Falciparum Assay Research Articles

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatment.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 (1), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Molecular Docking Studies, Synthesis and Biological Evaluation of Substituted Pyrimidine-2,4-diamines as Inhibitors of Plasmodium falciparum Dihydrofolate Reductase.

A series of 5-[(phenethylamino)methyl]pyrimidine-2,4-diamines were assessed in silico as potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), synthesised and tested for inhibitory activity against PfDHFR in vitro. The compounds displayed promising inhibitory activity against both wild-type (Ki 1.3-243 nM) and quadruple mutant (Ki 13-208 nM) PfDHFR in the biochemical enzyme assay, but were less potent in the whole-cell P. falciparum assay (IC50 (TM4/8.2) 0.4-28 μM; IC50 (V1S) 3.7-54 μM). Further investigation into the pharmacokinetic properties of these compounds may guide the development of more potent analogues.

Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon.

Malaria elimination efforts in Peru have dramatically reduced the incidence of cases in the Amazon Basin. To achieve the elimination, the detection of asymptomatic and submicroscopic carriers becomes a priority. Therefore, efforts should focus on tests sensitive enough to detect low-density parasitemia, deployable to resource-limited areas and affordable for large screening purposes. In this study, we assessed the performance of the Malachite-Green LAMP (MG-LAMP) using heat-treated DNA extraction (Boil & Spin; B&S MG-LAMP) on 283 whole blood samples collected from 9 different sites in Loreto, Peru and compared its performance to expert and field microscopy. A real-time PCR assay was used to quantify the parasite density. In addition, we explored a modified version of the B&S MG-LAMP for detection of submicroscopic infection in 500 samples and compared the turnaround time and cost of the MG-LAMP with microscopy. Compared to expert microscopy, the genus B&S MG-LAMP had a sensitivity of 99.4% (95%CI: 96.9%- 100%) and specificity of 97.1% (95%CI: 91.9%- 99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%- 100%) and specificity of 99.2% (95.5%- 100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%- 100%) and specificity of 99.3% (95%CI: 97.3%- 99.8%). The modified genus B&S MG-LAMP assay detected eight submicroscopic malaria cases (1.6%) which the species-specific assays did not identify. The turnaround time of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. The modified B&S MG-LAMP offers a simple and sensitive molecular test of choice for the detection of submicroscopic infections that can be used for mass screening in resources limited facilities in endemic settings nearing elimination and where a deployable test is required.

A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping.

BackgroundCRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection.MethodsWe developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance.FindingsThe suite of Plasmodium assays achieved analytical sensitivities ranging from 2•5-18•8 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities.InterpretationThese novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance.FundingNational Institutes of Health (T32GM007092, R21AI148579, K24AI134990, R01AI121558, UL1TR002489, P30CA016086)

Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays

BackgroundMultiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA.MethodsRecombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement.ResultsTotal IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 μg/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20–30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass.ConclusionsEven when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely.

Organometallic Conjugates of the Drug Sulfadoxine for Combatting Antimicrobial Resistance.

Fourteen novel arene RuII , and cyclopentadienyl (Cpx ) RhIII and IrIII complexes containing an N,N'-chelated pyridylimino- or quinolylimino ligand functionalized with the antimalarial drug sulfadoxine have been synthesized and characterized, including three by X-ray crystallography. The rhodium and iridium complexes exhibited potent antiplasmodial activity with IC50 values of 0.10-2.0 μm in either all, or one of the three Plasmodium falciparum assays (3D7 chloroquine sensitive, Dd2 chloroquine resistant and NF54 sexual late stage gametocytes) but were only moderately active towards Trichomonas vaginalis. They were active in both the asexual blood stage and the sexual late stage gametocyte assays, whereas the clinical parent drug, sulfadoxine, was inactive. Five complexes were moderately active against Mycobacterium tuberculosis (IC50 <6.3 μm), while sulfadoxine showed no antitubercular activity. An increase in the size of both the Cpx ligand and the aromatic imino substituent increased hydrophobicity, which resulted in an increase in antiplasmodial activity.

Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay: Development, Validation, and Evaluation

Decreasing malaria transmission warrants the search for highly sensitive point-of-care diagnostics, especially in resource-limited settings. The direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA) is a simplified PCR-based technique with a lateral flow readout that does not require sample preparation. Two duplex db-PCR-NALFIAs were developed: a pan-Plasmodium/Plasmodium falciparum (pan/P. falciparum) and a pan-Plasmodium/Plasmodium vivax (pan/P. vivax) assay. Confirmed positive samples (n=61) and negative controls (n=40) were used for laboratory validations. A prospective field evaluation of the pan/P. falciparum assay was performed in Kenya (n=300). In the laboratory validation, sensitivity and specificity of the pan/P. falciparum assay were 100% (95% CI, 94.1%-100%) and 100% (95% CI, 91.2%-100%), respectively. Sensitivity and specificity of the pan/P. vivax assay were 100% (95% CI, 94.1%-100%) and 97.5% (95% CI, 86.8%-99.9%), respectively. In Kenya, sensitivity of the pan/P. falciparum db-PCR-NALFIA was 97.2% (95% CI, 93.0%-99.2%) and specificity was 74.2% (95% CI, 67.0%-81.0%) compared with reference standard microscopy. When using real-time quantitative PCR as a reference standard, sensitivity was 84.5% (95% CI, 78.7%-89.3%) and specificity was 85.4% (95% CI, 77.1%-91.6%). Db-PCR-NALFIA is a sensitive, specific, and easy method for the detection and species differentiation of Plasmodium. This test is especially of interest for malaria control or elimination programs in low-transmission settings that require accurate detection of low parasite densities.

Biopreservation of RBCs for invitro Plasmodium falciparum culture.

Biopreservation of RBCs for invitro Plasmodium falciparum culture.

Effects of the angiotensin II Ala-scan analogs in erythrocytic cycle of Plasmodium falciparum (in vitro) and Plasmodium gallinaceum (ex vivo)

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala4]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala8]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala5]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control, except [Ala6]-Ang II, which was able to reduce the parasitemia about 49%. This approach provides insight for understanding the importance of each amino acid on the native Ang II sequence and provides a new direction for the design of potential chemotherapeutic agents without pressor activity.

Multiplex qPCR for Detection and Absolute Quantification of Malaria

We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R2 values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.

Antiplasmodial potential of medicinal plant extracts from Malaiyur and Javadhu hills of South India

The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest anti-malarial drug, coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the urgent need to tap the potential of plants for novel anti-malarials. The present study investigates the anti-malarial activity of the methanolic extracts of 13 medicinal plants from the Malaiyur and Javadhu hills of South India against blood stage CQ-sensitive (3D7) and CQ-resistant (INDO) strains of P. falciparum in culture using the fluorescence-based SYBR Green I assay. Sorbitol-synchronized parasites were incubated under normal culture conditions at 2% hematocrit and 1% parasitemia in the absence or presence of increasing concentrations of plant extracts. CQ and artemisinin were used as positive controls, while 0.4% DMSO was used as the negative control. The cytotoxic effects of extracts on host cells were assessed by functional assay using HeLa cells cultured in RPMI containing 10% fetal bovine serum, 0.21% sodium bicarbonate and 50 μg/mL gentamycin (complete medium). Plant extracts (bark methanol extracts of Annona squamosa (IC(50), 30 μg/mL), leaf extracts of Ocimum gratissimum (IC(50), 32 μg/mL), Ocimum tenuiflorum (IC(50), 31 μg/mL), Solanum torvum (IC(50), 31 μg/mL) and Justicia procumbens (IC(50), 63 μg/mL), showed moderate activity. The leaf extracts of Aristolochia indica (IC(50), 10 μg/mL), Cassia auriculata (IC(50), 14 μg/mL), Chrysanthemum indicum (IC(50), 20 μg/mL) and Dolichos biflorus (IC(50), 20 μg/mL) showed promising activity and low activity was observed in the flower methanol extracts of A. indica , leaf methanol extract of Catharanthus roseus, and Gymnema sylvestre (IC(50), >100 μg/mL). These four extracts exhibited promising IC(50) (μg/mL) of 17, 24, 19 and 24 respectively also against the CQ resistant INDO strain of P. falciparum. The high TC(50) in mammalian cell cytotoxicity assay and the low IC(50) in anti-malarial P. falciparum assay indicates selectivity and good resistance indices in the range of 0.9-1.7 for leaf extracts of A. indica, C. auriculata, C. indicum and D. biflorus suggests that these may serve as anti-malarial agents even in their crude form. These results indicate a possible explanation of the traditional use of some of these medicinal plants against malaria or malaria-like conditions.