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 languageEnglish
Article numbere0009199
JournalPLoS Neglected Tropical Diseases
Issue number3
StatePublished - 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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