Plasmodium Falciparum Histidine-rich Protein Research Articles

Ultrafast Electrothermal Flow‐Enhanced Magneto Biosensor for Highly Sensitive Protein Detection in Whole Blood

AbstractCurrent diagnostic tests for sensitive protein detection rely on immunological techniques, such as ELISA, which require sample purification, multiple washing steps and lengthy incubation, hindering their use for rapid testing. Here, we report a simple electrothermal flow‐enhanced biosensor for ultrafast, high sensitivity measurements of protein biomarkers in whole blood. Magnetic nanobeads dually‐labeled with a detection antibody and enzyme reporter are used to form immunocomplexes with the target protein, which are readily transported to the sensor via magnetic concentration. The incorporation of electrothermal flows enhances immunocomplex formation, allowing for rapid and sensitive detection without requiring blood purification or lengthy incubation. Proof of concept was carried out using Plasmodium falciparum histidine‐rich protein 2 (PfHRP2), a malaria parasite biomarker, which could be detected at concentrations as low as 5.7 pg mL−1 (95 fM) in whole blood in 7 min. The speed, sensitivity and simplicity of this device make it attractive for rapid diagnostic testing.

Automated Immunoassay Performed on a 3D Microfluidic Paper-Based Device for Malaria Detection by Ambient Mass Spectrometry.

Microfluidic paper-based analytical devices (μPADs) are emerging as a prominent platform for disease detection, specifically in developing countries. This paper device offer simplicity and affordability not typically seen in centralized laboratory settings. However, detection limits in μPADs are inadequate and often require test results to be read within a specific time interval to ensure accuracy. To overcome these challenges, we are developing an on-chip mass spectrometry (MS) detection strategy for immunoassays performed on paper substrates. Herein, we present our initial results from a proof-of-concept study toward the development of μPADs capable of storing immunoassay reagents within the confinements of the 3D device, automatic splitting of biofluid into four individual test zones, immuno-capture of the disease biomarker, and on-chip MS detection of the captured species. The reported study encourages the development of point-of-care and direct-to-customer testing using disposable μPADs to collect samples, followed by sensitive analysis using portable MSs. We demonstrate this capability using malaria Plasmodium falciparum histidine-rich protein 2 (PfHRP2) antigen detection.

Plasmodium falciparum histidine rich protein 2 (pfhrp2): an additional genetic marker suitable for anti-malarial drug efficacy trials

BackgroundGenotyping of the three Plasmodium falciparum polymorphic genes, msp1, msp2 and glurp, has been adopted as a standard strategy to distinguish recrudescence from new infection in drug efficacy clinical trials. However, the suitability of a particular gene is compromised in areas where its allelic variants distribution is significantly skewed, a phenomenon that might occur in isolated parasite populations or in areas of very low transmission. Moreover, observation of amplification bias has diminished the value of glurp as a marker.MethodsThe suitability of the polymorphic P. falciparum histidine-rich protein 2 (pfhrp2) gene was assessed to serve as an alternative marker using a PCR-sequencing or a PCR–RFLP protocol for genotyping of samples in drug efficacy clinical trials. The value of pfhrp2 was validated by side-by-side analyses of 5 admission-recrudescence sample pairs from Yemeni malaria patients.ResultsThe outcome of the single pfhrp2 gene discrimination analysis has been found consistent with msp1, msp2 and glurp pool genotyping analysis for the differentiation of recrudescence from new infection.ConclusionThe findings suggest that under the appropriate circumstances, pfhrp2 can serve as an additional molecular marker for monitoring anti-malarials efficacy. However, its use is restricted to endemic areas where only a minority of P. falciparum parasites lack the pfhrp2 gene.

Prevalence of Infection and Malaria Parasite Density among Under Five Children: A Case Study of Dunukofia Rural Community in Anambra State, Nigeria

Aim: Malaria still remains an overwhelming cause of morbidity and mortality among children under five years of age, especially in sub-Saharan Africa. The study was carried out to evaluate malaria prevalence amongst children less than five years old. 
 Study Design: A cross sectional study was carried out. The study adopted a retrospective descriptive survey using the hospital records and diagnostic cross sectional survey by examination of blood samples across three variables: gender, age group and mosquito net usage.
 Duration: The study was done in 2021 from the month of March to April in the rural community. 
 Methodology: Parasitological diagnosis was with Plasmodium falciparum histidine-rich protein 2-based malaria Rapid Diagnostic Test (RDT) and microscopy of giemsa-stained blood smears. Demographic information was collected using questionnaire.
 Results: Three hundred (300) children aged less than five years malaria infection status was investigate, 174 (58.00% ) of them were females while 126 (42.00%. ) were males. Twenty one percent (21.00%) of the respondents are <1 year, 23.33% (70) of them are between the ages of 2 to 3 years, while 55.67% (167) were 4 years and above. Current malaria prevalence was higher with microscopy (67.33%) than that of RDT (23.33%). Also, previous RDT results showed that there was a higher prevalence (73.56%) of malaria parasites in females than males (58.73%). The microscopy results showed that males had a higher prevalence (38.10%) of malaria parasites than females (12.64%). Overall gender result also revealed that males had a higher prevalence (96.83%) of malaria parasites than females (86.21%). There was a significant difference in the prevalence result with gender (P<0.05). Females had higher parasite density (28.05±15.390) than males (23.22±19.171), there was no significant difference (P>0.05). It further revealed that children from 4 years and above had higher intensity (29.68±17.357) while those of 1 year and below had the least (14.89±16.069). However, there was no significant difference in the malaria parasite among the age groups of patients (P>0.05).
 Conclusion: Prevalence of malaria parasitaemia was still high in Dunukaofia, Anamba State, Nigeria despite various control measures and interventions put in place by WHO.

Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation

AbstractPlasmodium falciparum-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) not only to disrupt the barrier-permeability function of endothelial cells but also to inhibit the antiinflammatory signaling function of AT. Here we investigated the mechanisms of the proinflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration, and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation, and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of P falciparum-derived HRPII in both cellular and animal models.

Genetic variation of Plasmodium falciparum histidine-rich protein 2 and 3 in Assosa zone, Ethiopia: its impact on the performance of malaria rapid diagnostic tests

BackgroundRapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes.MethodsA cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced.ResultsThe PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50–99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/μl.ConclusionThe findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A–EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A–EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.

A Comprehensive Analysis of the Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) in the Brazilian Amazon

Early diagnosis and treatment are fundamental to the control and elimination of malaria. In many endemic areas, routine diagnosis is primarily performed microscopically, although rapid diagnostic tests (RDTs) provide a useful point-of-care tool. Most of the commercially available RDTs detect histidine-rich protein 2 (HRP2) of Plasmodium falciparum in the blood of infected individuals. Nonetheless, parasite isolates lacking the pfhrp2 gene are relatively frequent in some endemic regions, thereby hampering the diagnosis of malaria using HRP2-based RDTs. To track the efficacy of RDTs in areas of the Brazilian Amazon, we assessed pfhrp2 deletions in 132 P. falciparum samples collected from four malaria-endemic states in Brazil. Our findings show low to moderate levels of pfhrp2 deletion in different regions of the Brazilian Amazon. Overall, during the period covered by this study (2002-2020), we found that 10% of the P. falciparum isolates were characterized by a pfhrp2 deletion. Notably, however, the presence of pfhrp2-negative isolates has not been translated into a reduction in RDT efficacy, which in part may be explained by the presence of polyclonal infections. A further important finding was the discrepancy in the proportion of pfhrp2 deletions detected using two assessed protocols (conventional PCR versus nested PCR), which reinforces the need to perform a carefully planned laboratory workflow to assess gene deletion. This is the first study to perform a comprehensive analysis of PfHRP2 sequence diversity in Brazilian isolates of P. falciparum. We identified 10 PfHRP2 sequence patterns, which were found to be exclusive of each of the assessed regions. Despite the small number of PfHRP2 sequences available from South America, we found that the PfHRP2 sequences identified in Brazil and neighboring French Guiana show similar sequence patterns. Our findings highlight the importance of continuously monitoring the occurrence and spread of parasites with pfrhp2 deletions, while also taking into account the limitations of PCR-based testing methods associated with accuracy and the complexity of infections.

Prevalence of Pfhrp2 and Pfhrp3 Gene Deletions in Plasmodium Falciparum Isolates and their Performance of Hrp2 Based Malaria Rapid Diagnostic Tests in Three Districts of Ghana

Malaria rapid diagnostic tests (MRDTs) are important for malaria disease management. However, the performance of the RDTs is affected when the targeted antigens in the parasite have a variation or are altogether absent. The most common parasite target antigen in RDTs, Plasmodium falciparum histidine-rich protein 2 (HRP2), has been reported to be absent in some P. falciparum parasites. 371 patient samples, from Akuapem North (58.5%), Atiwa East (21.3%), and from New Juaben (20.2%), were used in the study. PCR provided the highest number, 14.8% (55/371), of positive detections for falciparum infections. Microscopy detected parasites in 20/261 (7.7%) samples, and the minimum parasite density by microscopy was 430 parasites/µL. Out of the 371 samples, 27 (7.3%) were positive by RDT. The highest RDT positivity rate, 13.3% (10/75), was observed at New Juaben. False-negative RDT results were obtained in 43/55 (78.2%) of the negative branded RDT kits. Only two microscopies positive sample were RDT positive. Using 18SrDNA PCR, 55 (14.8%) samples were positive for P. falciparum. In Akuapem North, 79.2 % (19/24) of the PCR positive samples had P. falciparum parasites that lacked exon 2 of PFHRP2. An overall RDT positivity rate of 7.3% (27/371) and false-negative rate of 78.2% (43/55) were observed for the study sites. Plasmodium falciparum parasite populations with deletions of the PFHRP2 and PFHRP3 genes are present in Ghana. There is an urgent need to investigate the prevalence and geographic distribution of these parasites. Keywords: Histidine Rich Protien (HRP), Malaria Rapid Diagnostic Test (MRDT), Malaria, Rapid Diagnostic Test (RDT).

PfHRP2 detection using plasmonic optrodes: performance analysis

BackgroundEarly malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs).MethodsThis study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface.ResultsTheir performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection.ConclusionsAs malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

BackgroundMalaria diagnosis in many malaria-endemic countries relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia.MethodsPfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n = 951) and pregnant women at first antenatal care (ANC) visit (n = 87) enrolled in the Saint Joseph’s Catholic Hospital (Monrovia) from March to July 2019 were used to assess the frequency of false-negative RDT results. True–false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy results. Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3.ResultsOne-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and decreased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n = 5) and 8% (n = 7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the 16 RDT-negative but microscopy-positive samples which were available for molecular testing. Among the 14 samples that were positive by qPCR but negative by RDT and microscopy, 3 only amplified pfldh, and among these 3 all were positive for pfhrp2 and pfhrp3.ConclusionThere is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates that these deletions are not expected to affect the performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

Prevalence of Plasmodium falciparum isolates lacking the histidine rich protein 2 gene among symptomatic malaria patients in Kwilu Province of the Democratic Republic of Congo

BackgroundMalaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where Plasmodium falciparum Histidine Rich Protein 2-based rapid diagnostic tests (PfHRP2-based RDTs) are widely used. However, in the last decade, the accuracy of PfHRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the P. falciparum histidine rich protein 2 (pfhrp2) gene, resulting in false-negative results. In the Democratic Republic of Congo (D.R. Congo), little is known about the prevalence of the pfhrp2 gene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu Province of the D.R. Congo.MethodsWe used secondary data from a prospective health facility-based cross-sectional study conducted in 2018. Blood was collected for microscopy, PfHRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of pfhrp2 gene deletions. Fischer’s exact and the Kruskal–Wallis tests were applied to look for associations between potential explanatory variables and the pfhrp2 gene deletion with a level of statistical significance set at P < 0.05.ResultsOf the 684 enrolled symptomatic patients, 391 (57.7%) were female. The majority (87.7%) reported the presence of mosquito breeding sites within the household’s compound, and fever was the most reported symptom (81.6%). The overall prevalence of the pfhrp2 gene deletion was 9.2% (95% CI: 6.7%–12.1%). The deletion of the pfhrp2 gene was associated with health zone of origin (P = 0.012) and age (P = 0.019). Among false-negative PfHRP2-RDT results, only 9.9% were due to pfhrp2 gene deletion.ConclusionsP. falciparum isolates with pfhrp2 gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting PfHRP2 and parasite lactate dehydrogenase (pLDH) antigens could limit the spread of deleted isolates.Graphic

A comparative study of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) blood levels and peripheral blood parasitemia as parameters of disease severity in individuals with imported falciparum malaria

BackgroundIn falciparum malaria the total parasite biomass can be estimated by blood levels of histidine-rich protein 2 (PfHRP2), a Plasmodium falciparum-specific protein, which has been widely studied in malaria-endemic regions. This study investigates the usefulness of PfHRP2 as marker for disease severity in imported falciparum malaria. MethodsA retrospective cohort analysis was done in 145 patients with imported falciparum malaria. Associations between PfHRP2, malaria disease severity and classic parameters of disease severity were examined by statistical analyses. Patients with different travel purposes were examined in two groups: visiting friends and relatives (VFRs) and other travel purposes (mainly tourists). ResultsHigh PfHRP2 levels were clearly associated with disease severity. VFRs status showed to be an independent determinant protecting against severe malaria. At similar PfHRP2 levels VFRs patients had significantly lower levels of peripheral blood parasitemia compared to other patients. ConclusionOur study confirms the association between PfHRP2 and disease severity in patients with imported falciparum malaria, but for proper interpretation of PfHRP2 levels as disease severity marker in travellers, the possible presence of pre-existing acquired anti-malarial immunity should be taken into account as the correlation between PfHRP2 levels and disease severity differed significantly between VFRs patients and patients with other travel purposes.

Prevalence of Malaria among Children under Five Years in the Niger Delta Region of Nigeria

Background: Malaria still remains an overwhelming cause of morbidity and mortality among children under five years of age, especially in sub-Saharan Africa. The aim of this study was to determine the prevalence of Plasmodium parasitemia among children below 5 years of age in Akwa-Ibom, Delta and Rivers States, located in the Niger Delta region of Nigeria.&#x0D; Methodology: A cross sectional study of 2630 children, selected by multistage sampling from Akwa-Ibom, Delta and Rivers States of Nigeria between April and June 2019. Parasitological diagnosis was by Plasmodium falciparum histidine-rich protein 2-based malaria rapid diagnostic test (RDT) and microscopy of giemsa-stained blood smears. Demographic information was collected using soft copies of pretested interviewer-administered questionnaires via the Open Data Kit application installed on android phones. Data analysis was performed using the statistical software SPSS version 25. Frequency, percentages, and Chi-square test were used to interpret data at a confidence interval of 95% and a p-value less than 0.05 was regarded as statistically significant.&#x0D; Results: A total of 2630 children less than 5 years old were included in the study, 1016 from Akwa-Ibom and 807 each from Delta and Rivers states respectively. Malaria parasitaemia was detected in 230 (8.6%) and 198 (7.4%) children by RDT and microscopy, respectively. RDT used in this survey has high diagnostic accuracy (98.8%) compared to microscopy.&#x0D; Conclusion: The study shows a decline in the prevalence of malaria in children under 5 years. It also demonstrates the reliability of the RDTs in the diagnosis of malaria. The use of RDTs is thus further recommended especially in peripheral centers where the access to skilled microscopists and laboratory infrastructure may be lacking.

Detecting Plasmodium falciparum in community surveys: a comparison of Paracheck Pf\xae Test and ICT Malaria Pf\xae Cassette Test to polymerase chain reaction in Mutasa District, Zimbabwe

BackgroundMicroscopy and rapid diagnostic tests (RDTs) are the main techniques used to diagnose malaria. While microscopy is considered the gold standard, RDTs have established popularity as they allow for rapid diagnosis with minimal technical skills. This study aimed to compare the diagnostic performance of two Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs (Paracheck Pf® Test (Paracheck) and Malaria Pf™ ICT (ICT)) to polymerase chain reaction (PCR) in a community survey.MethodsA cross-sectional study was conducted between October 2012 and December 2014 in Mutasa District, Manicaland Province, eastern Zimbabwe. Households were randomly selected using satellite imagery, and 224 households were visited. Residents present in the household on the date of the visit were recruited for the study. Participants of all age groups from the selected households were screened with Paracheck and ICT RDTs in parallel. Dried blood spots (DBS) and thin and thick smears were collected. Parasite DNA extracted from the DBS was subjected to nested PCR targeting the Plasmodium cytochrome b mitochondrial gene. Data analysis was performed using the Cohen’s Kappa test to determine the interrater agreement and the sensitivity and specificity of the diagnostic test were reported.ResultsResults from a total of 702 participants were analysed. Most were females, 397 (57%), and the median age of participants was 21 years with an interquartile range of 9–39 years. Of those who were screened, 8 (1.1%), 35 (5.0%), and 21 (2.9%) were malaria parasite positive by microscopy, RDT and PCR, respectively. Paracheck and ICT RDTs had a 100% agreement. Comparing RDT and PCR results, 34 participants (4.8%) had discordant results. Most of the discordant cases were RDT positive but PCR negative (n = 24). Half of those RDT positive, but PCR negative individuals reported anti-malarials to use in the past month, which is significantly higher than reported anti-malarial drug use in the population (p < 0.001). The participant was febrile on the day of the visit, but relying on PfHRP2-based RDT would miss this case. Among the diagnostic methods evaluated, with reference to PCR, the sensitivity was higher with the RDT (52.4%) while specificity was higher with the microscopy (99.9%). The positive predictive value (PPV) was higher with the microscopy (87.5%), while the negative predictive values were similar for both microscopy and RDTs (98%). Overall, a strong correlated agreement with PCR was observed for the microscopy (97.9%) and the RDTs (95.2%).ConclusionsParacheck and ICT RDTs showed 100% agreement and can be used interchangeably. As malaria transmission declines and Zimbabwe aims to reach malaria elimination, management of infected individuals with low parasitaemia as well as non-P. falciparum infection can be critical.

Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage

BackgroundHaem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.MethodsThe CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites.ResultsPfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1).ConclusionOur data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.Graphical

High prevalence and extended deletions in Plasmodium falciparum hrp2/3 genomic loci in Ethiopia.

Deletions in Plasmodium falciparum histidine rich protein 2(pfhrp2) gene threaten the usefulness of the most widely used HRP2-based malaria rapid diagnostic tests (mRDTs) that cross react with its structural homologue, PfHRP3. Parasites with deleted pfhrp2/3 genes remain undetected and untreated due to 'false-negative' RDT results. As Ethiopia recently launched malaria elimination by 2030 in certain selected areas, the availability of RDTs and the scale of their use have rapidly increased in recent years. Thus, it is important to explore the presence and prevalence of deletion in the target genes, pfhrp2 and pfhrp3. From a total of 189 febrile patients visited Adama Malaria Diagnostic centre, sixty-four microscopically-and polymerase chain reaction (PCR)-confirmed P. falciparum clinical isolates were used to determine the frequency of pfhrp2/3 gene deletions. Established PCR assays were applied to DNA extracted from blood spotted onto filter papers to amplify across pfhrp2/3 exons and flanking regions. However, analysis of deletions in pfhrp2, pfhrp3 and flanking genomic regions was successful for 50 of the samples. The pfhrp2 gene deletion was fixed in the population with all 50(100%) isolates presenting a deletion variant. This deletion extended downstream towards the Pf3D7 0831900 (MAL7PI.230) gene in 11/50 (22%) cases. In contrast, only 2/50 (4%) of samples had deletions for the Pf3D7 0831700 (MALPI.228) gene, upstream of pfhrp2. Similarly, the pfhrp3 gene was deleted in all isolates (100%), while 40% of the isolates had an extension of the deletion to the downstream flanking region that codes for Pf3D7 13272400 (MAL13PI.485).The pfhrp3 deletion also extended upstream to Pf3D7 081372100 (MAL13PI.475) region in 49/50 (95%) of the isolates, exhibiting complete absence of the locus. Although all samples showed deletions of pfhrp2 exon regions, amplification of an intron region was successful in five cases. Two different repeat motifs in the intron regions were observed in the samples tested. Pfhrp2/3 gene deletions are fixed in Ethiopia and this will likely reduce the effectiveness of PfHRP2-based mRDTs. It will be important to determine the sensitivity PfHRP 2/3-based RDTs in these populations and conduct a countrywide survey to determine the extent of these deletions and its effect on routine RDT-based malaria diagnosis.

Impact of Plasmodium falciparum gene deletions on malaria rapid diagnostic test performance

BackgroundMalaria rapid diagnostic tests (RDTs) have greatly improved access to diagnosis in endemic countries. Most RDTs detect Plasmodium falciparum histidine-rich protein 2 (HRP2), but their sensitivity is seriously threatened by the emergence of pfhrp2-deleted parasites. RDTs detecting P. falciparum or pan-lactate dehydrogenase (Pf- or pan-LDH) provide alternatives. The objective of this study was to systematically assess the performance of malaria RDTs against well-characterized pfhrp2-deleted P. falciparum parasites.MethodsThirty-two RDTs were tested against 100 wild-type clinical isolates (200 parasites/µL), and 40 samples from 10 culture-adapted and clinical isolates of pfhrp2-deleted parasites. Wild-type and pfhrp2-deleted parasites had comparable Pf-LDH concentrations. Pf-LDH-detecting RDTs were also tested against 18 clinical isolates at higher density (2,000 parasites/µL) lacking both pfhrp2 and pfhrp3.ResultsRDT positivity against pfhrp2-deleted parasites was highest (> 94%) for the two pan-LDH-only RDTs. The positivity rate for the nine Pf-LDH-detecting RDTs varied widely, with similar median positivity between double-deleted (pfhrp2/3 negative; 63.9%) and single-deleted (pfhrp2-negative/pfhrp3-positive; 59.1%) parasites, both lower than against wild-type P. falciparum (93.8%). Median positivity for HRP2-detecting RDTs against 22 single-deleted parasites was 69.9 and 35.2% for HRP2-only and HRP2-combination RDTs, respectively, compared to 96.0 and 92.5% for wild-type parasites. Eight of nine Pf-LDH RDTs detected all clinical, double-deleted samples at 2,000 parasites/µL.ConclusionsThe pan-LDH-only RDTs evaluated performed well. Performance of Pf-LDH-detecting RDTs against wild-type P. falciparum does not necessarily predict performance against pfhrp2-deleted parasites. Furthermore, many, but not all HRP2-based RDTs, detect pfhrp2-negative/pfhrp3-positive samples, with implications for the HRP2-based RDT screening approach for detection and surveillance of HRP2-negative parasites.

Genetic variations in histidine-rich protein 2 and histidine-rich protein 3 of Myanmar Plasmodium falciparum isolates

BackgroundMalaria rapid diagnostic tests (RDTs) are precious tools to diagnose malaria. Most RDTs used currently are based on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in a patient’s blood. However, concern has been raised in recent years that deletion of pfhrp2 in the parasite could affect the accuracy of PfHRP2-based RDTs. In addition, genetic variation in pfhrp2 might influence the accuracy and sensitivity of RDTs. In this study, the genetic variation in pfhrp2 and pfhrp3 in Myanmar P. falciparum isolates was analysed.MethodsBlood samples were collected from malaria patients who were infected with P. falciparum in Mandalay, Naung Cho, Tha Beik Kyin, and Pyin Oo Lwin, Upper Myanmar between 2013 and 2015. The pfhrp2 and pfhrp3 were amplified by nested polymerase chain reaction (PCR), cloned and sequenced. Genetic variation in Myanmar pfhrp2 and pfhrp3 was analysed using the DNASTAR program. Comparative analysis of Myanmar and global pfhrp2 and pfhrp3 isolates was also performed.ResultsOne-hundred and two pfhrp2 and 89 pfhrp3 were amplified from 105 blood samples, of which 84 pfhrp2 and 56 pfhrp3 sequences were obtained successfully. Myanmar pfhrp2 and pfhrp3 showed high levels of genetic variation with different arrangements of distinct repeat types, which further classified Myanmar pfhrp2 and pfhrp3 into 76 and 47 haplotypes, respectively. Novel amino acid changes were also found in Myanmar pfhrp2 and pfhrp3, but their frequencies were very low. Similar structural organization was shared by Myanmar and global pfhrp2 and pfhrp3, and differences in frequencies of repeat types and lengths were also observed between and among global isolates.ConclusionLength polymorphisms and amino acid substitutions generated extensive genetic variation in Myanmar pfhrp2 and pfhrp3. Comparative analysis revealed that global pfhrp2 and pfhrp3 share similar structural features, as well as extensive length polymorphisms and distinct organizations of repeat types. These results provide a better understanding of the genetic structure of pfhrp2 and pfhrp3 in global P. falciparum populations and suggest useful information to develop RDTs with improved quality.

Microneedle-based skin patch for blood-free rapid diagnostic testing

Rapid diagnostic tests are one of the most commonly used tests to detect and screen for infectious diseases in the developing world. While these tests are simple, inexpensive, and readily available, they rely on finger-prick blood sampling, which requires trained medical personnel, poses risks of infection, and can complicate cooperation in young children, asymptomatic individuals, and communities with blood taboos. Here, we report a novel microneedle-based skin patch for the rapid detection of protein biomarkers in dermal interstitial fluid. Sample collection is facilitated by a hydrophilic hollow microneedle array that autonomously extracts and transports interstitial fluid to an antibody-based lateral flow test strip via surface tension for colorimetric antigen detection. We employ a simple gold enhancement treatment to enhance the detection sensitivity of this colloidal gold-based lateral flow assay and elucidate the underlying mechanism of this enhancement mechanism through experimental investigation. For proof-of-concept, this device was used to detect Plasmodium falciparum histidine-rich protein 2, a biomarker for malaria infection, which could be detected at concentrations as low as 8 ng/mL. Each test can be completed in <20 min and requires no equipment. To the best of our knowledge, this work is the first demonstration of a microneedle-based lateral flow assay for rapid protein detection in dermal interstitial fluid. In addition to its simplicity, minimally invasive nature, and low cost, this diagnostic device can be readily adapted to detect other protein biomarkers in interstitial fluid, making it a promising tool for point-of-care testing.