A Multidisciplinary Approach to Improving Diagnostics for Neurological Toxoplasmosis

Toxoplasma gondii is an obligate intracellular parasite that infects up to a third of the world’s population. It is an infection of minimal clinical consequence for most immunocompetent individuals. However, in the immunosuppressed, most classically those persons with HIV and a CD4 count less than 100, a severe neurological syndrome can develop. Neurological toxoplasmosis most commonly develops from reactivation of a prior infection due to insufficient immune system management of T. gondii. Diagnosis of neurological toxoplasmosis is challenging. It is clinically difficult to identify because of its similar presentation to several other neurological opportunistic infections common to persons living with HIV. Specific pathogen testing for neurological toxoplasmosis is of variable success, with increasing sensitivity as the invasiveness of the biological specimen collection and infrastructure requirements increases. Thus, serology from serum, is of limited utility because many persons are asymptomatically infected with T. gondii years prior to reactivation of the infection. Quantitative polymerase chain reaction (qPCR) in blood has poor sensitivity (1-50%); while qPCR in CSF has improved but still limited sensitivity (35- 70%). Finally stereotactic brain biopsy and visualization of T. gondii in the specimen is the gold standard of diagnosis- but hardly employed because of the risk to the patient. This thesis focuses on improving diagnostic options for neurological toxoplasmosis. Our first study attempts to use a non-invasive biological specimen (urine) to identify cases of neurological toxoplasmosis in a cohort of 216 HIV positive individuals. Through this work we successfully develop a western blot assay for two T. gondii antigens, surface antigen one (SAG1) and dense granule one (GRA1). GRA1 is demonstrated to correlate with neurological toxoplasmosis, while SAG1 does not. Our second study attempts to move the western blot urine diagnostic from the first study into a low resource friendly format- a sandwich lateral flow assay. Focusing solely on the inclusion of GRA1 in this assay, we initially develop an assay that could detect recombinant GRA1 but fails to detect GRA1 in patient samples. Several hypotheses are explored to evaluate the properties of GRA1 and what drove the difference in assay outcomes between the recombinant and patient derived GRA1. Findings include confirmation of: an N-linked glycan on GRA1; GRA1’s binding partner surface antigen two A; modification of GRA1’s secondary structure in response to changes in pH; and identification of the immunodominant region of GRA1. Our third study focuses on improving the clinical definition for neurological toxoplasmosis while exploring Next Generation Sequencing (NGS) as an alternative to specific pathogen testing for not just neurological toxoplasmosis but other opportunistic infections. Though we were not successful in improving the current clinical definition for neurological toxoplasmosis; NGS was able to identify several opportunistic infections. Overall, these studies enhance our understanding of the challenges and areas for future advancement in the diagnosis of neurological toxoplasmosis.