Vivax Lactate Dehydrogenase Research Articles

Characterisation of Plasmodium vivax lactate dehydrogenase dynamics in P. vivax infections

Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of P. vivax. It is widely used as a diagnostic biomarker and a measure of total-body parasite biomass in vivax malaria. However, the dynamics of PvLDH remains poorly understood. Here, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated key biological parameters characterising in vivo PvLDH dynamics based on longitudinal data of parasitemia and PvLDH concentration collected from P. vivax-infected humans, with the estimates informed by the ex vivo data as prior knowledge in a Bayesian hierarchical framework. We found that the in vivo accumulation rate of intraerythrocytic PvLDH peaks at 10–20 h post-invasion (late ring stage) with a median estimate of intraerythrocytic PvLDH mass at the end of the life cycle to be 9.4 × 10−3ng. We also found that the median estimate of in vivo PvLDH half-life was approximately 21.9 h. Our findings provide a foundation with which to advance our quantitative understanding of P. vivax biology and will facilitate the improvement of PvLDH-based diagnostic tools.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Symptomatic Plasmodium vivax Infection in Rwanda.

We report a Plasmodium vivax infection in a Rwandan child misdiagnosed with Plasmodium falciparum and administered artemether-lumefantrine. Antigen detection revealed an absence of P falciparum histidine-rich protein 2 (HRP2) and presence of Plasmodium vivax lactate dehydrogenase. Nested and real-time polymerase chain reactions verified that the sample only contained P vivax deoxyribonucleic acid.

Electrochemical immunosensor for detection of Plasmodium vivax lactate dehydrogenase.

BACKGROUND Malaria is a disease that affects many tropical and subtropical countries, including Brazil. The use of tests for malaria detection is one of the fundamental strategies recommended by the World Health Organization for the control and eradication of the disease. The lack of diagnostic tests leads to an increase in transmission and non-reporting cases.OBJECTIVES This work described an electrochemical immunosensor for detecting Plasmodium vivax lactate dehydrogenase antigen (Ag-PvLDH).METHODS The device has developed by immobilising egg yolk IgY antibodies (Ab-PvLDH) on a gold electrode surface using cysteamine as linker. The immunosensor fabrication was followed by differential pulse voltammetry, and contact angle measurements were performed to characterise the modified gold electrode surface.FINDINGS The results for Ag-PvLDH determination exhibit a linear response at 10-50 µg mL-1 concentration range, with a limit of detection of 455 ng mL-1. The excellent selectivity of the device was confirmed.MAIN CONCLUSIONS The developed immunosensor showed a good performance, therefore, it can be considered an alternative test to detect malaria caused by P. vivax.

Missed Plasmodium falciparum and Plasmodium vivax Mixed Infections in Ethiopia Threaten Malaria Elimination.

ABSTRACT.Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia. This study investigated whether mixed infections were missed by microscopy from a 2017 therapeutic efficacy study at two health facilities in Ethiopia. All patients (N = 304) were initially classified as having single-species P. falciparum (n = 148 samples) or P. vivax infections (n = 156). Dried blood spots were tested for Plasmodium antigens by bead-based multiplex assay for pan-Plasmodium aldolase, pan-Plasmodium lactate dehydrogenase, P. vivax lactate dehydrogenase, and histidine-rich protein 2. Of 304 blood samples, 13 (4.3%) contained both P. falciparum and P. vivax antigens and were analyzed by polymerase chain reaction for species-specific DNA. Of these 13 samples, five were confirmed by polymerase chain reaction for P. falciparum/P. vivax co-infection. One sample, initially classified as P. vivax by microscopy, was found to only have Plasmodium ovale DNA. Plasmodium falciparum/P. vivax mixed infections can be missed by microscopy even in the context of a therapeutic efficacy study with multiple trained readers.

Considerations for quality assurance of multiplex malaria antigen detection assays with large sample sets

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.

Fast Aptamer Generation Method Based on the Electrodynamic Microfluidic Channel and Evaluation of Aptamer Sensor Performance.

We demonstrate for the first time a fast aptamer generation method based on the screen-printed electrodynamic microfluidic channel device, where a specific aptamer selectively binds to a target protein on channel walls, following recovery and separation. A malaria protein as a model target, Plasmodium vivax lactate dehydrogenase (PvLDH) was covalently bonded to the conductive polymer layer formed on the carbon channel walls to react with the DNA library in a fluid. Then, the AC electric field was symmetrically applied on the channel walls for inducing the specific binding of the target protein to DNA library molecules. In this case, the partitioning efficiency between PvLDH and DNA library in the channel was attained to be 1.67 × 107 with the background of 5.56 × 10-6, which was confirmed using the quantitative polymerase chain reaction (qPCR). The selectively captured DNAs were isolated from the protein and separated in situ to give five aptamers with different sequences by one round cycle. The dissociation constants (Kd) of the selected aptamers were determined employing both electrochemical impedance spectroscopy (EIS) and the fluorescence method. The sensing performance of each aptamer was evaluated for the PvLDH detection after individual immobilization on the screen-printed array electrodes. The most sensitive aptamer revealed a detection limit of 7.8 ± 0.4 fM. The sensor reliability was evaluated by comparing it with other malaria sensors.

Evolution of abiotic cubane chemistries in a nucleic acid aptamer allows selective recognition of a malaria biomarker

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.

Capture and Detection of Plasmodium vivax Lactate Dehydrogenase in a Bead-Based Multiplex Immunoassay.

Laboratory detection of malaria antigens has proved valuable for research and epidemiological purposes. We recently developed a bead-based multiplex antigen assay for pan-Plasmodium and Plasmodium falciparum targets. Here, we report integration of a Plasmodium vivax-specific target to this multiplex panel: P. vivax lactate dehydrogenase (PvLDH). Within the multiplex panel, assay signal for purified PvLDH antigen titrated into the single-digit picogram range. Against a panel of polymerase chain reaction (PCR)-confirmed samples from acute P. vivax infections (n = 36), sensitivity was 91.7% in using PvLDH detection for identifying the presence of parasites. Specificity against a panel of persons with no Plasmodium infection (n = 44) was 100%, and specificity against a panel of PCR-confirmed P. falciparum, Plasmodium malariae, or Plasmodium ovale infections (n = 164) was 90.2%. Addition of this PvLDH capture and detection system into the multiplex antigen panel will now allow for sensitive screening for species identification of both P. falciparum and P. vivax in the laboratory.

A novel fluorescence probe of Plasmodium vivax lactate dehydrogenase based on adenosine monophosphate protected bimetallic nanoclusters

Specific detection of Plasmodium vivax lactate dehydrogenase (PvLDH), an important biomarker of malaria, remains a significant challenge. Herein, adenosine monophosphate protected gold-silver bimetallic nanoclusters, Au–AgNCs@AMP were used as a specific and sensitive fluorescence probe to detect PvLDH. After optimizing, a linear response was shown over a wide concentration range (10–100 nM) and an extremely low limit of detection (LOD) at 0.10 nM (3.7 ng mL−1) was achieved finally. Albeit the method was able to detect PvLDH sensitively, it could not discriminate different types of LDHs. Consequently, Al3+ was employed as an “assistant agent”, which induced an assay capacity to discriminate PvLDH from other LDHs. The bimetallic nanoclusters inhibited the activity of PvLDH, suggesting it bound near the active site of PvLDH with high affinity. Zeta potential and UV–vis absorption experiments showed that electrostatic interaction was the main driving force for the interaction between the nanoclusters and PvLDH. Through chemical modification it indicated free thiol groups in PvLDH played an implant role in the interaction. Overall, the fluorescence enhancement and blue-shift were attributed to assembly-induced emission enhancement (AIEE) and hydrophobic transfer. The present study provides a simple, robust, and easy-to-perform approach to detect PvLDH with high sensitivity and selectivity, with significant potential for malaria diagnosis in the developing world.

Development of a film-based immunochromatographic microfluidic device for malaria diagnosis.

In this study, a novel film-based immunochromatographic microfluidic device (IMD) has been developed for malaria diagnosis. A microfluidic channel was patterned on a polyethylene terephthalate (PET) double-sided adhesive film using a plotting cutter and was assembled with a polycarbonate (PC) film. The PC film used for the probe immobilization layer was activated using oxygen plasma treatment to modify the film surface with avidin-biotin linker to immobilize a capture antibody. A fluorescent labeled Pan type mAb conjugate was prepared for signal indicator after undergoing a sandwich enzyme-linked immunosorbent assay (ELISA). Target antigens include Plasmodium falciparum (P. falciparum) lactate dehydrogenase (LDH) and Plasmodium vivax (P. vivax) LDH which were injected into the sample inlet. Target antigens combined with the conjugate and then flowed to the detection chamber where two test dots and a control dot (Ctrl) exist. In the presence of P. falciparum LDH, three detection dots including test dot 1 (T1), test dot 2 (T2) and Ctrl revealed fluorescence signals where P. falciparum mAb, Pan type pLDH mAb and goat anti-mouse IgG were immobilized, respectively. When P. vivax LDH was present, T2 and Ctrl dots showed fluorescence signals while no signal was detected with the negative control. P. falciparum LDH and P. vivax LDH were successfully detected on the IMD with a detection limit of 50ng/mL and 100ng/mL, respectively. The IMD provides a point-of-care diagnosis platform which is able to analyze pathogenic bacteria and viruses that can be applied in the field of clinical diagnosis and food safety testing.

Aptamer-mediated Plasmodium-specific diagnosis of malaria

There is a critical need for better malaria rapid diagnostic tests to discriminate Plasmodium falciparum and Plasmodium vivax infection given the recent observation of HRP2 deletions in P. falciparum parasites. We previously identified a DNA aptamer, 2008s, that targets P. falciparum lactate dehydrogenase (PfLDH) and developed a sensitive aptamer-tethered enzyme capture (APTEC) assay. Here, we characterise two different LDH-binding DNA aptamers in their species-specific activities, then integrate within biochemical diagnostic assays and test in clinical samples. An enzyme-linked oligonucleotide assay demonstrated that aptamer pL1 bound with high affinity to both PfLDH and P. vivax lactate dehydrogenase (PvLDH), whereas aptamer 2008s was specific to PfLDH. An aptamer-tethered enzyme capture (APTEC) assay confirmed the specificity of 2008s in binding and capturing the enzyme activity of PfLDH which could be observed colorimetrically. In malaria patient samples, the 2008s APTEC assay was specific for P. falciparum blood samples and could discriminate against P. vivax blood samples. An aptamer for specific detection of falciparum malaria holds promise as a new strategy for species-specific malaria diagnosis rather than the conventional HRP2 immuno-assay.

Detection of Plasmodium Lactate Dehydrogenase Antigen in Buffer Using Aptamer-Modified Magnetic Microparticles for Capture, Oligonucleotide-Modified Quantum Dots for Detection, and Oligonucleotide-Modified Gold Nanoparticles for Signal Amplification.

To overcome the limitations associated with antibody-based sensors, we describe a proof-of-concept of an aptamer-based sandwich assay for detection of lactate dehydrogenase, an antigen associated with malaria. We show a detection limit of Plasmodium falciparum lactate dehydrogenase and Plasmodium vivax lactate dehydrogenase of 0.5 fmole in buffer, comparable to an antibody-based assay, using a magnetic particle-aptamer construct for capture and a quantum dot-aptamer construct for detection. We then demonstrate a detection limit of 10 amole (50-fold amplification) using oligonucleotide-functionalized gold nanoparticles to allow the conjugation of multiple quantum dots for each target antigen.

A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivaxdeveloped from two lactate dehydrogenase protein segments

BackgroundImmunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.MethodsTwo recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.Results“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.ConclusionBoth tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.

A colorimetric aptasensor for the diagnosis of malaria based on cationic polymers and gold nanoparticles

Malaria, a major burden of disease caused by parasites of the genus Plasmodium, is widely spread in tropical and subtropical regions. Here, we have successfully developed a diagnostic technique for malaria. The proposed method is based on the interaction among the Plasmodium lactate dehydrogenase (pLDH), which is a biomarker for malaria, and pL1 aptamer against Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum lactate dehydrogenase (PfLDH). In addition, the cationic polymers, poly(diallyldimethylammonium chloride) (PDDA) and poly(allylamine hydrochloride) (PAH), aggregate gold nanoparticles (AuNPs) that should be possible to observe the change in color from red to blue, which depends on the concentration of pLDH. Using this aptasensor, pLDH proteins were successfully detected with low detection limits. Moreover, the specificity test proved that the aptasenor is very specific in targeting proteins over other interfering proteins. In addition, the pLDH from infected blood samples of the two main species of malaria were also detected. The limits of detection for P. vivax were determined as 80 parasites/μl for PDDA and 74 parasites/μl for PAH. The aptasenor has great advantages that can simply and rapidly diagnose malaria. Thus, the developed aptasensor for detection of pLDH can offer an effective and sensitive diagnosis of malaria.

Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from Plasmodium vivax.

In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's. Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed. Observing increase of Km values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However kcat values were about the same throughout all mutant proteins. Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans.

Ethnopharmacology guided screening of traditional Indian herbs for selective inhibition of Plasmodium specific lactate dehydrogenase

Ethnopharmacological relevanceMedicinal plants traditionally used to treat malaria can provide quality leads towards identifying novel anti-malarial drugs. Here we combined this approach with target based drug discovery and explored Plasmodium specific lactate dehydrogenase (LDH) inhibitory activity of 8 Indian plants which are ethnically used to treat malaria. MethodsLDH from Indian Plasmodium falciparum and Plasmodium vivax strains, were cloned and expressed in Escherichia coli, followed by purification of recombinant enzymes (rPfLDH and rPvLDH respectively). Extracts of 8 plants in different organic and aqueous solvents, were screened for their inhibitory activity on rPfLDH, rPvLDH and mammalian LDHs. Phyllanthus amarus aqueous extract was further tested for in vitro parasiticidal activity. ResultsAqueous extract of Phyllanthus amarus Schum. and Thonn. and chloroform extract of Murraya koenigii (L.) Spreng. exhibited profound and exclusive inhibitory effect on Plasmodium falciparum LDH (IC50=11.2μg/ml±0.4) and Plasmodium vivax LDH (IC50=6.0μg/ml±0.6) respectively. Moreover, Phyllanthus amarus aqueous extract also demonstrated antiplasmodial activity in vitro, on Chloroquine sensitive and resistant strains of Plasmodium falciparum (IC50=7.1μg/ml±0.5 and 6.9μg/ml±0.7 respectively). ConclusionTarget specific screening of traditional herbs used in malaria treatment has proffered Phyllanthus amarus and Murraya koenigii extracts as hits which can optimistically provide novel antimalarial drugs.

Production of monoclonal antibodies for Plasmodium vivax lactate dehydrogenase and patient sera screening using sandwich ELISA

Malaria is a worldwide infectious disease. There are many diagnostic kits to detect malaria infection. However, the sensitivity of these diagnostic kits remains a problem. To develop a diagnostic kit for malaria that has high sensitivity, it is necessary to produce monoclonal antibodies (McAbs) with high affinity. The present study was undertaken to produce hybridoma cells that can be used to generate McAbs with high affinity and specificity against Plasmodium vivax lactate dehydrogenase (pvLDH). In this study, BALB/c mice were immunized with purified recombinant polypeptides that encode pvLDH. McAbs against pvLDH were produced according to the protocol of hybridoma technique using myeloma cells (SP2/0 cell lines). The McAbs were characterized by isotyping and by Western blot analysis. Two McAbs (D2H and D7E) against pvLDH antigen were obtained. The isotypes of D2H and D7E were IgG2b. They recognize 33 kDa proteins that were defined as pvLDH by Western blot analysis. In the affinity test, D2H and D7E showed positively optical density value until each McAbs were serially diluted at concentrations of 0.156 and 0.078 μg/ml, respectively. To evaluate sensitivity and specificity against clinical specimens of P. vivax, purified McAbs were tested with alkaline phosphatase-conjugated monoclonal antibodies and blood samples (n = 180) of P. vivax patients using the sandwich enzyme-linked immunosorbent assay, showing the 98% sensitivity. We suggest that McAbs produced in this study may be used for developing efficient and rapid diagnostic kits.

Anti-peptide antibodies in the diagnosis of malaria

Lactate dehydrogenase is one of the antigens targeted in lateral flow immunochromatographic rapid diagnostic tests for the diagnosis of malaria. Unique diagnostic peptide targets based on the respective amino-acid sequences to differentiate between P. falciparum and P. vivax lactate dehydrogenase were chosen, synthesized and coupled to rabbit albumin carrier. Anti-peptide antibodies were raised in chickens against the peptides and against each of the recombinant proteins and affinity purified. An antibody against a common epitope detected recombinant P. falciparum, P. vivax and P. yoelii and native P. falciparum and P. vivax protein lactate dehydrogenase. Antibodies against species specific lactate dehydrogenase epitopes differentiated between P. falciparum and P. vivax lactate dehydrogenase. The study supports an anti-peptide antibody approach for the design of malaria diagnostic reagents.

Analysis of active site loop amino acids of lactate dehydrogenase from Plasmodium vivax by site‐directed mutagenesis studies

AbstractThe spread of resistance of malaria parasites to conventional chemotherapies necessitates the development of new drugs. The present study analyzes the active site loop of the enzyme lactate dehydrogenase from Plasmodium vivax (PvLDH). A five–amino acids insertion was observed in the active site loop of PvLDH, as in other plasmodial LDHs, which is absent in human LDH and is therefore a potential novel target site for new antimalarial drugs. This study describes the mutagenic testing of overproduced Plasmodium vivax lactate dehydrogenase to evaluate the importance of the inserted amino acids in the active site loop, which were previously identified as important residues by crystal structure studies. Deletion of these 5 extra amino acids in the active site loop resulted in an inactive form of the protein, supporting the notion of using this site as a novel target for antimalarial drug design. Drug Dev. Res. 67:175–180, 2006 © 2006 Wiley‐Liss, Inc.