The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log ₁₀ inactivation is achieved with 4 mJ/cm ² UV and 10 mJ/cm ² low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log ₁₀ inactivation of T. gondii oocysts is achieved by 10-mJ/cm ² low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.

Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii which can lead to severe sequelae in the fetus during pregnancy. Definitive serologic diagnosis of the infection during gestation is mostly made by detecting T. gondii specific antibodies including immunoglobulin G (IgG) and immunoglobulin M (IgM) individually in a single serum using commercially available kits. The immunoglobulin A (IgA) test is used by some laboratories as an additional marker of an acute infection. Most of the commercial test results have failed to reach 100% of correlation with the reference method of Sabin Feldman dye test for the detection of Toxoplasma IgG antibodies. For Toxoplasma IgM and IgA antibodies, there is no reference method and their evaluations are done by comparing one assay to another. There is a need for multiplexed assay platforms as the serological diagnosis of T. gondii infection does not rely on a sole subtype of Ig detection. Here we describe the development of a plasmonic gold chip with vast fluorescence enhancement in the near-infrared (NIR) region for simultaneous detection of IgG, IgM and IgA antibodies against T. gondii in ∼ 1 microliter of serum or whole blood sample. By testing 168 samples on this platform, IgG antibodies detection sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) all afforded 100%. The IgM antibodies detection achieved 97.6% sensitivity and 96.9% specificity with 90.9% PPV and 99.2% NPV. Thus, the nanoscience based plasmonic gold platform enables a high performance, low cost and multiplexed assay requiring ultra-small blood volumes, paving the way to implement universal screening for toxoplasmosis infection during gestation.

Swimming in fecally-contaminated waterbodies can result in gastrointestinal infections. However, the pathogenic microorganisms responsible are not well understood because sporadic cases of illness are not reported completely, exposure information is often not collected, and epidemiology studies rely on self-reported symptoms. Noroviruses are considered a likely cause because they are found in high densities in sewage, resistant to wastewater treatment and survive in the environment. In this study, saliva samples were collected from subjects at a beach in Puerto Rico and tested for evidence of norovirus-specific IgG responses as an indicator of incident norovirus infection. Saliva samples were collected from 1298 participants using an oral swab. Samples were collected on the day of the beach visit (S1); after 10–12 days (S2); and after three weeks (S3). Saliva was tested for IgG responses to GI.1 and GII.4 noroviruses using a microsphere based multiplex salivary immunoassay. Immunoconversion was defined as a four-fold increase in median fluorescence intensity (MFI) from S1 to S2 with the S3 sample at least three times above the S1 MFI. Thirty-four subjects (2.6%) immunoconverted to GI.1 or GII.4 norovirus. Swimmers who immersed their head in water had a higher rate of immunoconversion (3.4%), compared to either non-swimmers (0.0%, p = 0.003) or waders and non-swimmers combined (0.4%, Odds Ratio: 5.07, 95% Confidence Interval:1.48–17.00). Immunoconversion was not associated with gastrointestinal symptoms. This is the first study to demonstrate an association between swimming at a beach impacted by fecal contamination and asymptomatic norovirus infection. The findings implicate recreational water as potentially important transmission pathway for norovirus infection.

Norovirus is one of the most common causes of gastroenteritis. Following infection, anti-norovirus salivary immunoglobulin G (IgG) rises steeply within 2 weeks and remains elevated for several months; this immunoconversion can serve as an indicator of infection. We used a multiplex salivary immunoassay to study norovirus infections among 483 visitors to a Lake Michigan beach in 2015. Saliva was collected on the day of the beach visit (S1); after 10–14 days (S2); and after 30–40 days (S3). Luminex microspheres were coupled to recombinant antigens of genogroup I (GI) and II (GII) noroviruses and incubated with saliva. Immunoconversion was defined as at least 4-fold increase in anti-norovirus IgG antibody response from S1 to S2 and a 3-fold increase from S1 to S3. Ten (2.1%) immunoconverted to either GI (2) or GII (8) norovirus. Among those who immunoconverted, 40% reported at least one gastrointestinal symptom and 33% reported diarrhea, compared to 15% (p = 0.06) and 8% (p = 0.04) among those who did not immunoconvert, respectively. The two participants who immunoconverted to GI norovirus both swallowed water during swimming (p = 0.08). This study demonstrated the utility of a non-invasive salivary immunoassay to detect norovirus infections and an efficient approach to study infectious agents in large cohorts.

In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.