RAPID IDENTIFICATION OF ETIOLOGICAL AGENTS OF RESPIRATORY TRACT INFECTIONS DURING THE COVID-19 PANDEMIC USING THE MULTIPLEX-PCR METHOD.

Objective. Most respiratory infections have similar symptoms, so it is clinically difficult to determine their etiology. This study aimed to show the importance of molecular diagnostics in identifying the etiological agent of respiratory infections, especially during the coronavirus disease 2019 (COVID-19) pandemic. Methods. A total of 849 samples from patients hospitalized at the University Clinical Center Kragujevac (from January 1 to August 1, 2022) were examined using automated multiplex-polymerase chain reaction (PCR) tests. The BioFire-FilmArray-Respiratory Panel 2.1 test was used for 742 nasopharyngeal swabs [identification of 19 viruses (including SARS-CoV-2) and four bacteria], while the BioFire-FilmArray-Pneumonia Panel was used [identification of 18 bacteria and nine viruses] (BioMerieux, Marcy l'Etoile, France) for 107 tracheal aspirates. The tests were performed according to the manufacturer's instructions, and the results were available within an hour. Results. In 582 (78.4%) samples, the BioFire-FilmArray-Respiratory Panel 2.1 plus test identified at least one pathogen. The rhinovirus (20.6%), SARS-CoV-2 (17.7%), influenza A (17.5%), respiratory syncytial virus (12.4%), and parainfluenza 3 (10.1%) were the most common. Other viruses were found less frequently, and Bordetella parapertussis was detected in one sample. In 85 (79.4%) samples, the BioFire-FilmArray-Pneumonia Panel test identified at least one bacterium or virus. The most prevalent bacteria were Staphylococcus aureus (42.4%), Haemophilus influenzae (41.2%), Streptococcus pneumoniae (36.5%), Moraxella catarrhalis (22.3%), and Legionella pneumophila (2.4%). Among viruses, rhinovirus (36.5%), adenovirus (23.5%), influenza A (11.8%), and the genus Coronavirus (4.7%), were detected. Conclusion. Multiplex-PCR tests improved the implementation of therapeutic and epidemiological measures, preventing the spread of the COVID-19 infection and Legionnaires' disease.Copyright © 2022, Serbian Medical Society. All rights reserved.

Programmable polymer materials empowered by DNA nanotechnology

Over the past century, synthetic polymers have had a transformative impact on human life, replacing nature-derived materials in many areas. Yet, despite their many advantages, the structure and function of synthetic polymers still appear rudimentary compared to biological matter: cells use dynamic self-assembly to construct complex materials and operate sophisticated macromolecular devices. The field of DNA nanotechnology has demonstrated that synthetic DNA molecules can be programmed to undergo predictable self-assembly, offering unparalleled control over the formation and dynamic properties of artificial nanostructures. Intriguingly, the principles of DNA nanotechnology can be applied to the engineering of soft programmable materials, bringing the abilities of synthetic polymers closer to their biological counterparts. In this perspective, we discuss the unique features of DNA-functionalized polymer materials. We describe design principles that allow researchers to build complex supramolecular architectures with predictable and dynamically adjustable material properties. Finally, we highlight two key application areas where this biologically inspired material class offers particularly promising opportunities: (1) as dynamic matrices for 3D cell and organoid culture and (2) as smart materials for nucleic acid sequencing and pathogen detection.

Molecular Diagnostics on Smartphone

During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.

DEVELOPMENT OF A TEST SYSTEM AND A METHOD FOR DETECTING RIBONUCLEIC ACID OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 USING REAL-TIME POLYMERASE CHAIN REACTION

Given the rapid spread of coronavirus disease 2019 (COVID-19) globally, test systems are needed for its diagnosis, timely treatment, and introduction of quarantine measures. In the shortest possible time, a diagnostic system based on real-time reverse-transcription polymerase chain reaction to detect the ribonucleic acid of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal and oropharyngeal smears was developed and registered. The method determines the nucleocapsid and small-membrane protein genes and the human PGK1 gene, acting as internal control reactions. The nucleotide sequences of SARS-CoV-2 were analyzed, and primers were selected. The conditions for carrying out real-time reverse-transcription polymerase chain reaction and the composition of a set of reagents were set. The diagnostic sensitivity and specificity of the kit were tested on biological samples, with the addition of inactivated SARSCoV-2. The high analytical characteristics of the developed set of reagents were demonstrated, with a sensitivity of at least 103 GE/mL and a specificity of 100%, and no false-positive or false-negative results were recorded. The high specificity of the test system was shown on a representative sample of genetic materials of respiratory viral pathogens. Clinical and laboratory tests of the diagnostic "SARS-CoV-2 test” were conducted in the N.F. Gamalei National Research Center for Epidemiology and Microbiology. A set of reagents for the detection of ribonucleic acid of SARS-CoV-2 through on real-time reverse-transcription polymerase chain reaction for in vitro diagnostics "SARS-CoV-2 test” was registered in the Russian Federation as a medical device (Registration certificate no. RZN 2020/10632, dated 06/03/2020). The article can be used under the CC BY-NC-ND 4.0 license © Authors, 2022.

X-ray study as an alternative method for visualizing changes in lungs, with infection caused by COVID-19.

Introduction. Despite the existence of generally accepted diagnostic protocols, when a new coronavirus infection is suspected, in some cases, it is increasingly difficult to detect changes in the lung tissue in a timely manner due to the heavy workload of the main method of radiation diagnostics - computed tomography. Purpose of the study. To determine the effectiveness of the appointment of an X-ray examination as first-line metgod, as well as to carry out a comparative analysis of the use of radiation diagnostics methods - computed tomography and radiography in relation to the diagnostic sensitivity to changes in lung tissue when a person is infected with the SARS-COV-2 virus. Materials and methods. 150 patients (63.0 +/- 8.4 years) with confirmed coronavirus infection were examined. Each of the participants underwent X-ray examination and computed tomography of the chest organs. The percentage of subjects studied for each of the degrees of severity of lung damage was determined to identify the proportion of involvement of lung tissue in the pathological process in the bulk of the examined individuals. Results. Of the 150 patients, changes in the lung tissue during chest X-ray were detected in 97 (65%), respectively, in 53 (35%), pathological changes in the lungs were not visualized. When examining patients by computed tomography, changes in the lungs were detected in 143 patients (95%), X-ray morphological changes were not detected in 7 subjects (5%). When detecting the volume of lung damage, it turned out that the majority of the subjects - 86 people (57%) - had the degree of damage CT-2. The degree of CT-1 and CT-3 was determined in 26 (17%) and 25 (17%) patients, respectively. CT-4 was observed in 6 patients (4%), and in 5% of cases, CT was not able to detect pathological changes in the lung tissue, the degree of CT-0 was established. Conclusion. In the assessment of viral lung damage, radiography takes a significant place, but in 35% of cases, radiographic examination failed to identify the existing pathological changes. CT of the chest organs confirms its value as the "gold standard" in the study of pulmonary pathology in coronavirus infection, but if it is impossible to perform it, radiography is recommended.Copyright © 2021 Vigne et Vin Publications Internationales. All rights reserved.

Nanobiosensors Potentialities for Monitoring SARS-CoV-2 in the Environment

The worldwide pandemic of coronavirus disease-2019 (COVID-19) is a devastating and distressing scenario that highlights humanity's inability to build fast diagnostic tools for emerging infectious diseases. However, the majority of existing approaches have a significant probability of false negatives, leading in patient diag-nostic errors and prolonging therapy. Nanoparticles have shown significant improve-ment and have the potential to be used as a platform for quickly and accurately identifying viral infection. The relevance of nanoparticles is potential platforms for COVID-19 diagnostics was emphasized in this research. In addition, nanomaterials have surface chemistry, which may be beneficial for the bioconjugation of molecules, large surface potential, and a significant amplification impact on signals. Due to various potential benefits, metallic nanomaterials like gold, silver nanoparticles, and carbon-based nanomaterials (carbon nanotube and graphene), nanogels, and photonic crystals are utilized for biosensing applications. In compared to traditional techniques for identifying severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), this study covers the most relevant aspects of nanobiosensor-based diagnostics tech-niques. Additionally, major potential challenges and prospects associated with the advancement of these distinct sensors for SARS-CoV-2 detection are discussed in detail. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022.

AI and the Infectious Medicine of COVID-19

Coinciding with the global pandemic of SARS-CoV-2 and the resulting global public health crisis caused by COVID-19, artificial intelligence methods started playing an ever more important role in Infectious Medicine. On one hand this was a result of a continuous digital transformation of Infectious Medicine-a trend started decades ago. On the other hand, the pandemic catalyzed the adoption of artificial intelligence and other digital and quantitative techniques by Infectious Medicine. In this chapter we review recent works touching upon aspects of COVID-19 patient journey and how it interconnects with big data and artificial intelligence. These include early and clinical research, epidemiology and detection, diagnostics, clinical care and decision support, as well as long-term care and prevention. We cross-compare the published works and assess their maturity. Finally, we provide a conclusion on the state of artificial intelligence in the Infectious Medicine of COVID-19 and attempt a future perspective. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022.

Emerging Invasive Group A Streptococcus M1UK Lineage Detected by Allele-Specific PCR, England, 20201.

Increasing reports of invasive Streptococcus pyogenes infections mandate surveillance for toxigenic lineage M1UK. An allele-specific PCR was developed to distinguish M1UK from other emm1 strains. The M1UK lineage represented 91% of invasive emm1 isolates in England in 2020. Allele-specific PCR will permit surveillance for M1UK without need for genome sequencing.

Raman and fourier transform infrared spectroscopy techniques for detection of coronavirus (COVID-19): a mini review.

Coronavirus pandemic has been a huge jeopardy to human health in various systems since it outbroke, early detection and prevention of further escalation has become a priority. The current popular approach is to collect samples using the nasopharyngeal swab method and then test for RNA using the real-time polymerase chain reaction, which suffers from false-positive results and a longer diagnostic time scale. Alternatively, various optical techniques, namely, optical sensing, spectroscopy, and imaging shows a great promise in virus detection. In this mini review, we briefly summarize the development progress of vibrational spectroscopy techniques and its applications in the detection of SARS-CoV family. Vibrational spectroscopy techniques such as Raman spectroscopy and infrared spectroscopy received increasing appreciation in bio-analysis for their speediness, accuracy and cost-effectiveness in detection of SARS-CoV. Further, an account of emerging photonics technologies of SARS-CoV-2 detection and future possibilities is also explained. The progress in the field of vibrational spectroscopy techniques for virus detection unambiguously show a great promise in the development of rapid photonics-based devices for COVID-19 detection.

Development of a universal real-time RT-PCR assay for detection of pan-SARS-coronaviruses with an RNA-based internal control.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for rapid diagnostic assays to prompt intensified virological monitoring both in human and wild animal populations. To date, there are no clinical validated assays for pan-SARS-coronavirus (pan-SARS-CoV) detection. Here, we suggest an innovative primer design strategy for the diagnosis of pan-SARS-CoVs targeting the envelope (E) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer-probe set targeting human ß2-microglobulin (B2M) as an RNA-based internal control for process efficacy. The universal RT-qPCR assay demonstrated no false-positive amplifications with other human coronaviruses or 20 common respiratory viruses, and its limit of detection (LOD) was 159.16 copies/ml at 95% detection probability. In clinical validation, the assay delivered 100% sensitive results in the detection of SARS-CoV-2-positive oropharyngeal samples (n = 120), including three variants of concern (Wuhan, Delta, and Omicron). Taken together, this universal RT-qPCR assay provides a highly sensitive, robust, and rapid detection of SARS-CoV-1, SARS-CoV-2, and animal-derived SARS-related CoVs.

Neurooncology: 2021 update.

This article briefly presents 10 topics that were selected by the author as 'top 10 discoveries' published in 2020 in the broader field of neurooncological pathology including neurosciences as well as clinical neurooncology of interest for neurooncological pathology. The selected topics concern new information on the molecular characteristics of gliomas (infratentorial IDH-mutant diffuse astrocytomas, pediatric low-grade gliomas, infant-type high-grade gliomas, hypermutation in gliomas), the immunological aspects of the brain tumor microenvironment (TME), the impact of the TME on preclinical glioma models, and the importance of lymphatic drainage on brain tumor surveillance. Furthermore, important papers were published on two 'new' genetic syndromes predisposing to medulloblastoma, on liquid biopsy-based diagnosis of central nervous system (CNS) tumors, and on the 'microbiome' in glioblastomas (and other cancers). In the last part of this review, a dozen of papers are given as examples of papers that did not make it to the top 10 list of the author, underscoring the subjective component in the selection process. Acknowledging that 2020 will be remembered as the year in which the world changed because of the COVID-19 pandemic, some of the consequences of this pandemic for neurooncological pathology are briefly discussed as well. Hopefully, this review forms an incentive to appreciate the wealth of information provided by the papers that were used as building blocks for the present manuscript.

Daily SARS-CoV-2 Nasal Antigen Tests Miss Infected and Presumably Infectious People Due to Viral Load Differences among Specimen Types.

In a recent household transmission study of SARS-CoV-2, we found extreme differences in SARS-CoV-2 viral loads among paired saliva, anterior nares swab (ANS), and oropharyngeal swab specimens collected from the same time point. We hypothesized these differences may hinder low-analytical-sensitivity assays (including antigen rapid diagnostic tests [Ag-RDTs]) by using a single specimen type (e.g., ANS) from reliably detecting infected and infectious individuals. We evaluated daily at-home ANS Ag-RDTs (Quidel QuickVue) in a cross-sectional analysis of 228 individuals and a longitudinal analysis (throughout infection) of 17 individuals enrolled early in the course of infection. Ag-RDT results were compared to reverse transcription-quantitative PCR (RT-qPCR) results and high, presumably infectious viral loads (in each, or any, specimen type). The ANS Ag-RDT correctly detected only 44% of time points from infected individuals on cross-sectional analysis, and this population had an inferred limit of detection of 7.6 × 106 copies/mL. From the longitudinal cohort, daily Ag-RDT clinical sensitivity was very low (<3%) during the early, preinfectious period of the infection. Further, the Ag-RDT detected ≤63% of presumably infectious time points. The poor observed clinical sensitivity of the Ag-RDT was similar to what was predicted based on quantitative ANS viral loads and the inferred limit of detection of the ANS Ag-RDT being evaluated, indicating high-quality self-sampling. Nasal Ag-RDTs, even when used daily, can miss individuals infected with the Omicron variant and even those presumably infectious. Evaluations of Ag-RDTs for detection of infected or infectious individuals should be compared with a composite (multispecimen) infection status to correctly assess performance. IMPORTANCE We reveal three findings from a longitudinal study of daily nasal antigen rapid diagnostic test (Ag-RDT) evaluated against SARS-CoV-2 viral load quantification in three specimen types (saliva, nasal swab, and throat swab) in participants enrolled at the incidence of infection. First, the evaluated Ag-RDT showed low (44%) clinical sensitivity for detecting infected persons at all infection stages. Second, the Ag-RDT poorly detected (≤63%) time points that participants had high and presumably infectious viral loads in at least one specimen type. This poor clinical sensitivity to detect infectious individuals is inconsistent with the commonly held view that daily Ag-RDTs have near-perfect detection of infectious individuals. Third, use of a combination nasal-throat specimen type was inferred by viral loads to significantly improve Ag-RDT performance to detect infectious individuals.

Design and implementation of a digital site-less clinical study of serial rapid antigen testing to identify asymptomatic SARS-CoV-2 infection.

Background: Rapid antigen detection tests (Ag-RDT) for SARS-CoV-2 with emergency use authorization generally include a condition of authorization to evaluate the test's performance in asymptomatic individuals when used serially. We aim to describe a novel study design that was used to generate regulatory-quality data to evaluate the serial use of Ag-RDT in detecting SARS-CoV-2 virus among asymptomatic individuals. Methods: This prospective cohort study used a siteless, digital approach to assess longitudinal performance of Ag-RDT. Individuals over 2 years old from across the USA with no reported COVID-19 symptoms in the 14 days prior to study enrollment were eligible to enroll in this study. Participants throughout the mainland USA were enrolled through a digital platform between October 18, 2021 and February 15, 2022. Participants were asked to test using Ag-RDT and molecular comparators every 48 hours for 15 days. Enrollment demographics, geographic distribution, and SARS-CoV-2 infection rates are reported. Key Results: A total of 7361 participants enrolled in the study, and 492 participants tested positive for SARS-CoV-2, including 154 who were asymptomatic and tested negative to start the study. This exceeded the initial enrollment goals of 60 positive participants. We enrolled participants from 44 US states, and geographic distribution of participants shifted in accordance with the changing COVID-19 prevalence nationwide. Conclusions: The digital site-less approach employed in the "Test Us At Home" study enabled rapid, efficient, and rigorous evaluation of rapid diagnostics for COVID-19 and can be adapted across research disciplines to optimize study enrollment and accessibility.

Solid-Phase Optical Sensing Techniques for Sensitive Virus Detection.

Viral infections can pose a major threat to public health by causing serious illness, leading to pandemics, and burdening healthcare systems. The global spread of such infections causes disruptions to every aspect of life including business, education, and social life. Fast and accurate diagnosis of viral infections has significant implications for saving lives, preventing the spread of the diseases, and minimizing social and economic damages. Polymerase chain reaction (PCR)-based techniques are commonly used to detect viruses in the clinic. However, PCR has several drawbacks, as highlighted during the recent COVID-19 pandemic, such as long processing times and the requirement for sophisticated laboratory instruments. Therefore, there is an urgent need for fast and accurate techniques for virus detection. For this purpose, a variety of biosensor systems are being developed to provide rapid, sensitive, and high-throughput viral diagnostic platforms, enabling quick diagnosis and efficient control of the virus's spread. Optical devices, in particular, are of great interest due to their advantages such as high sensitivity and direct readout. The current review discusses solid-phase optical sensing techniques for virus detection, including fluorescence-based sensors, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), optical resonators, and interferometry-based platforms. Then, we focus on an interferometric biosensor developed by our group, the single-particle interferometric reflectance imaging sensor (SP-IRIS), which has the capability to visualize single nanoparticles, to demonstrate its application for digital virus detection.

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes.

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

Digital PCR Discriminates between SARS-CoV-2 Omicron Variants and Immune Escape Mutations.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, mutations arise that will allow the virus to evade immune defenses and therapeutics. Assays that can identify these mutations can be used to guide personalized patient treatment plans. Digital PCR (dPCR) is a fast and reliable complement to whole-genome sequencing that can be used to discriminate single nucleotide polymorphisms (SNPs) in template molecules. Here, we developed a panel of SARS-CoV-2 dPCR assays and demonstrate its applications for typing variant lineages and therapeutic monoclonal antibody resistance. We first designed multiplexed dPCR assays for SNPs located at residue 3395 in the orf1ab gene that differentiate the Delta, Omicron BA.1, and Omicron BA.2 lineages. We demonstrate their effectiveness on 596 clinical saliva specimens that were sequence verified using Illumina whole-genome sequencing. Next, we developed dPCR assays for spike mutations R346T, K444T, N460K, F486V, and F486S, which are associated with host immune evasion and reduced therapeutic monoclonal antibody efficacy. We demonstrate that these assays can be run individually or multiplexed to detect the presence of up to 4 SNPs in a single assay. We perform these dPCR assays on 81 clinical saliva SARS-CoV-2-positive specimens and properly identify mutations in Omicron subvariants BA.2.75.2, BM.1.1, BN.1, BF.7, BQ.1, BQ.1.1, and XBB. Thus, dPCR could serve as a useful tool to determine if clinical specimens contain therapeutically relevant mutations and inform patient treatment. IMPORTANCE Spike mutations in the SARS-CoV-2 genome confer resistance to therapeutic monoclonal antibodies. Authorization for treatment options is typically guided by general trends of variant prevalence. For example, bebtelovimab is no longer authorized for emergency use in the United States due to the increased prevalence of antibody-resistant BQ.1, BQ.1.1, and XBB Omicron subvariants. However, this blanket approach limits access to life-saving treatment options to patients who are otherwise infected with susceptible variants. Digital PCR assays targeting specific mutations can complement whole-genome sequencing approaches to genotype the virus. In this study, we demonstrate the proof of concept that dPCR can be used to type lineage defining and monoclonal antibody resistance-associated mutations in saliva specimens. These findings show that digital PCR could be used as a personalized diagnostic tool to guide individual patient treatment.

Molecular detection of monkeypox and related viruses: challenges and opportunities.

The recent widespread emergence of monkeypox (mpox), a rare and endemic zoonotic disease by monkeypox virus (MPXV), has made global headlines. While transmissibility (R0 ≈ 0.58) and fatality rate (0-3%) are low, as it causes prolonged morbidity, the World Health Organization has declared monkeypox as a public health emergency of international concern. Thus, effective containment and disease management require quick and efficient detection of MPXV. In this bioinformatic overview, we summarize the numerous molecular tests available for MPXV, and discuss the diversity of genes and primers used in the polymerase chain reaction-based detection. Over 90 primer/probe sets are used for the detection of poxviruses. While hemagglutinin and A-type inclusion protein are the most common target genes, tumor necrosis factor receptor and complement binding protein genes are frequently used for distinguishing Clade I and Clade II of MPXV. Problems and possibilities in the detection of MPXV have been discussed.