Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics.

As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.

[Prediction of end of lockdown post-peak of cases in first wave of the COVID-19 pandemic in Chile]. / Predicción del fin del confinamiento después del máximo de casos en la primera ola de la pandemia COVID-19 en Chile.

INTRODUCTION: The results of mandatory confinement have been detrimental in several respects. Nonetheless, they have resulted in reducing the number of active cases of COVID-19. Chile has begun the de-escalation and needs to know the best time to end the restrictions. OBJECTIVE: We discuss the best conditions and guarantees for the end of compulsory confinement. METHODS: This study is based on a trend model with prediction estimation. The data of the variables of interest were subjected to linear regression studies to determine the curve that best explained the data. The coefficient of determination, the standard deviation of y in x, and the confidence interval of the observed curve were estimated. The trend curve was chosen in accordance with the regression estimates. OUTCOMES: It was found that all dependent variables tended to decrease over time in a quadratic fashion, except for the new cases variable. In general, the R2 and MAPE estimates are satisfactory, except for the variable number of PCR tests per day. CONCLUSIONS: Gradual and cautious steps should be taken before ending mandatory confinement. In the current de-escalator, daily PCR tests should be increased, maintaining vigilance on indicators of incidence, prevalence, and positivity of PCR tests. Evidence suggests with some degree of confidence that mandatory confinement could be safely lifted as of August 30, 2020. Long-term preparations must be made to contain future waves of new cases.

INTRODUCCIÓN: Los resultados del confinamiento obligatorio han sido perjudiciales en varios aspectos. No obstante, han surtido efecto en lograr el descenso de casos activos de COVID-19. Chile ha comenzado la desescalada y precisa conocer el mejor momento para poner fin a las restricciones. OBJETIVOS: Discutir las mejores condiciones y garantías para el fin del confinamiento obligatorio sobre la base de los casos nuevos, casos activos y positividad de exámenes de reacción en cadena de la polimerasa. MÉTODOS: Estudio basado en un modelo de tendencia con estimación de predicciones. Los datos de las variables de interés fueron sometidas a estudios de regresión lineal, con el objeto de determinar la curva que mejor explicaba los datos. Se estimó el coeficiente de determinación, la desviación estándar de y en x y el intervalo de confianza de la curva observada. Posteriormente, fue escogida la curva de tendencia en concordancia con las estimaciones de regresión. RESULTADOS: Se encontró que todas las variables dependientes tendían a disminuir con el tiempo de forma cuadrática, con excepción de la variable casos nuevos. En general, las estimaciones de coeficiente de determinación (R2) y error porcentual absoluto medio son satisfactorias, con excepción de la variable: número de exámenes de reacción en cadena de la polimerasa por día. CONCLUSIONES: Se deben tomar medidas graduales y cautelosas antes de poner fin al confinamiento obligatorio. En la actual desescalada, se deben aumentar los exámenes de reacción en cadena de la polimerasa diarios y mantener vigilancia en los indicadores de incidencia, prevalencia y positividad de dichos exámenes. La evidencia sugiere con cierto grado de confiabilidad que el confinamiento obligatorio podría levantarse de forma segura a contar del día 30 de agosto de 2020. Se deben hacer preparativos a largo plazo en contención de las futuras olas, es decir, una nueva alza de casos nuevos y activos luego del descenso.

Predicción del fin del confinamiento después del máximo de casos en la primera ola de la pandemia COVID-19 en Chile / Prediction of end of lockdown post-peak of cases in first wave of the COVID-19 pandemic in Chile

Introducción Los resultados del confinamiento obligatorio han sido perjudiciales en varios aspectos. No obstante, han surtido efecto en lograr el descenso de casos activos de COVID-19. Chile ha comenzado la desescalada y precisa conocer el mejor momento para poner fin a las restricciones. Objetivos Discutir las mejores condiciones y garantías para el fin del confinamiento obligatorio sobre la base de los casos nuevos, casos activos y positividad de exámenes de reacción en cadena de la polimerasa. Métodos Estudio basado en un modelo de tendencia con estimación de predicciones. Los datos de las variables de interés fueron sometidas a estudios de regresión lineal, con el objeto de determinar la curva que mejor explicaba los datos. Se estimó el coeficiente de determinación, la desviación estándar de y en x y el intervalo de confianza de la curva observada. Posteriormente, fue escogida la curva de tendencia en concordancia con las estimaciones de regresión. Resultados Se encontró que todas las variables dependientes tendían a disminuir con el tiempo de forma cuadrática, con excepción de la variable casos nuevos. En general, las estimaciones de coeficiente de determinación (R2) y error porcentual absoluto medio son satisfactorias, con excepción de la variable: número de exámenes de reacción en cadena de la polimerasa por día. Conclusiones Se deben tomar medidas graduales y cautelosas antes de poner fin al confinamiento obligatorio. En la actual desescalada, se deben aumentar los exámenes de reacción en cadena de la polimerasa diarios y mantener vigilancia en los indicadores de incidencia, prevalencia y positividad de dichos exámenes. La evidencia sugiere con cierto grado de confiabilidad que el confinamiento obligatorio podría levantarse de forma segura a contar del día 30 de agosto de 2020. Se deben hacer preparativos a largo plazo en contención de las futuras olas, es decir, una nueva alza de casos nuevos y activos luego del descenso.

Introduction The results of mandatory confinement have been detrimental in several respects. Nonetheless, they have resulted in reducing the number of active cases of COVID-19. Chile has begun the de-escalation and needs to know the best time to end the restrictions. Objective We discuss the best conditions and guarantees for the end of compulsory confinement. Methods This study is based on a trend model with prediction estimation. The data of the variables of interest were subjected to linear regression studies to determine the curve that best explained the data. The coefficient of determination, the standard deviation of y in x, and the confidence interval of the observed curve were estimated. The trend curve was chosen in accordance with the regression estimates. Outcomes It was found that all dependent variables tended to decrease over time in a quadratic fashion, except for the new cases variable. In general, the R2 and MAPE estimates are satisfactory, except for the variable number of PCR tests per day. Conclusions Gradual and cautious steps should be taken before ending mandatory confinement. In the current de-escalator, daily PCR tests should be increased, maintaining vigilance on indicators of incidence, prevalence, and positivity of PCR tests. Evidence suggests with some degree of confidence that mandatory confinement could be safely lifted as of August 30, 2020. Long-term preparations must be made to contain future waves of new cases.

Innovative and New Approaches to Laboratory Diagnosis of Zika and Dengue: A Meeting Report.

Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.

Transforming viral sequences to A (H1N1) flu diagnosis: the current status and future prospects of rtPCR based assays.

A variety of technologies can be used in the detection of contagious pathogens. In the early stage of an outbreak of a new infectious disease, rtPCR is advantageous over many other assays. The rtPCR can be developed either using low fidelity DNA polymerase or high fidelity DNA polymerase. The application of high fidelity DNA polymerase allows the shortening of assay development. In addition, the synthesized DNA template used as positive controls is suggested for shortening the time for assay development. Overall comparison of time required for assay development, specificity, and sensitivity for different types of molecular diagnostic technologies, it seems that early confirmation of viral infected patients will be diagnosed primarily with PCR or rtPCR-based assays presently and likely for the near future.

New developments in the diagnosis of avian influenza.

Avian influenza has become a serious concern from both veterinary and public health points of view. National and international organisations, veterinary health authorities, research institutions, diagnostic laboratories and field services make enormous efforts worldwide to detect, combat and prevent this important disease. Accordingly, the standard diagnostic protocols are being supported by a wide variety of molecular detection techniques, including improved polymerase chain reaction assays, microarray-based detection and characterisation methods, very rapid sequencing, simple pen-side tests and other on-site approaches. These recently developed 'closer to the field' methods allow rapid detection of influenza viruses and the identification of pathogenicity variants. However, in order to harmonise the diagnosis worldwide, attention has to be paid to the validation and standardisation of these technologies, to avoid erroneous interpretation of assay results, and, consequently, inappropriate epidemiological measures. This review gives an overview of the current and potential future developments related to avian influenza diagnostics.

The emergence of Clostridium difficile PCR ribotype 027 in Denmark--a possible link with the increased consumption of fluoroquinolones and cephalosporins?

Increasing rates of Clostridium difficile infection (CDI) with an unusual, severe course have been reported in several countries; this rise has partly been ascribed to the emergence of a virulent strain, C. difficile PCR ribotype 027 (CD027). An intriguing question is whether this could be related to increasing consumption of broadspectrum antibiotics. From 1997 to 2007, the number of hospital discharges in Denmark with the diagnosis enterocolitis caused by C. difficile increased from eight to 23 per 100,000 hospital discharges. This increase was proportional to a concomitant rise in the consumption of fluoroquinolones and cephalosporins. The first outbreak of CD027 in Denmark occurred from October 2006 to August 2007 and included 13 patients, most of them elderly, admitted to three hospitals in the same region. Most of the patients had overlapping periods of admission. All patients had been treated with broadspectrum antibiotics, in particular cephalosporins and fluoroquinolones, prior to positive culture of CD027. 30 days after confirmation of diagnosis, three of the 13 patients had died. Taken together, the data support the hypothesis that the increasing use of certain broadspectrum antibiotics may be related to a possible increase of C. difficile infection, and show that the specific contribution by CD027 in its emergence needs to be determined.

[Hypomethylation of 5'CpG island of insulin-like growth factor binding protein-related protein 1 is associated with its overexpression in colorectal cancer].

OBJECTIVE: To investigate the methylation status of 5'CpG island of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) in colorectal cancer and its relationship with gene expression and clinicopathologic parameters. METHODS: Semi-quantitative reverse transcription-PCR (RT-PCR) was used to detect the expression of IGFBP-rP1 in 46 cases of colorectal cancer and their matched normal mucosa. Methylation-specific PCR (MSP) was applied to evaluate the methylation status of 5'CpG island of IGFBP-rP1. Colon cancer cell lines LoVo and SW620 were treated with demethylation agent 5-aza-2'-deoxycytidine (5-aza-dC), followed by RT-PCR and MSP detection. RESULTS: At the mRNA level, the expression of IGFBP-rP1 was higher in colorectal cancer tissue than that in the matched normal mucosa (P < 0.05). IGFBP-rP1 was methylated in 28/46 (60.9%) cases of colorectal cancer and 37/46 (80.4%) matched normal mucosa samples (P < 0.05). A negative correlation was found between IGFBP-rP1 expression and its methylation status. The expression of IGFBP-rP1 was restored in LoVo and SW620 after treatment with 5-aza-dC and MSP confirmation of its demethylation status. No relationships was found between the methylation status and clinicopathologic parameters. CONCLUSIONS: IGFBP-rP1 expression is negatively correlated with its methylation status in colorectal cancer. DNA methylation is one of the mechanisms regulating the expression of IGFBP-rP1. Hypomethylation of IGFBP-rP1 gene with its overexpression plays an important role in the initiation and development of colorectal cancer.

Twenty-five years of quantitative PCR for gene expression analysis.

Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.

ZAG, a lipid mobilizing adipokine, is downregulated in human obesity

The main goal of this study was to compare the expression of Zinc-á2-glycoprotein(ZAG), a recently described adipokine, in obese and lean subjects. ZAG expressionwas determined by Real-time PCR analysis in subcutaneous abdominal adiposetissue of eighteen young men, 9 lean (BMI=23.1±0.4 kg/m2) and 9 obese (34.7±1.2kg/m2) with a similar habitual dietary intake of fat and physical activity, which wereassessed by validated methods. Our data revealed that ZAG gene was downregulated(–70%; p<0.05) in subcutaneous adipose tissue of obese compared to lean subjects.Moreover, statistically significant positive correlations between ZAG gene expressionand serum adiponectin (r=0.89; p<0.01) and a negative correlation with the plasmalevels of leptin (r=–0.82; p<0.05) and waist circumference (r=–0.64; p<0.05) werefound in obese subjects. Our data suggest that this novel adipokine could play a rolein human susceptibility to obesity related disorders and that upregulation of ZAGcould be a promising therapeutic target for metabolic syndrome treatment (AU)

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Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2.

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2.

PNA FISH: present and future impact on patient management.

Inappropriate and inaccurate antimicrobial therapy can lead to adverse patient outcomes and also the development of antimicrobial resistance. Peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) gives rapid reporting with highly sensitive and specific results to clinicians within 3 h after blood cultures turn positive, thereby offering targeted therapeutics where necessary. It is simple to establish compared with real-time PCR and has resulted in significant cost savings for hospitals. PNA FISH is a promising future technology for the microbiology laboratory that will impact on patient management and clinical guidelines. This article will review the clinical data supporting these new technologies.

The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections.

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.

Latest developments in in situ PCR.

Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to tissue preparations, fixation, mounting of slides, reverse transcription technique, primer design, target selection, size, and amplicon size, and thermocycler designs and the use, among many other fine details. This chapter describes the detail procedures that are used in the author's laboratory. It also discusses the variations and modification that can be used for the specific needs of an investigator. This protocol should serve as a primer for the investigators, and each researcher must use his or her variation according to their needs.

Novel retinoid targets in the mouse limb during organogenesis.

Bioactive retinoids are potent limb teratogens, upregulating apoptosis, decreasing chondrogenesis, and producing limb-reduction defects. To target the origins of these effects, we examined gene expression changes in the developing murine limb after 3 h of culture with teratogenic concentrations of vitamin A. Embryonic day 12 CD-1 limbs were cultured in the absence or presence of vitamin A (retinol acetate) at 1.25 and 62.5muM (n = 5). Total RNA was used to probe Atlas 1.2 cDNA arrays. Eighty-one genes were significantly upregulated by retinol exposure; among these were key limb development signaling molecules, extracellular matrix and adhesion proteins, oncogenes, and a large number of transcriptional regulators, including Eya2, Id3, Snail, and Hes1. To relate these expression changes to teratogenic outcome, the response of these four genes was assessed after culture with vitamin A and retinoid receptor antagonists that are able to rescue retinoid-induced malformations; expression levels were correlated with limb malformations. Lastly, pathways analysis revealed that a large number of the genes significantly affected by retinoid treatment are functionally linked through direct interactions. Several regulatory gene cascades emerged relevant to morphogenesis, cell-fate, and chondrogenesis; moreover, members of these cascades crosstalk with one other. These results indicate that retinoids act in a coordinated fashion to disrupt development at multiple levels. In sum, this work proposes several unifying mechanisms for retinoid-induced limb malformations, identifies novel retinoid targets, and highlights Eya2, Id3, Snail, and Hes1 as potential key teratogenic effectors.

Functional Genomics meets neurodegenerative disorders Part I: transcriptomic and proteomic technology.

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).

The power of real-time PCR.

In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene expression as a result of physiology, pathophysiology, or development. This method can be applied to model systems to measure responses to experimental stimuli and to gain insight into potential changes in protein level and function. Thus physiology can be correlated with molecular events to gain a better understanding of biological processes. For clinical molecular diagnostics, real-time PCR can be used to measure viral or bacterial loads or evaluate cancer status. Here, we discuss the basic concepts, chemistries, and instrumentation of real-time PCR and include present applications and future perspectives for this technology in biomedical sciences and in life science education.

Current issues for DNA microarrays: platform comparison, double linear amplification, and universal RNA reference.

DNA microarray technology has been widely used to simultaneously determine the expression levels of thousands of genes. A variety of approaches have been used, both in the implementation of this technology and in the analysis of the large amount of expression data. However, several practical issues still have not been resolved in a satisfactory manner, and among the most critical is the lack of agreement in the results obtained in different array platforms. In this study, we present a comparison of several microarray platforms [Affymetrix oligonucleotide arrays, custom complementary DNA (cDNA) arrays, and custom oligo arrays printed with oligonucleotides from three different sources] as well as analysis of various methods used for microarray target preparation and the reference design. The results indicate that the pairwise correlations of expression levels between platforms are relative low overall but that the log ratios of the highly expressed genes are strongly correlated, especially between Affymetrix and cDNA arrays. The microarray measurements were compared with quantitative real-time-polymerase chain reaction (QRT-PCR) results for 23 genes, and the varying degrees of agreement for each platform were characterized. We have also developed and tested a double amplification method which allows the use of smaller amounts of starting material. The added round of amplification produced reproducible results as compared to the arrays hybridized with single round amplified targets. Finally, the reliability of using a universal RNA reference for two-channel microarrays was tested and the results suggest that comparisons of multiple experimental conditions using the same control can be accurate.