Using Serum Specimens for Real-Time PCR-Based Diagnosis of Human Granulocytic Anaplasmosis, Canada.

Whole blood is the optimal specimen for anaplasmosis diagnosis but might not be available in all cases. We PCR tested serum samples collected in Canada for Anaplasma serology and found 84.8%-95.8% sensitivity and 2.8 average cycle threshold elevation. Serum can be acceptable for detecting Anaplasma spp. when whole blood is unavailable.

Proteomic Screen of Brain Glycoproteome Reveals Prion Specific Marker of Pathogenesis.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE-infected and aged-matched control mice using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Since the brain proteome is too complex to resolve all proteins using 2D-DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat-germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin-3 (CNN3), peroxiredoxin-6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE-infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.

Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.

BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

Endpoint Quaking-Induced Conversion: a Sensitive, Specific, and High-Throughput Method for Antemortem Diagnosis of Creutzfeldt-Jacob Disease.

The Prion Laboratory Section of the Public Health Agency of Canada supports heath care professionals dealing with patients suspected to have Creutzfeldt-Jakob disease (CJD) by testing cerebrospinal fluid (CSF) for protein markers of CJD. To better serve Canadian diagnostic requirements, a quaking-induced conversion (QuIC)-based assay has been added to the test panel. The QuIC tests exploit the ability of disease-associated prion protein, found in the CSF of a majority of CJD patients, to convert a recombinant prion protein (rPrP) into detectable amounts of a misfolded, aggregated form of rPrP. The rPrP aggregates interact with a specific dye, causing a measurable change in the dye's fluorescence emission spectrum. Optimal test and analysis parameters were empirically determined. Taking both practical and performance considerations into account, an endpoint QuIC (EP-QuIC) configuration was chosen. EP-QuIC uses a thermo-mixer to perform the shaking necessary to produce the quaking-induced conversions. Fluorescence readings are obtained from a microwell fluorescence reader only at the beginning and the end of EP-QuIC reactions. Samples for which the relative fluorescence unit ratio between the initial and final readings represent a ≥4 increase in signal intensity in at least two of the three replicates are classified as positive. A retrospective analysis of 91 CSF samples that included 45 confirmed cases of CJD and 46 non-CJD cases was used to estimate the performance characteristics of the EP-QuIC assay. The diagnostic sensitivity and specificity of the EP-QuIC test of this set of samples were 98 and 91%, respectively.

Molecular surveillance of microbial agents from cattle-attached and questing ticks from livestock agroecosystems of Antioquia, Colombia.

Ticks are obligate ectoparasites and vectors of pathogens affecting health, agriculture, and animal welfare. This study collected ticks from the cattle and questing ticks of 24 Magdalena Medio Antioquia region cattle farms. Genomic DNA was extracted from the specimens (individual or pools) of the 2088 adult ticks collected from cattle and 4667 immature questing ticks collected from pastures. The molecular detection of Babesia, Anaplasma, Coxiella and Rickettsia genera was performed by polymerase chain reaction amplification and subsequent DNA sequencing. In a total of 6755 Rhipicephalus microplus DNA samples, Anaplasma marginale was the most detected with a frequency of 2% (Confidence Interval- CI 1.68-2.36), followed by Babesia bigemina with 0.28% (CI 0.16-0.44), Coxiella spp. with 0.15% (CI 0.07-0.27), and Rickettsia spp. with 0.13% (CI 0.06-0.25). Molecular analysis of the DNA sequences obtained from the tick samples revealed the presence of Coxiella-like endosymbiont and R. felis. These results demonstrated the diversity of microorganisms present in R. microplus ticks predominantly associated with cattle and questing ticks from livestock agroecosystems, suggesting their role as reservoirs and potential biological vectors of these microorganisms on the studied sites. Also, it emphasizes the need to combine acarological surveillance with clinical diagnoses and control strategies on regional and national levels.

Stability of SARS-CoV-2 IgG in multiple laboratory conditions and blood sample types.

BACKGROUND: COVID-19 seroprevalence studies use serum/plasma samples to detect SARS-CoV-2 IgG. Data supporting alternate specimen types and freeze-thaw antibody stability is lacking. The stability of IgG and other immunoglobulins in multiple blood sample types stored in differing conditions and multiple freeze-thaw cycles (FTCs) was evaluated. MATERIALS AND METHODS: Serum, plasma, and heparinized-plasma samples were collected from COVID-19 recovered individuals. Samples underwent testing for SARS-CoV-2 antibodies upon collection, after each of 10-12 FTCs, and storage at -70°C, -20°C, 4°C, and room-temperature for 10-12 days using four high-throughput commercial assays, two rapid-test cassettes, a manual ELISA, and a surrogate neutralization assay. RESULTS: All three specimen types were collected from 34 COVID-19 recovered seropositive individuals (≥21 days post-symptoms). Using the Architect and Liaison assays, a positive qualitative SARS-CoV-2 IgG result was detected daily up to 12 FTCs and up to 10 days of storage at different temperatures. An additional 25 plasma samples consistently demonstrated detection of SARS-CoV-2 antibodies daily after 12 FTCs and storage at -20°C using two rapid test cassette assays (SD Biosensor and Hangzhou All Test), manual (Beijing Wantai) and surrogate neutralization (GenScript) ELISAs, and two high-throughput assays (Roche Elecsys nucleocapsid and spike). IgM antibodies were less frequently detected by one of the rapid test cassette assays. CONCLUSIONS: Serum, plasma, and heparinized-plasma constitute reliable samples for SARS-CoV-2 antibody detection. In particular, the IgG response was stable and reliably detected after multiple FTCs and storage at common laboratory conditions. IgM detection was variable due to the labile nature of this antibody class.