigens included Bm33 (10.9%), Bm14+ BmR1 (3.5%), and Wb123 (1.7%). Seroprevalence of Strongyloides antigen NIE was 16.8%, for Toxoplasma antigen SAG2A was 29.9%, and Giardia antigens GVSP3 + GVSP5 was 23.2%. Seroprevalence estimates for yaws Rp17 was 4.91%, for TmpA was 4.81%, and for combined seropositivity to both antigens was 1.2%. Seroprevalence estimates for trachoma Pgp3 + Ct694 were 4.5%. Age was a significant risk factors consistent among all antigens assessed, while other risk factors varied among the different antigens. Spatial heterogeneity of seroprevalence was observed more prominently in lymphatic filariasis and toxoplasmosis. Conclusions: Multiplex bead assays can be used to assess serological responses to numerous pathogens simultaneously to support infectious disease surveillance in rural communities, especially where prevalences estimates are lacking for neglected tropical diseases. Demographic and spatial data collected alongside serosurveys can prove useful in identifying risk factors associated with exposure and geographic distribution of transmission.

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Plasmodium vivax malaria serological exposure markers: Assessing the degree and implications of cross-reactivity with P. knowlesi.

Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivax serological markers, we assayed samples from human patients with P. knowlesi malaria. IgG antibody reactivity is high against P. vivax proteins with high sequence identity with their P. knowlesi ortholog. IgG reactivity peaks at 7 days post-P. knowlesi infection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivax proteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivax infections while reducing misclassification of recent P. knowlesi infections.

Diagnostic performance of a 5-plex malaria immunoassay in regions co-endemic for Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale.

Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan-Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform's performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.