Detection of toxoplasmic encephalitis in hiv positive patients in urine with hydrogel nanoparticles

Toxoplasmosis Working Group in Peru and Bolivia

doi:10.1371/journal.pntd.0009199

Detection of toxoplasmic encephalitis in hiv positive patients in urine with hydrogel nanoparticles

Toxoplasmosis Working Group in Peru and Bolivia

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

Background Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circum-stances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imag-ing technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Methology/principle findings Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopro-pylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substan-tially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurologi-cal symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/ 112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitor-ing (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Conclusion/significances Our results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

Original language	English
Article number	e0009199
Journal	PLoS Neglected Tropical Diseases
Volume	15
Issue number	3
DOIs	https://doi.org/10.1371/journal.pntd.0009199
State	Published - Mar 2021

Bibliographical note

Funding Information:
Robert H. Gilman supported staff salaries and laboratory supplies in this investigation with NIAID R01 (AI136722-01). Robert H. Gilman also supported the training of many of the Peruvian and Bolivian authors and working group members with 1D43TW010074-01. Natalie M. Bowman supported sample collection and was supported by a NIAID K23 award (AI113197), UNC CFAR P30 AI50410, Fogarty R25TW009340, and Burroughs Wellcome Fund/ASTMH Postdoctoral Fellowship in Tropical Infectious Diseases. Alessandra Luchini supported staff salaries and laboratory supplies in this investigation with NIAID R21 (AI138135). Lance A. Loitta supported staff salaries and laboratory supplies in this investigation with NICHD R21 (HD0974720). Hannah E. Steinberg was supported by NIH Fogarty International Center Grant #D43TW009340. Cusi Ferradas and Monica Diaz were supported by the Fogarty International Center of the National Institutes of Health under Award Number D43TW009343 and the University of California Global Health Institute. The funders did not play any role in the study design, data collection and analysis, the decision to publish or the preparation of manuscript.

Publisher Copyright:
© 2021 Steinberg et al.

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10.1371/journal.pntd.0009199

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title = "Detection of toxoplasmic encephalitis in hiv positive patients in urine with hydrogel nanoparticles",

abstract = "Background Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circum-stances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imag-ing technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Methology/principle findings Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopro-pylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substan-tially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurologi-cal symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/ 112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitor-ing (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Conclusion/significances Our results demonstrate nanoparticle technology{\textquoteright}s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.",

author = "{Toxoplasmosis Working Group in Peru and Bolivia} and Steinberg, {Hannah E.} and Bowman, {Natalie M.} and Andrea Diestra and Cusi Ferradas and Paul Russo and Clark, {Daniel E.} and Deanna Zhu and Ruben Magni and Edith Malaga and Monica Diaz and Viviana Pinedo-Cancino and Asayag, {Cesar Ramal} and Maritza Calder{\'o}n and Carruthers, {Vern B.} and Liotta, {Lance A.} and Gilman, {Robert H.} and Alessandra Luchini and Caryn Bern and Eduardo Ticona and Andres Lescano and Juan Jimenez and Mauricio Dorn and Kirwan, {Daniela E.} and Lilia Cabrera and Chasnamote, {Mar{\'i}a V{\'a}squez} and Langer, {Rafael Saavedra} and Urquizo, {Marilly Donayre} and Pinedo, {Linda Chanam{\'e}} and Jeroen Bok and Gaston Pinedo and Melanie Ayachi and Francesca Schiaffino and Renzo Gutierrez-Loli and Melissa Reimer-Mcatee and Meredith Holtz and Taryn Clark and Grace Trompeter and Jeong Choi and Omar Gandarilla and Enzo Fortuny and Anne Palumbo and Gerson Galdos and Roni Colanzi and Raquel Mugruza and Cesar Ticona and Paola Rondan and Aliki Traianou and Jonathan Juliano and Steven Meshnick and Faustino Torrico",

note = "Publisher Copyright: {\textcopyright} 2021 Steinberg et al.",

year = "2021",

month = mar,

doi = "10.1371/journal.pntd.0009199",

language = "Ingl{\'e}s",

volume = "15",

journal = "PLoS Neglected Tropical Diseases",

issn = "1935-2727",

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number = "3",

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TY - JOUR

T1 - Detection of toxoplasmic encephalitis in hiv positive patients in urine with hydrogel nanoparticles

AU - Toxoplasmosis Working Group in Peru and Bolivia

AU - Steinberg, Hannah E.

AU - Bowman, Natalie M.

AU - Diestra, Andrea

AU - Ferradas, Cusi

AU - Russo, Paul

AU - Clark, Daniel E.

AU - Zhu, Deanna

AU - Magni, Ruben

AU - Malaga, Edith

AU - Diaz, Monica

AU - Pinedo-Cancino, Viviana

AU - Asayag, Cesar Ramal

AU - Calderón, Maritza

AU - Carruthers, Vern B.

AU - Liotta, Lance A.

AU - Gilman, Robert H.

AU - Luchini, Alessandra

AU - Bern, Caryn

AU - Ticona, Eduardo

AU - Lescano, Andres

AU - Jimenez, Juan

AU - Dorn, Mauricio

AU - Kirwan, Daniela E.

AU - Cabrera, Lilia

AU - Chasnamote, María Vásquez

AU - Langer, Rafael Saavedra

AU - Urquizo, Marilly Donayre

AU - Pinedo, Linda Chanamé

AU - Bok, Jeroen

AU - Pinedo, Gaston

AU - Ayachi, Melanie

AU - Schiaffino, Francesca

AU - Gutierrez-Loli, Renzo

AU - Reimer-Mcatee, Melissa

AU - Holtz, Meredith

AU - Clark, Taryn

AU - Trompeter, Grace

AU - Choi, Jeong

AU - Gandarilla, Omar

AU - Fortuny, Enzo

AU - Palumbo, Anne

AU - Galdos, Gerson

AU - Colanzi, Roni

AU - Mugruza, Raquel

AU - Ticona, Cesar

AU - Rondan, Paola

AU - Traianou, Aliki

AU - Juliano, Jonathan

AU - Meshnick, Steven

AU - Torrico, Faustino

PY - 2021/3

Y1 - 2021/3

N2 - Background Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circum-stances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imag-ing technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Methology/principle findings Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopro-pylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substan-tially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurologi-cal symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/ 112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitor-ing (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Conclusion/significances Our results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

AB - Background Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circum-stances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imag-ing technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Methology/principle findings Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopro-pylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substan-tially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurologi-cal symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/ 112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitor-ing (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Conclusion/significances Our results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

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U2 - 10.1371/journal.pntd.0009199

DO - 10.1371/journal.pntd.0009199

M3 - Artículo

C2 - 33651824

AN - SCOPUS:85102965112

SN - 1935-2727

VL - 15

JO - PLoS Neglected Tropical Diseases

JF - PLoS Neglected Tropical Diseases

IS - 3

M1 - e0009199

ER -

Detection of toxoplasmic encephalitis in hiv positive patients in urine with hydrogel nanoparticles

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