Evidence for a role of Anopheles stephensi in the spread of drug- and diagnosis-resistant malaria in Africa.

Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case-control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April-July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito.

MOIRE: A software package for the estimation of allele frequencies and effective multiplicity of infection from polyallelic data.

Malaria parasite genetic data can provide insight into parasite phenotypes, evolution, and transmission. However, estimating key parameters such as allele frequencies, multiplicity of infection (MOI), and within-host relatedness from genetic data has been challenging, particularly in the presence of multiple related coinfecting strains. Existing methods often rely on single nucleotide polymorphism (SNP) data and do not account for within-host relatedness. In this study, we introduce a Bayesian approach called MOIRE (Multiplicity Of Infection and allele frequency REcovery), designed to estimate allele frequencies, MOI, and within-host relatedness from genetic data subject to experimental error. Importantly, MOIRE is flexible in accommodating both polyallelic and SNP data, making it adaptable to diverse genotyping panels. We also introduce a novel metric, the effective MOI (eMOI), which integrates MOI and within-host relatedness, providing a robust and interpretable measure of genetic diversity. Using extensive simulations and real-world data from a malaria study in Namibia, we demonstrate the superior performance of MOIRE over naive estimation methods, accurately estimating MOI up to 7 with moderate sized panels of diverse loci (e.g. microhaplotypes). MOIRE also revealed substantial heterogeneity in population mean MOI and mean relatedness across health districts in Namibia, suggesting detectable differences in transmission dynamics. Notably, eMOI emerges as a portable metric of within-host diversity, facilitating meaningful comparisons across settings, even when allele frequencies or genotyping panels are different. MOIRE represents an important addition to the analysis toolkit for malaria population dynamics. Compared to existing software, MOIRE enhances the accuracy of parameter estimation and enables more comprehensive insights into within-host diversity and population structure. Additionally, MOIRE's adaptability to diverse data sources and potential for future improvements make it a valuable asset for research on malaria and other organisms, such as other eukaryotic pathogens. MOIRE is available as an R package at https://eppicenter.github.io/moire/.

LFP Analysis of Brain Injured Anesthetized Animals Undergoing Closed-Loop Intracortical Stimulation.

Activity dependent stimulation (ADS) is a closed loop stimulation technique whose neurophysiological effects have not been deeply investigated. Here we explored how Local field Potentials (LFP) are impacted by a focal ischemic lesion and, subsequently, by ADS treatment. Intracortical microelectrode arrays were implanted in the rostral forelimb area (RFA) and in the primary somatosensory area (S1) of anaesthetized rats. An ischemic injury was induced in the caudal forelimb area through microinjections of Endothelin-1. The lesion induced an acute depressive trend in LFP power in RFA (evaluated in 6 bands of interest: Delta (1-4Hz), Theta (4-8Hz), Alpha (8-11Hz), Beta (11-30Hz), LowGamma (30-55Hz) and HighGamma (55-80)) followed by a noticeable significant rebound in both areas. Applying ADS induced an overall decrease of power. The lesion impacted the connectivity in a frequency specific manner, resulting in widespread increase in connectivity in Delta both between and within areas. Two hours after the lesion, without stimulation, correlated activity between areas increased in Beta and Gamma. After stimulation, inter-area connectivity increased in Delta, Theta and Alpha, while considerably dropping within RFA in highGamma. By computing phase-amplitude coupling, we found that the lesion produced an incremental increase in the coupling between (Theta) Alpha phase and (lowGamma) highGamma amplitude within RFA, while S1 had a more generalized increase. Likewise, coupling between Theta phase and lowGamma/highGamma amplitudes increased between areas after lesion. ADS induced a similar increase, but greater in magnitude both within and between RFA and S1. These results have important implications on the emerging field of closed-loop adaptive stimulation promoting ADS as an innovative tool for the treatment of neurological disorders.

Association between indoor residual spraying and pregnancy outcomes: a quasi-experimental study from Uganda.

BACKGROUND: Malaria is a risk factor for adverse pregnancy outcomes. Indoor residual spraying with insecticide (IRS) reduces malaria infections, yet the effects of IRS on pregnancy outcomes are not well established. We evaluated the impact of a large-scale IRS campaign on pregnancy outcomes in Eastern Uganda. METHODS: Birth records (n = 59 992) were obtained from routine surveillance data at 25 health facilities from five districts that were part of the IRS campaign and six neighbouring control districts ∼27 months before and ∼24 months after the start of the campaign (January 2013-May 2017). Campaign effects on low birthweight (LBW) and stillbirth incidence were estimated using the matrix completion method (MC-NNM), a machine-learning approach to estimating potential outcomes, and compared with the difference-in-differences (DiD) estimator. Subgroup analyses were conducted by HIV and gravidity. RESULTS: MC-NNM estimates indicated that the campaign was associated with a 33% reduction in LBW incidence: incidence rate ratio (IRR) = 0.67 [95% confidence interval (CI): 0.49-0.93)]. DiD estimates were similar to MC-NNM [IRR = 0.69 (0.47-1.01)], despite a parallel trends violation during the pre-IRS period. The campaign was not associated with substantial reductions in stillbirth incidence [IRRMC-NNM = 0.94 (0.50-1.77)]. HIV status modified the effects of the IRS campaign on LBW [ßIRSxHIV = 0.42 (0.05-0.78)], whereby HIV-negative women appeared to benefit from the campaign [IRR = 0.70 (0.61-0.81)], but not HIV-positive women [IRR = 1.12 (0.59-2.12)]. CONCLUSIONS: Our results support the effectiveness of the campaign in Eastern Uganda based on its benefit to LBW prevention, though HIV-positive women may require additional interventions. The IRS campaign was not associated with a substantively lower stillbirth incidence, warranting further research.

Inferring person-to-person networks of Plasmodium falciparum transmission: are analyses of routine surveillance data up to the task?

BACKGROUND: Inference of person-to-person transmission networks using surveillance data is increasingly used to estimate spatiotemporal patterns of pathogen transmission. Several data types can be used to inform transmission network inferences, yet the sensitivity of those inferences to different data types is not routinely evaluated. METHODS: The influence of different combinations of spatial, temporal, and travel-history data on transmission network inferences for Plasmodium falciparum malaria were evaluated. RESULTS: The information content of these data types may be limited for inferring person-to-person transmission networks and may lead to an overestimate of transmission. Only when outbreaks were temporally focal or travel histories were accurate was the algorithm able to accurately estimate the reproduction number under control, Rc. Applying this approach to data from Eswatini indicated that inferences of Rc and spatiotemporal patterns therein depend upon the choice of data types and assumptions about travel-history data. CONCLUSIONS: These results suggest that transmission network inferences made with routine malaria surveillance data should be interpreted with caution.

Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.

BACKGROUND: Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. METHODS: A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. RESULTS: Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. CONCLUSIONS: The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

Assessment of Plasmodium antigens and CRP in dried blood spots with multiplex malaria array.

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12639-020-01325-2) contains supplementary material, which is available to authorized users.

Entrainment of Network Activity by Closed-Loop Microstimulation in Healthy Ambulatory Rats.

As our understanding of volitional motor function increases, it is clear that complex movements are the result of the interactions of multiple cortical regions rather than just the output properties of primary motor cortex. However, our understanding of the interactions among these regions is limited. In this study, we used the activity-dependent stimulation (ADS) technique to determine the short/long-term effects on network activity and neuroplasticity of intracortical connections. ADS uses the intrinsic neural activity of one region to trigger stimulations in a separate region of the brain and can manipulate neuronal connectivity in vivo. Our aim was to compare single-unit neuronal activity within premotor cortex (rostral forelimb area, [RFA] in rats) in response to ADS (triggered from RFA) and randomly-generated stimulation in the somatosensory area (S1) within single sessions and across 21 consecutive days of stimulation. We examined firing rate and correlation between spikes and stimuli in chronically-implanted healthy ambulatory rats during spontaneous and evoked activity. At the end of the treatment, we evaluated changes of synaptophysin expression. Our results demonstrated the ability of ADS to modulate RFA firing properties and to promote synaptogenesis in S1, strengthening the idea that this Hebbian-inspired protocol can be used to modulate cortical connectivity.

Effects of tDCS on spontaneous spike activity in a healthy ambulatory rat model.

BACKGROUND: The neurophysiological effects of transcranial direct current stimulation (tDCS) are typically described with respect to changes in cortical excitability, defined by using transcranial magnetic stimulation pulses to determine changes in motor evoked potentials. However, how individual cortical neurons change firing patterns under the influence of tDCS is largely unknown. While the relatively weak currents produced in the brain by tDCS may not be adequate to directly depolarize neuronal membranes, ongoing neuronal activity, combined with subthreshold changes in membrane polarization might be sufficient to alter the threshold for neural firing. OBJECTIVES: The purpose of this study was to determine the effects of tDCS on neurophysiological activity in motor cortex of freely moving, healthy rats. METHODS: In nine healthy, ambulatory rats, each studied under six different stimulation conditions varying in current intensity (maximum current density = 39.8 A/m2 at 0.4 mA) and polarity (anodal or cathodal), neural activity was analyzed in response to 20 min of tDCS applied through bone screws insulated from the overlying scalp. RESULTS: After analysis of 480 multi-unit channels that satisfied a rigid set of neurophysiological criteria, we found no systematic effect of tDCS stimulation condition on firing rate or firing pattern. Restricting the analysis to the most responsive units, subtle, but statistically significant changes occurred only in the highest intensity anodal condition. CONCLUSIONS: These results confirm that at current densities typically used in human or animal tDCS studies, observed effects of tDCS are likely to occur via mechanisms other than direct neuronal depolarization.

Confirmation of the absence of local transmission and geographic assignment of imported falciparum malaria cases to China using microsatellite panel.

BACKGROUND: Current methods to classify local and imported malaria infections depend primarily on patient travel history, which can have limited accuracy. Genotyping has been investigated as a complementary approach to track the spread of malaria and identify the origin of imported infections. METHODS: An extended panel of 26 microsatellites (16 new microsatellites) for Plasmodium falciparum was evaluated in 602 imported infections from 26 sub-Saharan African countries to the Jiangsu Province of People's Republic of China. The potential of the 26 microsatellite markers to assign imported parasites to their geographic origin was assessed using a Bayesian method with Markov Chain Monte Carlo (MCMC) as implemented in the program Smoothed and Continuous Assignments (SCAT) with a modification to incorporate haploid genotype data. RESULTS: The newly designed microsatellites were polymorphic and are not in linkage disequilibrium with the existing microsatellites, supporting previous findings of high rate of recombination in sub-Saharan Africa. Consistent with epidemiology inferred from patients' travel history, no evidence for local transmission was found; nearly all genetically related infections were identified in people who travelled to the same country near the same time. The smoothing assignment method assigned imported cases to their likely geographic origin with an accuracy (Angola: 59%; Nigeria: 51%; Equatorial Guinea: 40%) higher than would be achieved at random, reaching statistical significance for Angola and Equatorial Guinea. CONCLUSIONS: Genotyping using an extended microsatellite panel is valuable for malaria case classification and programme evaluation in an elimination setting. A Bayesian method for assigning geographic origin of mammals based on genetic data was adapted for malaria and showed potential for identification of the origin of imported infections.

Multiplex Human Malaria Array: Quantifying Antigens for Malaria Rapid Diagnostics.

Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT), which quantifies the antigens commonly used in RDTs-Plasmodium falciparum-specific histidine-rich protein 2 (HRP2), P. falciparum-specific lactate dehydrogenase (Pf LDH), Plasmodium vivax -specific LDH (Pv LDH), and Pan malaria lactate dehydrogenase (Pan LDH), and human C-reactive protein (CRP), a biomarker of severity in malaria. At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.

Active Case Finding for Malaria: A 3-Year National Evaluation of Optimal Approaches to Detect Infections and Hotspots Through Reactive Case Detection in the Low-transmission Setting of Eswatini.

BACKGROUND: Reactive case detection (RACD) is a widely practiced malaria elimination intervention whereby close contacts of index cases receive malaria testing to inform treatment and other interventions. However, the optimal diagnostic and operational approaches for this resource-intensive strategy are not clear. METHODS: We conducted a 3-year prospective national evaluation of RACD in Eswatini, a malaria elimination setting. Loop-mediated isothermal amplification (LAMP) was compared to traditional rapid diagnostic testing (RDT) for the improved detection of infections and for hotspots (RACD events yielding ≥1 additional infection). The potential for index case-, RACD-, and individual-level factors to improve efficiencies was also evaluated. RESULTS: Among 377 RACD events, 10 890 participants residing within 500 m of index cases were tested. Compared to RDT, LAMP provided a 3-fold and 2.3-fold higher yield to detect infections (1.7% vs 0.6%) and hotspots (29.7% vs 12.7%), respectively. Hotspot detection improved with ≥80% target population coverage and response times within 7 days. Proximity to the index case was associated with a dose-dependent increased infection risk (up to 4-fold). Individual-, index case-, and other RACD-level factors were considered but the simple approach of restricting RACD to a 200-m radius maximized yield and efficiency. CONCLUSIONS: We present the first large-scale national evaluation of optimal RACD approaches from a malaria elimination setting. To inform delivery of antimalarial drugs or other interventions, RACD, when conducted, should utilize more sensitive diagnostics and clear context-specific operational parameters. Future studies of RACD's impact on transmission may still be needed.

Differential Effects of Open- and Closed-Loop Intracortical Microstimulation on Firing Patterns of Neurons in Distant Cortical Areas.

Intracortical microstimulation can be used successfully to modulate neuronal activity. Activity-dependent stimulation (ADS), in which action potentials recorded extracellularly from a single neuron are used to trigger stimulation at another cortical location (closed-loop), is an effective treatment for behavioral recovery after brain lesion, but the related neurophysiological changes are still not clear. Here, we investigated the ability of ADS and random stimulation (RS) to alter firing patterns of distant cortical locations. We recorded 591 neuronal units from 23 Long-Evan healthy anesthetized rats. Stimulation was delivered to either forelimb or barrel field somatosensory cortex, using either RS or ADS triggered from spikes recorded in the rostral forelimb area (RFA). Both RS and ADS stimulation protocols rapidly altered spike firing within RFA compared with no stimulation. We observed increase in firing rates and change of spike patterns. ADS was more effective than RS in increasing evoked spikes during the stimulation periods, by producing a reliable, progressive increase in stimulus-related activity over time and an increased coupling of the trigger channel with the network. These results are critical for understanding the efficacy of closed-loop electrical microstimulation protocols in altering activity patterns in interconnected brain networks, thus modulating cortical state and functional connectivity.

Chronic stability of single-channel neurophysiological correlates of gross and fine reaching movements in the rat.

While substantial task-related neural activity has been observed during motor tasks in rodent primary motor cortex and premotor cortex, the long-term stability of these responses in healthy rats is uncertain, limiting the interpretability of longitudinal changes in the specific patterns of neural activity associated with learning or motor recovery following injury. This study examined the stability of task-related neural activity associated with execution of two distinct reaching tasks in healthy rodents. A novel automated rodent behavioral apparatus was constructed and rats were trained to perform a reaching task combining a 'gross' lever press and a 'fine' pellet retrieval. In each animal, two chronic microelectrode arrays were implanted in motor cortex spanning the caudal forelimb area (rodent primary motor cortex) and the rostral forelimb area (rodent premotor cortex). We recorded multiunit spiking and local field potential activity from 10 days to 7-10 weeks post-implantation to characterize the patterns of neural activity observed during each task component and analyzed the consistency of channel-specific task-related neural activity. Task-related changes in neural activity were observed on the majority of channels. While the task-related changes in multi-unit spiking and local field potential spectral power were consistent over several weeks, spectral power changes were more stable, despite the trade-off of decreased spatial and temporal resolution. These results show that neural activity in rodent primary and premotor cortex is associated with specific phases of reaching movements with stable patterns of task-related activity across time, establishing the relevance of the rodent for future studies designed to examine changes in task-related neural activity during recovery from focal cortical lesions.

Improving an open-source commercial system to reliably perform activity-dependent stimulation.

OBJECTIVE: Activity-dependent stimulation (ADS) is designed to strengthen the connections between neuronal circuits and therefore may be a promising tool for promoting neurophysiological reorganization following a brain injury. To successfully perform this technique, two criteria must be met: (1) spikes in the extracellular electrical field potential must be detected accurately at one site of interest, and (2) stimulation pulses generated at fixed (<1 ms jitter), low-latency (<10 ms) intervals relative to each detected spike must be delivered reliably to a second site of interest. Here, we aimed to improve noise rejection in a low-cost commercial system to reliably perform ADS in awake, behaving rats, while maintaining latency requirements. APPROACH: We implemented a spike detection state machine on a field-programmable gate array (FPGA). Because the accuracy of spike detection can be heavily reduced in awake and behaving animals due to biological artifacts such as movement and chewing, the state machine tracks candidate spike waveforms, checking them against multiple programmable thresholds and rejecting any spikes that fail to meet a programmed threshold criterion. MAIN RESULTS: A series of offline analyses showed that our implementation was able to appropriately trigger stimulation during epochs of biological artifacts with an overall accuracy between 72% and 97%, fixed computational latency of 167 µs, and an algorithmic latency of 300 µs to 800 µs. SIGNIFICANCE: Our improvements have been made open-source and are freely available to all scientists working on closed-loop neuroprosthetic devices. Importantly, the improvements are easily incorporated into existing workflows that utilize the Intan Stimulation and Recording Controller.

High Genetic Diversity of Plasmodium falciparum in the Low-Transmission Setting of the Kingdom of Eswatini.

BACKGROUND: To better understand transmission dynamics, we characterized Plasmodium falciparum genetic diversity in Eswatini, where transmission is low and sustained by importation. METHODS: Twenty-six P. falciparum microsatellites were genotyped in 66% of confirmed cases (2014-2016; N = 582). Population and within-host diversity were used to characterize differences between imported and locally acquired infections. Logistic regression was used to assess the added value of diversity metrics to classify imported and local infections beyond epidemiology data alone. RESULTS: Parasite population in Eswatini was highly diverse (expected heterozygosity [HE] = 0.75) and complex: 67% polyclonal infections, mean multiplicity of infection (MOI) 2.2, and mean within-host infection fixation index (FWS) 0.84. Imported cases had comparable diversity to local cases but exhibited higher MOI (2.4 vs 2.0; P = .004) and lower mean FWS (0.82 vs 0.85; P = .03). Addition of MOI and FWS to multivariate analyses did not increase discrimination between imported and local infections. CONCLUSIONS: In contrast to the common perception that P. falciparum diversity declines with decreasing transmission intensity, Eswatini isolates exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission. Estimates of malaria transmission intensity from genetic data need to consider the effect of importation, especially as countries near elimination.

FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Using parasite genetic and human mobility data to infer local and cross-border malaria connectivity in Southern Africa.

Local and cross-border importation remain major challenges to malaria elimination and are difficult to measure using traditional surveillance data. To address this challenge, we systematically collected parasite genetic data and travel history from thousands of malaria cases across northeastern Namibia and estimated human mobility from mobile phone data. We observed strong fine-scale spatial structure in local parasite populations, providing positive evidence that the majority of cases were due to local transmission. This result was largely consistent with estimates from mobile phone and travel history data. However, genetic data identified more detailed and extensive evidence of parasite connectivity over hundreds of kilometers than the other data, within Namibia and across the Angolan and Zambian borders. Our results provide a framework for incorporating genetic data into malaria surveillance and provide evidence that both strengthening of local interventions and regional coordination are likely necessary to eliminate malaria in this region of Southern Africa.

Simultaneous Quantification of Plasmodium Antigens and Host Factor C-Reactive Protein in Asymptomatic Individuals with Confirmed Malaria by Use of a Novel Multiplex Immunoassay.

Malaria rapid diagnostic tests (RDTs) primarily detect Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, P. vivax LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria. The assay also measures C-reactive protein (CRP) levels as an indicator of inflammation. Validation was conducted with clinical specimens from 397 asymptomatic donors from Myanmar and Uganda, confirmed by PCR for infection, and from participants in induced blood-stage malaria challenge studies. The assay lower limits of detection for HRP2, pan LDH, P. vivax LDH, and CRP were 0.2 pg/ml, 9.3 pg/ml, 1.5 pg/ml, and 26.6 ng/ml, respectively. At thresholds for HRP2, pan LDH, and P. vivax LDH of 2.3 pg/ml, 47.8 pg/ml, and 75.1 pg/ml, respectively, and a specificity ≥98.5%, the sensitivities for ultrasensitive PCR-confirmed infections were 93.4%, 84.9%, and 48.9%, respectively. Plasmodium LDH (pLDH) concentration, in contrast to that of HRP2, correlated closely with parasite density. CRP levels were moderately higher in P. falciparum infections with confirmed antigenemia versus those in clinical specimens with no antigen. The 4-plex array is a sensitive tool for quantifying diagnostic antigens in malaria infections and supporting the evaluation of new ultrasensitive RDTs.

Genetic Evidence of Focal Plasmodium falciparum Transmission in a Pre-elimination Setting in Southern Province, Zambia.

BACKGROUND: Southern Province, Zambia has experienced a dramatic decline in Plasmodium falciparum malaria transmission in the past decade and is targeted for elimination. Zambia's National Malaria Elimination Program recommends reactive case detection (RCD) within 140 m of index households to enhance surveillance and eliminate remaining transmission foci. METHODS: To evaluate whether RCD captures local transmission, we genotyped 26 microsatellites from 106 samples collected from index (n = 27) and secondary (n = 79) cases detected through RCD in the Macha Hospital catchment area between January 2015 and April 2016. RESULTS: Participants from the same RCD event harbored more genetically related parasites than those from different RCD events, suggesting that RCD captures, at least in part, infections related through local transmission. Related parasites clustered in space and time, up to at least 250 m from index households. Spatial analysis identified a putative focal transmission hotspot. CONCLUSIONS: The current RCD strategy detects focal transmission events, although programmatic guidelines to screen within 140 m of index households may fail to capture all secondary cases. This study highlights the utility of parasite genetic data in assessing programmatic interventions, and similar approaches may be useful to malaria elimination programs seeking to tailor intervention strategies to the underlying transmission epidemiology.