ABSTRACT
ObjectivesThe objective of the current study was to develop a lower-cost and scalable protocol to identify and monitor SARS-CoV-2 variants in Paraguay by pairing real-time RT-PCR detection of spike mutations with amplicon Sanger sequencing and whole-genome Nanopore sequencing. Methods201 acute-phase nasopharyngeal samples from SARS-CoV-2-positive individuals were tested with two rRT-PCRs: 1) N2RP assay to confirm SARS-CoV-2 RNA detection (CDC N2 target), and 2) the Spike SNP assay to detect mutations in the spike receptor binding domain. The assay was performed with probes to identify mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). ResultsAll samples were positive for SARS-CoV-2 in the N2RP assay (mean Ct, 20.8; SD 5.6); 198/201 (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%) and most consistent with P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%); and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). Results were confirmed by Sanger sequencing in 181/181 samples (100%) with high-quality amplicon sequences, and variant calls were consistent with Nanopore sequencing in 29/29 samples. ConclusionsThe Spike SNP assay provides accurate detection of mutations associated with SARS-CoV-2 variants. This can be implemented in laboratories performing rRT-PCR to improve population-level surveillance for these mutations and inform the judicious use of scarce sequencing resources.
ABSTRACT
BackgroundIsothermal amplification-based tests were developed as rapid, low-cost, and simple alternatives to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests for SARS-COV-2 detection. MethodsClinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP COVID-19 detection test and OptiGene COVID-19 Direct Plus RT-LAMP test) was compared to clinical RT-PCR assays using different sampling strategies. A total of 1378 participants were tested across four study sites. ResultsCompared to standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic collected site. Sensitivity for nasopharyngeal was 65.4% (range across study sites:52.8%-79.8%), mid-turbinate 88.2%, saliva 55.1% (range across study sites:42.9%-77.8%) and anterior nares 66.7% (range across study sites:63.6%-76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall 82.7%) and those with a higher viral load (overall 92.4% for ct[≤]25). Sensitivity and specificity of the OptiGene Direct Plus RT-LAMP test, conducted at a single study-site, were 25.5% and 100%, respectively. ConclusionsThe Atila iAMP COVID test with mid-turbinate sampling is a rapid, low-cost assay for detecting SARS-COV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-COV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption.
ABSTRACT
Penile squamous cell carcinoma (PSCC) accounts for over 95% of penile malignancies and causes significant mortality and morbidity in developing countries. Molecular mechanisms and therapies of PSCC are understudied, owing to scarcity of laboratory models. Herein, we describe a genetically engineered mouse model of PSCC, by co-deletion of Smad4 and Apc in the androgen-responsive epithelium of the penis. Mouse PSCC fosters an immunosuppressive microenvironment with myeloid-derived suppressor cells (MDSCs) as a dominant population. Preclinical trials in the model demonstrate synergistic efficacy of immune checkpoint blockade with the MDSC-diminishing drugs cabozantinib or celecoxib. A critical clinical problem of PSCC is chemoresistance to cisplatin, which is induced by Pten deficiency on the backdrop of Smad4/Apc co-deletion. Drug screen studies informed by targeted proteomics identify a few potential therapeutic strategies for PSCC. Our studies have established what we believe to be essential resources for studying PSCC biology and developing therapeutic strategies.
Subject(s)
Carcinoma, Squamous Cell/therapy , Immunotherapy/methods , Penile Neoplasms/therapy , Animals , Carcinoma, Squamous Cell/metabolism , Cell Line , Cisplatin/pharmacology , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Myeloid-Derived Suppressor Cells/cytology , Myeloid-Derived Suppressor Cells/metabolism , Penile Neoplasms/metabolism , Proteomics , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Tissue Array Analysis , Transcriptome/geneticsABSTRACT
Se plantea la hipotesis de que con la aplicacion de una variante del sistema de reforzamiento por fichas, podria incrementarse la participacion de los pacientes en las actividades desarrolladas en una comunidad terapeutica para psicoticos y alcoholicos, y de que su aplicacion era factible dentro de la estructura organizativa mantenida en los tres ultimos anos de trabajo. Para lograr estos objetivos se Ilevo a cabo una investigacion desarrollada en tres etapas de tres meses cada una, y se controlo durante las mismas el comportamiento de los pacientes en cuanto a: asistencia diaria a la reunion matutina; cumplimiento del horario establecido para levantarse; y otros indicadores utilizados como control por no comtemplarse en el sistema de gratificaciones. Durante la primera etapa se mantuvo la estructura anterior, basada exclusivamente en gratificaciones morales; durante la segunda, se ensayaron diferentes tipos de premios, donde se incluyeron excursiones, premios en efectivo y articulos de artesania, asi como pases especiales a los pacientes destacados. En la tercera etapa se establecio el otorgamiento del pase de fin de semana mediante la acumulacion de puntos ganados por conductas positivas y anotados mediante gomigrafo en una tarjeta individual que se renovaria semanalmente. Los resultados evidenciaron un incremento importante de los indicadores contemplados en el sistema de gratificaciones, y poca modificacion en aquellos que se utilizaron como controles y para valorar la influencia indirecta de otras conductas positivas. Se considero que sua resultados apoyan las dos hipotesis planteadas, y que el pase de fin de semana otorgado mediante puntos acumulados constituye el premio de interes general mas adecuado. Se concluye que la experiencia acumulada hizo incorporar dicho sistema a la estructura comunitaria del servicio docente 'Rogelio Paredes', del Hospital Psiquiatrico de La Habana, y anadirlo al sitema previo de estimulaciones morales, exclusivamente.
