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Coronavirus disease (COVID) serological tests are essential to determine the overall seroprevalence of a population and to facilitate exposure estimates within that population. We performed a head-to-head assessment of enzyme immu...
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Coronavirus disease (COVID) serological tests are essential to determine the overall seroprevalence of a population and to facilitate exposure estimates within that population. We performed a head-to-head assessment of enzyme immunoassays (EIAs) and point-of-care lateral flow assays (POCTs) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. Demographics, symptoms, comorbidities, treatment, and mortality of patients whose sera were used were also reviewed. Six EIAs (Abbott, Affinity, Bio-Rad, DiaSorin, Euroimmun, and Roche) and six POCTs (BTNX, Biolidics, Deep Blue, Genrui, Getein BioTech, and Innovita) were evaluated for the detection of SARS-CoV-2 antibodies in known COVID-19-infected individuals. ABSTRACT Coronavirus disease (COVID) serological tests are essential to determine the overall seroprevalence of a population and to facilitate exposure estimates within that population. We performed a head-to-head assessment of enzyme immunoassays (EIAs) and point-of-care lateral flow assays (POCTs) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. Demographics, symptoms, comorbidities, treatment, and mortality of patients whose sera were used were also reviewed. Six EIAs (Abbott, Affinity, Bio-Rad, DiaSorin, Euroimmun, and Roche) and six POCTs (BTNX, Biolidics, Deep Blue, Genrui, Getein BioTech, and Innovita) were evaluated for the detection of SARS-CoV-2 antibodies in known COVID-19-infected individuals. Sensitivity of EIAs ranged from 50 to 100%, with only four assays having overall sensitivities of >95% after 21?days after symptom onset. Notably, cross-reactivity with other respiratory viruses (parainfluenza virus [PIV-4] [ n ?=?5], human metapneumovirus [hMPV] [ n ?=?3], rhinovirus/enterovirus [ n ?=?1], CoV-229E [ n ?=?2], CoV-NL63 [ n ?=?2], and CoV-OC43 [ n ?=?2]) was observed; however, overall specificity of EIAs was good (92 to 100%; all but one assay had specificity above 95%). POCTs were 0 to 100% sensitive >21?days after onset, with specificity ranging from 96 to 100%. However, many POCTs had faint banding and were often difficult to interpret. Serology assays can detect SARS-CoV-2 antibodies as early as 10?days after symptom onset. Serology assays vary in their sensitivity based on the marker (IgA/IgM versus IgG versus total) and by manufacturer; however, overall only 4 EIAs and 4 POCTs had sensitivities of >95% >21?days after symptom onset. Cross-reactivity with other seasonal coronaviruses is of concern. Serology assays should not be used for the diagnosis of acute infection but rather in carefully designed serosurveys to facilitate understanding of seroprevalence in a population and to identify previous exposure to SARS-CoV-2.
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Congenital infections with SARS-CoV-2 are uncommon. We describe two confirmed congenital SARS-CoV-2 infections using descriptive, epidemiologic and standard laboratory methods and in one case, viral culture. Clinical data were obt...
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Congenital infections with SARS-CoV-2 are uncommon. We describe two confirmed congenital SARS-CoV-2 infections using descriptive, epidemiologic and standard laboratory methods and in one case, viral culture. Clinical data were obtained from health records. Nasopharyngeal (NP) specimens, cord blood and placentas when available were tested by reverse transcriptase real-time PCR (RT-PCR). Electron microscopy and histopathological examination with immunostaining for SARS-CoV-2 was conducted on the placentas. For Case 1, placenta, umbilical cord, and cord blood were cultured for SARS-CoV-2 on Vero cells. This neonate was born at 30 weeks, 2 days gestation by vaginal delivery. RT-PCR tests were positive for SARS-CoV-2 from NP swabs and cord blood; NP swab from the mother and placental tissue were positive for SARS-CoV-2. Placental tissue yielded viral plaques with typical morphology for SARS-CoV-2 at 2.8 × 102 pfu/mL confirmed by anti-spike protein immunostaining. Placental examination revealed chronic histiocytic intervillositis with trophoblast necrosis and perivillous fibrin deposition in a subchorionic distribution. Case 2 was born at 36 weeks, 4 days gestation. RT-PCR tests from the mother and infant were all positive for SARS-CoV-2, but placental pathology was normal. Case 1 may be the first described congenital case with SARS-CoV-2 cultivated directly from placental tissue.
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Risk factors for nosocomial COVID-19 outbreaks continue to evolve. The aim of this study was to investigate a multi-ward nosocomial outbreak of COVID-19 between 1st September and 15th November 2020, occurring in a setting without ...
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Risk factors for nosocomial COVID-19 outbreaks continue to evolve. The aim of this study was to investigate a multi-ward nosocomial outbreak of COVID-19 between 1st September and 15th November 2020, occurring in a setting without vaccination for any healthcare workers or patients.Outbreak report and retrospective, matched case–control study using incidence density sampling in three cardiac wards in an 1100-bed tertiary teaching hospital in Calgary, Alberta, Canada. Patients were confirmed/probable COVID-19 cases and contemporaneous control patients without COVID-19. COVID-19 outbreak definitions were based on Public Health guidelines. Clinical and environmental specimens were tested by RT-PCR and as applicable quantitative viral cultures and whole genome sequencing were conducted. Controls were inpatients on the cardiac wards during the study period confirmed to be without COVID-19, matched to outbreak cases by time of symptom onset dates, age within ± 15 years and were admitted in hospital for at least 2 days. Demographics, Braden Score, baseline medications, laboratory measures, co-morbidities, and hospitalization characteristics were collected on cases and controls. Univariate and multivariate conditional logistical regression was used to identify independent risk factors for nosocomial COVID-19.The outbreak involved 42 healthcare workers and 39 patients. The strongest independent risk factor for nosocomial COVID-19 (IRR 3.21, 95% CI 1.47–7.02) was exposure in a multi-bedded room. Of 45 strains successfully sequenced, 44 (97.8%) were B.1.128 and differed from the most common circulating community lineages. SARS-CoV-2 positive cultures were detected in 56.7% (34/60) of clinical and environmental specimens. The multidisciplinary outbreak team observed eleven contributing events to transmission during the outbreak.Transmission routes of SARS-CoV-2 in hospital outbreaks are complex; however multi-bedded rooms play a significant role in the transmission of SARS-CoV-2.
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Objectives: Although the passage of blood in stools in children represents a medical emergency, children seeking emergency department (ED) care remain poorly characterized. Our primary objective was to compare clinical characteris...
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Objectives: Although the passage of blood in stools in children represents a medical emergency, children seeking emergency department (ED) care remain poorly characterized. Our primary objective was to compare clinical characteristics and etiologic pathogens in children with acute diarrhea with and without caregiver-reported hematochezia. Secondary objectives were to characterize interventions and resource utilization.Methods: We conducted a secondary analysis of the Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE) database. Children <18 years presenting to two pediatric EDs within a 24-hour period and <7 days of symptoms were consecutively recruited.Results: Of 1,061 participants, 115 (10.8%) reported hematochezia at the enrollment visit at which time those with hematochezia, compared to those without, had more diarrheal episodes/24-hour period (9 vs. 6; difference: 2; 95% confidence interval [CI]: 2.0, 4.0; p < 0.001), and were less likely to have experienced vomiting (54.8% vs. 80.2%; difference: -25.4; 95% CI: -34.9, -16.0; p < 0.001). They were more likely to receive intravenous fluids (33.0% vs. 17.9%; difference: 15.2; 95% CI: 6.2, 24.1; p < 0.001) and require repeat health care visits (45.5% vs. 34.7%; difference: 10.7; 95% CI: 0.9, 20.6; p = 0.03). A bacterial pathogen was identified in 33.0% of children with hematochezia versus 7.9% without (difference: 25.1; 95% CI: 16.3, 33.9; p < 0.001); viruses were detected in 31.3% of children with hematochezia compared to 72.3% in those without (difference: -41.0%, 95% CI: -49.9, -32.1; p < 0.001). Conclusion: In children with acute diarrhea, caregiver report of hematochezia, compared to the absence of hematochezia, was associated with more diarrheal but fewer vomiting episodes, and greater resource consumption. The former group of children was also more likely to have bacteria detected in their stool.
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During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, curre...
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During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii , an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii , immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.
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Advances in diagnostic microbiology allow for the rapid identification of a broad range of enteropathogens; such knowledge can inform care and reduce testing. We conducted a randomized, unblinded trial in a tertiary-care pediatric...
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Advances in diagnostic microbiology allow for the rapid identification of a broad range of enteropathogens; such knowledge can inform care and reduce testing. We conducted a randomized, unblinded trial in a tertiary-care pediatric emergency department. Participants had stool (and rectal swabs if stool was not immediately available) tested using routine microbiologic approaches or by use of a device (BioFire FilmArray gastrointestinal panel), which identifies 22 pathogens with a 1-h instrument turnaround time. Participants were 6 months to <18.0 years and had acute bloody diarrhea. Primary outcome was performance of blood tests within 72 h. From 15 June 2018 through 7 May 2022, 60 children were randomized. Patients in the BioFire FilmArray arm had a reduced time to test result (median 3.0 h with interquartile range [IQR] of 3.0 to 4.0 h, versus 42.0 h (IQR 23.5 to 47.3 h); difference of ?38.0 h, 95% confidence interval [CI] of ?41.0 to ?22.0 h). Sixty-five percent (20/31) of participants in the BioFire FilmArray group had a pathogen detected—most frequently enteropathogenic Escherichia coli (19%), Campylobacter (16%), and Salmonella (13%). Blood tests were performed in 52% of children in the BioFire FilmArray group and 62% in the standard-of-care group (difference of ?10.5%, 95% CI of ?35.4% to 14.5%). There were no between-group differences in the proportions of children administered intravenous fluids, antibiotics, hospitalized, or who had diagnostic imaging performed. Testing with the BioFire FilmArray reduced the time to result availability by 38 h. Although statistical significance was limited by study power, BioFire FilmArray use was not associated with clinically meaningful reductions in health care utilization or improved outcomes. IMPORTANCE Advances in diagnostic microbiology now allow for the faster and more accurate detection of an increasing number of pathogens. We determined, however, that in children with acute bloody diarrhea, these advances did not necessarily translate into improved clinical outcomes. While a greater number of pathogens was identified using a rapid turnaround multiplex stool diagnostic panel, with a reduction in the time to stool test result of over 1.5 days, this did not alter the practice of pediatric emergency medicine physicians, who continued to perform blood tests on a large proportion of children. While our conclusions may be limited by the relatively small sample size, targeted approaches that educate clinicians on the implementation of such technology into clinical care will be needed to optimize usage and maximize benefits.
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Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typ...
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Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.
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NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, par...
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NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.
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Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inf...
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Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inflammatory cytokine production in response to pathogenic molecules, such as ExoS, which is a TLR2 and TLR4 agonist produced by the opportunistic pathogen Pseudomonas aeruginosa. We reasoned that responses to ExoS, a protein, might differ from canonical TLR agonists such as LPS. Stimulating the expression of mCD14 with vitamin D3 enhanced the response to ExoS and LPS. Also, blocking anti-CD14 antibody or removing mCD14 using PLC reduced responses to ExoS and LPS. Furthermore, CD14-deficient cells were unable to bind and respond to ExoS, which was restored by stable transfection of mCD14, indicating that mCD14 was required for the response to ExoS. However, addition of sCD14 to culture enhanced responsiveness to LPS but not ExoS. Moreover, the addition of serum did not alter the response to ExoS but enhanced the response to LPS. Despite differences of adaptor molecule use between ExoS and LPS, lipid antagonists that compete for LPS binding to CD14 also inhibited the response to ExoS. These results highlight a fundamental difference between TLR agonists in their requirements for CD14 and serum components. These results suggest that understanding the dissimilarities and targeting overlapping sites of interaction on CD14 may yield a synergistic, clinical benefit during infections where a variety of TLR agonists are present.
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Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4+ T cells with IL-2 occurs...
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Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4+ T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4+ T cells is defective during HIV infection. This is because CD4+ T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rβ, produce granulysin, and kill C. neoformans . The increased expression of IL-2Rβ precedes granulysin, and preventing the increased expression of IL-2Rβ using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rβ, blocking subsequent signaling by IL-2 using IL-2Rβ-specific blocking Abs abrogates expression of granulysin. Finally, CD4+ T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rβ and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rβ, which in turn is required for production of granulysin. These results provide a mechanism to explain the “late” production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4+ T cells in HIV-infected patients.
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