A countrywide survey of hrp2/3 deletions and kelch13 mutation co-occurrence in Ethiopia.

Malaria elimination relies on detection of Plasmodium falciparum Histidine-Rich Proteins 2/3 (HRP2/3) through rapid diagnostic tests (RDTs) and treatment with artemisinin-combination therapies (ACTs). Data from the Horn of Africa suggest increasing hrp2/3 gene deletions and ACT partial resistance kelch13 (k13) mutations. To assess this, 233 samples collected during a national survey from 7 regions of Ethiopia were studied for hrp2/3 deletions by droplet digital dPCR and k13 mutations by DNA sequencing. Approximately 22% of the study population harbored complete hrp2/3 deletions by ddPCR. Thirty-two of 42 of k13 SNPs identified were R622I associated with ACT partial resistance. Both hrp2/3 deletions and k13 mutations associated with ACT partial resistance appear to be co-occurring especially in Northwest Ethiopia. Ongoing national surveillance relying on accurate laboratory methods are required to fully elaborate the genetic diversity of P. falciparum to inform public health policy makers.

Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2-based malaria RDTs in Northern Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools. METHODS: A cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA. RESULTS: Out of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections. CONCLUSION: These findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

BACKGROUND: False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. METHODS: This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. RESULTS: A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. CONCLUSION: Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions in Malaria-Hyperendemic Region, South Sudan.

Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Plasmodium falciparum histidine-rich protein 2 and 3 genes deletion in global settings (2010-2021): a systematic review and meta-analysis.

BACKGROUND: The usefulness of histidine-rich protein-2/3 (HRP2/3)-based rapid diagnostic tests of malaria due to Plasmodium falciparum has been threatened by the appearance of mutant PfHRP2/3 genes. This study was undertaken to determine the global pooled estimates of PfHRP2/3gene deletions. METHODS: Relevant publications were identified from electronic databases such as; PubMed, EMBASE, and MEDLINE online. Besides, all the relevant literatures were retrieved through Google and Google Scholar. STATA software was used for data analysis. The pooled estimates were calculated using random effect model. The summary estimates were presented using forest plots and tables. RESULTS: A total of 27 studies were included in the systematic review. However, only 24 and 17 studies were included for PfHRP2 and 3 gene deletion meta-analysis, respectively. The prevalence of PfHRP2 gene deletion across the individual studies ranged from the highest 100% to the lowest 0%. However, the meta-analysis result showed that the global pooled prevalence of PfHRP2 and PfHRP3 gene deletions were 21.30% and 34.50%, respectively. The pooled proportion of PfHRP2 gene deletion among false negative PfHRP2-based RDTs results was found to be 41.10%. The gene deletion status was higher in South America and followed by Africa. The pooled estimate of PfHRP2 gene deletion among studies, which did not follow the WHO PfHRP2/3 gene deletion analysis protocol was higher than their counter parts (21.3% vs 10.5%). CONCLUSIONS: This review showed that there is a high pooled prevalence of PfHRP2/3 gene deletions in Plasmodium falciparum confirmed isolates and also a high proportion of their deletions among false-negative malaria cases using PfHRP2-based RDT results. Hence, malaria diagnosis based on PfHRP2-based rapid tests seems to be less sensitive and warrants further evaluation of PfHRP2/3 gene deletions.

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

BACKGROUND: Rapid 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. METHODS: The 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. RESULTS: Using 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%. CONCLUSIONS: The 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.

Performance of antigen detection for HRP2-based malaria rapid diagnostic tests in community surveys: Tanzania, July-November 2017.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance. METHODS: A community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose-response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD). RESULTS: From the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT (4.3 ng/mL; 95% CI 3.4-5.4) when compared to pLDH/HRP2 dual target tests (5.4 ng/mL; 4.5-6.3), though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL. CONCLUSIONS: Both HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection.

Screening strategies and laboratory assays to support Plasmodium falciparum histidine-rich protein deletion surveillance: where we are and what is needed.

Rapid diagnostic tests (RDTs) detecting Plasmodium falciparum histidine-rich protein 2 (HRP2) have been an important tool for malaria diagnosis, especially in resource-limited settings lacking quality microscopy. Plasmodium falciparum parasites with deletion of the pfhrp2 gene encoding this antigen have now been identified in dozens of countries across Asia, Africa, and South America, with new reports revealing a high prevalence of deletions in some selected regions. To determine whether HRP2-based RDTs are appropriate for continued use in a locality, focused surveys and/or surveillance activities of the endemic P. falciparum population are needed. Various survey and laboratory methods have been used to determine parasite HRP2 phenotype and pfhrp2 genotype, and the data collected by these different methods need to be interpreted in the appropriate context of survey and assay utilized. Expression of the HRP2 antigen can be evaluated using point-of-care RDTs or laboratory-based immunoassays, but confirmation of a deletion (or mutation) of pfhrp2 requires more intensive laboratory molecular assays, and new tools and strategies for rigorous but practical data collection are particularly needed for large surveys. Because malaria diagnostic strategies are typically developed at the national level, nationally representative surveys and/or surveillance that encompass broad geographical areas and large populations may be required. Here is discussed contemporary assays for the phenotypic and genotypic evaluation of P. falciparum HRP2 status, consider their strengths and weaknesses, and highlight key concepts relevant to timely and resource-conscious workflows required for efficient diagnostic policy decision making.

Surveillance of pfhrp2 and pfhrp3 gene deletions among symptomatic Plasmodium falciparum malaria patients in Central Vietnam.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) remain the main point-of-care tests for diagnosis of symptomatic Plasmodium falciparum malaria in endemic areas. However, parasites with gene deletions in the most common RDT target, histidine rich protein 2 (pfhrp2/HRP2), can produce false-negative RDT results leading to inadequate case management. The objective of this study was to determine the prevalence of hrp2/3 deletions causing false-negative RDT results in Vietnam (Gia Lai and Dak Lak provinces). METHODS: Individuals presenting with malaria symptoms at health facilities were screened for P. falciparum infection using light microscopy and HRP2-RDT (SD Bioline Malaria Antigen Pf/Pv RDT, Abbott). Microscopically confirmed P. falciparum infections were analysed for parasite species by 18S rRNA qPCR, and pfhrp2 and pfhrp3 exon2 deletions were investigated by nested PCR. pfhrp2 amplicons were sequenced by the Sanger method and HRP2 plasma levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The prevalence of false-negative RDT results among symptomatic cases was 5.6% (15/270). No pfhrp2 and pfhrp3 deletions were identified. False-negative RDT results were associated with lower parasite density (p = 0.005) and lower HRP2 plasma concentrations (p < 0.001), as compared to positive RDT. CONCLUSIONS: The absence of hrp2/3 deletions detected in this survey suggests that HRP2-based malaria RDTs remain effective for the diagnosis of symptomatic P. falciparum malaria in Central Vietnam.

The in-vivo dynamics of Plasmodium falciparum HRP2: implications for the use of rapid diagnostic tests in malaria elimination.

BACKGROUND: Rapid diagnostic tests (RDTs) that rely on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) have become key tools for diagnosing P. falciparum infection. The utility of RDTs can be limited by PfHRP2 persistence, however it can be a potential benefit in low transmission settings where detection of persistent PfHRP2 using newer ultra-sensitive PfHRP2 based RDTs can serve as a surveillance tool to identify recent exposure. Better understanding of the dynamics of PfHRP2 over the course of a malaria infection can inform optimal use of RDTs. METHODS: A previously published mathematical model was refined to mimic the production and decay of PfHRP2 during a malaria infection. Data from 15 individuals from volunteer infection studies were used to update the original model and estimate key model parameters. The refined model was applied to a cohort of patients from Namibia who received treatment for clinical malaria infection for whom longitudinal PfHRP2 concentrations were measured. RESULTS: The refinement of the PfHRP2 dynamic model indicated that in malaria naïve hosts, P. falciparum parasites of the 3D7 strain produce 33.6 × 10-15 g (95% CI 25.0-42.1 × 10-15 g) of PfHRP2 in vivo per parasite replication cycle, with an elimination half-life of 1.67 days (95% CI 1.11-3.40 days). The refined model included these updated parameters and incorporated individualized body fluid volume calculations, which improved predictive accuracy when compared to the original model. The performance of the model in predicting clearance of PfHRP2 post treatment in clinical samples from six adults with P. falciparum infection in Namibia improved when using a longer elimination half-life of 4.5 days, with 14% to 67% of observations for each individual within the predicted range. CONCLUSIONS: The updated mathematical model can predict the growth and clearance of PfHRP2 during the production and decay of a mono-infection with P. falciparum, increasing the understanding of PfHRP2 antigen dynamics. This model can guide the optimal use of PfHRP2-based RDTs for reliable diagnosis of P. falciparum infection and re-infection in endemic settings, but also for malaria surveillance and elimination programmes in low transmission areas.

In vitro transcytosis of Helicobacter pylori histidine-rich protein through gastric epithelial-like cells and the blood-brain barrier.

Recent epidemiological studies have supported the correlation between Helicobacter pylori infection and the development of Alzheimer's disease. HpHpn, a histidine-rich H. pylori protein, forms amyloid-like oligomers; it may be a pathogenic factor for Alzheimer's disease progression. HpHpn may also be transported from the gastric epithelium to the brain. However, HpHpn is secreted from H. pylori on the outer surface of gastric epithelia; therefore, the hypothesized movement of HpHpn across the gastric epithelium to the blood remains controversial. Here, we found the HpHpn showed acidic pH-dependent cellular uptake and subsequent secretion in human gastric epithelial-like carcinoma cells. Furthermore, HpHpn exhibited in vitro permeability across the blood-brain barrier. Although further in vivo experiments are required, our findings suggest that in vitro transcytosis of HpHpn in gastric epithelial cells and the blood-brain barrier may provide new insights into the correlation between H. pylori infections and Alzheimer's disease progression.

The Discovery and Function of Filaggrin.

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the 'histidine-rich protein'. Steinert and Dale named the protein 'filaggrin' in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, 'profilaggrin,' was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into 'building blocks' that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean's team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.

Malaria hemozoin: A target for point-of-care diagnosis.

The challenges in malaria diagnosis continue to threaten the malaria elimination goal in India and other malaria-endemic countries. A rapid diagnostic test (RDT) kit is widely used in resource-constrained areas where microscopy and molecular methods are not easily deployable. Considering the problems associated with the currently available RDT kit, such as histidine-rich protein 2 gene deletion and prolonged stability of the protein in the blood, it suggests that new potential biomarkers are urgently needed. Hemozoin (Hz) is an important biomarker for malaria diagnosis, which is the by-product of a detoxification mechanism in the malaria parasite. This article highlights the importance of "Hz" for point-of-care malaria diagnosis when India and other countries are moving toward the goal of malaria elimination.

Plasma Plasmodium falciparum Histidine-rich Protein 2 Concentrations in Children With Malaria Infections of Differing Severity in Kilifi, Kenya.

BACKGROUND: Most previous studies support a direct link between total parasite load and the clinical severity of Plasmodium falciparum malaria infections. METHODS: We estimated P. falciparum parasite loads in 3 groups of children with malaria infections of differing severity: (1) children with World Health Organization-defined severe malaria (n = 1544), (2) children admitted with malaria but without features of severity (n = 200), and (3) children in the community with asymptomatic parasitemia (n = 33). RESULTS: Peripheral parasitemias were highest in those with uncomplicated malaria (geometric mean [GM] parasite count, 111 064/µL; 95% confidence interval, CI, 86 798-141 819/µL), almost 3 times higher than in those with severe malaria (39 588/µL; 34 990-44 791/µL) and >100 times higher than in those with asymptomatic malaria (1092/µL; 523-2280/µL). However, the GM P. falciparum histidine-rich protein 2 (PfHRP2) values (95% CI) increased with severity, being 7 (4-12) ng/mL in asymptomatic malaria, 843 (655-1084) ng/mL in uncomplicated malaria, and 1369 (1244-1506) ng/mL in severe malaria. PfHRP2 concentrations were markedly lower in the subgroup of patients with severe malaria and concomitant invasive bacterial infections of blood or cerebrospinal fluid (GM concentration, 312 ng/mL; 95% CI, 175-557 ng/mL; P < .001) than in those without such infections (1439 ng/mL; 1307-1584; P < .001). CONCLUSIONS: The clinical severity of malaria infections related strongly to the total burden of P. falciparum parasites. A quantitative test for plasma concentrations of PfHRP2 could be useful in identifying children at the greatest clinical risk and identifying critically ill children in whom malaria is not the primary cause.

Plasma MicroRNA Profiling of Plasmodium falciparum Biomass and Association with Severity of Malaria Disease.

Severe malaria (SM) is a major public health problem in malaria-endemic countries. Sequestration of Plasmodium falciparum-infected erythrocytes in vital organs and the associated inflammation leads to organ dysfunction. MicroRNAs (miRNAs), which are rapidly released from damaged tissues into the host fluids, constitute a promising biomarker for the prognosis of SM. We applied next-generation sequencing to evaluate the differential expression of miRNAs in SM and in uncomplicated malaria (UM) in children in Mozambique. Six miRNAs were associated with in vitro P. falciparum cytoadhesion, severity in children, and P. falciparum biomass. Relative expression of hsa-miR-4497 quantified by TaqMan-quantitative reverse transcription PCR was higher in plasma of children with SM than those with UM (p<0.048) and again correlated with P. falciparum biomass (p = 0.033). These findings suggest that different physiopathological processes in SM and UM lead to differential expression of miRNAs and suggest a pathway for assessing their prognostic value malaria.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.

BACKGROUND: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. METHODS: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. RESULTS: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. CONCLUSIONS: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

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

BACKGROUND: Malaria 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. METHODS: Blood 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. RESULTS: One-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. CONCLUSION: Length 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.

Plasmodium falciparum histidine-rich protein 2 diversity in Ghana.

BACKGROUND: In the absence of microscopy, Plasmodium falciparum histidine-rich proteins 2 (PfHRP2)-based rapid diagnostic tests (RDTs) are recommended for the diagnosis of falciparum malaria, particularly in endemic regions. However, genetic variability of the pfhrp2 gene threatens the usefulness of the test due to its impact on RDT sensitivity. This study aimed to investigate the diversity of pfhrp2 in malaria cases among children in Ghana. METHODS: A cross-sectional study was conducted at the Adidome Government Hospital in the Volta Region of Ghana. A total of 50 children with mean age of 6.6 ± 3.5 years and diagnosed falciparum malaria were included. Blood samples were collected for complete blood count, malaria parasite identification and counting using auto analyzer and microscopy, respectively. DNA was isolated from blood-spotted Whatman filters, amplified and sequenced. Nucleotide sequences were translated in silico to corresponding amino acids and the deduced amino acids sequences were analyzed for diversity using Mega X. RESULTS: The number of repeats and number of each repeat within PfHRP2 varied between isolates. Twelve rare PfHRP2 repeat types, two of which are previously unreported, were identified in this study. The HRP2 sequence obtained in this study shared high similarities with isolates from Kenya. Using Baker's regression model, Group B was the highest occurring type (58.0%). Screening of all sequences for epitopes recognized by PfHRP2-specific monoclonal antibodies (mAbs), the predominant motif was AHHAADAHH, which is recognized by the C1-13 mAbs. CONCLUSION: This study reports diversity of P. falciparum HRP2 in samples from Ghanaian children with symptomatic malaria. The findings of this study highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability.

Community-based surveys for Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in selected regions of mainland Tanzania.

BACKGROUND: Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania. METHODS: A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes. RESULTS: Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes. CONCLUSIONS: This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.