CONTRIBUTIONS TO UNRAVELLING THE NATURE OF THE FALSE POSITIVITY IN CIRCUMSPOROZOITE Plasmodium falciparum ELISA

INTRODUCTION: Plasmodium falciparum malaria, a vector-borne disease caused by the bite of Anopheles mosquitoes is a public health problem worldwide. Vector control remains as an efficient method to block malaria transmission. Vector incrimination, being a crucial factor for efficient vector control, nowadays mainly relies on detection of the parasite’s circumsporozoite protein in ELISA. However, false positivity in P. falciparum Circumsporozoite ELISA has been reported in Africa and Southeast Asia. The principal objective of this research is to determine the nature of the agent that provokes false positivity in Pf CSP ELISA through in silico analysis of Pf CSP, Next Generation Sequencing of bacterial 16s rDNA, detection of Microsporidia parasites in mosquitoes and isolation of the protein responsible for false positivity. MATERIALS AND METHODS: In silico analysis of Pf CSP was performed through BLAST search. Primers Bakt_341F/Bakt_805R attached to Illumina® adaptors were used to amplify bacterial 16s rDNA in 10 false positive samples from Vietnam and Cambodia, 1 laboratory reared An. stephensi and 2 true negative mosquitoes. Samples were identified into 16s rDNA database from GenBank with the Galaxy workflow modified for metagenomic studies. Cross-reaction of Microsporidia in Pf CSP ELISA was evaluated with Aedes aegypti infected with Vavraia culicis and Edhazardia aedis. Microsporidia were detected by specific amplification of 18s rDNA. Protein capture from 6 false positive samples was carried out using Dynabeads® M270 Epoxy antibody coupling kit and capture monoclonal antibodies 2A10. Isolated protein was separated through SDS-PAGE and identified by MALDI TOF/TOF analysis. RESULTS: In silico analysis of Pf CSP demonstrates some degree of identity with the domain Bacteria and a disordered NANPL region present in the Microsporidia E. aedis. Amplification and sequencing of 16s rDNA of false positive samples did not indicate a single species of bacteria present in the majority of false positive samples analyzed and absent in negative controls. Microsporidia did not show cross reactivity in Pf CSP ELISA and only one sample was found positive in head/thorax and abdomen for detection of Microsporidia by PCR. MALDI TOF/TOF analysis showed that actin cross- reacted with capture monoclonal antibodies in Pf CSP ELISA. DISCUSSION: In silico and laboratory analysis can not confirm Microsporidia, nor one single bacterial IV species as the cross-reactive protein. The remarkable diversity of species supports the hypothesis that more than one bacterial species could cross react with monoclonal antibodies in Pf CSP ELISA. The finding that the actin protein can be recognized by anti-Pf CSP monoclonal antibodies supports the animal origin theory of the causative agent of false positivity. Identification of this agent remains unclear, nevertheless important insights are provided towards a feasible explanation of false positivity in Pf CSP ELISA

Development of a nanobody-based amperometric immunocapturing assay for sensitive and specific detection of Toxocara canis excretory-secretory antigen

Introduction Human Toxocariasis (HT) is a zoonosis that, despite of its wide distribution around the world, remains poorly diagnosed. The identification of specific IgG immunoglobulins against the Toxocara canis Excretory-Secretory antigen (TES), a mix of glycoproteins that the parasite releases during its migration to the target organs in infected patients, is currently the only laboratory tool to detect the disease. The main drawbacks of this test are the inability to distinguish past and active infections together with lack of specificity. These factors seriously hamper the diagnosis, follow-up and control of the disease. Aim To develop an amperometric immunocapturing diagnostic assay based on single domain immunoglobulins from camelids (nanobodies) for specific and sensitive detection of TES. Methods After immunization of an alpaca (Vicugna pacos) with TES, RNA from peripheral blood lymphocytes was used as template for cDNA amplification with oligo dT primers and library construction. Isolation and screening of TES-specific nanobodies were carried out by biopanning and the resulting nanobodies were expressed in Escherichia coli. Two-epitopes amperometric immunocapturing assay was designed using paramagnetic beads coated with streptavidin and bivalent nanobodies. Detection of the system was carried out with nanobodies chemically coupled to horseradish peroxidase. The reaction was measured by amperometry and the limit of detection (LOD) was compared to conventional sandwich ELISA. Results We obtained three nanobodies that specifically recognize TES with no-cross reactivity to antigens of Ascaris lumbricoides and A. suum. The LOD of the assay using PBST20 0.05% as diluent was 100 pg/ml, 10 times more sensitive than sandwich ELISA. Conclusion Sensitive and specific detection of TES for discrimination of active and past infections is one of the most difficult challenges of T. canis diagnosis. The main advantage of our system is the use of two different nanobodies that specifically recognize two different epitopes in TES with a highly sensitive and straightforward readout. Considering that the amounts of TES available for detection in clinical samples are in the range of picograms or a few nanograms maximum, the LOD found in our experiments suggests that the test is potentially useful for the detection of clinically relevant cases of HT

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Nanobody-Based Immunosensor Detection Enhanced by Photocatalytic-Electrochemical Redox Cycling

peer reviewedDetection of antigenic biomarkers present in trace amounts is of crucial importance for medical diagnosis. A parasitic disease, human toxocariasis, lacks an adequate diagnostic method despite its worldwide occurrence. The currently used serology tests may stay positive even years after a possibly unnoticed infection, whereas the direct detection of a re-infection or a still active infection remains a diagnostic challenge due to the low concentration of circulating parasitic antigens. We report a time-efficient sandwich immunosensor using small recombinant single-domain antibodies (nanobodies) derived from camelid heavy-chain antibodies specific to Toxocara canis antigens. An enhanced sensitivity to pg/mL levels is achieved by using a redox cycle consisting of a photocatalytic oxidation and electrochemical reduction steps. The photocatalytic oxidation is achieved by a photosensitizer generating singlet oxygen (1O2) that, in turn, readily reacts with p-nitrophenol enzymatically produced under alkaline conditions. The photooxidation produces benzoquinone that is electrochemically reduced to hydroquinone, generating an amperometric response. The light-driven process could be easily separated from the background, thus making amperometric detection more reliable. The proposed method for detection of the toxocariasis antigen marker shows superior performances compared to other detection schemes with the same nanobodies and outperforms by at least two orders of magnitude the assays based on regular antibodies, thus suggesting new opportunities for electrochemical immunoassays of challenging low levels of antigens

Evolving trends in the management of acute appendicitis during COVID-19 waves. The ACIE appy II study

Background: In 2020, ACIE Appy study showed that COVID-19 pandemic heavily affected the management of patients with acute appendicitis (AA) worldwide, with an increased rate of non-operative management (NOM) strategies and a trend toward open surgery due to concern of virus transmission by laparoscopy and controversial recommendations on this issue. The aim of this study was to survey again the same group of surgeons to assess if any difference in management attitudes of AA had occurred in the later stages of the outbreak. Methods: From August 15 to September 30, 2021, an online questionnaire was sent to all 709 participants of the ACIE Appy study. The questionnaire included questions on personal protective equipment (PPE), local policies and screening for SARS-CoV-2 infection, NOM, surgical approach and disease presentations in 2021. The results were compared with the results from the previous study. Results: A total of 476 answers were collected (response rate 67.1%). Screening policies were significatively improved with most patients screened regardless of symptoms (89.5% vs. 37.4%) with PCR and antigenic test as the preferred test (74.1% vs. 26.3%). More patients tested positive before surgery and commercial systems were the preferred ones to filter smoke plumes during laparoscopy. Laparoscopic appendicectomy was the first option in the treatment of AA, with a declined use of NOM. Conclusion: Management of AA has improved in the last waves of pandemic. Increased evidence regarding SARS-COV-2 infection along with a timely healthcare systems response has been translated into tailored attitudes and a better care for patients with AA worldwide

"Estudios moleculares de Plamodium falciparum y P. vivax en la Provincia de Sucumbios, Ecuador"

Ecuador official treatment for cases of complicated malaria, caused by Plasmodium falciparum is based on the adminstration of sulfadoxine / pyrimethamine combined with artesunate, while infections by P. are tratads vivax with chloroquine and primaquine.El tratamiento oficial en Ecuador para los casos de malaria no-complicados causados por Plasmodium falciparum está basado en la adminstración de Sulfadoxina/Pirimetamina combinada con Artesunato, mientras que las infecciones por P. vivax son tratads con Cloroquina y Primaquina

Development of innovative nanobody-based strategies to improve the diagnosis of human toxocariasis

Human toxocariasis (HT) is a zoonotic disease caused by the infection of the larval stage of the dog roundworm Toxocara canis. Detection of active HT infections is elusive due to the lack of sensitivity and specificity of current laboratory tools. In this thesis, we introduce a sensitive and specific nanobody-based sandwich ELISA to detect the T. canis excretory-secretory antigen (TES) with a limit of detection of 0.650 ng/ml in serum spiked with TES. This ELISA employs bivalent biotinylated nanobodies as capturing agent and nanobodies chemically coupled to horseradish peroxidase for detection. Higher sensitivity was achieved converting the ELISA into an electrochemical magnetosensor assay, obtaining a limit 10 pg/ml in serum spiked with TES. This assay was evaluated in serum samples from children of remote rural communities in the province of Esmeraldas (Ecuador). We found that 38% (33/87) sera were positive for TES. Positivity was significantly correlated with eosinophils >19% of total white blood cell count. Additionally, the test showed low cross-reactivity to other helminths. This is to our knowledge, the most sensitive and specific immunoassay to diagnose HT currently available. This research highlights the impressive versatility of nanobodies for the development of highly specific and sensitive immunoassays

Nanobody-based sandwich enzyme-linked immunosorbent assay for the detection of Toxocara canis excretory secretory proteins.

IntroductionHuman Toxocariasis (HT) is a neglecteddisease resulting from tissue invasion of L2larva fromToxocaracanis. The only laboratory diagnostic tool currently available is aserological test detecting IgG against the excretory secretoryproteins of the parasite (TES). This method is unable todistinguish between current and past infections. We developed asandwich enzyme-linked immunosorbent assay (ELISA) takingadvantage of the inherent features of specific single variabledomain fragments of camelids (nanobodiesâ).Methods and MaterialsAn alpaca was immunized with125lg of TES 5 times in intervals of 7 days. One week after thelast immunization, peripheral blood was extracted andnanobodyâsequences were amplified through reverse-transcription polymerase chain reaction (RT-PCR) from bloodlymphocytes. Selection of binders was performed by biopanningbased on phage display. A sandwich ELISA was set up with acapturing nanobodyâcloned in pHEN6c vector and a detectionnanobodyâin vector pBAD17 containing an AviTagTMforin vivobiotinylation. Cross-reactivity was tested with excretoryantigens ofAscaris lumbricoidesandA. suum. Immunocapturingwith paramagnetic beads was used to isolate the specific fractionof TES recognized in the sandwich ELISA.ResultsNanobodyâsequences were present in 3x108transformants. 84% of them contained a plasmid encoding 20different nanobody sequences. The combination 1TCE39 and1TCE52 had the best Optical Density (OD) signal in sandwichELISA with no cross-reactivity withA. lumbricoidesorA. suum.The detection limit using ELISA sandwich format in negativesamples spiked with TES was 40 ng/ml. Immunocapturingdemonstrated that the epitopes recognised by the sandwichELISA are located in the 120 kDa fraction of TES.ConclusionsTests to diagnose active HT are currently notavailable, hampering the estimation of the real prevalence of thedisease and its control. Nanobodyâ-based sandwich ELISAprovides an innovative approach to detect TES ofT. canisinclinical samples.DisclosureThis project is funded by the ResearchFoundation Flanders (FWO

Selection and characterization of anti-NDM-1 nanobodies

Selection and characterization of anti-NDM-1 nanobodies Cawez Frédéric1, Mercuri Paola Sandra1, Piccirilli Alessandra3, Perilli Mariagrazia3, Morales Yánez Francisco2, Dumoulin Mireille2 & Galleni Moreno1 Biological Macromolecules1, NEPTUN2, Center for Protein Engineering, InBioS, University of Liege, Belgium; Dept of Biotechnological and Applied Clinical Sciences3 , University of L'Aquila, Italy. The misuses of beta-lactam antibiotics lead to the selection of multidrug-resistant (MDR) bacteria which are unaffected by the presence antibiotics. Therefore, it is essential to develop new rapid diagnostic assays in order to circumvent the nosocomial infections. In addition, it is essential to develop new inhibitor's scaffolds able to block the beta-lactamase activity. To develop new inhibitors, one strategy consists to select VHH antibodies that serve to the development of new beta-lactamase inhibitors by peptidomimetics

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies

peer reviewedBackground: The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Methods: Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Results: Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Conclusion: Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Keywords: Cell biology tools; diagnostics; immune libraries; nanobodies; research tools; single domain antibodies; therapeutics