Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody.

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6-200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%-102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Targeted Combination of Poly(ADP-ribose) Polymerase Inhibitors and Immune Checkpoint Inhibitors Lacking Evidence of Benefit: Focus in Ovarian Cancer.

The emergence of targeted therapeutics in ovarian cancer, particularly poly (ADP-ribose) polymerase inhibitors (PARPi's), has created additional opportunities for patients seeking frontline and recurrent disease management options. In particular, PARPi's have shown clinical benefits in BRCA mutant and/or homologous recombination deficient (HRD) ovarian cancer. Until recently, response was thought to be limited in BRCA wild-type, homologous recombination proficient (HRP) cancers. Therefore, attempts have been made at combination therapy involving PARPi to improve patient outcomes. Additionally, immune checkpoint inhibitors (ICIs) have demonstrated underwhelming results involving ovarian cancer. Many are searching for reliable biomarkers of immune response to increase efficacy of ICI therapy involving ovarian cancer. In this review, we examine the evidence supporting the combination of PARPi and ICIs in ovarian cancer, which is still lacking.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

A new nanobody-enzyme fusion protein-linked immunoassay for detecting antibodies against influenza A virus in different species.

Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion-based cELISA is an ideal method for monitoring IAV infection in different species.

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans.

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper.

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Assessing the histidine-rich protein 2/3 gene deletion in Plasmodium falciparum isolates from Burkina Faso.

BACKGROUND: Dual hrp2/hrp3 genes deletions in P. falciparum isolates are increasingly reported in malaria-endemic countries and can produce false negative RDT results leading to inadequate case management. Data on the frequency of hrp2/hrp3 deleted parasites are rarely available and it has become necessary to investigate the issue in Burkina Faso. METHODS: Plasmodium falciparum-positive dried blood spots were collected during a cross-sectional household survey of the malaria asymptomatic children from Orodara, Gaoua, and Banfora. Amplicons from the target regions (exon 2 of hrp2 and hrp3 genes) were generated using multiplexed nested PCR and sequenced according to Illumina's MiSeq protocol. RESULTS: A total of 251 microscopically positive parasite isolates were sequenced to detect hrp2 and hrp3 gene deletions. The proportion of RDTs negative cases among microscopy positive slides was 12.7% (32/251). The highest prevalence of negative RDTs was found in Orodara 14.3% (5/35), followed by Gaoua 13.1%(24/183), and Banfora 9.1% (3/33). The study found that 95.6% of the parasite isolates were wild type hrp2/ hrp3 while 4.4% (11/251) had a single hrp2 deletion. Of the 11 hrp2 deletion samples, 2 samples were RDT negative (mean parasitaemia was 83 parasites/ µL) while 9 samples were RDT positive with a mean parasitaemia of 520 parasites /µL (CI95%: 192-1239). The highest frequency of hrp2 deletion 4/35 (11.4%) was found in Orodara, while it was similar in the other two sites (< 3.5%). No single deletion of the hrp3 or dual deletion hrp2/3 gene was detected in this study. CONCLUSION: These results demonstrate that P. falciparum isolates lacking hrp2 genes are present in 4.4% of samples obtained from the asymptomatic children population in three sites in Burkina Faso. These parasites are circulating and causing malaria, but they are also still detectable by HRP2-based RTDs due to the presence of the intact pfhrp3 gene.

Therapeutic efficacy of artesunate-amodiaquine and artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Chad: clinical and genetic surveillance.

BACKGROUND: Artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) are the currently recommended first-and second-line therapies for uncomplicated Plasmodium falciparum infections in Chad. This study assessed the efficacy of these artemisinin-based combinations, proportion of day 3 positive patients, proportions of molecular markers associated with P. falciparum resistance to anti-malarial drugs and variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single-arm prospective study assessing the efficacy of AS-AQ and AL at three sites (Doba, Kelo and Koyom) was conducted between November 2020 to January 2021. Febrile children aged 6 to 59 months with confirmed uncomplicated P. falciparum infection were enrolled sequentially first to AS-AQ and then AL at each site and followed up for 28 days. The primary endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR). Samples collected on day 0 were analysed for mutations in pfkelch13, pfcrt, pfmdr-1, pfdhfr, pfdhps genes and deletions in pfhrp2/pfhrp3 genes. RESULTS: By the end of 28-day follow-up, per-protocol PCR corrected ACPR of 97.8% (CI 95% 88.2-100) in Kelo and 100% in Doba and Kayoma were observed among AL treated patients. For ASAQ, 100% ACPR was found in all sites. All, but one patient, did not have parasites detected on day 3. Out of the 215 day 0 samples, 96.7% showed pfkelch13 wild type allele. Seven isolates carried nonsynonymous mutations not known to be associated artemisinin partial resistance (ART-R). Most of samples had a pfcrt wild type allele (79% to 89%). The most prevalent pfmdr-1 allele detected was the single mutant 184F (51.2%). For pfdhfr and pfdhps mutations, the quintuple mutant allele N51I/C59R/S108N + G437A/540E responsible for SP treatment failures in adults and children was not detected. Single deletion in the pfhrp2 and pfhrp3 gene were detected in 10/215 (4.7%) and 2/215 (0.9%), respectively. Dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, were observed in 5/215 (2.3%) isolates. CONCLUSION: The results of this study confirm that AS-AQ and AL treatments are highly efficacious in study areas in Chad. The absence of known pfkelch13 mutations in the study sites and the high parasite clearance rate at day 3 suggest the absence of ART-R. The absence of pfdhfr/pfdhps quintuple or sextuple (quintuple + 581G) mutant supports the continued use of SP for IPTp during pregnancy. The presence of parasites with dual pfhrp2/pfhrp3 deletions, potentially threatening the efficacy of HRP2-based RDTs, warrants the continued surveillance. Trial registration ACTRN12622001476729.

Fluorescent Oligomeric Nanoparticle from Diaminopyridine Compound Via Enzyme-Catalyzed Oxidation.

An oligomeric nanoparticle was obtained through an enzyme-catalyzed oxidation reaction using Horse Radish Peroxidase (HRP) with the 2,3-diaminopyridine (DAP) compound as the starting material. The structural characterizations of the synthesized oligomeric nanoparticles [[oligo(DAP)]Enz] were performed with 1H-NMR and FT-IR. The surface features were determined by scanning electron microscopy. The optical properties were determined by UV-Vis and fluorescence spectra. It was found that the obtained oligomeric nanoparticles had a different fluorescent character with 15.90% quantum yield from the monomer and emitted green color at 485 nm when excited with light at a wavelength of 420 nm. The electrochemical band gap of the oligomeric nanoparticles, whose electrochemical character was investigated by cyclic voltammetry, was calculated as 2.09 eV. The thermal stability of the oligomeric material was determined from the mass loss against increasing temperature. The monomer exhibited greater thermal stability in comparison to the polymer, both in terms of the temperature at which the maximum mass loss occurred and the residual amount after heating concluded.

Microgel-based etalon immunoassay for IgG detection.

We developed an immunoassay for mouse immunoglobulin (IgG) quantitation using poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgel-based etalon devices. To achieve this, a biotinylated primary antibody specific to mouse IgG was immobilized on the top Au layer of an etalon device via its interaction with a streptavidin-modified etalon surface. Mouse IgG captured on the etalon surface from the solution was quantified using an HRP-conjugated secondary antibody. HRP catalyzed the oxidation of 4-chloro-1-naphthol (4CN) to form insoluble 4-chloro-1-naphthon (4CNP), resulting in a concentration change of 4CN in solution. The etalon was able to detect the 4CN concentration change by monitoring the extent of the etalon's reflectance peak shift, which allows the quantitation of mouse IgG. The etalon-based assay can detect mouse IgG down to 0.018 nM with a linear range of 0.02-5 nM.

Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris.

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.

Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia.

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

Synthesis of Novel Polymer-Assisted Organic-Inorganic Hybrid Nanoflowers and Their Application in Cascade Biocatalysis.

This study reports on the synthesis of novel bienzyme polymer-assisted nanoflower complexes (PANF), their morphological and structural characterization, and their effectiveness as cascade biocatalysts. First, highly porous polyamide 6 microparticles (PA6 MP) are synthesized by activated anionic polymerization in solution. Second, the PA6 MP are used as carriers for hybrid bienzyme assemblies comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). Thus, four PANF complexes with different co-localization and compartmentalization of the two enzymes are prepared. In samples NF GH/PA and NF GH@PA, both enzymes are localized within the same hybrid flowerlike organic-inorganic nanostructures (NF), the difference being in the way the PA6 MP are assembled with NF. In samples NF G/PAiH and NF G@PAiH, only GOx is located in the NF, while HRP is preliminary immobilized on PA6 MP. The morphology and the structure of the four PANF complexes have been studied by microscopy, spectroscopy, and synchrotron X-ray techniques. The catalytic activity of the four PANF was assessed by a two-step cascade reaction of glucose oxidation. The PANF complexes are up to 2-3 times more active than the free GOx/HRP dyad. They also display enhanced kinetic parameters, superior thermal stability in the 40-60 °C range, optimum performance at pH 4-6, and excellent storage stability. All PANF complexes are active for up to 6 consecutive operational cycles.

Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme-Antibody Conjugation Reaction and Quality Control of a Final Product.

The multi-detection size exclusion chromatography (SEC) has been recognized as an advanced analytical technique for the characterization of macromolecules and process control, as well as the manufacturing and formulation of biotechnology products. It reveals reproducible molecular characterization data, such as molecular weight and its distribution, and the size, shape, and composition of the sample peaks. The aim of this work was to investigate the potential and suitability of the multi-detection SEC as a tool for surveillance over the molecular processes during the conjugation reaction between the antibody (IgG) and horseradish peroxidase (HRP), and demonstrate the plausibility of its application in the quality control of the final product, the IgG-HRP conjugate. Guinea pig anti-Vero IgG-HRP conjugate was prepared using a modified periodate oxidation method, based on periodate oxidation of the carbohydrate side chains of HRP, followed by the formation of Schiff bases between the activated HRP and amino groups of the IgG. The quantitative molecular characterization data of the starting samples, intermediates, and final product were obtained by multi-detection SEC. Titration of the prepared conjugate was performed by the ELISA and its optimal working dilution was determined. This methodology proved to be a promising and powerful technology for the IgG-HRP conjugate process control and development, as well as for the quality control of the final product, as verified by the analysis of several commercially available reagents.

Performance of a Histidine Rich Protein-2 Based (First Response) and a p-Lactate Dehydrogenase-based (Optimal) Rapid Diagnostic Test for Diagnosis of Malaria in Patients With Pediatric Sickle Cell Disease.

BACKGROUND: Rapid diagnostic tests (RDTs) have been extensively evaluated and play an important role in malaria diagnosis. However, the accuracy of RDTs for malaria diagnosis in patients with sickle cell disease (SCD) is unknown. METHODS: We compared the performance of a histidine rich protein 2 (HRP-2)-based RDT (First Response) and a lactate dehydrogenase (LDH)-based RDT (Optimal) with routine microscopy as reference standard in 445 children with SCD and an acute febrile illness in Accra, Ghana. RESULTS: The overall sensitivity, specificity, and positive and negative predictive values of the HRP-2-based RDTs were 100%, 95.7%, 73.8%, and 100%, respectively. Comparable values for the LDH-based RDTs were 91.7%, 99.5%, 95.7%, and 99.0%, respectively. A total of 423 results were true in both tests, 1 result was false in both tests, 16 results were false in the HRP-2 test only, and 5 were false in the LDH test only (McNemar test, P = .03). At follow-up, 73.7% (28/38), 52.6% (20/38), 48.6% (17/35), and 13.2% (5/38) of study participants were HRP-2 positive on days 14, 28, 35, and 42, respectively, compared with 0%, 2.6% (1/38), 2.9% (1/35), and 2.6% (1/38) for LDH. CONCLUSION: The HRP2-based RDT fulfilled World Health Organization criteria for malaria diagnosis in patients with SCD and may provide diagnostic evidence for treatment to begin in cases in which treatment would otherwise have begun presumptively based on symptoms, whereas LDH-based RDTs may be more suitable as a confirmatory test in low-parasitemic subgroups, such as patients with SCD.

A novel function of HRP-3 in regulating cell cycle progression via the HDAC-E2F1-Cyclin E pathway in lung cancer.

To improve the poor survival rate of lung cancer patients, we investigated the role of HDGF-related protein 3 (HRP-3) as a potential biomarker for lung cancer. The expression of endogenous HRP-3 in human lung cancer tissues and xenograft tumor models is indicative of its clinical relevance in lung cancer. Additionally, we demonstrated that HRP-3 directly binds to the E2F1 promoter on chromatin. Interestingly, HRP-3 depletion in A549 cells impedes the binding of HRP-3 to the E2F1 promoter; this in turn hampers the interaction between Histone H3/H4 and HDAC1/2 on the E2F1 promoter, while concomitantly inducing Histone H3/H4 acetylation around the E2F1 promoter. The enhanced Histone H3/H4 acetylation on the E2F1 promoter through HRP-3 depletion increases the transcription level of E2F1. Furthermore, the increased E2F1 transcription levels lead to the enhanced transcription of Cyclin E, known as the E2F1-responsive gene, thus inducing S-phase accumulation. Therefore, our study provides evidence for the utility of HRP-3 as a biomarker for the prognosis and treatment of lung cancer. Furthermore, we delineated the capacity of HRP-3 to regulate the E2F1 transcription level via histone deacetylation.

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015.

BACKGROUND: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. METHODS: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual's RDT results. Dose-response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. RESULTS: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). CONCLUSIONS: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

Gene Coverage Count and Classification (GC3): a locus sequence coverage assessment tool using short-read whole genome sequencing data, and its application to identify and classify histidine-rich protein 2 and 3 deletions in Plasmodium falciparum.

BACKGROUND: The ability of malaria rapid diagnostic tests (RDTs) to effectively detect active infections is being compromised by the presence of malaria strains with genomic deletions at the hrp2 and hrp3 loci, encoding the antigens most commonly targeted in diagnostics for Plasmodium falciparum detection. The presence of such deletions can be determined in publically available P. falciparum whole genome sequencing (WGS) datasets. A computational approach was developed and validated, termed Gene Coverage Count and Classification (GC3), to analyse genome-wide sequence coverage data and provide informative outputs to assess presence and coverage profile of a target locus in WGS data. GC3 was applied to detect deletions at hrp2 and hrp3 (hrp2/3) and flanking genes in different geographic regions and across time points. METHODS: GC3 uses Python and R scripts to extract locus read coverage metrics from mapped WGS data according to user-defined parameters and generates relevant tables and figures. GC3 was tested using WGS data for laboratory reference strains with known hrp2/3 genotypes, and its results compared to those of a hrp2/3-specific qPCR assay. Samples with at least 25% of coding region positions with zero coverage were classified as having a deletion. Publicly available sequence data was analysed and compared with published deletion frequency estimates. RESULTS: GC3 results matched the expected coverage of known laboratory reference strains. Agreement between GC3 and a hrp2/3-specific qPCR assay reported for 19/19 (100%) hrp2 deletions and 18/19 (94.7%) hrp3 deletions. Among Cambodian (n = 127) and Brazilian (n = 20) WGS datasets, which had not been previously analysed for hrp2/3 deletions, GC3 identified hrp2 deletions in three and four samples, and hrp3 deletions in 10 and 15 samples, respectively. Plots of hrp2/3 coding regions, grouped by year of sample collection, showed a decrease in median standardized coverage among Malawian samples (n = 150) suggesting the importance of a careful, properly controlled follow up to determine if an increase in frequency of deletions has occurred between 2007-2008 and 2014-2015. Among Malian (n = 90) samples, median standardized coverage was lower in 2002 than 2010, indicating widespread deletions present at the gene locus in 2002. CONCLUSIONS: The GC3 tool accurately classified hrp2/3 deletions and provided informative tables and figures to analyse targeted gene coverage. GC3 is an appropriate tool when performing preliminary and exploratory assessment of locus coverage data.

Comparison of two malaria multiplex immunoassays that enable quantification of malaria antigens.

BACKGROUND: Immunoassay platforms that simultaneously detect malaria antigens including histidine-rich protein 2 (HRP2)/HRP3 and Plasmodium lactate dehydrogenase (pLDH), are useful epidemiological tools for rapid diagnostic test evaluation. This study presents the comparative evaluation of two multiplex platforms in identifying Plasmodium falciparum with presence or absence of HRP2/HRP3 expression as being indicative of hrp2/hrp3 deletions and other Plasmodium species. Moreover, correlation between the malaria antigen measurements performed at these platforms is assessed after calibrating with either assay standards or international standards and the cross-reactivity among Plasmodium species is examined. METHODS: A 77-member panel of specimens composed of the World Health Organization (WHO) international Plasmodium antigen standards, cultured parasites for P. falciparum and Plasmodium knowlesi, and clinical specimens with mono-infections for P. falciparum, Plasmodium vivax, and Plasmodium malariae was generated as both whole blood and dried blood spot (DBS) specimens. Assays for HRP2, P. falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), and all human Plasmodium species Pan malaria pLDH (PanLDH) on the Human Malaria Array Q-Plex and the xMAP platforms were evaluated with these panels. RESULTS: The xMAP showed a higher percent positive agreement for identification of hrp2-deleted P. falciparum and Plasmodium species in whole blood and DBS than the Q-Plex. For whole blood samples, there was a highly positive correlation between the two platforms for PfLDH (Pearson r = 0.9926) and PvLDH (r = 0. 9792), moderate positive correlation for HRP2 (r = 0.7432), and poor correlation for PanLDH (r = 0.6139). In Pearson correlation analysis between the two platforms on the DBS, the same assays were r = 0.9828, r = 0.7679, r = 0.6432, and r = 0.8957, respectively. The xMAP HRP2 assay appeared to cross-react with HRP3, while the Q-Plex did not. The Q-Plex PfLDH assay cross-reacted with P. malariae, while the xMAP did not. For both platforms, P. knowlesi was detected on the PvLDH assay. The WHO international standards allowed normalization across both platforms on their HRP2, PfLDH, and PvLDH assays in whole blood and DBS. CONCLUSIONS: Q-Plex and xMAP show good agreement for identification of P. falciparum mutants with hrp2/hrp3 deletions, and other Plasmodium species. Quantitative results from both platforms, normalized into international units for HRP2, PfLDH, and PvLDH, showed good agreement and should allow comparison and analysis of results generated by either platform.