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The true severity of infection due to COVID-19 is under-represented because it is based on only those who are tested. Although nucleic acid amplifications tests (NAAT) are the gold standard for COVID-19 diagnostic testing, serological assays provide better population-level SARS-CoV-2 prevalence estimates. Implementing large sero-surveys present several logistical challenges within Canada due its unique geography including rural and remote communities. Dried blood spot (DBS) sampling is a practical solution but comparative performance data on SARS-CoV-2 serological tests using DBS is currently lacking. Here we present test performance data from a well-characterized SARS-CoV-2 DBS panel sent to laboratories across Canada representing 10 commercial and 2 in-house developed tests for SARS-CoV-2 antibodies. Three commercial assays identified all positive and negative DBS correctly corresponding to a sensitivity, specificity, positive predictive value, and negative predictive value of 100% (95% CI = 72.2, 100). Two in-house assays also performed equally well. In contrast, several commercial assays could not achieve a sensitivity greater than 40% or a negative predictive value greater than 60%. Our findings represent the foundation for future validation studies on DBS specimens that will play a central role in strengthening Canada’s public health policy in response to COVID-19.

With the need to expedite the detection of multiple sexually transmitted blood-borne infections (STBBIs), there is an increased demand for digital innovations and tests that improve the efficiency of point-of-care testing in outreach community settings. Multiplexed testing is being offered to at-risk populations by frontline health care professionals. With this in mind, we evaluated AideSmart! (for health aides or health care workers), an integrated app and platform solution that enables multiplexed STBBI screening at the point of care, for feasibility, preference, accuracy, and impact. With AideSmart!, we provided trained health care workers with the ability to perform rapid multiplexed tests; offer STBBI pre- or posttest counseling; maintain quality assurance of testing; facilitate linkages to care; and enhance health care provider–patient communication, data documentation, and patient engagement through the multiplexed STBBI screening and linkage process. We evaluated the performance of multiplexed tests. In a cross-sectional study conducted during the COVID-19 pandemic, we recruited at-risk populations in Canada from community-based organizations in Montreal, Quebec, and Saint John, New Brunswick, with unknown serostatus for HIV, hepatitis C virus (HCV), and syphilis. Following orientation and pretest counseling with AideSmart!, we performed multiplexed tests, simultaneously screening for HIV, HCV, and syphilis, using 2 rapid investigational multiplexed tests (Chembio for HIV and syphilis and MedMira for HIV, HCV, and syphilis) followed by confirmatory testing from local and national laboratories. Of the 401 participants, all (100%) accepted the AideSmart! multiplexed strategy: 59.4% (148/249) preferred multiplexed rapid tests over laboratory tests, and 56.6% (141/249) preferred receiving same-day test results. Rapid test results were obtained in 15 minutes (vs laboratory tests at 4-24 days). A total of 29 new infections (n=27, 93% HCV; n=1, 3% syphilis; and n=1, 3% HIV) were identified and treated within a week. Feasibility of the strategy (completion of testing and linkages to care) was at 76.1% (305/401). Health care professionals provided positive feedback and emphasized the importance of knowing one’s negative or positive serostatus, especially during a pandemic. Multiplexed rapid tests’ specificity (against laboratory reference standards) exceeded 98% (98.7%-100%) for all pathogens and devices. An electronic reader, used by the Chembio rapid test, enhanced sensitivity (HIV: 100%, 95% CI 79.4%-100%; syphilis: 86.8%, 95% CI 71.9%-95.6% [Chembio] vs HIV: 100%, 95% CI 78.2%-100%; HCV: 90.3%, 95% CI 80.1%-96.4%; and syphilis: 57.9%, 95% CI 40.8%-73.7% [MedMira]). The AideSmart! digital multiplexed rapid screening strategy for health care workers facilitated STBBI testing for multiple STBBIs and arranged for pre- or posttest counseling and rapid linkages with high feasibility and acceptability. Electronic readers enhanced the diagnostic performance of multiplexed biomarker tests. This study generated data in support of digital multiplexed strategies in digitally enabled settings for at-risk populations nationally and worldwide.

Objectives: Antibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. Methods: We developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD) and nucleocapsid (N). We automated a surrogate neutralisation (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results: Our single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for ≥ 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. Conclusions: Measuring antibodies to three viral antigens and identify neutralising antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardised serological assays, permitting inter-laboratory data comparison and aggregation.

Correction of: https://www.jmir.org/2024/1/e55075

Chronic wasting disease (CWD) is an emerging infectious prion disorder that is spreading rapidly in wild populations of cervids in North America. The risk of zoonotic transmission of CWD is as yet unclear but a high priority must be to minimize further spread of the disease. No simple diagnostic tests are available to detect CWD quickly or in live animals; therefore, easily accessible biomarkers may be useful in identifying infected animals. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that circulate in blood and are promising biomarkers for several infectious diseases. In this study we used next-generation sequencing to characterize the serum miRNA profiles of 35 naturally infected elk that tested positive for CWD in addition to 35 elk that tested negative for CWD. A total of 21 miRNAs that are highly conserved amongst mammals were altered in abundance in sera, irrespective of hemolysis in the samples. A number of these miRNAs have previously been associated with prion diseases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminative potential of these miRNAs as biomarkers for the diagnosis of CWD. We also determined that a subgroup of 6 of these miRNAs were consistently altered in abundance in serum from hamsters experimentally infected with scrapie. This suggests that common miRNA candidate biomarkers could be selected for prion diseases in multiple species. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pointed to a strong correlation for 3 of these miRNAs, miR-148a-3p, miR-186-5p, miR-30e-3p, with prion disease.

During rollout of mRNA-based COVID-19 vaccines, several jurisdictions extended the interval between the first and second doses to prioritize wider population access to limited vaccine supply. This study evaluated the effects of an extended dose interval on development of antibody and cell-mediated responses following the primary dose series and a subsequent booster dose. Blood samples were collected from mRNA COVID-19 vaccine recipients at baseline and longitudinally after each dose. Samples were analyzed for SARS-CoV-2-specific antibody titers, neutralizing antibodies and memory T cell responses. An extended dose interval was associated with improved breadth of neutralizing antibody responses against both ancestral and early SARS-CoV-2 variants, but not Omicron variants. Dose interval had no impact on the development of antigen-specific memory T cell responses, the memory or T helper phenotypes of responding T cells or cytokine production. The effects of the primary dose interval on immune outcomes were no longer evident after a third dose of mRNA vaccine. An extended primary dose interval resulted in short-term benefits to humoral immunity but these were transient in the context of subsequent exposures. However, in addition to the public health benefits of wider population access to vaccines, the short-term immunological benefits of extending the dose interval may have been sustained in the absence of boosters. These findings underscore the importance of evaluating dosing intervals during the development of future vaccine candidates.

End-stage renal disease (ESRD) is associated with an increased susceptibility to infectious diseases, including infection with . Mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites produced by many bacterial species, including Mtb, and may play an important role in providing protective immunity against tuberculosis infection in the lung. To date, little is known about MAIT cell frequency, phenotype, or function in ESRD patients. MAIT cells, identified by surface marker expression or MR1 tetramer binding, were characterized in 20 ESRD and 20 healthy control participants by multicolor flow cytometry. MAIT cell phenotype and cytokine production following PMA/ionomycin, IL-12/IL-18, or stimulation were determined. Monocyte phenotype and plasma C-reactive protein/inflammatory cytokine levels were quantified by flow cytometry, ELISA, and multiplex bead array. Peripheral blood MAIT cells were significantly depleted among ESRD patients compared to controls by both phenotypic and tetramer analysis and exhibited a loss of CXCR3 expression coupled to increased expression of CCR6 and CXCR6. ESRD was also associated with a shift in MAIT PMA-induced cytokine production away from IFNγ production and toward granulocyte macrophage-colony stimulating factor (GM-CSF) secretion, and a loss of -stimulated tumor necrosis factor α expression. Loss of IFNγ expression was associated with a combination of age, alterations in Tbet and Eomes expression, and inflammatory plasma cytokine levels. The loss of peripheral blood MAIT cells and associated shifts in tissue homing receptor expression and GM-CSF production may contribute to an immune environment that is permissive to bacterial replication, particularly in the lungs.

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck ( Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing ( TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.

The optimal 6-carboxy-X-rhodamine (ROX) concentration, which is used as a passive reference dye for real-time quantitative polymerase chain reaction (PCR) with molecular beacon chemistry, was determined with the Mx4000™ Multiplex Quantitative PCR System. Additionally, the effects of changing ROX concentrations on PCR reproducibility, Ct values, and efficiency were investigated with this system by using the PCR data obtained from amplification of the Escherichia coli shiga toxin 2 (stx2) gene and the Campylobacter jejuniluxS gene. This study indicated that different ROX concentrations influence many aspects of the real-time PCR reaction. ROX concentration variation could have consequences in the analysis of quantitative data and may lead to erroneous results. This study further indicated that the optimal ROX concentration is 60 nmol/L for real-time PCR, using molecular beacon chemistry for PCR assay of luxS and stx2 genes.

This study examines childhood cancer survival rates and prognostic factors related to survival in the majority Hispanic population of South Texas. The population-based cohort study used Texas Cancer Registry data (1995–2017) to examine survival and prognostic factors. Cox proportional hazard models and Kaplan-Meier survival curves were used for survival analyses. The 5-year relative survival rate for 7,999 South Texas cancer patients diagnosed at 0–19 years was 80.3% for all races/ethnicities. Hispanic patients had statistically significant lower 5-year relative survival rates than non-Hispanic White (NHW) patients for male and female together diagnosed at age≥5 years. When comparing survival among Hispanic and NHW patients for the most common cancer, acute lymphocytic leukemia (ALL), the difference was most significant in the 15–19 years age range, with 47.7% Hispanic patients surviving at 5 years compared to 78.4% of NHW counterparts. The multivariable-adjusted analysis showed that males had statistically significant 13% increased mortality risk than females [hazard ratio (HR): 1.13, 95% confidence interval (CI):1.01–1.26] for all cancer types. Comparing to patients diagnosed at ages 1–4 years, patients diagnosed at age < 1 year (HR: 1.69, 95% CI: 1.36–2.09), at 10–14 year (HR: 1.42, 95% CI: 1.20–1.68), or at 15–19 years (HR: 1.40, 95% CI: 1.20–1.64) had significant increased mortality risk. Comparing to NHW patients, Hispanic patients showed 38% significantly increased mortality risk for all cancer types, 66% for ALL, and 52% for brain cancer. South Texas Hispanic patients had lower 5-year relative survival than NHW patients especially for ALL. Male gender, diagnosis at age<1 year or 10–19 years were also associated with decreased childhood cancer survival. Despite advances in treatment, Hispanic patients lag significantly behind NHW patients. Further cohort studies in South Texas are warranted to identify additional factors affecting survival and to develop interventional strategies.