Histidine-rich Protein Research Articles

Hepatic HRC induces hepatocyte pyroptosis and HSCs activation via NLRP3/caspase-1 pathway.

The histidine-rich calcium-binding protein (HRC) is a regulator of Ca2 + homeostasis and it plays a significant role in liver fibrosis. Pyroptosis, a specific inflammatory cell death, can lead to hepatic stellate cells (HSCs) activation and liver fibrosis. However, the role of HRC in pyroptosis has not been explored. In this study, we demonstrated that HRC, mainly located in the hepatocyte, was over expressed in fibrotic liver tissues. We further found that enforced expression of HRC in hepatocytes induced pyroptosis and HMGB1 release, and subsequently led to HSCs activation by NLRP3/caspase-1 pathway. In addition, the proliferation and migration of HSCs were also enhanced by HRC overexpression in hepatocytes. Furthermore, NLRP3 inhibitor MCC950 and caspase-1 inhibitor VX-765 alleviated hepatic HRC-mediated hepatocytes pyroptosis and HSCs activation. This study demonstrated that hepatic HRC promoted HSCs activation by inducing hepatocyte pyroptosis, which suggests that HRC may be a promising therapeutic target to prevent liver fibrosis.

Performance Evaluation of Nested Polymerase Chain Reaction (Nested PCR), Light Microscopy, and Plasmodium falciparum Histidine-Rich Protein 2 Rapid Diagnostic Test (PfHRP2 RDT) in the Detection of Falciparum Malaria in a High-Transmission Setting in Southwestern Nigeria.

Malaria remains a major public health challenge worldwide. In order to ensure a prompt and accurate malaria diagnosis, the World Health Organization recommended the confirmatory parasitological diagnosis of malaria by microscopy and malaria rapid diagnostic test (RDT) prior to antimalarial administration and treatment. This study was designed to evaluate the performance of nested polymerase chain reaction (nested PCR), light microscopy, and Plasmodium falciparum histidine-rich protein 2 rapid diagnostic test (PfHRP2 RDT) in the detection of falciparum malaria in Akure, Nigeria. A cross-sectional and hospital-based study involving 601 febrile volunteer participants was conducted in Akure, Nigeria. Approximately 2-3 mL venous blood samples were obtained from each study participant for parasitological confirmation by microscopy and PfHRP2-based malaria RDT. Thick and thin films were prepared and viewed under the light microscope for parasite detection, parasite density quantification, and species identification, respectively. Dry blood spot samples were prepared on 3MM Whatman filter paper for nested PCR. The overall prevalence of microscopy, PfHRP2 RDT, and nested PCR were 64.89% (390/601), 65.7% (395/601), and 67.39% (405/601), respectively. The estimates of sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and Youden's j index of microscopy and RDT were 96.30, 100.00, 100.00, 92.89, 97.50, 0.963, and 95.06, 94.90, 97.47, 90.29, 95.01, and 0.899, respectively. Malaria RDT recorded higher false negativity, compared microscopy (4.94% vs. 3.70%). A near perfect agreement was reported between microscopy and nested PCR, and between PfHRP2 RDT and nested PCR with Cohen's kappa (k) values of 0.94 and 0.88, respectively. This study revealed that PfHRP2 RDT and microscopy continues to remain sensitive and specific for falciparum malaria diagnosis in the study area.

Real-Time Infrared Spectroscopic Measurement of Natural Moisturizing Factor

Real-Time Infrared Spectroscopic Measurement of Natural Moisturizing Factor

Risk factors for acute kidney injury at presentation among children with CNS malaria: a case control study

BackgroundRecent research has established that acute kidney injury (AKI) is a common problem in severe paediatric malaria. Limited access to kidney diagnostic studies in the low resources settings where malaria is common has constrained research on this important problem.MethodsEnrolment data from an ongoing clinical trial of antipyretics in children with central nervous system (CNS) malaria, CNS malaria being malaria with seizures or coma, was used to identify risk factors for AKI at presentation. Children 2–11 years old with CNS malaria underwent screening and enrollment assessments which included demographic and anthropomorphic data, clinical details regarding the acute illness, and laboratory studies including creatinine (Cr), quantitative parasite count (qPC), quantitative histidine rich protein 2 (HRP2), lactate, and bilirubin levels. Children with a screening Cr > 106 µmol/l were excluded from the study due to the potential nephrotoxic effects of the study drug. To identify risk factors for AKI at the time of admission, children who were enrolled in the study were categorized as having AKI using estimates of their baseline (i.e. before this acute illness) kidney function and creatinine at enrollment applying the Kidney Disease: Improving Global Outcome (KDIGO) 2012 guidelines. Logistic regressions and a multivariate model were used to identify clinical and demographic risk factors for AKI at presentation among those children enrolled in the study.Results465 children were screened, 377 were age-appropriate with CNS malaria, 22 (5.8%) were excluded due to Cr > 106 µmol/l, and 209 were enrolled. Among the 209, AKI using KDIGO criteria was observed in 134 (64.1%). One child required dialysis during recovery. Risk factors for AKI in both the logistic regression and multivariate models included: hyperpyrexia (OR 3.36; 95% CI 1.39–8.12) and age with older children being less likely to have AKI (OR 0.72; 95% CI 0.62–0.84).ConclusionAKI is extremely common among children presenting with CNS malaria. Hyperpyrexia with associated dehydration may contribute to the AKI or may simply be a marker for a more inflammatory systemic response that is also affecting the kidney. Appropriate fluid management in children with CNS malaria and AKI may be challenging since generous hydration to support kidney recovery could worsen malaria-induced cerebral oedema in this critically ill population.Trial registrationhttps://clinicaltrials.gov/ct2/show/NCT03399318

Proteomic Analysis of Urine from Patients with Plasmodium vivax Malaria Unravels a Unique Plasmodium vivax Protein That Is Absent from Plasmodium falciparum.

Five species of Plasmodium cause malaria in humans and two of them, P. vivax and P. falciparum, pose the greatest threat. Rapid antigen detection tests (RADT) have been used for many years to diagnose and distinguish malaria caused by these two parasites. P. falciparum malaria can single-handedly be diagnosed using an RADT, which detects the unique P. falciparum specific histidine-rich protein 2 (HRP2). Unfortunately, there is no RADT that can single-handedly diagnose P. vivax malaria because no specific marker of this parasite has yet been described. Here, we report the discovery of a unique P. vivax protein (Vir14, NCBI Reference Sequence: XP_001612449.1) that has no sequence similarity with proteins of P. falciparum and no significant similarities with proteins of other species of Plasmodium. We propose that this protein could be an outstanding candidate molecule for the development of a promising RADT that can single-handedly and specifically diagnose P. vivax malaria.

Genetic deletions and high diversity of Plasmodium falciparum histidine-rich proteins 2 and 3 genes in parasite populations in Ghana.

Rapid diagnostic tests (RDTs) are used to diagnose malaria in Ghana and other malaria endemic countries. Plasmodium falciparum histidine-rich protein 2 (PFHRP2) based RDTs are widely used, however the occurrence of deletions of the pfhrp2 gene in some parasites have resulted in false negative test results. Monoclonal antibodies of PFHRP2 cross reacts with PFHRP3 because they share structural similarities and this complements the detection of the parasites by RDT. These two genes were investigated in Ghanaian P. falciparum parasite population to detect deletions and the polymorphisms in exon 2 of the pfhrp2 and pfhrp3 genes. Parasite isolates (2,540) from children ≤ 12 years with uncomplicated malaria from 2015 to 2020 transmission seasons were used. Both genes were amplified using nested PCR and negative results indicated the presence of the deletion of genes. Amplified genes were sequenced for the detection of the amino acid repeats. Deletions were observed in 30.7% (780/2,540) and 17.2% (438/2,540) of the samples for pfhrp2 and pfhrp3 respectively with increasing trends over the three time periods (χ2 -10.305, p = 0.001). A total of 1,632 amplicons were sequenced for each gene, analysis was done on 1,124 and 1,307 good quality sequences for pfhrp2 and pfhrp3 respectively. Pfhrp2 repeat polymorphisms were dominantly of types 2 (AHHAHHAAD) and 7 (AHHAAD) with large numbers of variants. A novel variant of type 14 (AHHANHATD) was seen for pfhrp2. For the pfhrp3 repeat types, 16 (AHHAAN), 17 (AHHDG) and 18 (AHHDD) were the dominant types observed. Variants of type 16 (AHHAAH) and (AHHASH) were also dominant. Repeat types 1, 2, 3, 4, 5, 6, 7, 8, 11, 13, 15, 16, and 19 were observed be shared by both genes. The haplotype diversity of both genes ranged between 0.872 and 1 indicating high diversity of the polymorphisms in the isolates. The implication of the findings of the frequencies of the pfhrp2 and pfhrp3 deletions as well as the variants of the main epitopes of the monoclonal antibodies for the RDT (types 2 and 7) in our isolates is an indication of decreased sensitivity of the RDTs in diagnosing malaria infections in Ghana.

Malaria Diagnosis Using Paper-Based Immunoassay for Clinical Blood Sampling and Analysis by a Miniature Mass Spectrometer.

In this work, we have developed a paper-based microfluidic device capable of remote biofluid collection followed by an analysis of the dried clinical samples using a miniature mass spectrometer. We have evaluated a portable mass spectrometer as a possible surveillance platform by analyzing the clinical malaria samples (whole blood) collected from Ghana. We synthesized pH-sensitive ionic probes and coupled them with monoclonal antibodies specific to the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) malaria antigen. We then used the antibody-ionic probe conjugates in a paper-based immunoassay to capture PfHRP2 antigen from untreated whole blood. After the immunoassay, the bound ionic probes were cleaved, and the released mass tags were analyzed through an on-chip paper spray mass spectrometry strategy. During process optimization, we determined the detection limit for PfHRP2 in untreated human serum to be 0.216 nmol/L when using the miniature mass spectrometer. This sensitivity is comparable to the World Health Organization's suggested threshold of 0.227 nmol/L for PfHRP2, proving that our method will be applicable to diagnose symptomatic malaria infection (≥200 parasites per μL blood). The paper device can be stored at room temperature for at least 25 days without affecting the clinical outcome, with each stored paper chip offering good repeatability and reproducibility (RSD = 4-12%). The stability and sensitivity of the developed paper-based immunoassay platform will allow miniature mass spectrometers to be used for point-of-care malaria detection as well as in large-scale surveillance screening to aid eradication programs.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions and Relatedness to Other Global Isolates, Djibouti, 2019-2020.

Deletions of pfhrp2 and paralogue pfhrp3 (pfhrp2/3) genes threaten Plasmodium falciparum diagnosis by rapid diagnostic test. We examined 1,002 samples from suspected malaria patients in Djibouti City, Djibouti, to investigate pfhrp2/3 deletions. We performed assays for Plasmodium antigen carriage, pfhrp2/3 genotyping, and sequencing for 7 neutral microsatellites to assess relatedness. By PCR assay, 311 (31.0%) samples tested positive for P. falciparum infection, and 296 (95.2%) were successfully genotyped; 37 (12.5%) samples were pfhrp2+/pfhrp3+, 51 (17.2%) were pfhrp2+/pfhrp3–, 5 (1.7%) were pfhrp2–/pfhrp3+, and 203 (68.6%) were pfhrp2–/pfhrp3–. Histidine-rich protein 2/3 antigen concentrations were reduced with corresponding gene deletions. Djibouti P. falciparum is closely related to Ethiopia and Eritrea parasites (pairwise GST 0.68 [Ethiopia] and 0.77 [Eritrea]). P. falciparum with deletions in pfhrp2/3 genes were highly prevalent in Djibouti City in 2019–2020; they appear to have arisen de novo within the Horn of Africa and have not been imported.

Factors Affecting the Performance of HRP2-Based Malaria Rapid Diagnostic Tests.

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.

Application of egg yolk IgY on carboxylated polypyrrole films for impedimetric detection of PfHRP2 antigen

This paper described an impedimetric immunosensor for detecting Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Antibodies from egg yolk (Ab-PfHRP2, IgY type) were linked covalently to the screen-printed gold electrodes (SPGE) surface modified with a thin film of Poly-pyrrole-pyrrole 3 carboxylic acid (P(Py-Py3COOH) to develop the sensing platform. The fabrication steps were followed by microscopic (scanning electron microscopy), spectroscopic (RAMAN spectroscopy and Energy-dispersive X-ray spectroscopy), and electrochemical (electrochemical impedance spectroscopy (EIS) and cyclic voltammetry) techniques. The determination of Ag-PfHRP2 was performed by EIS, and the BSA(bovine serum albumin)/Ab-PfHRP2(IgY)/P(Py-Py3COOH)/SPGE immunosensor recorded a linear response at 100–1000 ng mL−1 concentration range, with a limit of detection (LOD) of 27.47 ng mL−1. Its performance was confirmed by Enzyme-Linked Immuno-Sorbent Assay. The fabricated device uses a simple strategy of IgY immobilization, showing high sensitivity and good selectivity, and can be considered an alternative for carrying out malaria tests.

Comparative Assessment of the Sensitivity of Ten Commercial Rapid Diagnostic Test Kits for the Detection of Plasmodium.

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.

Detection of Malaria Parasite Protein in Urine of Patients with Acute Uncomplicated Malaria Using Rapid Diagnostic Test Kits

Objectives: The invasive nature of the current malaria diagnostic techniques impairs compliance to diagnosis, especially for on-field detection. Adapting non-invasive methods of biological sample collection for rapid diagnosis of malaria infections may provide a more efficient approach to case management and epidemiological studies of malaria. This study was designed to evaluate the detection of Plasmodium falciparum Histidine-rich Protein II (PfHRP-2) in urine samples and optimization as diagnostic markers for P. falciparum infection. 
 Methods: One hundred (100) microscopically confirmed patients with Plasmodium falciparum infection and 25 P. falciparum negative controls were recruited for the study. Blood samples of all participants were tested for the presence of PfHRP-2 using Rapid Diagnostic Test (RDT) kits. In addition, urine samples of the confirmed malaria-infected patients were analyzed for PfHRP-2 using the CareStartTM and Global Devices (USA) Malaria kits. The diagnostic performances of the RDT kits were evaluated.
 Results: Overall, the two brands of malaria rapid diagnostics demonstrated 71% sensitivity (95%CI=62.1-79.9%) and 96% specificity (95%CI=88.3-103.7%) for PfHRP-2 detection in urine. 
 The sensitivities of the tests in urine at asexual parasitemia ≤ 2000 μL-1 and asexual parasitemia > 2000 μL-1 were 69.6% (95%CI=56.3-82.9%) and 72.2% (95%CI=60.3-84.2%) respectively. Global Devices and CareStartTM kits had individual sensitivities of 80% (95%CI= 65.7-94.3%) and 67.1% (95%CI= 56.1-78.1%) respectively for PfHRP-2 detection in urine (P= 0.072).
 Conclusion: Findings revealed that urine-based RDTs have limited capacities for malaria diagnosis due to their low sensitivity and require more optimizations to meet required diagnostic standards. J Microbiol Infect Dis 2022; 12(3):97-107.

Genetic Sequence Variation in the Plasmodium falciparum Histidine-Rich Protein 2 Gene from Field Isolates in Tanzania: Impact on Malaria Rapid Diagnosis.

Malaria rapid diagnosis test (RDT) is crucial for managing the disease, and the effectiveness of detection depends on parameters such as sensitivity and specificity of the RDT. Several factors can affect the performance of RDT. In this study, we focused on the pfhrp2 sequence variation and its impact on RDTs targeted by antigens encoded by Plasmodium falciparum histidine-rich protein 2 (pfhrp2). Field samples collected during cross-sectional surveys in Tanzania were sequenced to investigate the pfhrp2 sequence diversity and evaluate the impact on HRP2-based RDT performance. We observed significant mean differences in amino acid repeats between current and previous studies. Several new amino acid repeats were found to occur at different frequencies, including types AAY, AHHAHHAAN, and AHHAA. Based on the abundance of types 2 and 7 amino acid repeats, the binary predictive model was able to predict RDT insensitivity by about 69% in the study area. About 85% of the major epitopes targeted by monoclonal antibodies (MAbs) in RDT were identified. Our study suggested that the extensive sequence variation in pfhrp2 can contribute to reduced RDT sensitivity. The correlation between the different combinations of amino acid repeats and the performance of RDT in different malaria transmission settings should be investigated further.

Evaluation of HRP2 and pLDH-based rapid diagnostic tests for malaria and prevalence of pfhrp2/3 deletions in Aweil, South Sudan

BackgroundRapid diagnostic tests (RDT) for malaria are the primary tool for malaria diagnosis in sub-Saharan Africa but the utility of the most commonly used histidine-rich protein 2 (HRP2) antigen-based tests is limited in high transmission settings due to the long duration of positivity after successful malaria treatment. HRP2 tests are also threatened by the emergence of Plasmodium that do not carry pfhrp2 or pfhrp 3 genes. Plasmodium lactate dehydrogenase (pLDH)-based tests are promising alternatives, but less available. This study assessed the performances of HRP2 and pLDH(pan) tests under field conditions.MethodsThe study performed a prospective facility-based diagnostic evaluation of two malaria RDTs in Aweil, South Sudan, during the high transmission season. Capillary blood by fingerprick was collected from 800 children under 15 years of age with fever and no signs of severity. SD Bioline HRP2 and CareStart pLDH(pan) RDTs were performed in parallel, thick and thin smears for microscopy were examined, and dried blood was used for PCR testing.ResultsUsing microscopy as the gold standard, the sensitivity of both tests was estimated at > 99%, but the specificity of each was lower: 55.0% for the pLDH test and 61.7% for the HRP2 test. When using PCR as the gold standard, the sensitivity of both tests was lower than the values assessed using microscopy (97.0% for pLDH and 96.5% for HRP2), but the specificity increased (65.1% for pLDH and 72.9% for HRP2). Performance was similar across different production lots, sex, and age. Specificity of both the pLDH and HRP2 tests was significantly lower in children who reported taking a therapeutic course of anti-malarials in the 2 months prior to enrollment. The prevalence of pfhrp2/3 deletions in the study population was 0.6%.ConclusionsThe low specificity of the pLDH RDT in this setting confirms previous results and suggests a problem with this specific test. The prevalence of pfhrp2/3 deletions in the study area warrants continued monitoring and underscores the relevance of assessing deletion prevalence nationally. Improved malaria RDTs for high-transmission environments are needed.

A comparison of peripheral blood smear microscopy and detection of histidine-rich protein 2 in blood in the diagnosis of malaria in pregnancy.

Detection of malaria in pregnancy poses a huge challenge in Sub-Saharan Africa. Accurate diagnosis enables timely and appropriate clinical management. This study aimed to compare the accuracy of peripheral venous blood smear microscopy (PVBSM) and a rapid diagnostic test detecting histidine-rich protein 2 in the blood (RDT-HRP2) with placental histology as the control in the diagnosis of malaria in pregnancy in Nigerian women. This was a comparative, cross-sectional study conducted between January and July 2017. Asymptomatic pregnant women who presented to the labor ward and who gave informed consent had peripheral venous blood samples as well as placental tissue obtained following delivery. The blood samples obtained were tested for malaria parasites using PVBSM and RDT-HRP2 in blood, while the placenta was subjected to histology. A total of 326 patients participated in the study. The prevalence of malaria in pregnancy was 13.8%, 17.8%, and 32.8% using PVBSM, RDT-HRP2, and placental histology. The sensitivity, specificity, and accuracy of PVBSM were 40.2%, 99.1%, and 79.8% and the corresponding values for RDT-HRP2 were 49.5%, 97.7%, and 81.9%. The positive predictive value was 95.6% for PVBSM and 91.4% for RDT-HRP2 and the negative predictive value was 77.2% for PVBSM and 79.9% for RDT- HRP2. The diagnostic performances of both tests were comparable; however, RDT-HRP2 had a higher sensitivity and accuracy than PVBSM for the diagnosis of malaria in pregnancy.

Phase 3 Evaluation of an Innovative Simple Molecular Test for the Diagnosis of Malaria and Follow-Up of Treatment Efficacy in Pregnant Women in Sub-Saharan Africa (Preg-Diagmal).

The malaria parasite Plasmodium falciparum (Pf) can sequester in the placenta resulting in low density of peripheral parasitemia and consequently in false negative malaria diagnosis (by microscopy) in pregnant women. Moreover, the use of rapid diagnostic tests (RDTs) in diagnostic strategies, including those for the detection of a malaria infection during pregnancy, is constrained by either persistent malaria antigen (histidine-rich protein 2; HRP2) after successful treatment, leading to false positive test results, or by false negative results as previously mentioned due to parasite sequestration (which is further exacerbated due to the low limited of detection [LoD] of conventional RDTs) or to HRP2 deletion. Recently, a direct blood polymerase chain reaction combined with a nucleic acid lateral flow immunoassay (dbPCR-NALFIA) has been developed, which circumvents these challenges and has demonstrated its diagnostic potential in phase 1 and 2 studies. The PREG-DIAGMAL trial presented in this manuscript will assess the diagnostic performance of dbPCR-NALFIA for the diagnostic of malaria in pregnant women and its potential to monitor treatment efficacy in these subjects. The work is ancillary embedded in an ongoing EDCTP funded trial, the PyraPreg project (PACTR202011812241529) in which the safety and efficacy of a newly registered Artemisinin-Based Combination (Pyronaridine-Artesunate) is being evaluated in pregnant women. This is a Phase 3 diagnostic evaluation conducted in 2 African countries: Democratic Republic of the Congo (DRC) and Burkina Faso. Pregnant women fulfilling the inclusion criteria of the PyraPreg study will be also invited to participate in the PREG-DIAGMAL study. Diagnostic accuracy will be assessed following the WHO/TDR guidelines for the evaluation of diagnostics and reported according to STARD principles. Due to the lack of a 100% specific and sensitive standard diagnostic test for malaria, the sensitivity and specificity of the new test will be compared to the available diagnostic practice in place at the selected settings (microscopy and/or RDT) and to quantitative PCR as the reference test. This phase 3 diagnostic study is designed towards the evaluation of the performance of a new diagnostic tool for the screening of malaria and the monitoring of treatment in pregnant women under real conditions life. If successful, the dbPCR-NALFIA could be a valuable tool to add to the diagnostic arsenal for malaria, in particular during pregnancy. Trial registration: Pan African Clinical Trial Registry database (PACTR202203780981413). Registered on 17 March 2022.

Phase 3 evaluation of an innovative simple molecular test for the diagnosis of malaria in different endemic and health settings in sub-Saharan Africa (DIAGMAL).

Rapid Diagnostic Tests (RDTs) have become the cornerstone for the management of malaria in many endemic settings, but their use is constrained for several reasons: (i) persistent malaria antigen (histidine-rich protein 2; HRP2) leading to false positive test results; (ii) hrp2 deletions leading to false negative PfHRP2 results; and (iii) limited sensitivity with a detection threshold of around 100 parasites/μl blood (pLDH- and HRP2-based) leading to false negative tests. Microscopy is still the gold standard for malaria diagnosis, and allows for species determination and quantitation, but requires trained microscopists, maintained microscopes and has detection limit issues. Consequently, there is a pressing need to develop and evaluate more sensitive and accurate diagnostic tests. To address this need we have developed a direct on blood mini PCR-NALFIA test that combines the benefits of molecular biology with low infrastructural requirements and extensive training. This is a Phase 3 diagnostic evaluation in 5 African countries. Study sites (Sudan, Ethiopia, Burkina, Kenya and Namibia) were selected to ensure wide geographical coverage of Africa and to address various malaria epidemiological contexts ranging from high transmission to near elimination settings with different clinical scenarios and diagnostic challenges. Study participants will be enrolled at the study health facilities after obtaining written informed consent. Diagnostic accuracy will be assessed following the WHO/TDR guidelines for the evaluation of diagnostics and reported according to STARD principles. Due to the lack of a 100% specific and sensitive standard diagnostic test for malaria, the sensitivity and specificity of the new test will be compared to the available diagnostic practices in place at the selected sites and to quantitative PCR as the reference test. This phase 3 study is designed to validate the clinical performance and feasibility of implementing a new diagnostic tool for the detection of malaria in real clinical settings. If successful, the proposed technology will improve the diagnosis of malaria. Enrolment started in November 2022 (Kenya) with assessment of long term outcome to be completed by 2023 at all recruitment sites. Pan African Clinical Trial Registry (www.pactr.org) PACTR202202766889963 on 01/02/2022 and ISCRTN (www.isrctn.com/) ISRCTN13334317 on 22/02/2022.

Molecular Dynamics Investigation into pH Dependent Metal Binding of the Intrinsically Disordered Worm Jaw Protein, Nvjp-1.

Sclerotization of the Nereis virens jaw is mediated by metal binding to the histidine-rich jaw protein, Nvjp-1. Previous studies showed that the mechanical properties of Nvjp-1 hydrogels could be modulated with zinc binding as well as the associated anion. Here, we show that the mechanical properties of Nvjp-1 hydrogels can be modulated by pH and that zinc binding to Nvjp-1 is stable at both acidic and alkaline pH conditions. To probe the mechanism of Zn2+ binding to Nvjp-1 at different pH conditions, we utilized all atom molecular dynamics simulations employing a polarizable force field. At low pH conditions, polar residues predominantly interacted with Zn2+, with at most two residues interacting with a given zinc ion. Surprisingly, little to no Zn2+ binding was observed with the abundant Nvjp-1 acidic residues, which form salt-bridges with the protonated histidines to effectively block their binding to Zn2+ ions. As the pH was shifted to alkaline conditions, Zn2+ binding residues reconfigured to form additional coordination bonds with histidine, resulting in a reduction in the radius of gyration that correlated with hydrogel sclerotization. Furthermore, acetate ions were shown to facilitate the capture of zinc ions through association with protonated histidines at low pH, freeing acidic residues to interact with Zn2+ ions and increasing the number of Zn2+ ions that diffuse into the Nvjp-1 interior. Thus, these studies provide valuable molecular insights into how amino acid residues in Nvjp-1 manage metal salt binding and coordination in hydrogels as a function of the pH and ionic environments.

Expansion of nickel binding- and histidine-rich proteins during gastric adaptation of Helicobacter species.

Acquisition and homeostasis of essential metals during host colonization by bacterial pathogens rely on metal uptake, trafficking, and storage proteins. How these factors have evolved within bacterial pathogens is poorly defined. Urease, a nickel enzyme, is essential for Helicobacter pylori to colonize the acidic stomach. Our previous data suggest that acquisition of nickel transporters and a histidine-rich protein (HRP) involved in nickel storage in H. pylori and gastric Helicobacter spp. have been essential evolutionary events for gastric colonization. Using bioinformatics, proteomics, and phylogenetics, we extended this analysis to determine how evolution has framed the repertoire of HRPs among 39 Epsilonproteobacteria; 18 gastric and 11 non-gastric enterohepatic (EH) Helicobacter spp., as well as 10 other Epsilonproteobacteria. We identified a total of 213 HRPs distributed in 22 protein families named orthologous groups (OGs) with His-rich domains, including 15 newly described OGs. Gastric Helicobacter spp. are enriched in HRPs (7.7±1.9HRPs/strain) as compared to EH Helicobacter spp. (1.9±1.0HRPs/strain) with a particular prevalence of HRPs with C-terminal histidine-rich domains in gastric species. The expression and nickel-binding capacity of several HRPs was validated in five gastric Helicobacter spp. We established the evolutionary history of new HRP families, such as the periplasmic HP0721-like proteins and the HugZ-type heme oxygenases. The expansion of histidine-rich extensions in gastric Helicobacter spp. proteins is intriguing but can tentatively be associated with the presence of the urease nickel enzyme. We conclude that this HRP expansion is associated with unique properties of organisms that rely on large intracellular nickel amounts for their survival.

Neurotransmitters and molecular chaperones interactions in cerebral malaria: Is there a missing link?

Plasmodium falciparum is responsible for the most severe and deadliest human malaria infection. The most serious complication of this infection is cerebral malaria. Among the proposed hypotheses that seek to explain the manifestation of the neurological syndrome in cerebral malaria is the vascular occlusion/sequestration/mechanic hypothesis, the cytokine storm or inflammatory theory, or a combination of both. Unfortunately, despite the increasing volume of scientific information on cerebral malaria, our understanding of its pathophysiologic mechanism(s) is still very limited. In a bid to maintain its survival and development, P. falciparum exports a large number of proteins into the cytosol of the infected host red blood cell. Prominent among these are the P. falciparum erythrocytes membrane protein 1 (PfEMP1), P. falciparum histidine-rich protein II (PfHRP2), and P. falciparum heat shock proteins 70-x (PfHsp70-x). Functional activities and interaction of these proteins with one another and with recruited host resident proteins are critical factors in the pathology of malaria in general and cerebral malaria in particular. Furthermore, several neurological impairments, including cognitive, behavioral, and motor dysfunctions, are known to be associated with cerebral malaria. Also, the available evidence has implicated glutamate and glutamatergic pathways, coupled with a resultant alteration in serotonin, dopamine, norepinephrine, and histamine production. While seeking to improve our understanding of the pathophysiology of cerebral malaria, this article seeks to explore the possible links between host/parasite chaperones, and neurotransmitters, in relation to other molecular players in the pathology of cerebral malaria, to explore such links in antimalarial drug discovery.