Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis.

Metagenomic sequencing has emerged as a transformative tool in infectious disease diagnosis, offering a comprehensive and unbiased approach to pathogen detection. Leveraging international standards and guidelines is essential for ensuring the quality and reliability of metagenomic sequencing in clinical practice. This review explores the implications of international standards and guidelines for the application of metagenomic sequencing in infectious disease diagnosis. By adhering to established standards, such as those outlined by regulatory bodies and expert consensus, healthcare providers can enhance the accuracy and clinical utility of metagenomic sequencing. The integration of international standards and guidelines into metagenomic sequencing workflows can streamline diagnostic processes, improve pathogen identification, and optimize patient care. Strategies in implementing these standards for infectious disease diagnosis using metagenomic sequencing are discussed, highlighting the importance of standardized approaches in advancing precision infectious disease diagnosis initiatives.

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann-Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal-Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples.

Exploring the Role of Circulating Cell-Free RNA in the Development of Colorectal Cancer.

Circulating tumor RNA (ctRNA) has recently emerged as a novel and attractive liquid biomarker. CtRNA is capable of providing important information about the expression of a variety of target genes noninvasively, without the need for biopsies, through the use of circulating RNA sequencing. The overexpression of cancer-specific transcripts increases the tumor-derived RNA signal, which overcomes limitations due to low quantities of circulating tumor DNA (ctDNA). The purpose of this work is to present an up-to-date review of current knowledge regarding ctRNAs and their status as biomarkers to address the diagnosis, prognosis, prediction, and drug resistance of colorectal cancer. The final section of the article discusses the practical aspects involved in analyzing plasma ctRNA, including storage and isolation, detection technologies, and their limitations in clinical applications.

Cell-free circulating tumor RNAs in plasma as the potential prognostic biomarkers in colorectal cancer.

Background: Cell free RNA (cfRNA) contains transcript fragments from multiple cell types, making it useful for cancer detection in clinical settings. However, the pathophysiological origins of cfRNAs in plasma from colorectal cancer (CRC) patients remain unclear. Methods: To identify the tissue-specific contributions of cfRNAs transcriptomic profile, we used a published single-cell transcriptomics profile to deconvolute cell type abundance among paired plasma samples from CRC patients who underwent tumor-ablative surgery. We further validated the differentially expressed cfRNAs in 5 pairs of CRC tumor samples and adjacent tissue samples as well as 3 additional CRC tumor samples using RNA-sequencing. Results: The transcriptomic component from intestinal secretory cells was significantly decreased in the in-house post-surgical cfRNA. The HPGD, PACS1, and TDP2 expression was consistent across cfRNA and tissue samples. Using the Cancer Genome Atlas (TCGA) CRC datasets, we were able to classify the patients into two groups with significantly different survival outcomes. Conclusions: The three-gene signature holds promise in applying minimal residual disease (MRD) testing, which involves profiling remnants of cancer cells after or during treatment. Biomarkers identified in the present study need to be validated in a larger cohort of samples in order to ascertain their possible use in early diagnosis of CRC.

The clinical characteristics of pediatric patients infected by SARS-CoV-2 Omicron variant and whole viral genome sequencing analysis.

Pediatric population was generally less affected clinically by SARS-CoV-2 infection. Few pediatric cases of COVID-19 have been reported compared to those reported in infected adults. However, a rapid increase in the hospitalization rate of SARS-CoV-2 infected pediatric patients was observed during Omicron variant dominated COVID-19 outbreak. In this study, we analyzed the B.1.1.529 (Omicron) genome sequences collected from pediatric patients by whole viral genome amplicon sequencing using Illumina next generation sequencing platform, followed by phylogenetic analysis. The demographic, epidemiologic and clinical data of these pediatric patients are also reported in this study. Fever, cough, running nose, sore throat and vomiting were the more commonly reported symptoms in children infected by Omicron variant. A novel frameshift mutation was found in the ORF1b region (NSP12) of the genome of Omicron variant. Seven mutations were identified in the target regions of the WHO listed SARS-CoV-2 primers and probes. On protein level, eighty-three amino acid substitutions and fifteen amino acid deletions were identified. Our results indicate that asymptomatic infection and transmission among children infected by Omicron subvariants BA.2.2 and BA.2.10.1 are not common. Omicron may have different pathogenesis in pediatric population.

The diagnostic significance of CDH17-positive circulating tumor cells in patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths in Hong Kong. We tested the hypothesis that circulating tumor cell (CTC) analysis by ARB101 antibody could be used as a tool for CRC detection, progression, and therapy response. RESEARCH METHODS: ARB101 antibody was used for investigation of CDH17 expression in formalin-fixed, paraffin-embedded (FFPE) tissue sections and circulating tumor cells (CTCs) of CRC patients. RESULTS: Using ARB101, highest sensitivity was observed in 98/100 (98%) colorectal cancer tissue compared to 72/100 gastric cancer (72%) and 27/32 pancreatic cancer (84%). Immunoreactivity of CDH17 was significantly higher in distant metastatic (tumor-node-metastasis [TNM] stage IV) than non-distant metastatic (TNM stage I to III) CRC. ARB101 antibody also manifested the higher sensitivity than c-erbB2 (8%) and epidermal growth factor receptor (EGFR)-targeting antibodies (37%) with the significance (p < 0.0001). ARB101 positive CTCs were detected in 64/83 (77%) TNM stage I to IV CRC patients. Furthermore, ARB101 positive CTCs detected in TNM stage I to III CRC patients before and after surgical operation are statistically significant (p < 0.0001). CONCLUSIONS: CTC detection by ARB101 antibody could serve as a potential non-invasive approach for CRC detection, progression, and monitoring of treatment response.

Targeted Sequencing Approach and Its Clinical Applications for the Molecular Diagnosis of Human Diseases.

The outbreak of COVID-19 has positively impacted the NGS market recently. Targeted sequencing (TS) has become an important routine technique in both clinical and research settings, with advantages including high confidence and accuracy, a reasonable turnaround time, relatively low cost, and fewer data burdens with the level of bioinformatics or computational demand. Since there are no clear consensus guidelines on the wide range of next-generation sequencing (NGS) platforms and techniques, there is a vital need for researchers and clinicians to develop efficient approaches, especially for the molecular diagnosis of diseases in the emergency of the disease and the global pandemic outbreak of COVID-19. In this review, we aim to summarize different methods of TS, demonstrate parameters for TS assay designs, illustrate different TS panels, discuss their limitations, and present the challenges of TS concerning their clinical application for the molecular diagnosis of human diseases.

The current and future applications of in situ hybridization technologies in anatomical pathology.

INTRODUCTION: In situ hybridization (ISH) plays an important role in the field of molecular diagnostics, especially in an anatomical pathology laboratory. ISH is a technique that can detect the targeted DNA or RNA sequences in tissue sections from frozen or fixed materials with labeled DNA or RNA probes. Radioactive and non-radioactive probes are the two major probes that can be used to label the targeted nucleic acids. AREAS COVERED: Two decades after the Human Genome Project, ISH has not only simply been applied to identify the chromosomal location of a human gene but has also been extensively applied to gene expressions studies and utilized for clinical diagnosis, especially for the determination of biomarkers for breast and ovarian cancers - human epidermal growth factor receptor 2. Duchenne muscular dystrophy, Cri-du-chat syndrome, Angelman syndrome, PraderWilli syndrome, cystic fibrosis, and trisomy are diseases that can also be detected by ISH. In this review, the basic principles, historical development, advantages and disadvantages, enhancement in reporting molecules and probes, advancement in detection methods, in situ PCR, clinical applications and novel applications of ISH will be discussed. EXPERT OPINION: With the advancement in ISH technologies and appropriate training, diagnosis can be improved in Anatomical Pathology.

An update on COVID-19 pandemic: the epidemiology, pathogenesis, prevention and treatment strategies.

INTRODUCTION: To date, the transmission of Coronavirus Disease-2019 (COVID-19) is still uncontrollable with the fact that the numbers of confirmed and death cases are still increasing. Up to 1st October 2020, 33,842,281 confirmed cases and 1,010,634 confirmed deaths have been reported to the World Health Organization from 216 different countries, areas and territories. Despite the urgent demand for effective treatment strategies, there is still no specific antiviral treatment for COVID-19 and the treatment guidelines for COVID-19 vary between countries. AREA COVERED: In this article, we summarized the current knowledge on COVID-19 and the pandemic worldwide. Moreover, the epidemiology, pathogenesis, prevention and different treatment options will be discussed so that we shall prepare ourselves better to fight with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). EXPERT OPINION: The situation of the COVID-19 pandemic is still unpredictable. There is no effective vaccine or specific anti-viral drug to treat serve COVID-19 patients. Combination therapies have shown promising clinical improvement. Repurposing FDA-approved drugs might be one of possible treatment options. Without specific treatment and vaccines for COVID-19, the most effective way to prevent from being infected is to generate an ecosystem with effective protection, precautions and preventive measures.

The diagnostic methods in the COVID-19 pandemic, today and in the future.

INTRODUCTION: The emergence of anovel coronavirus identified in patients with unknown cause of acute respiratory disease in Wuhan, China at the end of 2019 has caused aglobal outbreak. The causative coronavirus was later named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease caused by SARS-CoV-2 was named as Coronavirus Disease-2019 (COVID-19). As of 10 August 2020, more than 19,718,030 confirmed cases and 728,013 deaths have been reported. COVID-19 is spread via respiratory droplets which are inhaled into the lungs. AREAS COVERED: In this article, we summarized the knowledge about the causative pathogen of COVID-19 and various diagnostic methods in this pandemic for better understanding of the limitations and the nuances of virus testing for COVID-19. EXPERT OPINION: In this pandemic, rapid and accurate identification of COVID-19 patients are critical to break the chain of infection in the community. RT-PCR provides a rapid and reliable identification of SARS-CoV-2 infection. In the future, molecular diagnostics will still be the gold standard and next-generation sequencing can help us to understand more on the pathogenesis and detect novel mutations. It is believed that more sophisticated detection methods will be introduced to detect SARS-CoV-2 as earliest as possible.

Current and future molecular diagnostics of gastric cancer.

Introduction: Gastric cancer (GC) is the fifth most common cancer and confers the second-highest mortality among other cancers. Improving the survival rates of GC patients requires prompt and accurate diagnosis and effective treatment which is often preceded by the poorly understood pathogenic mechanisms. Area covered: This literature review aims to summarize current understanding of genetic and molecular alterations that promote carcinogenesis including (1) activation of oncogenes, (2) overexpression of growth factors, receptors and matrix metalloproteinases, (3) inactivation of tumor suppressor genes, DNA repair genes, and cell adhesion molecules and (4) alterations of cell-cycle regulators that regulate biological characteristics of cancer cells. Moreover, the significance of molecular biomarkers such as micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) and advanced molecular techniques including droplet digital polymerase chain reaction (ddPCR), quantitative PCR (qPCR) and next-generation sequencing (NGS) are also discussed. Expert opinion: A GC-specific panel of biomarkers based on the NGS or ddPCR has the potential for diagnosis, prognosis, and monitoring treatment response in GC patients. Despite the requirements for validation in larger population in clinical studies, race-specific differences in the gene panel have also to be examined by performing the clinical trials in subjects with different races.

Application of NanoString technologies in companion diagnostic development.

Introduction: NanoString nCounter technology, a novel molecular assay, is gaining prevalent use in clinical settings as it can overcome some common constraints that are associated with the use of polymerase chain reaction (PCR). Compared to PCR, NanoString technology does not involve any amplification steps, which significantly minimizes the chance of contamination. NanoString measures the number of mRNA transcripts directly by 'molecular counting', as up to 800 colored probes can be run simultaneously in a single reaction. Areas covered: This manuscript reviews the principle of NanoString and covers the main applications of NanoString in companion diagnostics with a focus on cancer immunotherapy and disease prognosis estimation. This review has also taken a step in the direction of personalized medicine, with the application of NanoString on the realm of companion diagnostics. Expert opinion: NanoString is going to take a vital role in companion diagnostics and personalized medicine, owing to its simple and easy to use characteristics. Yet, the use of NanoString requires normalization of expression level, which is represented by the copy number of respective mRNA, with a reference gene. Furthermore, difficulty in probe design, which demands prior knowledge of known sequence, has also been a limitation of NanoString.