Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles.

BACKGROUND: Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging 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-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially 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 neurological 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-monitoring (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.

Cristaluria por sulfadiazina en paciente con toxoplasmosis diseminada / Sulphadiazine crystaluria in a patient with disseminated toxoplasmosis

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Toward detection of toxoplasmosis from urine in mice using hydro-gel nanoparticles concentration and parallel reaction monitoring mass spectrometry.

Diagnosis of clinical toxoplasmosis remains a challenge, thus limiting the availability of human clinical samples. Though murine models are an approximation of human response, their definitive infection status and tissue availability make them critical to the diagnostic development process. Hydrogel mesh nanoparticles were used to concentrate antigen to detectable levels for mass spectrometry. Seven Toxoplasma gondii isolates were used to develop a panel of potential peptide sequences for detection by parallel reaction monitoring (PRM) mass spectrometry. Nanoparticles were incubated with decreasing concentrations of tachyzoite lysate to explore the limits of detection of PRM. Mice whose toxoplasmosis infection status was confirmed by quantitative real-time PCR had urine tested by PRM after hydrogel mesh concentration for known T. gondii peptides. Peptides from GRA1, GRA12, ROP4, ROP5, SAG1, and SAG2A proteins were detected by PRM after nanoparticle concentration of urine, confirming detection of T. gondii antigen in the urine of an infected mouse.

[Real-time fluorescence quantitative polymerase chain reaction detection for Toxoplasma gondii B1 gene in mice urine].

A pair of specific primers and a TaqMan probe were designed based on the sequence of Toxoplasma gondii B1 gene from GenBank database. Total DNA of T. gondii was extracted from fresh mice urine. DNA fragment of B1 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. Following identification, the positive recombinant plasmid was used as reference template to generate standard curve and melt curve. Sensitivity, reproducibility, linear range and stability of reference plasmids were determined. The sensitivity of this method was 10(4) copies/ml. The coefficient of variation (cv) of intra-assay and inter-assay were 2.42% and 4.18%, respectively. Linear range was (10(3)-10(7)) copies/ml. The specificity was 100%. The reference materials were stable. Real-time FQ-PCR of T. gondii DNA in mice urine has been constructed, which is a convenient, sensitive and reliable method for quantifying T. gondii DNA in mice urine.

Development of membrane-based tests for the detection of urinary antigens and antibodies in human toxoplasmosis: preliminary studies in Ghanaian patients.

Two membrane-based ELISA systems were used in detecting Toxoplasma antigens and anti-Toxoplasma antibodies in urine samples collected from 54 ophthalmology (22 suggestive active and 32 suggestive past infection) patients and 26 pregnant women attending obstetrics/gynaecology clinic (OGP), suspected of toxoplasmosis by eye examination, past medical records and questionnaire, respectively, in Ghana from mid-February to April 2002. The antigen detecting ELISA was able to demonstrate antigen in 100% (22/22) ophthalmology (active infection) and 62.5% (20/32) ophthalmology (past infection) patients, and 42% (11/26) of OGP which included 3 that were sero-negative prior to and during this study, giving an overall prevalence of 66.3% (53/80). The urinary antigen positive samples also included 6 that were negative for both the Dye Test (DT) and latex agglutination test (LAT). Antigen was not detected in the urine of 22 normal (sero-negative for antibodies to Toxoplasma) individuals. The membrane-based urinary antibody detecting sandwich ELISA also detected anti-Toxoplasma antibodies in 100% (22/22) of ophthalmology (active infection) and 81.3% (26/32) of ophthalmology (past infection) patients, a total of 89% (48/54); and 80.8% (21/26) of OGP with an overall prevalence of 86.3% (69/80), including 7 ophthalmology patients' samples that were sero-negative for both DT and LAT. Antibody sero-positivity of the samples was determined by DT as 87% (47/54) in ophthalmology patients and 73.1% (19/26) in pregnant women, LAT as 85.2% (46/54) and 65.4% (17/26), and an overall prevalence as 82.5% (66/80) and 78.8% (63/80), respectively. The membrane-based ELISA systems appear promising but need to be investigated further for its efficacy as reliable diagnostic tests.

Toxoplasmosis surveillance during pregnancy and quality assurance of methods in Hungary.

In Hungary, screening programs have been performed for the early detection of toxoplasmosis in pregnant women in three different counties. The results of a screening program performed in the town of Szeged are discussed in details. The pregnant women are screened by serological and molecular biological methods (anti-Toxoplasma CFT, IgG, IgM, anti-P30 IgA ELISA, IgG avidity test, PCR amplification). The women are first screened within the first 16 weeks of gestation. Seronegative cases are retested for seroconversion in every second month. Appropriate treatment is immediately started both in the mothers suspicious of acute toxoplasmosis and in their offspring. The urine samples of the babies are examined by nested PCR specific to B1 gene of Toxoplasma gondii. No cases of congenital toxoplasmosis have been detected among the screened and treated children so far. Thus, we consider the program as highly successful for screening of congenital toxoplasmosis. To insure the quality of the applied laboratory diagnostic methods, the QualiCont Company organizes two quality control investigations yearly in the laboratories involved.

[Acute toxoplasmosis. Laboratory diagnosis]. / Toxoplasmosis aguda. Diagnóstico de laboratorio.

BACKGROUND: The microbiological diagnosis of acute toxoplasmosis in immunosuppressed patients, cannot, usually, be done through serological techniques. The methods for isolation or direct detection of parasite are the only ones that might be helpful to determine an earlier diagnosis, on these patients. METHODS AND RESULTS: The use of urine in 3 patients with acute toxoplasmosis, allowed us to isolate the parasite after inoculation of the sample in human embryonic fibroblast cell line (MRC-5). CONCLUSION: In our opinion urine could be an adequate sample for isolation or detection of Toxoplasma gondii in immunosuppressed patients, and an alternative to serological methods, as well.