Detection In Clinical Specimens Research Articles

Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Wastewater-based SARS-CoV-2 surveillance and sequencing

Though most often associated with poliovirus surveillance, wastewater-based surveillance (WBS) can be employed for viruses shed in human excreta (faeces, urine, skin, sputum, blood) that may enter the wastewater system, including SARS-CoV-2. WBS has been widely adopted during the COVID-19 pandemic, to complement clinical surveillance in monitoring community burden and implementing timely public health interventions. As wastewater is a non-biased, composite sample, it can provide population-level health information in near real-time, in a cost-effective manner compared to similarly scaled clinical surveillance. In many instances, data gathered from wastewater, including viral loads (quantified by RT-qPCR) and variant detections (determined through partial or whole genome sequencing), have been predictive of what is observed eventually in clinical cases. Newly emergent lineages, including the recent BA.2.86 variant, can and have been detected in wastewater samples prior to their detection in clinical specimens. There remain many challenges to wastewater genomic analysis including the presence of RT-qPCR inhibitors, degraded nucleic acid and the lack of consistent or standardised methodology between reporting labs. The wide adoption of WBS practices provides an excellent opportunity to expand this method for surveillance of other pathogens of public health importance. Herein, a broad overview of the WBS field will be provided including discussion on its advantages and applications, challenges, and how it is being utilised to characterise circulating SARS-CoV-2 lineages through sequencing.

Tracing the origin of SARS-CoV-2 omicron-like spike sequences detected in an urban sewershed: a targeted, longitudinal surveillance study of a cryptic wastewater lineage

The origin of novel SARS-CoV-2 spike sequences found in wastewater, without corresponding detection in clinical specimens, remains unclear. We sought to determine the origin of one such cryptic wastewater lineage by tracking and characterising its persistence and genomic evolution over time. We first detected a cryptic lineage, WI-CL-001, in municipal wastewater in Wisconsin, USA, in January, 2022. To determine the source of WI-CL-001, we systematically sampled wastewater from targeted sub-sewershed lines and maintenance holes using compositing autosamplers. Viral concentrations in wastewater samples over time were measured by RT digital PCR. In addition to using metagenomic 12s rRNA sequencing to determine the virus's host species, we also sequenced SARS-CoV-2 spike receptor binding domains, and, where possible, whole viral genomes to identify and characterise the evolution of this lineage. We traced WI-CL-001 to its source at a single commercial building. There we detected the cryptic lineage at concentrations as high as 2·7× 109 genome copies per L. The majority of 12s rRNA sequences detected in wastewater leaving the identified source building were human. Additionally, we generated over 100 viral receptor binding domain and whole-genome sequences from wastewater samples containing the cryptic lineage collected over the 13consecutive months this virus was detectable (January, 2022, to January, 2023). These sequences contained a combination of fixed nucleotide substitutions characteristic of Pango lineage B.1.234, which circulated in humans in Wisconsin at low levels from October, 2020, to February, 2021. Despite this, mutations in the spike gene and elsewhere resembled those subsequently found in omicron variants. We propose that prolonged detection of WI-CL-001 in wastewater indicates persistent shedding of SARS-CoV-2 from a single human initially infected by an ancestral B.1.234 virus. The accumulation of convergent omicron-like mutations in WI-CL-001's ancestral B.1.234 genome probably reflects persistent infection and extensive within-host evolution. People who shed cryptic lineages could be an important source of highly divergent viruses that sporadically emerge and spread. The Rockefeller Foundation, Wisconsin Department of Health Services, Centers for Disease Control and Prevention, National Institute on Drug Abuse, and the Center for Research on Influenza Pathogenesis and Transmission.

Impact of swab removal in the detection of SARS-CoV-2 weakly-positive specimens.

Removing the swab after collection can speed up diagnosis and improve the quality of laboratory procedures. This study investigates the impact of swab removal on SARS-CoV-2 detection in clinical specimens with a focus on high Cycle threshold (Ct) samples (Cts≥32). The method assessed pairs of SARS-CoV-2 samples mimicking combined throat and nose swabs and tested them on two real-time-PCR platforms; the Applied Biosystems 7500 and the Abbott Alinity. Swab removal did not significantly affect detection rates of SARS-CoV-2 samples with Ct values<32, regardless of the PCR platform. However, reduced reproducibility was seen at the endpoint limit of detection of the platforms, which meant that fewer samples with Ct values≥32 were detected in the swab removal group.

Latest Advances in Laboratory Detection of Mycoplasma genitalium.

Mycoplasma genitalium is an important sexually transmitted pathogen affecting both men and women. Its extremely slow growth in vitro and very demanding culture requirements necessitate the use of molecular-based diagnostic tests for its detection in clinical specimens. The recent availability of U.S. Food and Drug Administration (FDA)-cleared commercial molecular-based assays has enabled diagnostic testing to become more widely available in the United States and no longer limited to specialized reference laboratories. Advances in the knowledge of the epidemiology and clinical significance of M. genitalium as a human pathogen made possible by the availability of molecular-based testing have led to updated guidelines for diagnostic testing and treatment that have been published in various countries. This review summarizes the importance of M. genitalium as an agent of human disease, explains the necessity of obtaining a microbiological diagnosis, describes currently available diagnostic methods, and discusses how the emergence of antimicrobial resistance has complicated treatment alternatives and influenced the development of diagnostic tests for resistance detection, with an emphasis on developments over the past few years.

A performance comparison between fluorescent immunoassay and immunochromatography for rapid dengue detection in clinical specimens

Dengue virus (DENV 1–4) infection has been a global health threat where no specific treatment is currently available. Therefore, a rapid and accurate diagnosis is critical for an appropriate management as it could reduce the burden of severe clinical manifestation. Currently, dengue immunochromatography (IC) is commonly used to primarily differentiate acute febrile illnesses. Fluorescent immunoassay (FIA) utilized a highly sensitive detection system and claimed 70–100% sensitivity and 83.5–91.7% specificity for dengue infection in a preliminary report. This report recruited samples with acute febrile illnesses sent for dengue screening and tested IC and FIA in parallel. The performance of both tests was verified by a definitive diagnosis retrieved from combinatorial reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) for IgM and IgG confirmation tests. Results showed that the viral nonstructural protein (NS1) performance of FIA was slightly higher than IC with the sensitivity, specificity, PPV, NPV, agreement, kappa, and its standard error at 79.11, 92.28, 86.81, 87.31, 352 (87.13%), 0.725 ± 0.035, respectively; whereas those of the IC were at 76.58, 92.28, 86.43, 85.98, 348 (86.14%), 0.703 ± 0.037, respectively. Moreover, the IgM and IgG performance of FIA had higher specificity, PPV, and agreement than the IgM IC performance, suggesting that the FIA was more specific but less sensitive for antibody detection. No correlation was observed in IgM and IgG levels of ELISA and FIA assays. In conclusion, the FIA and IC were highly sensitive, specific, and substantially agreed in NS1 detection but moderately agreed in IgM and IgG detection.

Ionic Liquid-Functionalized ZrO2/Reduced Graphene Oxide Nanocomposites for Carcinoembryonic Antigen Electrochemical Detection

Herein, we develop an electrochemical immunosensor based on a zirconia-reduced graphene oxide-1-ethyl-3-methylimidazolium tetrafluoroborate (EMIM.BF4) ionic liquid (ZrO2-rGO-IL) nanocomposite for the detection of a carcinoembryonic antigen (CEA) breast cancer biomarker. The characterization results of the synthesized ZrO2-rGO-IL nanocomposite show that it has high electroconductivity, oxygen functionalities, and a larger active surface area, which favors high loading of anti-CEA antibodies. The fabricated BSA/anti-CEA/ZrO2-rGO-IL/GCE immunosensor can detect CEA with high sensitivity and selectivity in a broad detection range between 25.0 fg mL–1 and 25.0 ng mL–1 with an ultralow limit of detection (LOD) of 1.23 fg mL–1 through differential pulse voltammetry (DPV). In addition, we tested the CEA in serum samples having linear detection ranges from 100.0 fg mL–1 to 5.0 ng mL–1 with an LOD of 2.25 fg mL–1. Subsequently, the incubation time and selectivity of the immunosensor show a great affinity for CEA. Therefore, the proposed immunosensor can be a promising candidate for CEA detection in clinical specimens. Prospectively, the synthesized nanocomposite will have the potential for the effective detection of other cancer biomarkers.

LEGIONNAIRES DISEASE: A CASE WITH SEVERE NEUROLOGIC AND RENAL INVOLVEMENT

LEGIONNAIRES DISEASE: A CASE WITH SEVERE NEUROLOGIC AND RENAL INVOLVEMENT

Second near-infrared fluorescent dye for lateral flow immunoassays rapid detection of influenza A/B virus

Sensitive and rapid diagnostic point of care testing (POCT) system is of great significance to prevent and control human virus infection. Here reported an immunochromatographic strip technology. The second near-infrared (NIR-II) fluorescent dye encapsulated into polystyrene (PS) nanoparticles, was integrated into a lateral flow assay platform to achieve excellent detection of influenza A/B. This surface-functionalized and mono-dispersed PS nanoparticles has been conjugated with influenza nucleoprotein monoclonal antibody as targets for influenza antigen-detection. This assay achieved the detection limit of 0.015 ng/mL for influenza A nucleoprotein and 4.3*10−5 HAU/mL (102.08 TCID50/mL) influenza A virus (influenza B: 0.037 ng/mL, 9.7*10−7 HAU/mL (100.43 TCID50/mL)). Compared with an Au-based lateral flow test strip, the strip's sensitivity is about 16-fold higher than it. Strip detection properties remain stable for 6 months under 4 °C to 30 °C storage. The assay's intra assay variation is 5.14% and the inter assay variation is 7.74%. Other potential endogenous and exogenous interfering substances (whole blood, nasal mucin, saliva, antipyretics, antihistamines and neuraminidase inhibitors) showed negative results, which verified the excellent specificity of this method. This assay was successfully applied to the POCT quantitative detection of influenza A/B virus, the sensitivity to influenza A and B viruses was 70% and 87.5% respectively, and the specificity was 100%. Therefore, these microspheres can be used as an effective material for rapid POCT detection in clinical specimens.

Novel nanostructure-coupled biosensor platform for one-step high-throughput quantification of serum neutralizing antibody after COVID-19 vaccination

COVID-19 vaccination efficacy depends on serum levels of the neutralizing antibodies (NAs) specific to the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Therefore, a high-throughput rapid assay capable of measuring the total SARS-CoV-2 NA level is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, vaccine development, and assessment. Here, we developed a novel nanoplasmonic immunosorbent assay (NanoPISA) platform for one-step rapid quantification of SARS-CoV-2 NAs in clinical serum samples for high-throughput evaluation of COVID-19 vaccine effectiveness. The NanoPISA platform enhanced by the use of nanoporous hollow gold nanoparticle coupling was able to detect SARS-CoV-2 NAs with a limit of detection of 0.2 pM within 15 min without washing steps. The one-step NanoPISA for SARS-CoV-2 NA detection in clinical specimens yielded good results, comparable with those obtained in the gold-standard seroneutralization test and the surrogate virus-neutralizing enzyme-linked immunosorbent assay. Collectively, the one-step NanoPISA might be a rapid and high-throughput NA-quantification platform for evaluating the effectiveness of COVID-19 vaccines.

Impact of the Nucleic Acid Extraction Method and the RT-qPCR Assay on SARS-CoV-2 Detection in Low-Viral Samples.

COVID-19 was initially reported in China at the end of 2019 and soon thereafter, in March 2020, the WHO declared it a pandemic. Until October 2021, over 240 million COVID-19 cases were recorded, with 4.9 mln deaths. In order to stop the spread of this disease, it is crucial to monitor and detect any infected person. The etiologic agent of COVID-19 is a novel coronavirus called SARS-CoV-2. The gold standard for the detection of the virus is the RT-qPCR method. This study evaluated two RNA extraction methods and four commercial RT-qPCR assays routinely used in diagnostic laboratories for detecting SARS-CoV-2 in human specimens from the upper respiratory tract. We analyzed a panel of 70 clinical samples with varying RNA loads. Our study demonstrated the significant impact of the diagnostic methods selected by the laboratory on the SARS-CoV-2 detection in clinical specimens with low viral loads.

Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV)

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

Detection of SARS-CoV-2 by real-time PCR under challenging pre-analytical conditions reveals independence of swab media and cooling chain

With global demand for SARS-CoV-2 testing ever rising, shortages in commercially available viral transport media pose a serious problem for laboratories and health care providers. For reliable diagnosis of SARS-CoV-2 and other respiratory viruses, executed by Real-time PCR, the quality of respiratory specimens, predominantly determined by transport and storage conditions, is crucial. Therefore, our aim was to explore the reliability of minimal transport media, comprising saline or the CDC recommended Viral Transport Media (HBSS VTM), for the diagnosis of SARS-CoV-2 and other respiratory viruses (influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus) compared to commercial products, such as the Universal Transport Media (UTM). We question the assumptions, that the choice of medium and temperature for storage and transport affect the accuracy of viral detection by RT-PCR. Both alternatives to the commercial transport medium (UTM), HBSS VTM or saline, allow adequate detection of SARS-CoV-2 and other respiratory viruses, regardless of storage temperatures up to 28 °C and storage times up to 28 days. Our study revealed the high resilience of SARS-CoV-2 and other respiratory viruses, enabling proper detection in clinical specimens even after long-time storage at high temperatures, independent of the transport medium’s composition.

Identification of chondroitin polymerizing factor (CHPF) as tumor promotor in cholangiocarcinoma through regulating cell proliferation, cell apoptosis and cell migration

ABSTRACT Cholangiocarcinoma (CCA) is a variety of biliary epithelial tumors involving intrahepatic, perihilar and distal bile duct. It is the most common malignant bile duct tumor in the liver and the second most common primary liver cancer, whose molecular mechanism not fully understood. Specifically, the relationship between CCA and chondroitin polymerizing factor (CHPF) is still not clear. In this study, detection of clinical specimens was performed to preliminarily study the role of CHPF in CCA. CCA cells with CHPF knockdown were constructed for in vitro study, which was also used in the construction of mice xenograft model for investigating the role of CHPF in the development of CCA. The results demonstrated that CHPF was significantly upregulated in CCA tissues compared with normal tissues. High expression of CHPF was correlated with more advanced tumor grade. Moreover, knockdown of CHPF significantly inhibited cell proliferation, cell migration, promoted cell apoptosis and arrest cell cycle in G2 phase in vitro, as well as suppressed tumor growth in vivo. In conclusion, CHPF was identified as a tumor promotor in the development and metastasis of CCA, which may provide a novel therapeutic target for the targeted therapy against CCA.

Functional Characterization of Circulating Mumps Viruses with Stop Codon Mutations in the Small Hydrophobic Protein.

Between 2015 and 2017, routine molecular surveillance in the United States detected multiple mumps viruses (MuVs) with mutations in the small hydrophobic (SH) gene compared to a reference virus of the same genotype. These mutations include an unusual pattern of uracil-to-cytosine hypermutations and other mutations resulting in the generation of premature stop codons or disruption of the canonical stop codon. The mumps virus SH protein may serve as a virulence factor, based on evidence that it inhibits apoptosis and innate immune signaling in vitro and that recombinant viruses that do not express the SH protein are attenuated in an animal model. In this study, mumps viruses bearing variant SH sequences were isolated from contemporary outbreak samples to evaluate the impact of the observed mutations on SH protein function. All isolates with variant SH sequences replicated in interferon-competent cells with no evidence of attenuation. Furthermore, all SH-variant viruses retained the ability to abrogate induction of NF-κB-mediated innate immune signaling in infected cells. Ectopic expression of variant mumps SH genes is consistent with findings from infection experiments, indicating that the observed abrogation of signaling was not mediated by other viral factors that may modulate innate immune signaling. Molecular surveillance is an important public health tool for monitoring the diversity of circulating mumps viruses and can provide insights into determinants of disease. These findings, in turn, will inform studies employing reverse genetics to elucidate the specific mechanisms of MuV pathogenesis and potential impacts of observed sequence variants on infectivity, fitness, and virulence.IMPORTANCE Mumps virus (MuV) outbreaks occur in the United States despite high coverage with measles, mumps, rubella (MMR) vaccine. Routine genotyping of laboratory-confirmed mumps cases has been practiced in the United States since 2006 to enhance mumps surveillance. This study reports the detection of unusual mutations in the small hydrophobic (SH) protein of contemporary laboratory-confirmed mumps cases and is the first to describe the impact of such mutations on SH protein function. These mutations are predicted to profoundly alter the amino acid sequence of the SH protein, which has been shown to antagonize host innate immune responses; however, they were neither associated with defects in virus replication nor attenuated protein function in vitro, consistent with detection in clinical specimens. A better understanding of the forces governing mumps virus sequence diversity and of the functional consequences of mutations in viral proteins is important for maintaining robust capacity for mumps detection and disease control.

A Cell-Based Capture Assay for Rapid Virus Detection.

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

Abstract 5741: Highly specific copy number variant-flagging algorithm using next-generation sequencing in cancer-predisposition genes

Abstract Background: Hereditary cancer accounts for 5% to 10% of all cancers. In addition to sequence variants, copy number variants (CNVs) are a cause of inherited cancer syndromes. Next-generation sequencing (NGS) technology enables simultaneous interrogation of multiple genes in a cost-effective manner. However, accurate CNV detection by NGS remains challenging owing to bias and noise that distort the association between copy number and read-depth. We previously developed and validated an NGS-based CNV detection algorithm using 18 CNV-positive and 85 CNV-negative specimens; the algorithm yielded 100% sensitivity and 98% specificity. In this study, we evaluated the performance of the algorithm on a large group of clinical specimens. Methods: Retrospective CNV analysis was performed on 19,722 specimens submitted for a 34-gene cancer predisposition panel (n=5,726) or 4 sub-panels: BRCA1/2 (n=13,156), a 5-gene Lynch syndrome panel (n=553), a 13-gene colorectal cancer panel (n=205), or a 14-gene women's health panel (n=82). For NGS, target genes were captured using an RNA-bait hybridization method and sequenced on an Illumina NextSeq 500 instrument. CNV-positive specimens were identified using a CNV-flagging algorithm developed in-house that is based on the well-established NGS read-depth approach. All specimens flagged as having CNVs by NGS were reflexed to array comparative genomic hybridization (aCGH) using a custom-designed Agilent array for confirmation. Although highly reliable and widely used, aCGH can only simultaneously process a limited number of specimens. Results: Among the 19,722 clinical specimens, the NGS CNV flagging algorithm identified 485 CNVs; 235 were positive by aCGH and 250 were negative; thus, specificity of the algorithm alone was 98.7% (19,472/19,722) and improved to 100% when aCGH was incorporated. Of 235 aCGH-confirmed CNVs, 224 (95.3%) were pathogenic or likely pathogenic. Approximately 1% of specimens tested by a 34-gene panel (60/5,726) was CNV-positive. BRCA1 (20% of positives) was the most frequently mutated gene followed by CHEK2 (15%), ATM (10%), and PALB2 (10%). From the 4 sub-panels, the 5-gene lynch panel had highest positive CNV rate (4%, 22/553), followed by 13-gene colorectal panel (2%, 4/205), and BRCA1/2 test (1%, 149/13,156). Conclusions: The developed CNV-flagging algorithm enabled CNV detection of clinical specimens, in a variety of panels, with high specificity. Supplementation of aCGH confirmation improved CNV detection specificity to 100%. Our data underscore the important clinical utility of NGS-based CNV detection in the molecular diagnosis of hereditary cancer. Citation Format: Sun Hee Rosenthal, Ke Zhang, Yan Liu, Renius Owen, Alla Smolgovsky, Arlene Buller-Burckle, Felicitas Lacbawan. Highly specific copy number variant-flagging algorithm using next-generation sequencing in cancer-predisposition genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5741.

Enteric Virome and Carcinogenesis in the Gut.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths in both the USA and the world. Recent research has demonstrated the involvement of the gut microbiota in CRC development and progression. Microbial biomarkers of disease have focused primarily on the bacterial component of the microbiome; however, the viral portion of the microbiome, consisting of both bacteriophages and eukaryotic viruses, together known as the virome, has been lesser studied. Here we review the recent advancements in high-throughput sequencing (HTS) technologies and bioinformatics, which have enabled scientists to better understand how viruses might influence the development of colorectal cancer. We discuss the contemporary findings revealing modulations in the virome and their correlation with CRC development and progression. While a variety of challenges still face viral HTS detection in clinical specimens, we consider herein numerous next steps for future basic and clinical research. Clinicians need to move away from a single infectious agent model for disease etiology by grasping new, more encompassing etiological paradigms, in which communities of various microbial components interact with each other and the host. The reporting and indexing of patient health information, socioeconomic data, and other relevant metadata will enable identification of predictive variables and covariates of viral presence and CRC development. Altogether, the virome has a more profound role in carcinogenesis and cancer progression than once thought, and viruses, specific for either human cells or bacteria, are clinically relevant in understanding CRC pathology, patient prognosis, and treatment development.

Platinum ions mediate the interactions between DNA and carbon quantum dots: diagnosis of MRSA infections.

In this study, we have developed a rapid and cost-effective method employing platinum ion (Pt4+)-capped fluorescent carbon quantum dots (CQDs) coupled with loop-mediated isothermal amplification (LAMP) to detect dual MRSA genes. We synthesized nitrogen- and chlorine-co-doped fluorescent CQDs (CQDSPDs) from spermidine trihydrochloride via a simple one-step pyrolysis. The CQDSPDs capped with Pt4+ ions through the cooperative coordination of the amine and chlorine groups on the surface of CQDs facilitated the double-stranded DNA (dsDNA)-induced fluorescence quenching of CQDs, and enabled the construction of the CQDSPDs/Pt4+ probe for the detection of as few as 10 copies of the MRSA gene (mecA and femA). The sensitivity and specificity of the CQDSPDs/Pt4+ probe for MRSA detection in clinical specimens (n = 24) were 94% and 86%, respectively. Our results reveal that the CQDSPDs/Pt4+ probe has great potential for the diagnosis of antibiotic-resistant superbugs with high sensitivity, specificity, and agreement.

Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing.

Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.