- Email: [email protected]
A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid
Accepted Manuscript A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid Agnes Fleury, Patricia Sastre, Edda Sciutto, Silvia Correia, Alejandro Monedero, Andrea Toledo, Maricela Hernandez, Leslie J.S. Harrison, R. Michael E. Parkhouse PII:
S0014-4894(16)30277-6
DOI:
10.1016/j.exppara.2016.10.016
Reference:
YEXPR 7326
To appear in:
Experimental Parasitology
Received Date: 7 March 2016 Revised Date:
13 September 2016
Accepted Date: 26 October 2016
Please cite this article as: Fleury, A., Sastre, P., Sciutto, E., Correia, S., Monedero, A., Toledo, A., Hernandez, M., Harrison, L.J.S., Parkhouse, R.M.E., A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.10.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Research brief
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A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using
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cerebrospinal fluid
5 Agnes Fleury, Patricia Sastre, Edda Sciutto, Silvia Correia, Alejandro Monedero, Andrea Toledo,
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Maricela Hernandez, Leslie J.S. Harrison, R. Michael E. Parkhouse.
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Agnes Fleury
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Unidad de Neuroinflamación (Instituto de Investigaciones Biomédicas-UNAM, Instituto
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Nacional de Neurología y Neurocirugia, Facultad de Médicina-UNAM), Insurgentes Sur 3877,
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Colonia La Fama, delegación Tlalpan, Mexico D.F. / Neurocysticercosis clinic, Instituto Nacional
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de Neurología y Neurocirugia, México D.F.
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[email protected]
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Patricia Sastre
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Inmunología y Genética Aplicada S.A. (INGENASA), C/Hermanos García Noblejas 39
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28037 Madrid, Spain
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[email protected]
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20 Edda Sciutto
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Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional
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Autónoma de México, México, D.F. 04510, Mexico
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[email protected]
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Sílvia Correia
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Instituto Gulbenkian de Ciência
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2780-156 Oeiras,
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Portugal
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[email protected]
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Alejandro Monedero
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Inmunología y Genética Aplicada S.A. (INGENASA), C/Hermanos García Noblejas 39
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28037 Madrid, Spain
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(+34) 913680501 1
ACCEPTED MANUSCRIPT [email protected]
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Andrea Toledo
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Unidad de Neuroinflammation (Instituto de Investigaciones Biomédicas- UNAM, Instituto
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Nacional de Neurología y Neurocirugia, Facultad de Médicina-UNAM), Insurgentes Sur 3877,
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Colonia La Fama, delegación Tlalpan, Mexico D.F.
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[email protected]
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42 Marisela Hernandez
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Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional
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Autónoma de México, México, D.F. 04510, Mexico
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[email protected]
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47 Leslie J. S. Harrison
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University of Edinburgh, Royal (Dick) School of Veterinary Science
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Easter Bush Veterinary Centre, Easter Bush, ROSLIN, Midlothian, Scotland, UK, EH259RG
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[email protected]
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52 R. Michael E. Parkhouse
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Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal
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[email protected]
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(+351 21 4407939)
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Corresponding author
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ACCEPTED MANUSCRIPT ABSTRACT (94)
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A lateral flow assay (LFA) for the diagnosis and monitoring of extraparenchymal
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neurocysticercosis, has been developed. The assay is based on the use of the monoclonal
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antibody HP10, and when applied to cerebrospinal fluid, correctly identified 34 cases of active
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extraparenchymal neurocysticercosis, but was negative with 26 samples from treated and
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cured neurocysticercosis patients and with 20 samples from unrelated neurological diseases.
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There was complete agreement between the HP10 Ag-ELISA results and the HP10-LFA. The
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HP10-LFA thus has utility for diagnosis and treatment of extraparenchymal neurocysticercosis,
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frequently a more dangerous form of the infection.
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6 keywords – extraparenchymal neurocysticercosis; diagnosis; lateral flow assay; Taenia; HP10;
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NTD
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2919 words (all including legends) 2355 words (text)
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ACCEPTED MANUSCRIPT 1. Introduction
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Neglected Tropical diseases (NTD’s), now recognized by the World Health Organization as a
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serious global health issue, include most of the common parasitic diseases (WHO report,
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2016). Due to their disproportionate prevalence in the poorest of the world’s population, their
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control and elimination have received low priority. Many of these diseases, for example;
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malaria, echinococosis, Taenia solium cysticercosis, and toxoplasmosis have a neurological
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involvement, (Carpio et al, 2016) and this aspect of their clinical and economic impact has
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received even less attention. Typically, parasite infections of the nervous system are “silent”,
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without the classical neurological symptoms (e.g. headache, epilepsy, coma) appearing long
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after the initial invasion of the brain and, importantly, when considerable, sometimes
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irreversible, damage has occurred (Carpio et al, 2016). Thus, early and reliable confirmatory
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diagnosis of these parasite infections, subsequent to clinical and radiological examination, is an
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essential tool in the control and treatment of parasites invading the nervous system.
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This is particularly true in the case of neurocysticercosis (NCC), typically caused by invasion of
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the central nervous system and eyes by the metacestode of Taenia solium, and considered to
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be one of the most important NTD’s with neurological impact (Carpio et al, 2016). Imaging
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studies (Computed Tomography (CT) scan and Magnetic Resonance Imaging (MRI)) are
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considered, by neurologists, as the “gold standard” for NCC diagnosis. Moreover, reliable
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diagnosis of extraparenchymal NCC requires MRI special sequences (FIESTA), not yet
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universally available (Carrillo Mezo et al., 2015). Although, these tools permit the diagnosis in
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most of the cases, particularly when parasites are located in the parenchyma (Fleury et al
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2011, 2013), unfortunately, patients’ access to CT, and MRI, in endemic countries is frequently
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limited or financially impossible. As a result, there has been considerable investigation into the
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development of serological and molecular biological procedures for the diagnosis of NCC
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(Wilkins, 2013). In addition to the commercially available western blot (EITB) and antibody
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tests (WHO, 2015; 2016), a variety of recombinant metacestode antigens and synthetic
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peptides has been tested as targets for detection of antibodies (Deckers and Dorny, 2010;
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Ferrer et al, 2007). Antibody detection, however, is not definitive proof of a current, infection
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with living metacestodes, as antibodies can persist for months, even years, after elimination of
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the parasite. As an alternative to the antibody assays, diagnosis of active cysticercosis through
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the detection of molecules actively secreted by viable metacestodes and found in the
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cerebrospinal fluid (CSF) and serum of NCC patients, has been evaluated for diagnosis and
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follow-up of NCC (Harrison et al, 1989; Brandt et al, 1992, Garcia et al, 1998; Garcia et al, 2000;
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Garcia et al, 2002, Nguekan et al, 2003; Rodriguez et al 2009, Fleury et al 2013). In systematic
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ACCEPTED MANUSCRIPT investigations, testing paired serum and CSF samples from clinically characterised NCC
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patients, the HP10 Ag-ELISA, which detects the viable metacestode surface/secreted HP10
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antigen (Harrison et al, 1989) was demonstrated to function well for extraparenchymal NCC,
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but was often low or negative for parenchymal NCC (Bobes et al, 2006; Fleury et al, 2007,
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2013).
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From the practical point of view, however, extraparenchymal NCC often has a much more
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threatening pathogenesis than parenchymal (Bobes et al, 2006; Fleury et al, 2007, 2011, 2013)
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and, importantly, imaging studies are less sensitive for the detection of extraparenchymal NCC
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(Graeff-Teixeira et al, 2009; Fleury et al, 2011). A particularly useful application of the HP10 Ag-
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ELISA is the follow-up of extraparenchymal NCC patients after albendazole/praziquantel
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treatment as it helps determine the efficacy of cysticidal treatment. Thus effective drug
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treatment is clearly established by the decreased parasite antigen levels in CSF, whereas
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ineffective treatment, which occurs in a significant number of cases, is revealed by the
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continued presence of the secreted parasite product in the CSF or serum (Garcia et al, 2000;
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Cardenas et al, 2010).
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In this communication we report the successful adaptation of the HP10 Ag-ELISA to a lateral
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flow assay format (HP10 Ag-LFA), using CSF samples from clinically and imagenologically
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characterized patients, and so provide a preliminary validation of its application for the
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clinically important management of extraparenchymal NCC.
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1. Materials and Methods
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1.1 Samples
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Samples of CSF were collected by lumbar puncture from patients attending the Instituto
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Nacional de Neurologia y Neurocirugía (INNN), Mexico City. The study was approved by and
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carried out under the guidelines of the Ethical Committee of the INNN, D.F., México. All
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patients provided written informed consent for the collection of samples and subsequent
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analysis. Diagnosis of viable extraparenchymal NCC was based on clinical manifestations
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(intracranial hypertension, focal deficit, or affection of cranial nerves and intracranial
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hypertension), imaging studies (MRI with FIESTA sequences showing cystic images located in
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subarachnoid cisterns or ventricular system) and cytochemical analysis of CSF showing an
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increased cellular count (>30 cells/mm3) and protein concentrations (>80mg/ml). CSF samples
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from inactive NCC were from patients who had previously presented active extraparenchymal
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NCC, were treated with the cysticidal drug albendazole, and who were demonstrated to lack
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ACCEPTED MANUSCRIPT cysts by MRI 3-7 months after their treatment. In addition, their neurological symptoms had
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improved (eg. headache, epilepsy, coma) and the CSF samples used to evaluate the HP10 Ag-
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LFA were normal (cells<6/mm3 and protein concentration <40mg/ml). A total of 20 control CSF
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samples were collected from patients with other neurological diseases. Their diagnosis, in
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addition to negative MRI studies for NCC, were as follows: multiple sclerosis (10), optical
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neuritis (2), myelomalacia (2), non-NCC related epilepsy (1), headache (1), tumor (1), subdural
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hematoma (1). Diagnosis of NCC was considered negative based on a combination of MRI
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results, CSF cytochemical analysis and negativity of HP10. All MRI studies were interpreted in a
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double-blind manner by a certified neuroradiologist with extensive experience in NCC
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diagnosis.
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2.2. Lateral Flow ASSAY methodology (HP10-LFA)
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All the CSF samples were assayed by ELISA for the presence of the HP10 secreted metacestode
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antigen (Harrison et al, 1989; Fleury et al, 2013) and tested in the current report in the HP10
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Ag-LFA, immunochromatographic system.
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In brief, the LFA test to detect the HP10 antigen was based on the use of different coloured
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carboxyl-modified latex microspheres, which were covalently linked to the specific target
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proteins or antibodies. Specifically, for the test line, the purified HP10 monoclonal antibody
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was conjugated to red latex particles using EDC (1-ethyl-3-(3-dimethylaminopropyl)
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carbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) to activate the surface of the
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beads (Hermanson, 2013). For the control line, blue latex particles were similarly coated with
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the control protein. Next, the red test and blue control latex particles were diluted in Tris/HCl
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25mM pH 9.5 buffer containing humidity preservatives and blocking agents (3.0% (w/v) bovine
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serum albumin, 1.5% (w/v) casein, 0.35% (w/v) sucrose, 1% (w/v) Tween 20, 0.095% (w/v)
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sodium azide) and the mixture dispensed onto the rayon conjugate pad, using a Matrix 1600
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dispenser (Kinematic Automation, Inc.). For the test capture line, the HP10 MAb was diluted at
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0.6 mg/ml in buffer Tris/HCl 20 mM pH 7.5, containing 5.0% (w/v) sucrose and 0.095% (w/v)
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sodium azide as preservative. The anti-control protein IgG monoclonal antibody used as the
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control line capture reagent was diluted at 1 mg/ml in the same buffer. Both test and control
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capture reagents were dispensed in two parallel lines on nitrocellulose membrane at 1 µl/cm.
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Finally, a master card was assembled on a plastic backing with adhesive (Lohmann, The
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Bonding Engineers) and nitrocellulose membrane (Millipore, Ltd) as follows; the conjugated
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pad (Operon, Ltd), sample pad (GFA/D, Operon, Ltd) and absorbent pad (Ahlstrom, Ltd) were
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pasted and covered with a protector film. The master card was then cut to 4.2 mm width strips
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plastic housings (Figure 1 shows a schematic diagram of the lateral flow cassette design). The
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shelf life of the cassettes, as determined experimentally, is one year.
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For the test procedure, for highest sensitivity 80 µl of the CSF sample was added to the round
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window of the LFA device, followed by 120 µl of running buffer (Tris/HCl pH 7.5, NaCl, casein
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and NaN3 as preservative). In the case of a positive sample, the antigen captured by the red
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HP10-latex particle migrates across the test strip by capillarity and is captured by the HP10
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MAb printed on the membrane capture line, resulting in the appearance of a red test line (T
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line). The appearance of a blue control line in the control line (C line) results from capture of
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the blue control protein-coated latex particles by the anti-control protein antibody, and
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indicates that the chromatography has been correctly performed. The entire test procedure is
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complete in 10 minutes. An example of the HP10 Ag-LFA with test positive and negative
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control samples, is presented in Figure 2.
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3. Results
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The results, clearly demonstrate the usefulness of the system for the diagnosis of
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extraparenchymal NCC, showing complete agreement between the HP10 Ag-ELISA, the HP10
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Ag-LFA and the clinical diagnosis (Table 1). Thus all 34 of the CSF samples from cases of active
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parenchymal NCC were positive in both the HP10 Ag-ELISA and the HP10 Ag-LFA, whereas
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respectively 26 and 20 samples from inactive and other neurological conditions respectively,
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were negative. In this study, therefore, there was 100% sensitivity/specificity.
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Assay reproducibility proved good, since testing a pool of CSF samples prepared from HP10 Ag-
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ELISA positive CSF’s, identical positive results were obtained with six consecutive HP10 Ag-LFA
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tests.
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Although the manufacturers (INGENESA Ltd) recommend reading the HP10 Ag-LFA after 10
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minutes, with samples giving an optical density of 0.89-1.70 in the HP10 Ag-ELISA, the positive
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red band was apparent after 2-4 minutes. In other unpublished observations, samples with
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HP10 Ag-ELISA optical densities of 0.4-0.6 may take longer for the appearance of the red
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positive line.
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In summary, cases of NCC, clearly positive by clinical and HP10 Ag-ELISA were confirmed by the
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HP10 Ag-LFA. There were no false positive in the negative controls. The method is extremely
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simple and compatible with “bedside” application by medical staff immediately the collection
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of a CSF sample.
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These results confirm that the presence of the HP10 secreted metacestode glycoprotein in CSF
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is highly indicative of the presence of extraparenchymal cysts. As the MRI analysis was not
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quantitative, unfortunately, we cannot estimate the minimum number of cysts detectable by
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the HP10 Ag-LFA. As can be seen (Table 1) a positive Hp10 Ag-LFA result was obtained with CFS
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samples giving an optical density of 0.89 - 1.7. In other experiments, however, a positive result
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was obtained with CSF samples giving an optical density of 0.25 in the HP10 Ag-ELISA.
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However, a study in Peru showed that the HP10 Ag-ELISA was only negative in NCC patients
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with just a single viable cyst (Garcia et al, 2000).
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Thus, in the hospital situation, where the neurologist routinely takes CSF samples from
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patients exhibiting neurological symptoms, the ease and rapidity of the HP10 Ag-LFA should
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provide a valuable tool for both diagnosis and follow up of patients following drug treatment
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for NCC, and may reduce the need for repetitive and expensive imaging procedures. Further
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exhaustive evaluation is therefore our next priority as is a similar testing of paired serum and
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CSF samples. Clearly, this technology would have a major advantage if applied to serum
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samples. The, previously observed agreement between the HP10 Ag-ELISA in CSF, serum and
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MRI evaluation (Fleury et al, 2007 and 2013) is encouraging in this respect. Finally, as the
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target recognised by the HP10 monoclonal antibody ia a carbohydrate epitope shared by both
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T. solium and Taenia saginata, the HP10 Ag-LFA should also be validated for the diagnosis of
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bovine cysticercosis.
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References
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Bobes, R.J., Hernandez, M., Marquez, C., Fragoso, G., Garcia, E., Parkhouse, R.M.E., Harrison,
238
L.J.S., Sciutto, e., Fleury, A., 2006, Subarachnoid and intraventricular human
240
SC
M AN U
TE D
EP
AC C
239
RI PT
216
neurocysticercosis: application of an antigen detection assay for the diagnosis and follow-up. Trop. Med Int. Health. 11, 943-950.
241
Brandt, J.R., Geerts, S., De Deken, R., Kumar V., Ceulemans F., Brijs L., Falla N., 1992. A
242
monoclonal antibody-based ELISA for the detection of circulating excretory-secretory
243
antigens in Taenia saginata cysticercosis. Int. J. Parasitol. 22, 471-477.
244
Cárdenas G., Carrillo-Mezo R., Jung H., Sciutto E., Hernandez J.L., Fleury A., 2010.
245
Subarachnoidal Neurocysticercosis non-responsive to cysticidal drugs: a case series.
246
B.M.C. Neurol. 4, 10-16.
8
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Carpio A., Matthew L. R., Parkhouse, R.M.E. Short, B., Dua, T., 2016 Parasitic diseases of the
248
central nervous system: lessons for clinicians and policy makers, Expert Review of
249
Neurotherapeutics, 16:4, 401-414, DOI: 10.1586/14737175.2016.1155454 Carrillo Mezo R., Lara García J., Arroyo M., Fleury A., 2015. Relevance of 3D magnetic
251
resonance imaging sequences in diagnosing basal subarachnoid neurocysticercosis.
252
Acta. Trop. 152, 60-65.
253 254
RI PT
250
Deckers N., Dorny P., 2010. Immunodiagnosis of Taenia solium taeniosis/cysticercosis. Trends Parasitol. 26, 137-144.
Ferrer E., González L.M., Martínez-Escribano J.A., González-Barderas M.E., Cortéz M.M., Dávila
256
I., Harrison L.J.S., Parkhouse R.M.E., Gárate T., 2007. Evaluation of recombinant HP6-
257
Tsag, an 18 kDa Taenia saginata oncospheral adhesion protein, for the diagnosis of
258
cysticercosis. Parasitol. Res. Aug. 101, 517-525.
SC
255
Fleury, A., Hernandez, M., Avila, M., Cardenas, G., Bobes, R.J., Huerta, M., Fragoso, G., Uribe-
260
Campero, L., Harrison, L.J.S., Parkhouse, R.M.E., Sciutto, E., 2007, Detection of HP10
261
antigen in serum for diagnosis and follow-up of subarachnoid and intraventricular
262
human neurocysticercosis. J. Neurol, Neurosurg. Psychiatry. 78, 970-974.
M AN U
259
Fleury A., Carrillo-Mezo R., Flisser A., Sciutto E., Corona T. 2011., Subarachnoid basal
264
neurocysticercosis: a focus on the most severe form of the disease. Expert Rev. Anti.
265
Infect. Ther. 9, 123-133.
TE D
263
266
Fleury A., Garcia E., Hernández M., Carrillo R., Govezensky T., Fragoso G., Sciutto E., Harrison
267
L.J.S., Parkhouse R.M.E., 2013. Neurocysticercosis: HP10 antigen detection is useful for
268
the follow-up of the severe patients. PLoS Negl. Trop. Dis. 7, e2096. Garcia, H.H., Harrison, L.J.S., Parkhouse, R.M.E., Montenegro, T., Martinez S.M., Tsang, V.C.W.,
270
Gilamn, R.H., and the Cysticercosis Working Group in Peru., 1998. A specific antigen-
271
detection ELISA for the diagnosis of human neurocysticercosis. Trans. Roy, Soc. Trop.
AC C
272
EP
269
Med Hyg. 92, 411-414.
273
Garcia H.H., Parkhouse R.M.E, Gilman R.H., Montenegro T., Bernal T., Martinez S.M., Gonzalez
274
A.E., Tsang V.C., Harrison L.J.S.; Cysticercosis Working Group in Peru., 2000. Serum
275 276
antigen detection in the diagnosis, treatment, and follow-up of neurocysticercosis patients. Trans. R. Soc. Trop. Med. Hyg. 94, 673-676.
277
Garcia, H.H., Gonzalez, A.E., Gilman, R.H., Bernal, T., Rodriguez, S., Pretell, E.J., Azcurra, O.,
278
Parkhouse, R.M.E., Tsang, V.C., Harrison, L.J.S.; Cysticercosis Working Group in Peru.
279
2002. Circulating parasite antigen in patients with hydrocephalus secondary to
280
neurocysticercosis. Am. J. Trop. Med. Hyg. 66, 427-430.
9
ACCEPTED MANUSCRIPT 281
Graeff-Teixeira
C.,
da
Silva
A.C.,
Yoshimura
K.,
2009.
Update
on
eosinophilic
282
meningoencephalitis and its clinical relevance. Clin. Microbiol. Rev. 22, 322-348
283
Harrison L.J.S., Joshua G.W., Wright S.H., Parkhouse R.M.E., 1989. Specific detection of
284
circulating surface/secreted glycoproteins of viable cysticerci in Taenia saginata
285
cysticercosis. Parasite Immunol. 11, 351-370.
287
Hemanson, G.T., 2013. Microparticles and Nanoparticles. In: Bioconjugate Techniques (Third Edition), Academic Press, pp. 549-587.
RI PT
286
288
Nguekam, Zoli A.P., Ongolo-Zogo P., Dorny P., Brandt J., Geerts S. 2003. Follow-up of
289
neurocysticercosis patients after treatment using an antigen detection ELISA. Parasite.
290
10, 65-68.
Rodriguez S., Dorny P., Tsang V.C., Pretell E.J., Brandt J., Lescano A.G., Gonzalez A.E., Gilman
292
R.H., Garcia H.H.; Cysticercosis Working Group in Peru., 2009. Detection of Taenia
293
solium antigens and anti-T. solium antibodies in paired serum and cerebrospinal fluid
294
samples from patients with intraparenchymal or extraparenchymal neurocysticercosis.
295
J. Infect. Dis. 199, 1345-1352.
M AN U
SC
291
296
WHO, 2015, Third report on neglected tropical diseases: WHO/HTM/NTD/2015. Geneva, 2015.
297
WHO, 2016, Taenia solium taeniasis/cysticercosis diagnostic tools: report of a stakeholder meeting, Geneva, 17-18 December 2015
299
http://www.who.int/iris/handle/10665/206543
302 303 304
36.
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Wilkins P.P. 2013., Immunodiagnosis of CNS parasitic infections. Handb Clin Neurol. 114, 23-
AC C
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Figure and Table captions
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Figure 1. The HP10 lateral flow assay, a schematic diagram
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The red carboxyl latex microparticles covalently coated with MAb HP10 and the blue carboxyl
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latex microparticles covalently coated with the control protein are indicated by the , respectively, and the control protein by CP.
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and
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Example of a positive (red band) HP10 Ag-LFA. The blue band is the negative control.
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Table 1. High level of agreement in the detection of HP10 antigen by the HP10 Ag-ELISA and
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the HP10 Ag-LFA in 80 clinically verified neurological cases (details indicated in the Materials
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and Methods). The assays were done with cerebrospinal fluid sampled during the clinical
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workup of each case. In all of the neurocysticercosis (NCC) cases, all of the observed the Taenia
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solium cysts had an extraparenchymal location. Since all the samples that were positive in the
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HP10 Ag-ELISA were also positive in the HP10 Lateral Flow Assay, the unweighted Kappa
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coefficient was 1.0.
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Figure 1.
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Table 1.
343 HP10 Ag-ELISA No. positives
Optical densities group mean +/- sd (range)
No. positive
34
34
1.75 ± 0.89 (0.45-3.2)
34
26
0
0.07 ± 0.01 (0.01 – 0.1)
0
20
0
0.06 ± 0.01 (0.04 – 0.09)
0
Number
Active extraparenchymal NCC Inactive NCC Other neurological
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Samples
Cases
HP10 Ag-LFA
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No. positives
Optical densities group mean +/- sd (range)
No. positive
34
34
1.75 ± 0.89 (0.45-3.2)
34
26
0
0.07 ± 0.01 (0.01 – 0.1)
0
20
0
0.06 ± 0.01 (0.04 – 0.09)
0
Samples
Active extraparenchymal NCC Inactive extraparenchymal NCC Other neurological
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HP10 Lateral Flow
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Neurocysticercosis (NCC) the invasion of the central nervous system by the metacestodes of Taenia solium, is one of the most important parasite infections with neurological impact, responsible for an estimated 30% of preventable cases of epilepsy and 2.8 million disability adjusted life years. NCC is a ‘silent’ infection with the classical neurological symptoms appearing long after the initial invasion of the brain, when considerable damage has been done. Thus early diagnosis is essential to treatment. The HP10 antigen assay allows, in a simple ELISA procedure (HP10 Ag-ELISA), the detection, in cerebrospinal fluid (CSF), of HP10 antigen, which is secreted by viable metacestodes. Furthermore, the HP10-Ag-ELISA preferentially detects, the more dangerous, extraparenchymally located metacestodes and has now been developed into the form of a rapid Lateral Flow Assay (HP10-LFA). The HP10-LFA was positive for 34/34 CSF samples from cases of active (viable) extraparenchymal NCC and negative for a total of 46 negative control CSF samples. The ease and rapidity of the HP10-LFA should provide a valuable tool for diagnosis and follow-up of NCC cases at hospital/clinic level.
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S0014-4894(16)30277-6
DOI:
10.1016/j.exppara.2016.10.016
Reference:
YEXPR 7326
To appear in:
Experimental Parasitology
Received Date: 7 March 2016 Revised Date:
13 September 2016
Accepted Date: 26 October 2016
Please cite this article as: Fleury, A., Sastre, P., Sciutto, E., Correia, S., Monedero, A., Toledo, A., Hernandez, M., Harrison, L.J.S., Parkhouse, R.M.E., A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.10.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Research brief
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A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using
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cerebrospinal fluid
5 Agnes Fleury, Patricia Sastre, Edda Sciutto, Silvia Correia, Alejandro Monedero, Andrea Toledo,
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Maricela Hernandez, Leslie J.S. Harrison, R. Michael E. Parkhouse.
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Agnes Fleury
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Unidad de Neuroinflamación (Instituto de Investigaciones Biomédicas-UNAM, Instituto
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Nacional de Neurología y Neurocirugia, Facultad de Médicina-UNAM), Insurgentes Sur 3877,
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Colonia La Fama, delegación Tlalpan, Mexico D.F. / Neurocysticercosis clinic, Instituto Nacional
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de Neurología y Neurocirugia, México D.F.
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[email protected]
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Patricia Sastre
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Inmunología y Genética Aplicada S.A. (INGENASA), C/Hermanos García Noblejas 39
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28037 Madrid, Spain
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[email protected]
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20 Edda Sciutto
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Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional
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Autónoma de México, México, D.F. 04510, Mexico
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[email protected]
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Sílvia Correia
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Instituto Gulbenkian de Ciência
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2780-156 Oeiras,
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Portugal
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[email protected]
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Alejandro Monedero
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Inmunología y Genética Aplicada S.A. (INGENASA), C/Hermanos García Noblejas 39
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28037 Madrid, Spain
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(+34) 913680501 1
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Andrea Toledo
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Unidad de Neuroinflammation (Instituto de Investigaciones Biomédicas- UNAM, Instituto
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Nacional de Neurología y Neurocirugia, Facultad de Médicina-UNAM), Insurgentes Sur 3877,
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Colonia La Fama, delegación Tlalpan, Mexico D.F.
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[email protected]
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42 Marisela Hernandez
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Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional
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Autónoma de México, México, D.F. 04510, Mexico
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[email protected]
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47 Leslie J. S. Harrison
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University of Edinburgh, Royal (Dick) School of Veterinary Science
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Easter Bush Veterinary Centre, Easter Bush, ROSLIN, Midlothian, Scotland, UK, EH259RG
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[email protected]
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52 R. Michael E. Parkhouse
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Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal
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[email protected]
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(+351 21 4407939)
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Corresponding author
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ACCEPTED MANUSCRIPT ABSTRACT (94)
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A lateral flow assay (LFA) for the diagnosis and monitoring of extraparenchymal
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neurocysticercosis, has been developed. The assay is based on the use of the monoclonal
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antibody HP10, and when applied to cerebrospinal fluid, correctly identified 34 cases of active
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extraparenchymal neurocysticercosis, but was negative with 26 samples from treated and
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cured neurocysticercosis patients and with 20 samples from unrelated neurological diseases.
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There was complete agreement between the HP10 Ag-ELISA results and the HP10-LFA. The
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HP10-LFA thus has utility for diagnosis and treatment of extraparenchymal neurocysticercosis,
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frequently a more dangerous form of the infection.
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6 keywords – extraparenchymal neurocysticercosis; diagnosis; lateral flow assay; Taenia; HP10;
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NTD
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2919 words (all including legends) 2355 words (text)
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ACCEPTED MANUSCRIPT 1. Introduction
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Neglected Tropical diseases (NTD’s), now recognized by the World Health Organization as a
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serious global health issue, include most of the common parasitic diseases (WHO report,
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2016). Due to their disproportionate prevalence in the poorest of the world’s population, their
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control and elimination have received low priority. Many of these diseases, for example;
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malaria, echinococosis, Taenia solium cysticercosis, and toxoplasmosis have a neurological
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involvement, (Carpio et al, 2016) and this aspect of their clinical and economic impact has
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received even less attention. Typically, parasite infections of the nervous system are “silent”,
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without the classical neurological symptoms (e.g. headache, epilepsy, coma) appearing long
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after the initial invasion of the brain and, importantly, when considerable, sometimes
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irreversible, damage has occurred (Carpio et al, 2016). Thus, early and reliable confirmatory
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diagnosis of these parasite infections, subsequent to clinical and radiological examination, is an
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essential tool in the control and treatment of parasites invading the nervous system.
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This is particularly true in the case of neurocysticercosis (NCC), typically caused by invasion of
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the central nervous system and eyes by the metacestode of Taenia solium, and considered to
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be one of the most important NTD’s with neurological impact (Carpio et al, 2016). Imaging
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studies (Computed Tomography (CT) scan and Magnetic Resonance Imaging (MRI)) are
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considered, by neurologists, as the “gold standard” for NCC diagnosis. Moreover, reliable
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diagnosis of extraparenchymal NCC requires MRI special sequences (FIESTA), not yet
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universally available (Carrillo Mezo et al., 2015). Although, these tools permit the diagnosis in
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most of the cases, particularly when parasites are located in the parenchyma (Fleury et al
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2011, 2013), unfortunately, patients’ access to CT, and MRI, in endemic countries is frequently
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limited or financially impossible. As a result, there has been considerable investigation into the
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development of serological and molecular biological procedures for the diagnosis of NCC
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(Wilkins, 2013). In addition to the commercially available western blot (EITB) and antibody
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tests (WHO, 2015; 2016), a variety of recombinant metacestode antigens and synthetic
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peptides has been tested as targets for detection of antibodies (Deckers and Dorny, 2010;
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Ferrer et al, 2007). Antibody detection, however, is not definitive proof of a current, infection
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with living metacestodes, as antibodies can persist for months, even years, after elimination of
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the parasite. As an alternative to the antibody assays, diagnosis of active cysticercosis through
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the detection of molecules actively secreted by viable metacestodes and found in the
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cerebrospinal fluid (CSF) and serum of NCC patients, has been evaluated for diagnosis and
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follow-up of NCC (Harrison et al, 1989; Brandt et al, 1992, Garcia et al, 1998; Garcia et al, 2000;
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Garcia et al, 2002, Nguekan et al, 2003; Rodriguez et al 2009, Fleury et al 2013). In systematic
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patients, the HP10 Ag-ELISA, which detects the viable metacestode surface/secreted HP10
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antigen (Harrison et al, 1989) was demonstrated to function well for extraparenchymal NCC,
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but was often low or negative for parenchymal NCC (Bobes et al, 2006; Fleury et al, 2007,
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2013).
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From the practical point of view, however, extraparenchymal NCC often has a much more
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threatening pathogenesis than parenchymal (Bobes et al, 2006; Fleury et al, 2007, 2011, 2013)
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and, importantly, imaging studies are less sensitive for the detection of extraparenchymal NCC
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(Graeff-Teixeira et al, 2009; Fleury et al, 2011). A particularly useful application of the HP10 Ag-
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ELISA is the follow-up of extraparenchymal NCC patients after albendazole/praziquantel
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treatment as it helps determine the efficacy of cysticidal treatment. Thus effective drug
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treatment is clearly established by the decreased parasite antigen levels in CSF, whereas
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ineffective treatment, which occurs in a significant number of cases, is revealed by the
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continued presence of the secreted parasite product in the CSF or serum (Garcia et al, 2000;
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Cardenas et al, 2010).
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In this communication we report the successful adaptation of the HP10 Ag-ELISA to a lateral
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flow assay format (HP10 Ag-LFA), using CSF samples from clinically and imagenologically
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characterized patients, and so provide a preliminary validation of its application for the
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clinically important management of extraparenchymal NCC.
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1. Materials and Methods
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1.1 Samples
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Samples of CSF were collected by lumbar puncture from patients attending the Instituto
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Nacional de Neurologia y Neurocirugía (INNN), Mexico City. The study was approved by and
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carried out under the guidelines of the Ethical Committee of the INNN, D.F., México. All
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patients provided written informed consent for the collection of samples and subsequent
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analysis. Diagnosis of viable extraparenchymal NCC was based on clinical manifestations
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(intracranial hypertension, focal deficit, or affection of cranial nerves and intracranial
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hypertension), imaging studies (MRI with FIESTA sequences showing cystic images located in
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subarachnoid cisterns or ventricular system) and cytochemical analysis of CSF showing an
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increased cellular count (>30 cells/mm3) and protein concentrations (>80mg/ml). CSF samples
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from inactive NCC were from patients who had previously presented active extraparenchymal
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NCC, were treated with the cysticidal drug albendazole, and who were demonstrated to lack
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improved (eg. headache, epilepsy, coma) and the CSF samples used to evaluate the HP10 Ag-
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LFA were normal (cells<6/mm3 and protein concentration <40mg/ml). A total of 20 control CSF
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samples were collected from patients with other neurological diseases. Their diagnosis, in
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addition to negative MRI studies for NCC, were as follows: multiple sclerosis (10), optical
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neuritis (2), myelomalacia (2), non-NCC related epilepsy (1), headache (1), tumor (1), subdural
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hematoma (1). Diagnosis of NCC was considered negative based on a combination of MRI
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results, CSF cytochemical analysis and negativity of HP10. All MRI studies were interpreted in a
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double-blind manner by a certified neuroradiologist with extensive experience in NCC
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diagnosis.
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2.2. Lateral Flow ASSAY methodology (HP10-LFA)
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All the CSF samples were assayed by ELISA for the presence of the HP10 secreted metacestode
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antigen (Harrison et al, 1989; Fleury et al, 2013) and tested in the current report in the HP10
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Ag-LFA, immunochromatographic system.
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In brief, the LFA test to detect the HP10 antigen was based on the use of different coloured
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carboxyl-modified latex microspheres, which were covalently linked to the specific target
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proteins or antibodies. Specifically, for the test line, the purified HP10 monoclonal antibody
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was conjugated to red latex particles using EDC (1-ethyl-3-(3-dimethylaminopropyl)
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carbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) to activate the surface of the
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beads (Hermanson, 2013). For the control line, blue latex particles were similarly coated with
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the control protein. Next, the red test and blue control latex particles were diluted in Tris/HCl
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25mM pH 9.5 buffer containing humidity preservatives and blocking agents (3.0% (w/v) bovine
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serum albumin, 1.5% (w/v) casein, 0.35% (w/v) sucrose, 1% (w/v) Tween 20, 0.095% (w/v)
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sodium azide) and the mixture dispensed onto the rayon conjugate pad, using a Matrix 1600
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dispenser (Kinematic Automation, Inc.). For the test capture line, the HP10 MAb was diluted at
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0.6 mg/ml in buffer Tris/HCl 20 mM pH 7.5, containing 5.0% (w/v) sucrose and 0.095% (w/v)
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sodium azide as preservative. The anti-control protein IgG monoclonal antibody used as the
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control line capture reagent was diluted at 1 mg/ml in the same buffer. Both test and control
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capture reagents were dispensed in two parallel lines on nitrocellulose membrane at 1 µl/cm.
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Finally, a master card was assembled on a plastic backing with adhesive (Lohmann, The
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Bonding Engineers) and nitrocellulose membrane (Millipore, Ltd) as follows; the conjugated
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pad (Operon, Ltd), sample pad (GFA/D, Operon, Ltd) and absorbent pad (Ahlstrom, Ltd) were
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pasted and covered with a protector film. The master card was then cut to 4.2 mm width strips
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plastic housings (Figure 1 shows a schematic diagram of the lateral flow cassette design). The
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shelf life of the cassettes, as determined experimentally, is one year.
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For the test procedure, for highest sensitivity 80 µl of the CSF sample was added to the round
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window of the LFA device, followed by 120 µl of running buffer (Tris/HCl pH 7.5, NaCl, casein
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and NaN3 as preservative). In the case of a positive sample, the antigen captured by the red
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HP10-latex particle migrates across the test strip by capillarity and is captured by the HP10
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MAb printed on the membrane capture line, resulting in the appearance of a red test line (T
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line). The appearance of a blue control line in the control line (C line) results from capture of
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the blue control protein-coated latex particles by the anti-control protein antibody, and
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indicates that the chromatography has been correctly performed. The entire test procedure is
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complete in 10 minutes. An example of the HP10 Ag-LFA with test positive and negative
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control samples, is presented in Figure 2.
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3. Results
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The results, clearly demonstrate the usefulness of the system for the diagnosis of
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extraparenchymal NCC, showing complete agreement between the HP10 Ag-ELISA, the HP10
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Ag-LFA and the clinical diagnosis (Table 1). Thus all 34 of the CSF samples from cases of active
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parenchymal NCC were positive in both the HP10 Ag-ELISA and the HP10 Ag-LFA, whereas
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respectively 26 and 20 samples from inactive and other neurological conditions respectively,
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were negative. In this study, therefore, there was 100% sensitivity/specificity.
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Assay reproducibility proved good, since testing a pool of CSF samples prepared from HP10 Ag-
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ELISA positive CSF’s, identical positive results were obtained with six consecutive HP10 Ag-LFA
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tests.
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Although the manufacturers (INGENESA Ltd) recommend reading the HP10 Ag-LFA after 10
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minutes, with samples giving an optical density of 0.89-1.70 in the HP10 Ag-ELISA, the positive
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red band was apparent after 2-4 minutes. In other unpublished observations, samples with
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HP10 Ag-ELISA optical densities of 0.4-0.6 may take longer for the appearance of the red
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positive line.
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In summary, cases of NCC, clearly positive by clinical and HP10 Ag-ELISA were confirmed by the
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HP10 Ag-LFA. There were no false positive in the negative controls. The method is extremely
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simple and compatible with “bedside” application by medical staff immediately the collection
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of a CSF sample.
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These results confirm that the presence of the HP10 secreted metacestode glycoprotein in CSF
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is highly indicative of the presence of extraparenchymal cysts. As the MRI analysis was not
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quantitative, unfortunately, we cannot estimate the minimum number of cysts detectable by
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the HP10 Ag-LFA. As can be seen (Table 1) a positive Hp10 Ag-LFA result was obtained with CFS
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samples giving an optical density of 0.89 - 1.7. In other experiments, however, a positive result
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was obtained with CSF samples giving an optical density of 0.25 in the HP10 Ag-ELISA.
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However, a study in Peru showed that the HP10 Ag-ELISA was only negative in NCC patients
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with just a single viable cyst (Garcia et al, 2000).
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Thus, in the hospital situation, where the neurologist routinely takes CSF samples from
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patients exhibiting neurological symptoms, the ease and rapidity of the HP10 Ag-LFA should
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provide a valuable tool for both diagnosis and follow up of patients following drug treatment
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for NCC, and may reduce the need for repetitive and expensive imaging procedures. Further
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exhaustive evaluation is therefore our next priority as is a similar testing of paired serum and
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CSF samples. Clearly, this technology would have a major advantage if applied to serum
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samples. The, previously observed agreement between the HP10 Ag-ELISA in CSF, serum and
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MRI evaluation (Fleury et al, 2007 and 2013) is encouraging in this respect. Finally, as the
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target recognised by the HP10 monoclonal antibody ia a carbohydrate epitope shared by both
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T. solium and Taenia saginata, the HP10 Ag-LFA should also be validated for the diagnosis of
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bovine cysticercosis.
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References
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Bobes, R.J., Hernandez, M., Marquez, C., Fragoso, G., Garcia, E., Parkhouse, R.M.E., Harrison,
238
L.J.S., Sciutto, e., Fleury, A., 2006, Subarachnoid and intraventricular human
240
SC
M AN U
TE D
EP
AC C
239
RI PT
216
neurocysticercosis: application of an antigen detection assay for the diagnosis and follow-up. Trop. Med Int. Health. 11, 943-950.
241
Brandt, J.R., Geerts, S., De Deken, R., Kumar V., Ceulemans F., Brijs L., Falla N., 1992. A
242
monoclonal antibody-based ELISA for the detection of circulating excretory-secretory
243
antigens in Taenia saginata cysticercosis. Int. J. Parasitol. 22, 471-477.
244
Cárdenas G., Carrillo-Mezo R., Jung H., Sciutto E., Hernandez J.L., Fleury A., 2010.
245
Subarachnoidal Neurocysticercosis non-responsive to cysticidal drugs: a case series.
246
B.M.C. Neurol. 4, 10-16.
8
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Carpio A., Matthew L. R., Parkhouse, R.M.E. Short, B., Dua, T., 2016 Parasitic diseases of the
248
central nervous system: lessons for clinicians and policy makers, Expert Review of
249
Neurotherapeutics, 16:4, 401-414, DOI: 10.1586/14737175.2016.1155454 Carrillo Mezo R., Lara García J., Arroyo M., Fleury A., 2015. Relevance of 3D magnetic
251
resonance imaging sequences in diagnosing basal subarachnoid neurocysticercosis.
252
Acta. Trop. 152, 60-65.
253 254
RI PT
250
Deckers N., Dorny P., 2010. Immunodiagnosis of Taenia solium taeniosis/cysticercosis. Trends Parasitol. 26, 137-144.
Ferrer E., González L.M., Martínez-Escribano J.A., González-Barderas M.E., Cortéz M.M., Dávila
256
I., Harrison L.J.S., Parkhouse R.M.E., Gárate T., 2007. Evaluation of recombinant HP6-
257
Tsag, an 18 kDa Taenia saginata oncospheral adhesion protein, for the diagnosis of
258
cysticercosis. Parasitol. Res. Aug. 101, 517-525.
SC
255
Fleury, A., Hernandez, M., Avila, M., Cardenas, G., Bobes, R.J., Huerta, M., Fragoso, G., Uribe-
260
Campero, L., Harrison, L.J.S., Parkhouse, R.M.E., Sciutto, E., 2007, Detection of HP10
261
antigen in serum for diagnosis and follow-up of subarachnoid and intraventricular
262
human neurocysticercosis. J. Neurol, Neurosurg. Psychiatry. 78, 970-974.
M AN U
259
Fleury A., Carrillo-Mezo R., Flisser A., Sciutto E., Corona T. 2011., Subarachnoid basal
264
neurocysticercosis: a focus on the most severe form of the disease. Expert Rev. Anti.
265
Infect. Ther. 9, 123-133.
TE D
263
266
Fleury A., Garcia E., Hernández M., Carrillo R., Govezensky T., Fragoso G., Sciutto E., Harrison
267
L.J.S., Parkhouse R.M.E., 2013. Neurocysticercosis: HP10 antigen detection is useful for
268
the follow-up of the severe patients. PLoS Negl. Trop. Dis. 7, e2096. Garcia, H.H., Harrison, L.J.S., Parkhouse, R.M.E., Montenegro, T., Martinez S.M., Tsang, V.C.W.,
270
Gilamn, R.H., and the Cysticercosis Working Group in Peru., 1998. A specific antigen-
271
detection ELISA for the diagnosis of human neurocysticercosis. Trans. Roy, Soc. Trop.
AC C
272
EP
269
Med Hyg. 92, 411-414.
273
Garcia H.H., Parkhouse R.M.E, Gilman R.H., Montenegro T., Bernal T., Martinez S.M., Gonzalez
274
A.E., Tsang V.C., Harrison L.J.S.; Cysticercosis Working Group in Peru., 2000. Serum
275 276
antigen detection in the diagnosis, treatment, and follow-up of neurocysticercosis patients. Trans. R. Soc. Trop. Med. Hyg. 94, 673-676.
277
Garcia, H.H., Gonzalez, A.E., Gilman, R.H., Bernal, T., Rodriguez, S., Pretell, E.J., Azcurra, O.,
278
Parkhouse, R.M.E., Tsang, V.C., Harrison, L.J.S.; Cysticercosis Working Group in Peru.
279
2002. Circulating parasite antigen in patients with hydrocephalus secondary to
280
neurocysticercosis. Am. J. Trop. Med. Hyg. 66, 427-430.
9
ACCEPTED MANUSCRIPT 281
Graeff-Teixeira
C.,
da
Silva
A.C.,
Yoshimura
K.,
2009.
Update
on
eosinophilic
282
meningoencephalitis and its clinical relevance. Clin. Microbiol. Rev. 22, 322-348
283
Harrison L.J.S., Joshua G.W., Wright S.H., Parkhouse R.M.E., 1989. Specific detection of
284
circulating surface/secreted glycoproteins of viable cysticerci in Taenia saginata
285
cysticercosis. Parasite Immunol. 11, 351-370.
287
Hemanson, G.T., 2013. Microparticles and Nanoparticles. In: Bioconjugate Techniques (Third Edition), Academic Press, pp. 549-587.
RI PT
286
288
Nguekam, Zoli A.P., Ongolo-Zogo P., Dorny P., Brandt J., Geerts S. 2003. Follow-up of
289
neurocysticercosis patients after treatment using an antigen detection ELISA. Parasite.
290
10, 65-68.
Rodriguez S., Dorny P., Tsang V.C., Pretell E.J., Brandt J., Lescano A.G., Gonzalez A.E., Gilman
292
R.H., Garcia H.H.; Cysticercosis Working Group in Peru., 2009. Detection of Taenia
293
solium antigens and anti-T. solium antibodies in paired serum and cerebrospinal fluid
294
samples from patients with intraparenchymal or extraparenchymal neurocysticercosis.
295
J. Infect. Dis. 199, 1345-1352.
M AN U
SC
291
296
WHO, 2015, Third report on neglected tropical diseases: WHO/HTM/NTD/2015. Geneva, 2015.
297
WHO, 2016, Taenia solium taeniasis/cysticercosis diagnostic tools: report of a stakeholder meeting, Geneva, 17-18 December 2015
299
http://www.who.int/iris/handle/10665/206543
302 303 304
36.
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Wilkins P.P. 2013., Immunodiagnosis of CNS parasitic infections. Handb Clin Neurol. 114, 23-
AC C
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Figure and Table captions
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Figure 1. The HP10 lateral flow assay, a schematic diagram
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The red carboxyl latex microparticles covalently coated with MAb HP10 and the blue carboxyl
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latex microparticles covalently coated with the control protein are indicated by the , respectively, and the control protein by CP.
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and
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Example of a positive (red band) HP10 Ag-LFA. The blue band is the negative control.
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Table 1. High level of agreement in the detection of HP10 antigen by the HP10 Ag-ELISA and
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the HP10 Ag-LFA in 80 clinically verified neurological cases (details indicated in the Materials
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and Methods). The assays were done with cerebrospinal fluid sampled during the clinical
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workup of each case. In all of the neurocysticercosis (NCC) cases, all of the observed the Taenia
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solium cysts had an extraparenchymal location. Since all the samples that were positive in the
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HP10 Ag-ELISA were also positive in the HP10 Lateral Flow Assay, the unweighted Kappa
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coefficient was 1.0.
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Figure 1.
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Table 1.
343 HP10 Ag-ELISA No. positives
Optical densities group mean +/- sd (range)
No. positive
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1.75 ± 0.89 (0.45-3.2)
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26
0
0.07 ± 0.01 (0.01 – 0.1)
0
20
0
0.06 ± 0.01 (0.04 – 0.09)
0
Number
Active extraparenchymal NCC Inactive NCC Other neurological
SC
conditions
RI PT
Samples
Cases
HP10 Ag-LFA
344
AC C
EP
TE D
M AN U
345
13
ACCEPTED MANUSCRIPT HP10 Ag-ELISA No.
No. positives
Optical densities group mean +/- sd (range)
No. positive
34
34
1.75 ± 0.89 (0.45-3.2)
34
26
0
0.07 ± 0.01 (0.01 – 0.1)
0
20
0
0.06 ± 0.01 (0.04 – 0.09)
0
Samples
Active extraparenchymal NCC Inactive extraparenchymal NCC Other neurological
AC C
EP
TE D
M AN U
SC
conditions
RI PT
Cases description
HP10 Lateral Flow
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Bullet Points
•
RI PT
SC
•
M AN U
•
TE D
•
EP
•
Neurocysticercosis (NCC) the invasion of the central nervous system by the metacestodes of Taenia solium, is one of the most important parasite infections with neurological impact, responsible for an estimated 30% of preventable cases of epilepsy and 2.8 million disability adjusted life years. NCC is a ‘silent’ infection with the classical neurological symptoms appearing long after the initial invasion of the brain, when considerable damage has been done. Thus early diagnosis is essential to treatment. The HP10 antigen assay allows, in a simple ELISA procedure (HP10 Ag-ELISA), the detection, in cerebrospinal fluid (CSF), of HP10 antigen, which is secreted by viable metacestodes. Furthermore, the HP10-Ag-ELISA preferentially detects, the more dangerous, extraparenchymally located metacestodes and has now been developed into the form of a rapid Lateral Flow Assay (HP10-LFA). The HP10-LFA was positive for 34/34 CSF samples from cases of active (viable) extraparenchymal NCC and negative for a total of 46 negative control CSF samples. The ease and rapidity of the HP10-LFA should provide a valuable tool for diagnosis and follow-up of NCC cases at hospital/clinic level.
AC C
•