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1.
Viruses ; 15(7)2023 07 15.
Article in English | MEDLINE | ID: mdl-37515241

ABSTRACT

The unprecedented COVID-19 pandemic posed major challenges to local, regional, and global economies and health systems, and fast clinical diagnostic workflows were urgently needed to contain the spread of SARS-CoV-2. Here, we describe the platform and workflow established at the Cornell COVID-19 Testing Laboratory (CCTL) for the high-throughput testing of clinical samples from the university and the surrounding community. This workflow enabled efficient and rapid detection and the successful control of SARS-CoV-2 infection on campus and its surrounding communities. Our cost-effective and fully automated workflow enabled the testing of over 8000 pooled samples per day and provided results for over 2 million samples. The automation of time- and effort-intensive sample processing steps such as accessioning and pooling increased laboratory efficiency. Customized software applications were developed to track and store samples, deconvolute positive pools, track and report results, and for workflow integration from sample receipt to result reporting. Additionally, quality control dashboards and turnaround-time tracking applications were built to monitor assay and laboratory performance. As infectious disease outbreaks pose a constant threat to both human and animal health, the highly effective workflow implemented at CCTL could be modeled to establish regional high-capacity testing hubs for infectious disease preparedness and emergency response.


Subject(s)
COVID-19 , Communicable Diseases , Humans , COVID-19 Testing , COVID-19/diagnosis , SARS-CoV-2 , Clinical Laboratory Techniques/methods , Pandemics
2.
Microbiol Spectr ; 10(3): e0226421, 2022 06 29.
Article in English | MEDLINE | ID: mdl-35575498

ABSTRACT

In the present study, we assessed the diagnostic sensitivity and determined the viral RNA load and infectivity of SARS-CoV-2 in paired respiratory (nasopharyngeal and anterior nares) and oral samples (saliva and sublingual swab). Samples were collected from 77 individuals of which 75 were diagnosed with COVID-19 and classified as symptomatic (n = 29), asymptomatic (n = 31), or postsymptomatic (n = 15). Specimens were collected at one time point from each individual, between day 1 and 23 after the initial COVID-19 diagnosis, and included self-collected saliva (S), or sublingual (SL) swab, and bilateral anterior nares (AN) swab, followed by health care provider collected nasopharyngeal (NP) swab. Sixty-three specimen sets were tested using five assay/platforms. The diagnostic sensitivity of each assay/platform and specimen type was determined. Of the 63 specimen sets, SARS-CoV-2 was detected in 62 NP specimens, 52 AN specimens, 59 saliva specimens, and 31 SL specimens by at least one platform. Infectious SARS-CoV-2 was isolated from 21 NP, 13 AN, 12 saliva, and one SL specimen out of 50 specimen sets. SARS-CoV-2 isolation was most successful up to 5 days after initial COVID-19 diagnosis using NP specimens from symptomatic patients (16 of 24 positives, 66.67%), followed by specimens from asymptomatic patients (5 of 17 positives, 29.41%), while it was not very successful with specimens from postsymptomatic patients. Benefits of self-collected saliva and AN specimens balance the loss of sensitivity relative to NP specimens. Therefore, saliva and AN specimens are acceptable alternatives for symptomatic SARS-CoV-2 diagnostic testing or surveillance with increased sampling frequency of asymptomatic individuals. IMPORTANCE The dynamics of infection with SARS-CoV-2 have a significant impact on virus infectivity and in the diagnostic sensitivity of molecular and classic virus detection tests. In the present study we determined the diagnostic sensitivity of paired respiratory (nasopharyngeal and anterior nares swabs) and oral secretions (saliva and sublingual swab) and assessed infectious virus shedding patterns by symptomatic, asymptomatic, or postsymptomatic individuals. Understanding the diagnostic performance of these specimens and the patterns of infectious virus shedding in these bodily secretions provides critical information to control COVID-19, and may help to refine guidelines on isolation and quarantine of positive individuals and their close contacts identified through epidemiological investigations.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Humans , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva , Specimen Handling , Viral Load
3.
Arch Virol ; 166(9): 2551-2561, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34259914

ABSTRACT

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , Multiplex Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , Epidemiological Monitoring , Gene Expression , Humans , Multiplex Polymerase Chain Reaction/economics , Multiplex Polymerase Chain Reaction/instrumentation , Nasal Cavity/virology , Nasopharynx/virology , Phosphoproteins/genetics , Reproducibility of Results , Sensitivity and Specificity , Specimen Handling/methods , Viral Load
4.
Acta Neuropathol ; 114(5): 459-69, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17687559

ABSTRACT

The significance of caveolin-1, a major constituent of caveolae, and the tight junction proteins occludin and claudin-5 in early blood-brain barrier (BBB) breakdown was assessed by sequential demonstration of the expression of these proteins over a period of 12 h to 6 days post-lesion in the rat cortical cold injury model. Pial and intracerebral vessels of control rats showed punctuate endothelial immunoreactivity for caveolin-1 and caveolin-2, while claudin-5 and occludin were localized as longitudinal strands in endothelium. During the early phase of BBB breakdown following injury at 12 h and on day 2, western blot analyses detected a significant increase in caveolin-1 expression at the lesion site while immunohistochemistry showed that the caveolin-1 increase was localized to the endothelium of lesion vessels. Decreased expression of occludin occurred at the lesion site only on days 2 and 4 post-lesion while claudin-5 expression was decreased only on day 2. Dual labeling for fibronectin, a marker of BBB breakdown, and caveolin-1 or the tight junction proteins demonstrated that only lesion vessels with BBB breakdown showed a marked increase of caveolin-1, loss of occludin and reduced localization of claudin-5. The issue whether these alterations precede or follow BBB breakdown is uncertain; however, increased expression of caveolin-1 preceded the decreased expression of occludin and claudin-5. Thus caveolae and caveolin-1 have an important role in early BBB breakdown and could be potential therapeutic targets in the control of early brain edema.


Subject(s)
Blood-Brain Barrier/metabolism , Brain Edema/metabolism , Caveolin 1/metabolism , Membrane Proteins/metabolism , Tight Junctions/metabolism , Animals , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiopathology , Brain Edema/pathology , Brain Edema/physiopathology , Brain Injuries/metabolism , Brain Injuries/pathology , Brain Injuries/physiopathology , Caveolae/metabolism , Caveolae/pathology , Cell Membrane/metabolism , Cell Membrane/pathology , Cerebral Arteries/metabolism , Cerebral Arteries/pathology , Cerebral Arteries/physiopathology , Claudin-5 , Cold Temperature/adverse effects , Disease Models, Animal , Down-Regulation/physiology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Male , Occludin , Rats , Rats, Wistar , Time Factors , Up-Regulation/physiology
5.
Exp Mol Pathol ; 83(1): 65-72, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17335802

ABSTRACT

A systemic inflammatory response (SIR) occurs prior to and during the treatment of severe diabetic ketoacidosis (DKA). IL-1beta, TNF-alpha and C5b-9 are components of SIR and have been speculated to be involved in the clinical brain edema (BE) of DKA. We studied IL-1beta, TNF-alpha, C5b-9, inducible nitric oxide (iNOS), ICAM-1, IL-10 and Hsp70 expression in the brains of two patients who died as the result of clinical BE during the treatment of DKA. IL-1beta was strongly expressed in the choroid plexus epithelium (CPE) and ependyma, and to a lesser extent in the hippocampus, caudate, white matter radiation of the pons, molecular layer of the cerebellum and neurons of the cortical gray matter. TNF-alpha was expressed to a lesser extent than IL-1beta, and only in the CP. C5b-9, previously shown to be deposited on neurons and oligodendrocytes, was found on CPE and ependymal cells. iNOS and ICAM-1 had increased expression in the CPE and ependyma. Hsp70 and IL-10 were also expressed in the CPE of the case with the shorter duration of treatment. Our data demonstrate the presence of a multifaceted neuroinflammatory cytotoxic insult of the CPE, which may play a role in the pathophysiology of the fatal brain edema of DKA.


Subject(s)
Choroid Plexus/metabolism , Choroid Plexus/pathology , Diabetic Ketoacidosis/metabolism , Diabetic Ketoacidosis/pathology , Nervous System Diseases/metabolism , Nervous System Diseases/pathology , Adolescent , Apoptosis , CD59 Antigens/metabolism , Complement Membrane Attack Complex/metabolism , Epithelium/metabolism , Epithelium/pathology , Female , HSP70 Heat-Shock Proteins/metabolism , Humans , Immunohistochemistry , Inflammation/metabolism , Inflammation/pathology , Intercellular Adhesion Molecule-1/metabolism , Interleukin-1beta/metabolism , Nitric Oxide Synthase Type II/metabolism , Tumor Necrosis Factor-alpha/metabolism
6.
Lab Invest ; 85(10): 1189-98, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16056241

ABSTRACT

Normal intracerebral and pial vessels show constitutive expression of angiopoietin (Ang) 1 in endothelium while weak Ang2 immunoreactivity is present in occasional vessels. In the early phase postinjury, blood-brain barrier (BBB) breakdown at the lesion site is associated with decreased endothelial Ang1 and increased Ang2 expression, raising the possibility that Ang2 may have a role in early BBB breakdown. In order to determine whether Ang2 can cause BBB breakdown, the effect of recombinant Ang2 on cerebrovascular permeability to horseradish peroxidase (HRP) was studied in normal rat cortex. As hypothesized, Ang2 produced significant BBB breakdown to HRP as compared with vehicle-injected control rats. Since Ang2 is reported to have proapoptotic activity, the possibility that Ang2 may be associated with endothelial apoptosis was investigated in the rat cortical cold injury model over a period of 6 h to 6 days postinjury. Perilesion and pial vessels showed evidence of endothelial apoptosis as demonstrated by active Caspase-3 localization and TUNEL staining. Dual labeling for Ang proteins and active Caspase-3 demonstrated endothelial colocalization of Ang2 with active caspase-3. These data suggest that following injury, Ang2 may play a role in BBB breakdown of perilesional vessels, and it may also be a factor in endothelial cell apoptosis that occurs at days 1 and 2 following the injury.


Subject(s)
Angiopoietin-2/biosynthesis , Apoptosis/physiology , Blood-Brain Barrier/pathology , Endothelium, Vascular/pathology , Angiopoietin-1/analogs & derivatives , Angiopoietin-1/physiology , Animals , Brain Injuries/metabolism , Brain Injuries/pathology , Caspase 3 , Caspases/metabolism , Cerebral Cortex/blood supply , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , In Situ Nick-End Labeling , Male , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Rats , Rats, Inbred WF , Rats, Wistar
7.
Endocr Pathol ; 16(1): 67-73, 2005.
Article in English | MEDLINE | ID: mdl-16000849

ABSTRACT

Interaction of vascular endothelial growth factor (VEGF) with the angiopoietins (Ang) is an essential component of angiogenesis. Localization of VEGF in the anterior pituitary raises the possibility that Ang must be present in the pituitary gland as well. In this study Ang expression was detected in the normal rat pituitary gland at the gene level by reverse-transcriptase polymerase chain reaction and at the protein level by immunohistochemistry. The latter was analyzed by both light and confocal microscopy. Constitutive expression of Ang1, Ang2, and their receptor Tie2 was detected at both the mRNA and protein level in all the pituitary glands studied. Of interest was the localization of both Ang1 and Ang2 in scattered PAS positive adenohypophysial cells rather than in endothelial cells. Confocal microscopy showed colocalization of both Ang1 and Ang2 proteins within the same adenohypophysial cells. Dual immunostaining for Ang1 and the anterior pituitary hormones that show PAS positivity demonstrated colocalization of Ang1 with follicle stimulating hormone and luteinizing hormone. In the posterior pituitary, strong Ang1 signal observed in vascular endothelial cells masked the weak Ang2 signal, a pattern that is similar to that reported in brain endothelial cells. The presence of both angiopoietins and VEGF in the pituitary gland suggest that these ligands interact during angiogenesis as they are known to do in other systems to maintain the rich vascular network of the gland. This first report of angiopoietin localization in the rat pituitary gland opens a new line of investigation on angiogenesis in pituitary glands that will impact human endocrinology in the future.


Subject(s)
Angiopoietin-1/metabolism , Angiopoietin-2/metabolism , Pituitary Gland, Anterior/metabolism , Pituitary Gland, Posterior/metabolism , Angiopoietin-1/genetics , Angiopoietin-2/genetics , Animals , Biomarkers/metabolism , Fluorescent Antibody Technique, Indirect , Gene Expression , Immunoenzyme Techniques , Male , Microscopy, Confocal , Pituitary Gland, Anterior/cytology , Pituitary Gland, Posterior/cytology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction
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