Hard-to-reach communities represent Peru\'s main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ\'s Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

BACKGROUND: Dual hrp2/hrp3 genes deletions in P. falciparum isolates are increasingly reported in malaria-endemic countries and can produce false negative RDT results leading to inadequate case management. Data on the frequency of hrp2/hrp3 deleted parasites are rarely available and it has become necessary to investigate the issue in Burkina Faso.
METHODS: Plasmodium falciparum-positive dried blood spots were collected during a cross-sectional household survey of the malaria asymptomatic children from Orodara, Gaoua, and Banfora. Amplicons from the target regions (exon 2 of hrp2 and hrp3 genes) were generated using multiplexed nested PCR and sequenced according to Illumina\'s MiSeq protocol.
RESULTS: A total of 251 microscopically positive parasite isolates were sequenced to detect hrp2 and hrp3 gene deletions. The proportion of RDTs negative cases among microscopy positive slides was 12.7% (32/251). The highest prevalence of negative RDTs was found in Orodara 14.3% (5/35), followed by Gaoua 13.1%(24/183), and Banfora 9.1% (3/33). The study found that 95.6% of the parasite isolates were wild type hrp2/ hrp3 while 4.4% (11/251) had a single hrp2 deletion. Of the 11 hrp2 deletion samples, 2 samples were RDT negative (mean parasitaemia was 83 parasites/ μL) while 9 samples were RDT positive with a mean parasitaemia of 520 parasites /μL (CI95%: 192-1239). The highest frequency of hrp2 deletion 4/35 (11.4%) was found in Orodara, while it was similar in the other two sites (< 3.5%). No single deletion of the hrp3 or dual deletion hrp2/3 gene was detected in this study.
CONCLUSIONS: These results demonstrate that P. falciparum isolates lacking hrp2 genes are present in 4.4% of samples obtained from the asymptomatic children population in three sites in Burkina Faso. These parasites are circulating and causing malaria, but they are also still detectable by HRP2-based RTDs due to the presence of the intact pfhrp3 gene.

UNASSIGNED: Accurate diagnosis and timely treatment are crucial in combating malaria.
UNASSIGNED: We evaluated the diagnostic performance of three Rapid Diagnostic Tests (RDTs) in diagnosing febrile patients, namely: Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and LDH on separate bands), and SD Bioline Malaria Ag Pf (detecting HRP2). Results were compared to qPCR.
UNASSIGNED: Among 449 clinical patients, 45.7% (205/449) tested positive by qPCR for P. falciparum with a mean parasite density of 12.5parasites/μL. The sensitivity of the Biocredit RDT was 52.2% (107/205), NxTek RDT was 49.3% (101/205), and Bioline RDT was 40.5% (83/205). When samples with parasite densities lower than 20 parasites/uL were excluded (n=116), a sensitivity of 88.8% (79/89, NxTek), 89.9% (80/89, Biocredit), and 78.7% (70/89, Bioline) was obtained. All three RDTs demonstrated specificity above 95%. The limits of detection was 84 parasites/μL (NxTek), 56 parasites/μL (Biocredit, considering either HRP2 or LDH), and 331 parasites/μL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the LDH target, carried hrp2/3 deletions.
UNASSIGNED: The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies and both RDTs performed better than Bioline RDT.

The expression of genetic information is tightly controlled by chromatin regulatory proteins, including those in the heterochromatin gene repression family. Many of these regulatory proteins work together on the chromatin substrate to precisely regulate gene expression during mammalian development, giving rise to many different tissues in higher organisms from a fixed genomic template. Here we identify and characterize the interactions of two related heterochromatin regulatory proteins, heterochromatin protein 1 alpha (HP1α) and M-phase phosphoprotein 8 (MPP8), with hepatoma-derived growth factor-related protein 2 (HRP2). We find in biochemical experiments that HRP2 copurifies and co-sediments with heterochromatin-associated proteins, including HP1α and MPP8. Using the Chromatin in vivo Assay in multiple cell types, we demonstrate that HP1α-mediated gene repression dynamics are altered by the presence of HRP2. Furthermore, the knockout of HRP2 in MDA-MB-231 cells results in significant changes to chromatin structure and stability, which alter gene expression patterns. Here, we detail a mechanism by which HRP2 contributes to epigenetic transcriptional regulation through engagement with heterochromatin-associated proteins to stabilize the chromatin landscape and influence gene expression.

BACKGROUND: The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points.
METHODS: GC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates.
RESULTS: GC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007-2008 and 2014-2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002.
CONCLUSIONS: The GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.

BACKGROUND: Accurate detection of asymptomatic malaria parasitaemia in children living in high transmission areas is important for malaria control and reduction programmes that employ screen-and-treat surveillance strategies. Relative to microscopy and conventional rapid diagnostic tests (RDTs), ultrasensitive RDTs (us-RDTs) have demonstrated reduced limits of detection with increased sensitivity to detect parasitaemia in symptomatic individuals. In this study, the performance of the NxTek™ Eliminate Malaria P.f test was compared with traditional microscopy and quantitative polymerase chain reaction (qPCR) testing methods of detection for P. falciparum parasitaemia among asymptomatic children aged 7-14 years living in an area of high malaria transmission intensity in western Kenya.
METHODS: In October 2020, 240 healthy children without any reported malaria symptoms were screened for the presence of P. falciparum parasitaemia; 120 children were randomly selected to participate in a follow-up visit at 6-10 weeks. Malaria parasitaemia was assessed by blood-smear microscopy, us-RDT, and qPCR of a conserved var gene sequence from genomic DNA extracted from dried blood spots. Sensitivity, specificity, and predictive values were calculated for field diagnostic methods using qPCR as the gold standard. Comparison of detectable parasite density distributions and area under the curve were also calculated to determine the effectiveness of the us-RDT in detecting asymptomatic infections with low parasite densities.
RESULTS: The us-RDT detected significantly more asymptomatic P. falciparum infections than microscopy (42.5% vs. 32.2%, P = 0.002). The positive predictive value was higher for microscopy (92.2%) than for us-RDT (82.4%). However, false negative rates were high for microscopy and us-RDT, with negative predictive values of 53.7% and 54.6%, respectively. While us-RDT detected significantly more infections than microscopy overall, the density distribution of detectable infections did not differ (P = 0.21), and qPCR detected significantly more low-density infections than both field methods (P < 0.001, for both comparisons).
CONCLUSIONS: Us-RDT is more sensitive than microscopy for detecting asymptomatic malaria parasitaemia in children. Though the detectable parasite density distributions by us-RDT in our specific study did not significantly differ from microscopy, the additional sensitivity of the us-RDT resulted in more identified asymptomatic infections in this important group of the population and makes the use of the us-RDT advisable compared to other currently available malaria field detection methods.

The recent COVID-19 pandemic has profoundly impacted global malaria elimination programs, resulting in a sharp increase in malaria morbidity and mortality. To reduce this impact, unmet needs in malaria diagnostics must be addressed while resuming malaria elimination activities. Rapid diagnostic tests (RDTs), the unsung hero in malaria diagnosis, work to eliminate the prevalence of Plasmodium falciparum malaria through their efficient, cost-effective, and user-friendly qualities in detecting the antigen HRP2 (histidine-rich protein 2), among other proteins. However, the testing mechanism and management of malaria with RDTs presents a variety of limitations. This paper discusses the numerous factors (including parasitic, host, and environmental) that limit the performance of RDTs. Additionally, the paper explores outside factors that can hinder RDT performance. By understanding these factors that affect the performance of HRP2-based RDTs in the field, researchers can work toward creating and implementing more effective and accurate HRP2-based diagnostic tools. Further research is required to understand the extent of these factors, as the rapidly changing interplay between parasite and host directly hinders the effectiveness of the tool.

Malaria is one of the most common tropical diseases encountered by members of the French military who are deployed in operations under constrained conditions in malaria-endemic areas. Blood smear microscopy-the gold standard for malaria diagnosis-is often not available in such settings, where the detection of malaria relies on rapid diagnostic tests (RDTs). Ten RDTs (from Biosynex, Carestart, Humasis, SD Bioline, and CTK Biotech), based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) or lactate dehydrogenase (pLDH, PfLDH, or PvLDH), were assessed against 159 samples collected from imported malaria cases, including 79 P. falciparum, 37 P. vivax, 22 P. ovale, and 21 P. malariae parasites. Samples had been previously characterised using microscopy and real-time PCR. The overall sensitivities for the Plasmodium test ranged from 69.8% (111/159) to 95% (151/159). There was no significant difference for the specific detection of P. falciparum (96.2% to 98.7%, p = 0.845). No significant difference was found between sensitivities to P. vivax by pan LDH or pvLDH (81.1% (30/37) to 94.6% (35/37) (p = 0.845)). Some of the RDTs missed most of P. ovale and P. malariae, with sensitivities for all RDTs ranging respectively from 4.5% (1/22) to 81.8% (18/22) and 14.3% (3/21) to 95.2% (20/21). Carestart Malaria Pf/Pan (pLDH) Ag G0121, a pLDH-based RDT (PfLDH and pLDH), showed the highest sensitivities to P. falciparum (98.7%, 78/79), P. vivax (94.6%, 35/37), P. ovale (81.8%, 18/22), and P. malariae (95.2%, 20/21) and meets the requirements for military deployments in malaria-endemic areas.

BACKGROUND: Immunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. This study presents the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species. Moreover, correlation between the malaria antigen measurements performed at these platforms is assessed after calibrating with either assay standards or international standards and the cross-reactivity among Plasmodium species is examined.
METHODS: A 77-member panel of specimens composed of the World Health Organization (WHO) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens. Assays for HRP2, P. falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex and the xMAP platforms were evaluated with these panels.
RESULTS: The xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH (Pearson r = 0.9926) and PvLDH (r = 0. 9792), moderate positive correlation for HRP2 (r = 0.7432), and poor correlation for PanLDH (r = 0.6139). In Pearson correlation analysis between the two platforms on the DBS, the same assays were r = 0.9828, r = 0.7679, r = 0.6432, and r = 0.8957, respectively. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS.
CONCLUSIONS: Q-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform.

BACKGROUND: Malaria has been identified as a significant public health burden, exhibiting a high risk of death and morbidity. In sub-Saharan Africa, most young children attending primary healthcare facilities are commonly diagnosed with malaria. Thus, introduction of malaria rapid diagnostic test (mRDT) kits and effective antimalarials has substantially improved the management of malaria cases. However, healthcare worker confidence and adherence to procedures dependent on malaria test results remain variable in high-burden settings due to lacking alternative point-of-care tests to diagnose other causes of fever. In this study, we compared the results of malaria screenings using mRDT and microscopy in febrile children presenting at a primary health facility.
METHODS: This study was conducted at a primary health center in Owo, Ondo State, Nigeria. Children with fever were assessed for malaria by health staff and, where indicated, screened using Plasmodium falciparum histidine-rich protein-2 mRDT kits. Blood samples were collected on slides for microscopy and in hematocrit tubes for hematocrit determination simultaneously, whereas the mRDT test was done by routine health staff. Children found positive for malaria via mRDT were diagnosed as uncomplicated malaria cases and treated as outpatients using artemether-lumefantrine. Blood slides were read independently by two trained microscopists blinded to the mRDT results. The parasite densities were defined as average counts by both microscopists. We then assessed the sensitivity, specificity, and predictive value of mRDT for the diagnosis of malaria.
RESULTS: We compared the test results of 250 febrile children who are under 15 years old. The test positivity rates were 93.6% (234/250) and 97.2% (243/250) using microscopy and rapid RDTs, respectively. The sensitivity and specificity of mRDT compared to microscopy were 100.0% and 43.8%, respectively, with a positive predictive value of 96.3% (95% CI 93.1-98.3). The hematocrit value was <30% in 64% of the children.
CONCLUSIONS: As per our findings, mRDTs have correctly detected infections in febrile children. Healthcare workers and caregivers should be encouraged to act in accordance with the test results by means of regular feedback on the quality of mRDTs in use in malaria case management.